XXVIII International Conference on Animal Genetics in Göttingen - Germany

ISAG International Society for Animal Genetics

Proceedings 2002

ISBN 3-00-010597-2

28th ISAG Conference 2002

Contents

Invitation of Local Organizing Committee ...... 2

Greeting from the Minister for Science and Culture ...... 3

Members of Local Organizing Committee...... 4

Presentation of Guest Speakers ...... 5

Abstracts:

Section A: Genome technologies...... 26

Section B: MHC and Immunogenetic ...... 41

Section C: Functional Genomics ...... 47

Section D: Marker, Polymorphism and Biodiversity...... 78

Section E: Associations between markers and traits...... 147

Section F: Bioinformatics...... 178

Index of Authors...... 184

These proceedings are published under:

ISBN 3-00-010597-2

1

28th ISAG Conference 2002

Georg-August-University City of Göttingen

Dear colleagues, on behalf of the Local Organizing Committee, the International Society for Animal Genetics and the Georg-August-University of Göttingen, we would like to welcome all delegates and accompanying persons to the 28th International Conference on Animal Genetics in Göttingen. It is a great pleasure and honor for us that a conference of the ISAG will take place again in Göttingen after 17 years. The conference will be a unique blending of individual sessions and themes designed to provide you with an exceptional experience. The conference gathers a cadre of international experts in molecular genetics, molecular biology and related fields. These world-class presenters bring together a wealth of experience and knowledge for your benefit. Add to this the ability to share ideas and interact with a global network as well as to view the latest results in Animal Genetics from around the world and you have a conference that cannot be missed. The ideal forum to increase your personal knowledge and professional capabilities, the ISAG2002 conference will sure exceed your expectations. We wish you all pleasant, inspiring and motivating days in one of the oldest and most attractive University cities in Germany. Because of its central geographical position in Germany, Göttingen is also an extremely good starting point for pre- and post conference tours. We hope to see you in Göttingen at the International Conference and look forward to welcoming you to the "City of Science".

Bertram Brenig Jan-Nikolaus Meyer

2

28th ISAG Conference 2002

Greeting from the Minister for Science and Culture of Lower Saxony,

Thomas Oppermann

Only 17 years ago the term Animal Genetics was in public at most connected with the attempt to increase the milk production through breeding. Today it can be found in almost any news media, related to a variety of issues. “Dolly”, the name of the first ever sheep that had been cloned is just as well the label for a milestone in the history of modern science. For weeks on end “Dolly” made headlines and triggered controversial discussions about genetic engineering and biological sciences. But another problem just as well represents the new requirements Animal Genetics is facing today: The public world-wide was alarmed when BSE spread among the all over Europe. Suddenly, everybody was looking at this field of veterinary medicine, at Animal Genetics, as a source of hope and wishes as well as fear and worries. Considering this growth in importance, I am especially happy the 28th International Conference for Animal Genetics is taking place in Göttingen again after 17 years. Surely triggered by Animal Genetics, genetics has undergone quite an astonishing development during this time and is becoming one of the most important science branches in the 21st century. The genetic mapping of animals has progressed so far that in the years to come there could be enormous breakthroughs concerning the resistance to illnesses in animals and breeds. As well, the existing methods of animal breeding are going to change drastically due to the progress in Animal Genetics. The 28th International Conference for Animal Genetics is approaching the complexity of these issues from several sides. I am especially pleased that in addition to the participation of several veterinary and biological branches of science, bioethical aspects will be discussed as well. The general public will be expecting the new biological sciences to put up a critical reflection on this issue in particular. I’d like to thank the International Society for Animal Genetics (ISAG) for being host to this conference and the Institute of Veterinary Medicine and the Institute of Animal Breeding and Genetics of the Georg-August-Universität Göttingen and the Chairmen of the local organizing committee, Professor B. Brenig and Dr. Jan Meyer for preparing it and I wish all participants of this conference good progress and stimulating discussions.

3

28th ISAG Conference 2002

Local Organizing Committee 2002

Sabine Bramsmann Andreas Müller-Belecke Bertram Brenig Ina Pfeiffer Claus-Peter Czerny Henner Simianer Gabriele Hörstgen-Schwark Helge Täubert Christoph Knorr Clemens Wollny Ute Margan Secretary: Kaarina Hillmer Jan-N. Meyer Katharina Kurth Burchhard Möllers Treasurer: Monika Werst

Institute of Animal Breeding and Genetics, Albrecht-Thaer-Weg 3 D-37075 Göttingen, Germany Institute of Veterinary Medicine, Groner Landstraße 2, D-37073 Göttingen, Germany

Göttingens most famous monument „Gänseliesel“

4 28th ISAG Conference 2002

Gue t ea ers Topic

Prof. Dr. Adriano Aguzzi (Switzerland) The immunobiology of prion [email protected] diseases

Prof. Dr. Choy Leong Hew (Singapore) Biotechnology and Transgenesis [email protected] in fish

Dr. Bjorn Ingemarsson, PhD (Sweden) Comprehensive DNA analysis T [email protected] using Pyrosequencing M techno

Prof Dr. Dr. Bernhard Irrgang (Getmany) Ethical Issues of Genetic [email protected] ManipuJation of Lifestock

Prof Dr. Joan Lunney (USA) Can we use genomics to select for [email protected] healthier swine?

Prof. Dr. Paula Schneider (Brazil) Biodiversity and Conservation in [email protected] Amazon

Prof. Dr. Hans-J. Thiesen (Gennany) Proteornics hans-;[email protected]

Dr. Alain Vignal (France) SNP teclmology [email protected]

8 28th ISAG Conference 2002

Adriano Aguzzi studied medicine at the University of Freiburg (Gennany) fonn 1980-1986. He received his Ph.D. in 1986. From 1986 to 1989 he was resident in Neuropathology at the University Hospital of ZUrich (Switzerland). Since 1997 he is full professor of Neuropathology and Director of the Institute of Neuropathology at the University of ZUrich. He is president of the Swiss Society of Neuropathology and member of severa] international Societies. His research interests are in the area of prion diseases with special focus on their pathogenesis.

Choy Leong Hew is Head of the Department of Biological Sciences at the University of Singapore. He received his Ph.D. in 1970 at the University of British Columbia. From 1972 to 1974 he was a C.H. Best fellow at the Banting & Best Dept. of Medical Research of the University of Toronto (Canada). He was then working at the Memorial University of ~ Newfoundland as assistant professor until 1983. From 1983 to 1999 he was a visting professor at a variety of Universities in Canada and China. His main research areas are biology and biotechnology of antifreeze , transgenic fish, molecular endocrinology and proteomics.

Bjorn Ingemarsson is Director of Pyrosequencing AB's technical and scientific support function based m Uppsala. Prior to Jommg Pyrosequencing 3 years ago, he worked as a specialist in automated DNA analysis at Amersharn Pharmacia Biotech. He aquired his Ph.D. in plant physiology at the Stockholm University, where he thereafter continued research on the physiology, biochemistry and molecular biology of nitrogen assimilation in plants for several years.

Bernhard Irrgang studied Philosophy, Theology and German Philology at the University of Wiirzburg (Germany), Passau (Germany) and Munich (Germany). He received his Ph.D. in Philosophy in 1982 and Theology in 1991. From 1982 to 1992 he worked at different Universities in Germany. Currently, he is Director of the Institute for Philosophy and the Centre for Interdisciplinary Technical Research at the Technical University of Dresden (Gennany). Bernhard Irrgang main interests are in the area of medical and technical ethics, consequences of technology and philosophy of technology.

9 Joan K. Lunney studied Chemistry at the JOM Hopkins University (Baltimore, USA) and received her Ph.D. on studies on the regulation of serum glycoprotein homeostasis in 1976. From 1976 to 1979 she was guest postdoctoral research worker in the Immunology Branch (NCI, NIH, Bethesda, MD). From 1983 until 1995 she was working in different position in the Helminthic Diseases Laboratory at the USDA, ARS (Beltsvi11e, MD). Since 1995 she is GM15 Research Leader and Supervisory Research Immunogeneticist at the Immunology and Disease Resistance Laboratory, USDA, ARS (Beltsville, MD).

Maria-Paula Cruz Schneider is Director of the Laboratory of DNA

Polymorphisms at the University of Belem (P~ Brasil). She received her Ph.D. 1988 at the Federal University of Rio Grande do SuI (Brasil) working on polymorphisms in primates of the Amazon region. Her main interests are in the area of genetics and molecular genetics in domestic species.

Hans -J urgen Thiesen studied medicine at the University of Hamburg (Gennany) from 1976 to 1982. He received his Ph.D. in 1983 for studying the physiology of parathyroid hOffi1one. From 1985 to 1987 he was working at the EMBL followed by a fellowship the Basel Institute of Immunology. In 1996 he received a Chair in Immunology at the University of Rostock (Gennany). His main interests are focussed on technologies of functional genomics in a clinically-oriented environment. Hans-liirgen Thiesen is member of the Proteome-Center at which complex human diseases are analysed by microarray analysis.

Alain Vignal received his B.Sc. in Agronomic Sciences in 1981. From 1982-1987 he studied biochemistry, cellular genetics and molecular biology at the University of Paris. In his Ph.D. thesis at the INSERM (Laboratory of Jean-Pierre Carton) Alain Vignal analysed the human glycophorin A and B family. Between 1991 and 1993 he had a postdoctoral feJ]owship at the Genethon (Laboratory of Jean Weissenbach). During this period he was involved in the hwnan genome project, genotyping the CEPH reference families. Since 1994 he is at the INRA in Toulouse (France). He is responsible for the development of structural genornics in chicken and expert in the field of QTL mapping.

10 Plenary Session: Invited Speakers

POOl:

Comprehensive DNA analysis llsing Pyrosequencing™ technology

Bjorn Ingemarsson Pyrosequencing AB, Uppsa/a, Sweden

Pyrosequencing™ is a fast and accurate technology for analysis of short to medium length DNA sequences. It is a non-electrophoretic sequencing method based on luminometric real-time detection of pyrophosphate released upon nucleotide incorporation by DNA polymerase. Samples are sequenced and analyzed in a standard microtiter plate fonnat, without any need for labeled primers or nucleotides. The technique is well established for rapid genotyping of single nucleotide polymoqilisms (SNPs). High accuracy is achived by polymerase-catalyzed incorporation of nucleotides at the polymorphic positions as well as adjacent nucIeotides. The non-variant positions serve as internal controls and allow for automatic quality assessment of each analyzed sample. Single well rrultiplex genotyping on pooled simplex or multiplex peR products can be accurately perfonned for SNPs and insertion!deletion polymorphisms." The close correlation between nucleotide incorporation/pyrophosphate release and light detection, makes Pyrosequencing technology suitable for quantitative applications such as, allele frequency detennination, assessment of gene copy number, analysis of splice mutations and CpG methylation, genotyping in mixed cell populations or polyploidic genomes, as well as for determination of viral load The technology can also be used for sequence identification/verification and de novo sequencing of 20-50 nucIeotides in less than one hour. Hence, it can be used for a number of applications including, analysis of mutation hot spot areas, cDNA fe-sequencing, virus and bacteria identificationityping, as well as resistance typing. Pyrosequencing AB has developed complete systems for low to high troughput demands, which include instrument, optimized reagent kits and dedicated software.

11 Plenary Session: Invited Speakers

P002:

SNP technology

Alain Vignal Laboratoire de genetiqlle cellulaire, lnra, chemin de Borde-Rouge, Auzeville BP 27,31326 Castanet­ Tolosan cedex, France.

Although the highly informative, multi-allelic microsatellite markers have dominated for now over ten years the field of molecular genetic studies in human and in animals, either used as model organisms or studied for their interest in agriculture, an increasing importance is now given to SNPs (Single Nucleotide Polymorphisrns), that are merely di-allelic base substitutions of lower heterozygosity. This is due to several reasons, amongst which the main are that SNPs can be found at very high densities throughout the genome (1 SNP every 1300 bp, when two random human are compared), and that they can be found in exons of . The very high densities of SNPs in the genome make them ideal for association and short range haplotype studies, although this usually means that far more markers wil1 have to be used than for microsatellites. The coding SNPs, sometimes referred to as cSNPs, are often studied as candidate polymorphisms when they imply an amino acid change, although it is not always easy to distinguish between the real implication of the poJymorphism in the biological phenomenon studied and a simple allelic association. However, contrariwise to microsatellites, for which the genotyping technology has only evolved from manual scoring of alleles, seen as PCR products of varying sizes on acrylamide gels, to a :emi­ automated method using sequencing machines, the situation is not so clear concerning the genotyping of SNPs, for which a broad range of methodologies are available. Amongst these are: direct hybridisation of allele -specific oligonucleotides (ASO) on membranes or glass arrays containing PCR products of individuals to genotype; hybridisation of PCR products on chips containing oligonucleotides; single base primer extensions (SBE) whose products can be separated by various means such as gel electrophoresis, tagged arrays, tagged beads or matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF); oligonucleotide ligation assay (OLA); monitoring the exonuclease degradation of an internal allele -specific oligonucleotide during PCR; real time sequencing ... For small scale studies, the major concerns when choosing an SNP genotyping technology, will be the possibilities of using existing platforms available at hand, such as an automatic sequencer or a DNA spotter. For larger scale studies, according to the number of SNPs and of individuals to be genotyped, the choices will vary. Indeed, some technologies, such as those involving glass arrays or chips, will enable to study high numbers of SNPs in parallel, but only on a low number of individuals, and others, such as MALDI-TOF separation of SBE products, will be more efficient with a high number of samples, but will need more effort put into the development of each individual SNP. Efforts are still underway, to develop a very high throughput genotyping system, capable of generating the very high numbers of genotypes needed for the genome-wide association studies, such as envisaged in human genetics.

12 POD3:

Biotechnology and Transgenesis in fish

Choy L.Hew, Xiaobo Zhang, Canhua Huang, Qingsong Lin Department ofBiological Sciences and Tropical Marine Science Institute National University of Singapore. Singapore and

Garth Fletcher Ocean Science Center, Memorial University of Newfoundland, St John's, Canada

Food security will be a major chal1enge facing mankind as we enter the new mil1ennium. The challenge for the agricultural sector is to double food production by year 2025 and triples it by year 2050. It is anticipated that aquaculture and mariculture will become a major force to increase food production. However, new technologies will needed to be employed to improve the efficiency and many bottleneck issues facing the industries. Numerous platfonn biotechnologies that include molecular biology, genomics, cloning technologies, DNA chips and proteomics can improve the industry's output substantially. In this presentation, we will describe two specific approaches from our and other laboratories. A. Transgenesi s. Transgenic technology is the transfer of a foreign gene into a host organism enabling the host to acquire a new and inheritable trait. The technology is specific for the properties of the candidate gene and appears to be ~tter than the traditional selective breeding methods. We have attempted to generate transgenic fish that are freeze-tolerant, faster growing and disease resistant using the antifreeze protein, growth hormone or the pleurocidin genes from different fish species respectively. We have demonstrated that these transgenes can be stably integrated, inherited and expressed. There is also a strong correlation between genotypes and phenotypes. The faster growing salmon (OR transgenic) is now being commercialized. Some of the issues dealing with food safety and ecology will be commented. b. Proteomics. Proteomics provides a global and dynamic profiling of protein activities within a cell or organisms and is now a popular method in functional genomics to examine cellular activities as well as for biomarker identifications. We have used this approach successfully to characterize the viral proteins from shrimp white spot syndrome virus. More than 15 novel proteins have been isolated and these proteins are potential candidates for vaccine development to prevent this pandemic disease in the shrimp industry. (Supported by NSERC, Canada and A*STAR, Singapore)

13 Plenary Session: Invited Speakers

P004:

Biodiversity and Conservation in Amazon - Prospects of Habitat Fragmentation Studies

Maria Paula Cruz Schneider Departamento de Genetica, Universidade Federal do Para, Caixa Postal 8609, CEP 66750-900, Be/em-PA, Brazil. E-mail: paula@u(lJa.br.

Brazil is the country with the largest undisturbed Tropical rain forest in the world, with a1most half of the 6.5 million km2 of the Amazon river basin being found in its territory. Tropical rain forests are also found in Central America, Africa and Southeast Asia, and they are supposed to play a central role in environment and climate stability worldwide. Besides the forest, the Amazon region is fonned by a complex of ecosystems which includes cerrado and other types of vegetation totaling 5,029 millions 2 km , comprising around 10 to 20% of all animal and plant species of the planet. However, such biodiversity has been threatened by an accelerated rate of damaging and destruction of various habitats. Official reports alerted to the high level of deforestation and decline of biodiversity due to commercial logging, burning, construction of dams for hydroelectric plants, cattle ranching, and agriculture expansion. Thus the Brazilian Government, through its Ministry of Environment, provided incentives, by way of grants and scientific meetings, in order to elaborate a national policy for biodiversity based on the intentions of the 1992 UN Conference on Environment and Development (Earth Summit). The granted proposals would identifY environmental, social and economic conditions for conservation and sustainable use of animal and plant species, as well as partition of the benefits resulting from biodiversity usage. Priority areas for conservation and inventory of species were also planned to be defined. The research conducted by our group in Belem was always related to biodiversity and conservation issues. One of our main interests during two decades was the study of genetic polymorphism and molecular phylogeny of Neotropical primatesl- (3). The aim of assessment of the diversity of New World primates is to have at hand a list of Primates as a basis for conservation measures, and to stimulate further research into the systematics an taxonomy of the group. In fact, without the formal structure of names and an agreed system of usage, there can be no understanding of what exists to be conserved (4,5). More recently we focused our research on the effects of fragmentation of some Amazonian habitats on populations of silvery marmoset (Mico argentatus), howler monkey (Alouatta belzebul) and birds species (guara, Eudocimus ruber, and some species of migrating shorebirds). Studies of habitat fragmentation has clearly and easily evaluated effects on parameters such as species diversity, population density, and behavioral patterns, but there are as yet few data available on its influence on genetic variation in free-ranging primate populations (6,7). Inbreeding and loss ofgenetic variability are generally presumed to be the primary results of the fragmentation of continuous populations, although outbreeding depression may also be relevant in many cases (8,9). Preliminary data on the variabi1ity of DNA microsatellite markers in silvery marmoset popUlations at four sites in central Amazonia (10), representative of different degrees of habitat fragmentation, found a surprisingly high genetic variability among popUlations even in the smallest ones. However, the level of heterozygosity indicates that all populations are subject to inbreeding or genetic drift. Despite the preliminary nature of the data, the results show that the remnant populations of M. argentatus are quite genetically distinct. Direct evidence of inbreeding depression has been recorded in fragmented populations of golden lion tamarins (Leontopithecus rosalia), a species belonging to the same family as the silvery marmoset (Callitrichidae) (11), although no clear indications of this phenomenon were found in the M. argentatus population from the right bank of the Tapaj6s river. Whatever the exact effect on genetic parameters, habitat fragmentation, in particular the isolation of relatively small subsets of original popUlations, is likely to have highly deleterious implications for the long-term viability of remnant populations (12). A systematic amlysis of complementary ecological factors will be required before definitive guidelines can be drawn up for the management of rerrmant populations of silvery mannosets (10). Another research currently under way by our group aims to provide ecological and genetic data about the long-tenn effects fragmentation habitats created after building of a hydroelectric plant dam. For

14 this purpose we use a wide range primate species, the howler monkey (Alouatta belzebul), whose habitat was radically altered through the formation of thousands of islands along the Tocantins river banks after the flooding of 2,500 km 2 due to the construction of the Tucurui Hydroelectric plant (1984/1985), In order to obtain a more precise picture of how the genetic variability changed through time in this species, DNA microsatellite markers are being used in recent collected samples of the species, as well as in samples obtained 18 years ago, before the dam was built. Hence, the data gathered in that study may help to define viable population size and management in A, belzebuI, as well as in other mammal populations, after habitat fragmentation.

Acknowledgements The studies herein described were financed by the National Program for Biodiversity (PRONABIO) of the Brazilian Ministry of Environment, through BIRD and CNPq. Additional support was received from IBAMAISantarem and the Kapok Foundation. IBAMA authorized the collection of specimens through special licences. We also thank to MCTIPPG7 and to ELETRONORTE for financial support of the Tucurui Project.

REFERENCES

I.Schneider& Rosenberger, 1996. Molecules, morphology, and Platyrrhini systematics. In: Adaptative Radiations of Neotropical Primates, M. A. Norkonk, A.L. Rosenberger and P.A. Garber (Eds.), pp.3- 19. Plenum Press, N.Y 2.Schneider, H., Schneider., M. P. C., Sampaio, 1., Harada, M. L., Stanhope, M., Czesluniak, 1. and Goodman, M. 1993. Molecular phylogenetics of the New World monkeys (Platyrrhini, Primates) Molecular Phylogenetics and Evolution 2 (3): 225-242. 3.Schneider, H., Sampaio, 1., Harada, M. L., Barroso, C. M. L., Schneider., M. P. C., Cze1usniak,1. and Goodman, M. 1996. Molecular phylogeny of the New World monkeys (Platyrrhini, Primates) based on two unlinked nuclear genes: IRBP lntron 1 and epsilon-globin sequences. Am. 1. Phys. Anthropology. 100: 153-179 4. Collar (1997), N. 1. 1997. Taxonomy and conservation: Chicken and egg. Bull. B. O. C. 117 (2):122-136. 5.Rylands, A. Schneider, H., Lannguth, A. Mittermeier, R. A., Groves, C., Rodrigues-Luna E. 2000. An assessment of the diversity of New World primates. Neotropical Primates 8 (2). 6.Pope, T. R. 1996, Socioecology, population fragmentation, and patterns of genetic loss in endangered primates, in: 1. C. Avise and 1. L. Hamrick, eds., Conservation Genetics: Case Histories from Nature, Chapman and Hall, New York, pp. 119-159. 7.Pope, T. R. 1998, Genetic variation in remnant populations of the woolly spider monkey (Brachyteles arachnoides). Int. J. Primatol. 19: 95-109. 8.Templeton, A. R., 1986, Coadaptation and outbreeding depression, in: M. E. Soule, ed., Conservation Biology. The Science of Scarcity and Diversity, Sinauer Associates, Sunderland, pp. 105-116. 9.Dudash, M. R. and Fenster, C. B., 2000. Inbreeding and outbreeding depression in fragmented populations, in: A. G. Young and G. M. Clarke, eds., Genetics, Demography and Viability'\ of Fragmented Populations, Cambridge University Press, Cambridge, pp. 35-53. 10.Gon<;alves, E. C., Ferrari, S. F., Silva, A., Coutinho, P., Menezes, E., Schneider, M. P. C. 2002.Effects of habitat fragmenttion on the genetic variability of silvery marmosets, Mico argentatus. In L. K. Marsh, ed., Primates in fragments, Plenum Press, New York, in Press. I1.Dietz, J. M., Baker, A. J. and Bal1ou, J. A. 2000, Demographic evidence of inbreeding depression in wild golden lion tamarins, in: A. G. Young and G. M. Clarke, eds., Genetics, Demography and Viability of Fragmented Populations, Cambridge University Press, Cambridge, pp. 203-211. 12.Lacy, R. C. 1997, Importance of genetic variation to the viability of mammalian populations. J. Mammal. 78: 320-335.

15 Plenary Session: Invited Speakers

P005:

Can we use genomics to select for healthier swine?

Joan K. Lunney USDA, ARS, ANRI, Immunology and Disease Resistance Laboratory, Beltsville, MD USA

Genome mapping efforts in swine hive been targeted mainly to production traits, reproductive issues and meat quality. Early efforts did result in identifying genes encoding resistance to specific bacterial infections, e.g., K88R. Recent work has identified swine with improved, not complete, resistance to specific viral infections. However, identifYing genes encoding resistance for each of the large number of viral and bacterial infections that impact swine production is virtually impossible. Therefore our research has been aimed at defining immune properties that result in swine with improved production traits and survival capacity due to improved disease responses, targeting cells and genes associated with type 1 immunity. Edfors-Lilja, Andersson, et a1. mapped QTL associated with 'stress' (mixing and transport) induced alterations of porcine immune functions. Mallard, Wilkie, et a1. selected swine for high immune response. Delineation of traits most appropriate to targeting for "enhanced immune properties" associated with disease resistance as well as vaccine and immune responsiveness will be discussed. Functional genomic tools, real time peR and microarray analyses, as well as SNP studies, will help identify relevant genes, their regulators and best alleles. Major collaborative studies that integrate analyses for enhanced immune properties along with production traits are required to confinn the potential of using these genomic approaches to select for healthier swine.

Nomenclature: Functional genomics, disease resistance, immune responsiveness.

16 P006:

Proteomics

Hans-J. Thiesen Institut!iir Immunology, Schillingallee 70, D-J8055 Rostock

With the advent of complete genome sequences the analysis of gene functions is one of the scientific challenges in the post-genome-era. Great efforts have to be put forward on the analysis of complex gene and protein networks describing cel1ular as we]] as intercel1ular interactions. Corresponding research projects take advantage of novel enabling technologies i.e. chip technology and mass spectrometry.

Comparative Genomics Research projects studying processes in developmental biology and celluJar differentiation, demonstrated in the past as exemplified by the functional analysis of homeobox-genes how informative the comparative analysis of protein domains from drosophila to human sequences turned out to be (Comparative Genomics). The functional analysis of protein domains manifests that protein functions can be correlated with structural domains. Based on sequence information on evolutionarily conserved structural elements, proteins can be grouped, classified and assigned to functional groups enabling phenotype-genotype comparisons.

Functional genomics Currently, multiple genomes have already been completely sequenced. Nowadays, the elucidation of individual gene functions becomes one of the prominent enterprises in research. Hereto, new research disciplines evolved such as transcriptomics, proteomics and metabolomics that are combined by integrative bioinfonnatic platfonns (Figure I). Each of these disciplines is based on enabling technology platforms that requires specific instrumentation, expertise and the establishment of standard operating procedures (SOPs). How these technologies can be successfully integrated in one centre, has recently been realized by the Proteome Centre Rostock (www.proteome-alliance.de).

Definition Proteome: The expression "Proteome" firstly coined by Marc Wilkins in I 994 describes the attempt of describing the composition of all proteins in a qualitative and quantitative manner present within one entity (tissue, body fluid, organism) at specified physiological states and time points.

Comparison Genome and Proteome In contrast to a genome, the proteome is dynamic and presents specific physiological states under specified environmental conditions at specific time points. Furthermore, the proteome determines the functional state of living organisms. The genome just ensures that the proteome is functional. How the proteome of a cell operates, is based on environmental factors ie. such as light, energy, nutrition and physical activities. In multicellular organisms, the interplay and tum-over of cells within one orgarusa'l has to be taken into account as wen. Obviously, the proteome determines how the genetic infonnation is being used (Transcription factors regulate the retrieval of genetic information). Thus, one might even say the proteome determines under evolutionary aspects how the corresponding genome has been assembled. The genome probably ensures that the proteome remains operational in living organisms. In general, protein compositions have to be stabilized by replacing proteins according to their specific half-lives. Furthennore, perturbations of the proteome can then be introduced by changing the composition or the rate of protein renewal.

Proteome -analysis: With the development of the 2-dimensional gel electrophoresis by Patrick O'Farell and Joachim Klose in 1975, the basis for proteome analysis had been established. This technology was then extended by the engineering of sophisticated mass spectrometry tools such as MALDI-TOF- (Matrix-Assisted­ Laser-Desorption-Ionisation-Time-Of- Flight-) and electrospray mass spectrometry. In general, proteome analysis starts with the sampling of biological material under standardised conditions and ends with the computer analysis of mass spectrometric data that finally leads to the assignments of

17 Plenary Session: Invited Speakers identified proteins to pathways displaying functional activities. In particular, biological samples are collected under standardized conditions (SOPs) and prefractinated i.e. using gradient centrifugation, free-flow-electrophoresis, multidimensional protein identification technology (rnudPIT) followed by 2-dimensional gel electrophoresis. Either chromatographic1y separated protein fractions are directly subjected to liquid chromatographic mass spectrometry (Nano-electrospray-MS) or are further separated on 2D-gel-electrophoresis (2DE) using immobiJine strips or ampholyte gel electrophoresis. Proteins identified in 2DE to be differentially expressed are isolated by picking robots and digested by enzyme digestion (i.e. Trypsin). The obtained peptide fragments are then subjected to MALDI-TOF­ MS analysis for protein identification by mass finger printing.

Proteome analysis Initially, global proteomic approaches were performed followed by the analysis of subproteomes ( Organelles and protein complexes). Hereto, Cellzome (Heidelberg) developed technologies for detennining protein-protein interaction maps. MELTEC (Magdeburg) developed protocols to analyze the toponome of cells and tissues coined toponomics and Biovision in Harmover concentrates on studying the peptidome (peptidomics).

New Technologies Recently, fluorescence dyes have been introduced to visualize differentially expressed proteins (Pharmacia), or to meet the sensitivity of silver stains (SyproRuby). Microfluid-Chips are expected in the future to replace 2DE gels~ multidimensional protein identification technology (MudPIT) has been successfuI1y developed to approach proteins that do not separate we]] in conventional 2DE gels., lCAT methods are used for quantification purposes.

Bioinformatics Laboratory-Information-Management-Systems (LIMS) are essential to enable high-throughput­ analysis. Hereto, the Proteome-Center Rostock has established the "Proteobase-Data-Management­ System", in which clinical data, SOPs, RNA and protein expression data are going to be integrated.

The BMBF-Leitprojekt: In the BMBF-Leitprojekt "Proteom-Analyse des Menschen" (www.proteome-alliance.de). genome-, transcriptome- and proteome analyses are combined to elucidate common and disease specific mechanisms involved in the pathophysiology of autoimmune diseases. For instance, only a correlation coefficient of 0.2 was obtained once RNA and protein expression levels of synovial tissues were compared. That RNA- and protein expression levels are not well correlated is not surprising since numerous kinds of transcriptional and translational regulation are known to exist in the cell. In addition, mRNA molecules and proteins vary in their propensities to become degraded. This comparative analysis underlines the importance of determining RNA and proteome expression levels in order to validate the relevance of RNA and protein expression profiling experiments.

Services The Steinbeis-Transfer-Center for Proteome-Analysis offers services in protein and transcriptome analysis 41ttp:llwww.stw.de/stzJ424.htm) by making use of our Affymetrix Service 1 custom-made chip approach as well as the technology platform in proteomics run by Prof. Dr. M.O. Glocker.

Perspectives By bringing experts of experimental research together with mathematicians and engineers, a new discipline is about to be born coined system biology with the aim of developing new theoretical approaches and computational tools in order to analyze, to model, and to predict network behaviours of proteomes. Acknowledgement: This work is supported by the BMBF FKZ 01GG9831/3. We thank our experimental and clinical collaborators for their support. URLs: http://dip.doe-mbi.ucla.eduJ http://www.cgr.harvard.eduJresearchibiological.html http://yeast.cellzome.comlbrowsec.php

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Toponomies High Throughput

Peptidomics ICAT Proteine Com

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Animal Kingdom

19 Plenary Sesslon: Invited Speakers

P007:

The immunobiology of prion diseases

Adriano Aguzzi Institute ofNeuropathology, University Hospital Zurich. CH-809J Zurich, Switzerland

Mice deficient in the nonnal prion protein are resistant to exposure to prion infectivity, and expression of the nonnal prion protein by neurons is necessary for the development of Iistological damage I. But how do prions reach the brain after entering the body from peripheral sites? The first portal of entry in the gut may be represented by M-celIs2. Neuroinvasion, i.e. the process by which prions march through the body of the host towards the brain, is dependent upon expression of the nonnal prion protein in a non-hematopoietic extra cerebral site). We therefore developed the hypothesis that neuroinvasion takes place in two distinct steps: first the Iymphoreticular system is diffusely colonized by the agent, while at a later time infectivity progresses from lymphoreticular organs to the central 4 5 6 nervous system , probably via sympathetic nerves • . There is an absolute requirement for B­ 7 lymphocytes in peripheral prion pathogenesis • Surprisingly, the presence of the nonnal prion protein 8 is not necessary on B-Iymphocytes to enable them to support this process . The mechanism of action 9 of B lymphocytes may consist of presentation of lyrnphotoxin-B to follicular dendritic cel1s . This paves the way to post-exposure prophylaxis strategies lO that exploit the anti-prion effect of soluble lymphotoxin-B receptors. Why do follicular dendritic cells accumulate prions? We tested the hypothesis that prion uptake may be complement-mediated. Indeed, certain components of the complement system (Cl q, CR1/2) proved to play an inportant role in pathogenesis II. Final1y, we have found that transgenic expression of an anti-PrP antibody heavy chain suffices to confer to mice antiprion protection - a finding that may be relevant to the development of antiprion vaccines 12.

1. Brandner, S., Isenmann, S., Raeber, A' j Fischer, M., Sailer, A., Kobayashi, Y., Marino, S., Weissmann, C. & Aguzzi, A. Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379, 339-43 (1996). 2. Heppner, F. L., Christ, A. D., Klein, M. A., Prinz, M., Fried, M., Kraehenbuhl, 1. P. & Aguzzi, A. Transepithelial prion transport by M cells. Nat Med 7, 976-7 (2001). 3. Blattler, T., Brandner, S., Raeber, A. 1., Klein, M. A., VoigtUinder, T., Weissmann, C. & Aguzzi, A. PrP -expressing tissue required for transfer of scrapie infectivity from spleen to brain. Nature 389, 69-73 (1997). 4. Aguzzi, A. & Weissmann, C. Prion research: the next frontiers. Nature 389, 795-798 (1997). 5. Glatzel, M., Flechsig, E., Navarro, B., Klein, M. A., Paterna, J. C., Bueler, H. & Aguzzi, A. Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system. Proc Natl Acad Sci USA 97,442-7 (2000). 6. Glatzel, M., Heppner, F. L., Albers, K. M. & Aguzzi, A. Sympathetic innervation of lymphoreticular organs is rate limiting for prion neuroinvasion. Neuron 31, 25-34. (2001). 7. Klein, M. A., Frigg, R., Flechsig, E., Raeber, A. 1., Kalinke, V., Bluethmann, H., Bootz, F., Suter, M., Zinkemagel, R. M. & Aguzzi, A. A crucial role for B cells in neuroinvasive scrapie. Nature 390,687-90 (1997). 8. Klein, M. A., Frigg, R., Raeber, A. 1., Flechsig, E., Hegyi, 1., Zinkernagel, R. M., Weissmann, C. & Aguzzi, A. PrP expression in B lymphocytes 1s not required for prion neuroinvasion. Nat Med 4, 1429-33 (1998). 9. Montrasio, F., Frigg, R., Glatzel, M., Klein, M. A., Mackay, F., Aguzzi, A. & Weissmann, C. Impaired prion replication in spleens of mice lacking functional follicular dendritic cells. Science 288, 1257-9 (2000). 10. Aguzzi, A. & Collinge, 1. Post-exposure prophylaxis after accidental prion inoculation. Lancet 350, 1519-20 (1997). 11. Klein, M. A., Kaeser, P. S., Schwarz, P., Weyd, H., Xenarios, 1., ZinkernageI, R. M., Carroll, M . C., Verbeek, 1. S., Botto, M., Wa~ort, M. 1., Molina, H., Kalinke, U., Acha-Orbea, H. & Aguzzi, A. Complement facilitates early prion pathogenesis. Nat Med 7, 488-92. (2001). 12. Heppner, F. L., Musahl, C., Arrighi, 1., Klein, M. A., Rulicke, T., Oesch, B., Zinkernagel, R. M., KaIinke, U. & Aguzzi, A. Prevention of Scrapie Pathogenesis by Transgenic Expression of Anti-Prion Protein Anti bodies. Sc ie nce 294, 178-182 (200 I).

20 poos:

Ethical Issues of Genetic Manipulation of Lifestock

Prof. Dr. Dr. Bernhard Irrgang, TV Dresden, Institute ojPhilosophy, G-O 1062 Dresden; E-Mail: [email protected] Tel.. 0049-35 J -4633600 J www.tu-dresden.delphfiphldozentenlirrgang.htm

I would like to answer the question of ethical general conditions for genetic manipulation of life in two steps: (1) First it needs to be clarified why we have moral obligations towards live and how we can point them out; (2) Then the question of necessary special regulations for genetic manipulation needs to be answered. These issues should be discussed concerning the positions ofBiocentric and "Deep Ecology".

1) About the attempt of an Ethics of nature

Regarding the question of man's use of live in a morally acceptable way, various attitudes are possible: (l) Anthropocentric, (2) Pathocentric, and (3) Biocentric. From the ethical point of view it is important to know why we have the obligation of moral behaviour (for example consideration) towards living organisms. The classical Anthropocentric (I. Kant) considers cruel treatment of animals as an act against man's duties towards himself because through becoming insensible, morality will be weakened ore destroyed. For those who follow Pathocentric (P. Singer and M. St. Dawkins) in the tradition of Utilitarism, the ability of suffocation is the central criteria. Following Bentham's thesis they also include animals because of their ability of suffocation within the weighing of interests. But it is difficult to find out empirical criteria for animal suffocation for example the phenomenon of stress (measurement of heartbeat) ore the behaviour of an anima1 in painful situations. The scoop of interpretation is large. The members of Biocentric (A. Schweitzer, G. M. Teutsch, T. Regan) with their claim for an attitude of "respect flr live" avoid this problems. Modern biocentrical positions rely on the consciousness of animals and their interests. Many philosophers of Biocentric consider the difference between the human and the animal consciousness as gradual and not essencial, a problematic premise. They demand an equality of man and animal, as extensive as possible. O. Baffe takes a mediate position with his though of a (regarding the organisation of the sense organs and the central nervous system) hierarchical solidarity between man and animal. Interpreted as a priority criteria this leads to the demand of equal treatment of man and animal in comparable situations. Based upon this urgency criteria concrete demands for the use of nature ore animals can be made (lrrgang 1997, 172-182).

Within the field of Philosophy especially since Descartes the animal-man-difference and since Darwin the animal-man-transition is being discussed. The question of man-animal-comparison (Teutsch 19~, 133-135) is of primary importance for the personal status but also difficult: Until today there has been no real success in the exact definition of difference between man and animal (Brockhaus 1975, 110). The consideration would be widely extended for those animals to which we attribute the personal status and the individua1 interest of survival. Peter Singer discusses such a personal status for chimpanzees, gorillas, dolphins and whales. Traditionally the animal-man-difference and the personal status is considered to be located in the rationality and morality of man. If personality is being connected to criteria like feelings of pleasure and reluctance ore consciousness ore individuality, it is allowed to ask the question weather there can be something like an animal-person.

In deed, some common ideas about animals and their consciousness will have to be corrected. Griffin could make this plausible in many cases (Griffin 1990, 28-30). But there is no reliable criteria for ascribing consciousness to animals (Griffin 1990, 68). Nevertheless it is possible to barn something about their thinkjng from their ability to learn, to adjust and their communication behaviour. Even though I do not consider a animal-person from the ethical point of view, ethically relevant criteria for the consideration of animals are pan, their repertoire of behaviour, especially their communication

21 Plenary Session: Invited Speakers behaviour and the possibility of consciousness. Therefore a close inspection is necessary. In this sense I do not know any position which claims that the acting of animals is morally responsible. Therefore it would have to be proofed that a least some animals do act morally responsible and give reasons for their objectives.

The most radical ethical duties towards nature and live are being claimed by the "deep ecology~'. Already in 1972 when the Norwegian analytical Philosopher Arne Naess introduced the expression "Deep Ecology", it was the central objective to make a revolutionary change in the anthropocentric orientation of western Ethics and Politics. The utopical character of many of the deep ecology objectives is obvious. The basic attitude of society should be overcome with the interpretation constructions "the nature" and especially the system as a whole like landscape, ecosystems and biological species all of which are nothing purely existing but constructions which include human perception, cultural interpretation and scientific conventions (Bimbacher 1997, 12). Deep Ecology does support many of the objectives of the Reformism but it is also revolutionary and seeks a new Metaphysics, Epistemology, Cosmology and Enviromnental Ethics for the man-earth-system (Dervall 1997, 17). Following the current paradigm, nature is only a supply of resources which has to be produced in order to meet the pennanently growing needs of the permanently increasing number of people. Science and technology work hand in hand. Technology develops techniques to conduct natural processes. Changes become an end in itself. The new is higher regarded than the old, the currant generation higher than the future one i (Dervall 1997, 19f). Deep ecology puts it's main effort into finding and discussing alternatives to the conventional way of thinking of the modem West. That includes modem natural sciences and technology, especially genetic engineering and it's full technological handling of live. But did modem science bring such a big change into our relation to nature?

2) The claim for Biocentic and "Deep Ecology"

Because of the demoralisation of the definition of live in modem biology, modem biocentric positions ore the so called "deep ecology" rather follows the romantic - organic definition of live which is especial1y for Schopenhauer and in the philosophy of live connected to a (cultural-) pessimistic thinking. This background is also being established in the basic attitude of respect for every live as it has been formulated in the Ethics of Albert Schweitzer. But if one tries to live consequently following this attitude, huge problems come up. In it's close sense it is not possible to realise. Also, respect is a religious ore at least no specific moral attitude. One can have respect for virus but fight them with good reasons without guiltiness if they harm ore endanger man. Attitudes towards live which are similar to Biocentric can be fOlll1d in Jainism (Religion of he Jaina, India), in Buddhism (Eastern Asia) and in a slightly different way in Hinduism (India) in the Ahimsa-Corrunandrnent (prohibition of hanning any life).

Biocentric position demands protection of life by itself. Interventions in nature are actually Jrohibited and need to be justified. Gunther Altner claims: "The possibility of a self-recreational evolution for nature has to be preserved because this is it's freedom" (Altner 1987, 216). Like in the definition of the animal-person, the living nature is being personalised in order to ascribe moral values and moral rights to it. The first claim of biocentric positions is the protection of species: "The self-value of non­ human creatures shows itself in the unique developed nature, in it's natural interplay which again expresses itself in the typical community of species (biotopes) and in the relatively stable ecological balance" (Altner 1991, 217). A living nature is being imputed in which man does not exist. To give reasons for it's protection, nature is being personalised. But in the other hand, a more correct method would be the use of definition of species in the sense of ecological adjusting as an ethically relevant empirical criteria for the protection of species.

Further, biocentric positions ascribe consciousness to animals and often demand a general prohibition of killing animals. Tom Reagan regards the difference in consciousness of animal and man as only gradual and not essential. He ascribes a certain autonomy to animals because they can express preferences especially interests of wellbeing. The definition of interests is cormected to the definitions: selfishness, use in general, survival, egoism, protection of benefits and avoiding of damages, though calculated benefits on the basis of reason and needs as action motivation can most probably only be

22 ascribed to man, natural efforts towards damage prevention and protection of ones own life can be found also with other organisms.

3) Fairness towards every living being

The following critics against Biocentric and "Deep Ecology" have to be done: Although the fact of one origin of every living being, including man, gives a certain respects towards aJl fonns of life although not all of them are fundamentaIly equal. The needs of man are more differentiated than the ones of animals. As well animals do not have the reflective consciousness which enables man to make experiences and act responsible, to make claims ore judgements (Frey 1980, 120). Moral acting cannot be ascribed to animals. From the ethical point of view the self-value of nature cannot be the same as the one of man (Irrgang 1997, 211 f). This puts the personification of nature but not the use of biology in the sense of an ethically relevant empirical criteria into question. In the end, not the use of empirical results 1ike certain proofs of live but the use of ethical principals like justice ore damage prevention gives reasons for claims like prohibition of direct hanning of animals (animal prevention) and the protection of species (biodiverstiy).

That means that the principal of justice of life is fundamental for Bioethics of life. It applies to all living beings. Nobody seriously claims that animals can act morally responsible. At the most a moral­ like behaviour of animals is being described (Brockhaus 1975, Ill). Although Bioethics does not justify the concept of a morally responsible acting animal-person it does develop a ethically relevant empirical criteria for the consideration of animals: Ability to feel pain, behaviour repertoire, communication behaviour and the possibility of consciousness.

In order to avoid the naturalistic fallacy from evolutionary justified "similarities" to moral obligations (Irrgang 1997, 163-182), a gradualism has been developed on the base of an ecological conception of justice. The gradual urgency criteria of consideration of the concerned acting persons (Irrgang 1997, 204-212), considers as commandment of fairness ore justice with growing grade of urgency the balance and cycles of nature which are fundamental for dher general coherences of life, as well as issues of species protection not only for vertebrates, emotionality and ability to feel pain of organisms and the attempt of higher developed animals to lead a live without suffering, especially if we have a special responsibility for them as useful animals and that means not only to consider them as genus but as individuals and also the urgency criteria does ascribe a very high consideration to highly developed mammals like monkeys ore certain sea mammals (Irrgang 1997, 205).

This way a differentiated Ethics of protection of life on the basis of an ecological anthropocentric can be justified. Animals are to be protected in coherence to their communication ability and competence, pain and fOnTIS of consciousness. The unjustified harming of any parts of nature and especially highly developed animals does brake the ethical principal of justice. In order to measure weather we are hanning animals, ethically relevant criteria are needed. Obligations towards animals ore towards nature cannot be shown apodictically but they need an argument of convergence which considers most of the mentioned criteria as possible. In general these are ethically relevant empirical criteria which help to assess in which cases the obligation of the protection of animals does ore does not apply.

4) Biodiversity - an absolutely obligatory criteria?

Many people consider Biodiversity as diversity of life - in German mentioned as diversity of species - as an indicator which they suppose to know from direct [empirical] experience within a nature which is intact and meets the needs of humans. But it is unclear what diversity of life means and through which criteria and basis this diversity has to be seen. The problem already begins with the attempt to find a clear definition for the classical objects which are supposed to show the diversity of life, these are "species ", "gene" and "ecosystem". The differences in the terminology are signs for divergences regarding the in biological sciences recognised research procedures and their corresponding research objects. All together, throughout the whole earth age, it turned out that the palecrbiodiversity has neither through background events nor tluough exogenous catastrophes been extinguished nor damaged with lasting effect. Although living beings have died out not only as individuaJs but also as families ore orders but this has always been an accompanying phenomena for the development of life

23 Plenary Session: Invited Speakers on earth in which the decreasing of diversity was always been followed by an increase. Even though some species, families ore orders have disappeared from earth forever, it did not lead to a long-tenn damage of the world of organisms in total.

The micro-biological diversity research showed that only a very small number of currently living micro-organisms have been isolated given a valid description. Regarding this it has to be mentioned that micro-biologists do not demand the protection of single species but the protection of locations for the taking of specimen. Procedures of molecular-biology put main emphasis on genetic information. This is especially suitable as a base for procedures of proving of biodiversity. Significant parts of the biodiversity of plants cannot be just morphologically characterised on the genetic level. The objects of the biodiversity debates like gene, species ore ecosystem are no purely natural are scientific objects but are also determined by giving practical purposes (Jaruch et al. 2001, XXI till XXIX). The reasons for the widespread dying of ~ecies are shield tectonic, sea level and changing of magnetic fields as well as volcano and asteroids (Janich et aI. 2001, 70-99). The value of species can be differentiated as a self-value and an instrumental value. Biodiversity does have a certain production value, although the problem of value assessment of biodiversity has to be regarded in relation to the problem of prevention of nature.

Biodiversity and prevention of species are ethical claims which can be justified, especially considering our non-knowledge about most of the biological species and considering the caution regulation, but it is no absolute commandment. Above all a prohibition of an inter-species gene transfer cannot be justified on this base. In order to answer this question it needs the reflection of the specification of genetic acting in comparison to all other technological acting. Special regulations are only obligatory, if the genetic praxis does fundamentally differ from all the other forms of technical praxis.

5) Genetic Engineering as Laboratory Praxis

The praxis of genetic engineering does basically not differ from other technological praxis. The structure of technological acting gives reasons for the unavoidable trying out. This trying out can never be fully calculated although certain possibilities can in advance be excluded as being practically irrelevant through calculation. Technological acting is no blind use of nature ore objects. It is no accidental process but it is lead by an organised ore at least a heuristic process of searching and fmding. Useful animals and plants are no objects in the c10se sense but in order to deal with them, man first of all uses the natural processes of reproduction. Useful animals are no technological means, no tools are machines in the average sense but they also are no classical objects. The expanded definition of technology as handling of both technological and natural processes can avoid a to close understanding of technology in the sense of tool and object. It constitutes a concept of technological acting both from the point of view of the participant and of the observer (about the concept of instrumental understanding see Irrgang 1998a, 75-120, about technological acting see CoronaJlrrgang 1999, 166-212). The technological handling of a single and concrete technological problem takes place on the basis of a (implicit) technological regulation knowledge with the aid of means and / ore technological knowledge. This regulation knowledge depends on frameworks ore paradigms which often cannot be shown explicitly. But on the basis of a reflected handling knowledge they can be understood j put as subject and be criticised. I explained this more detailed in my book "Technische Kultur" (Irrgang 2001 a).

In the praxis of breeding organisms of one and the same kind are being crossed with a combination of genetic marks without having an exact knowledge of the genetic information. The definition of species can be regarded as a human interpretation construction, which although it might give some kind of infonnation about the biological reality it does give reasons for doubt. The gene transfer changes one ore several genetic marks of an organism although it does normally not change the species which in theory would be possible. The differences between teclmical and technological praxis that is differences between breeding and genetic engineering are in general the inserted laboratory procedures. These lead to better infonnation about details and causal paradigm of biological processes but not to information about general coherences. This is the difference between breeding praxis on farms and breeding research in labs, whereas the modem seed breeding companies are differing from university ore MPI- laboratories. The procedures of genetic engineering in the sense

24 of a technological praxis - a reflected strategy of attempts and errors, ethical evaluation included - does correspond with a concept of technological acting which reduces technology to objects. Technological acting which opens new technological possibilities opens a new range of possibilities using them in praxis which often were regarded as technological imperatives (this is possible and is sometimes being done) but actually it is the opposite: only after trying out you know what is possible and you can decide which action options one wants ore wants not to take. Technology as technological acting can be seen under several aspects: objectives, technological means, consequences ore interventions in nature can also be relevant. Especially the last point is of interest. Technology is not to be regarded as practical experimental science. On one hand experimental science also needs to give things a trial but on the other hand a lot of things work out in theory but not in praxis. Asking for the possibilities of trying out ore making experiments - at the end there are no other ways to handle technological action than to give technologies a try in praxis. The meaning of technological experiences can never be reduced to the pure observer position. Possibilities of making mistake and of making innovation depend on each other. Organised and rational experiments also includes responsibility and not to give it a try no matter what happens. This also has consequences for ethical evaluation of genetic engineering. Nevertheless, every form of selection includes certain risks of failure. Risks in the releasing of trans-genetic organisms cannot be totally excluded. No fonn of technological acting is without risks. Although, they do not depend on the method of gene transfer but on the nature of the changed organism and ecosystems into which it is being reJeased, which means they depend on the way of technological acting. A special Ethics for genetic engineering such as "Gene-ethics" is not necessary, but a differentiated ethical reflection within specified areas for a specialised form of technological practice is needed.

Literature:

Altner, Giinther 1987: Die Nutzungsziele def Gentechnologie unter der Perspektive von Umwelt- und Sozialvertfi:iglichkeit; in: V. Braun, D. Mieth, K. Steigleder (ed.); Ethische und rechtliche Fragen der Gentechnologie und der Reproduktionsmedizin; Munich, 213-223 Altner, Giinther 1992: Naturvergessenheit. Grundlagen einer umfassenden Bioethik; DalTIlstadt Bayertz, Kurt 1989: Entmoralisienmg des Lebendigen; in: Matthias Gatzemeier (ed.): Verantwortung in Wissenschaft und Technik; Mannheim, , Zurich, 220-238 Bimbacher, Dieter 1997: Okophilosophie, Brockhaus, Wilhelm 1975: (Ed.) Das Recht der Tiere in def Zivilisation. Eine Einfiihrung in Naturwissenschaft, Philosophie und Einzelfragen des Vegetarismus, Munich Corona, Nestor; Irrgang, Bernhard 1999: Technik als Geschick? Geschichtsphilosophie der Technik; Dettelbach Dervall, Bill 1997: "Tiefenokologie" - Eine okologische Ganzheitsprulosophie; in: D. Birnbacher, Okophilosophie, Stuttgart, 17-59 Frey, R. G. 1980: Interests and Rights. The case Against Animals; Oxford Griffin, Donald R. 1990: Wie Tiere denken. Ein Vorsto13 ins BewuI3tsein der Tiere; translation by Elisabeth M. Walther (1984); Munich Irrgang, Bernhard 1991 : Transgene Tiennodelle im biomedizinischen Experimept. Forschungsethische Ubedegungen; in: Forum fur interdiszipJinare Forschung 4 (1991), issue 1,54-66 Irrgang, Bernhard 1992: Ethische Aspekte der Biotechnik; in: H. Wilhelm Schaumann-Stiftung (ed.): Biologisch-technische Entwicklungen in der Tierproduktion (14. Hiilsenberger Gespdiche); Hamburg, 36-46 Irrgang, Bernhard 1996: Handbuch der angewandten Ethik; ed. by Julian Nida-Riimelin; article Gen­ Ethik; Stuttgart 1996, 510-551 Irrgang, Bernhard 1997: Forschungsethik Gentechnik und neue Biotechnologie. Grundlegung unter besonderer Beriicksichtigung von gentechnologischen Projekten an Pflanzen, Tieren und Mikroorganismen; Stuttgart Irrgang, Bernhard 1998a: Praktische Ethik aus hermeneutischer Perspektive; Paderbom Irrgang, Bernhard 1998b: Wozu konnen Klonierungsverfahren dienen: ethische Bewertungskriterien; in: Johannes S. Ach, Gerd Brudermiiller, Christa Runtenberg (ed.): Hello Dolly? Ober das Kionen; FrankfurtlM., 72-89

25 Plenary Session: Invited Speakers Irrgang, Bernhard 1998c: Lexikon def Bioethik; ed. by Wilhelm Korff, Lutwin Beck, Paul Mikat; Giiters]oh 1998; art. Bjozentrik (vol. I, 402-404); art, Pathozentrik (vol II. 2, 834-835); art. Physiozentrik (vol. III, 28-30); art. Theozentrik (vol. III, 526-528); art. Tierschutz (vol. III, 561-567) Irrgang, Bernhard 1998d: Am Ende der Anthropozentrik? Wie 1assen sich unsere Verpflichtungen gegentiber der Natur begrtinden; in. Gotthard Fuchs, Guido Knorzer (ed.): Tier, Gott, Mensch. Beschadigte Beziehungen; 1998, 13-32 Irrgang, Bernhard 1999a: Gentechnik in der Tierziichtung; in: Thomas Hausmanninger, Scheule, Rupert (ed.): ... geklont am 8. Schopfimgstag. Gentechnologie im interdisziphnaren Gesprach; Augsburg, 227-242 Irrgang, Bernhard 199b: - Nachha1tigkeit als Leitbild fur Griine Gentechnik? in: Dirk Harreus (ed.) Gentechnologie. Fakten und Meinungen zum Kemthema des 21. lahrhunderts; 1999, 146, 195- 210, 241f Irrgang, Bernhard 2000: Ethische Betrachtungen der Anwendung bio- und gentechnologischer Verfahren in der Tierzucht; in: Deutsche Gesellschaft :fur Ziichtungskunde e.V. (ed.) Bio- und Gentechnologie in der Rinderzucht - Risiko oder Chance? DGtZ-Schriftenreihe 17; Bonn, 56-66 Irrgang, Bernhard 2001a: Technische Kultur. Instrumentelles Verstehen und technisches Handeln; (Philosophie der Technik vol. 1); Paderbom Irrgang, Bernhard 2001b: Gefangen in Sachzwangen? Zur ethischen Dimension der Gestaltbarkeit der Biotechnologie; in: St. Heiden et al. (ed.): Biotechnologie als interdisziplinare Studie; Heidelberg / Berlin, 83-96 I rrgang , Bernhard 2002: Technische Praxis. Gestaltungsperspektiven technischer Entwicklung; (Philosopllle der Technik vol. 2); Paderborn 2002 Irrgang, Bernhard et al. 2000: Gentechnik in def Pflanzenzucht. Eine interdisziplinare Studie; Dettelbach lanich, P. et al. 2000 (ed.): Biodiversitat. Wissenschaftliche Grundlagen und gesetzliche Relevanz; Berlin et al.

Lenkl Hans; Maring, Matthias 2001: Advances and Problems in the Philosophie of Technology; Miinster Teutsch, Gotthard M. 1985: Lexikon def Umweltethik; Gotlingen Zimmerli, W. Ch. 1993: Die Bedeutung der empirischen Wissenschaften und der Technologie fur die Ethik; in: A. Hertz, W. Korff, T. Rendtorff, H. Ringeling (ed.): Handbuch der christlichen Ethik; Freiburg, Basel, Vienna, Aktualisierte Neuausgabe vol. I, 297-316

26 Section A: Genome technologies

Section A: MARIA BALLESTER1, ELENA IBÁÑEZ2, Genome technologies RAMONA N. PENA3, JOSEP MOLIST2, JOSEP SANTALÓ2, ARMAND SÁNCHEZ1, JOSEP M FOLCH1 A001 1 Mb resolution radiation hybrid map of the Departament de Ciència Animal i dels Aliments, canine genome Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain; 2 RICHARD GUYON1, LISA KIM2, CHRISTO- Departament de Biologia Cellular, Fisiologia i PHE HITTE1, TRAVIS LORENTZEN2, Immunologia, Facultat de Ciències, Universitat 1 2 Autònoma de Barcelona, Bellaterra 08193, Spain EDOUARD CADIEU , HEIDI G. PARKER , 3 PASCALE QUIGNON1, JENNIFER K. LOWE2, and Division of and Develop- CORINNE RENIER1, BORIS GELFENBEYN2, ment, Roslin Institute, Roslin EH25 9PS, Midlo- STEPHANIE GLOUX1, FRANÇOISE VIGN- thian, UK β AUX1, GREGORY MAHAIRAS3; CATHERINE Eight lines of transgenic mice for the goat - ANDRÉ1, FRANCIS GALIBERT1, ELAINE A. lactoglobulin gene (βLG), comprising a 410 bp- OSTRANDER2 long promoter, were generated to study the effect 1UMR 6061 CNRS, Génétique et développement, of promoter size on transgene expression, result- Faculté de Médecine, 35043 Rennes, France; ing in high levels of expression in a position inde- 2Clinical and Human Biology Divisions, Fred pendent manner. Homozygous animals for the Hutchinson Cancer Research Center, Seattle WA transgenic line Tg56 showed a distinct phenotype 98109-1024, USA; 3Department of Genome Sci- consisting in retarded growth, high mortality after ences, University of Washington, Seattle WA,USA birth and infertility in females. This line contained The purebred dog population consists of over 300 22 copies of the transgene integrated in a single genetic isolates termed breeds, representing a genomic position and expressed the goat β-LG at unique resource for dissecting the genetic basis of high levels in the mammary gland. In the present both simple and complex mammalian traits. To- work, we have analysed the transgene integration ward this end, we developped a comprehensive site to verify if the phenotype was produced by radiation hybrid map of the canine genome com- the disruption of a specific mouse gene. The posed of 3400 markers. The map was constructed “Vectorette system” (Genosys), which is based on using the RHDF5000-2 whole-genome radiation the Anchored PCR technique, was used to isolate hybrid panel and computed using the Multimap and sequence the flanking regions of the transgene and TSP/CONCORDE programs. This map pro- integration site. The 22 copies of the goat βLG are vides an average inter-marker distance of 1Mbase integrated in the intron 30 of the Phospholipase and a nearly complete coverage of each of the 40 C-β1 gene (PLC-β1). This gene is normally ex- chromosomes composing the canine karyotype. pressed at high levels in the mouse brain and its The inclusion of 1000 dog genes and ESTs allows protein is implicated in the transmembrane signal refined numbering and mapping of conserved transduction, catalyzing the hydrolysis of phos- segments between human and dog. Moreover, of phoinositide 4,5-biphosphate. An expression the 1,700 mapped microsatellites, approximately analysis of the PLC-β1 gene in the Tg56 line by 800 have HET or PIC values ≥0.5, thus providing RT-PCR and sequencing, showed that an hybrid a well characterized resource of highly poly- mRNA is transcribed. This hybrid mRNA con- morphic markers suitable for genome wide scans tains a truncated mouse PLC-β1 gene (from exon in genetic linkage studies with pedigrees of inte- 1 to 30) fused to all seven exons of the goat βLG rest. Finally, the 700 mapped BAC-ends constitute gene (including the polyadenylation signal). an initial framework of clones for anchoring a physical map and provide a format for positional A003 cloning studies. Overall this work defines a po- Cytogenetic studies on Assam local cattle(Bos werful resource for genetic studies in the canine Indicus) and Jersey bulls(Bos Taurus) system, and underscores the emerging power of canine genomics in the pursuit of the genes cau- ANJANA CHOUDHURY, R.N. GOSWAMI, sing human variation and disease. D. DAS http://www-recomgen.univ-rennes1.fr and Animal Husbandry & Veterinary Department, http://fhcrc.org/science/dog_genome/dog.html Govt. of Assam; Guwahati, Assam, India Studies on chromosomal architecture, karyotyping A002 of chromosomes and chromosomal abnormalities , Disruption of the PLC-β1 gene in a βLG trans- if any in 30 Assam Local cattle ( 15 males and 15 genic line affects growth and fertility in mice females ) and 12 Jersey bulls were undertaken. 26 Section A: Genome technologies

The diploid number of chromosomes was 60 irre- genes, Pax3), cell cycle regulators (cyclins) and spective of genetic groups and sexes. All the 58 several muscle specific genes. autosomes were acrocentric and X-chromosomes were submatacentric in both the genetic groups. A005 Existances of a distinct morphological difference Recombinant Bovine Growth Hormone and in Y- between Assam Local male and Peptidylglycine Monooxygenase Activity Jersey bulls was confirmed. The mean relative length of chromosomes with their standard errors PETER CREIGHTON, JOHN DONLON and coefficient of variation for both local cattle Department of Biochemistry, National University and jersey bulls were determined. No major of Ireland, Galway, Ireland chromosomal abnormalities could be detected in Carboxy-terminal amidation is a frequent post- animals of either genetic groups under study. translational modification required for biological For Correspondence: activity and stability of many bioactive peptides. Dr.(Ms.) Anjana Choudhury C/O. Dr. B.N.Saikia About half of all known neuropeptides and bioac- ,Assoc. Prof., Deptt. Of Animal Nutrition, tive peptides are C-terminally activated. Peptide College of Vety.Science, AAU, Post Box No.15, amidation, in vivo, is a two-step process with Khanapara, Guwahati-781022 hydroxylation of a glycine-extended peptide fol- lowed by its dismutation to release glyoxylate A004 with retention of the α-carbon glycine as the ami- Study of expressed sequences during somite de nitrogen. Peptidylglycine monooxygenase and limb development in chicken (Gallus gal- (PGM) is the enzyme involved in the first step of lus) the amidation process and the second step is ca- talysed by the enzyme peptidylamidoglycolate ERIKA C. JORGE, CLARISSA S. SILVA, lyase (PAL). Previous studies have shown that CLAUDIA B. MONTEIRO-VITORELLO, PGM isolated from bovine pituitary is identical to MATEUS PATRÍCIO, LUIZ L. COUTINHO somatotrophin or growth hormone (GH). Since Animal Production Departament – ESALQ - USP commercially produced recombinant bovine - Piracicaba – Brazil growth hormone does not show amidation activi- Embryonic development is marked by an intricate ty, we set out to express and purify recombinant succession of events that controls cell division, bovine growth hormone using non-denaturing determination and differentiation. We are par- methodologies that allow retention of the enzyme ticularly interested in the processes that control activity of GH. Our studies confirm that recom- myogenesis. Expressed Sequence Tags (ESTs) binant bovine growth hormone expressed in E.coli allow determination of gene expression profiles in shows peptidylglycine monooxygenase activity. any particular tissue under different conditions or status. In order to identify genes important for A006 muscle formation, two cDNA libraries from so- Sampling gene expression from mammary tis- mite and limb were prepared. Somites, containing sues by EST sequencing of ORESTES cDNA neural tube and notochord, were dissected from libraries from Holstein and Gir cattle. 290 stage 15 (E15) H&H embryos. Limb tissue was dissected from 60 embryos in three different ADILSON F. DA MOTA1,2,3, TAD S. stages of development (E21, E24 and E26, H&H). SONSTEGARD1, CURTIS P. VAN TASSELL1, mRNA was selected from total RNA and con- ERIN E. CONNOR1, ANTHONY V. CAPUCO1, verted into double-stranded cDNA by Super- MARIA APARECIDA P. BRITO2, MARCO A. ScriptRT II and oligo (dT) primer. A directional MACHADO2, MARIO L. MARINEZ2, LUIZ L. library was constructed and cloned in pSPORT 1 COUNTINHO2 vector. Clones were sequenced from its 5’ end, 1United States Dept. Of Agriculture, Agricultural using Big Dye Terminator Kit (Applied Biosys- Research Service, Beltsville, USA; 2EMBRAPA tems - Perkin Elmer) and T7 primer. Sequences Dairy Cattle Center, Juiz de Fora, Brazil; were analyzed with Phred/Phrap/Consed and (3)University of Sao Paulo, ESALQ, Piracicaba, Cap3. A total of 2328 clones from the limb library Brazil resulted in 527 singletons and 485 contigs while Even though more than 230,000 bovine expressed the 2383 clones from the somite library resulted in sequence tags (EST) are available in public 891 singletons and 411 contings. Chicken ESTs databases, additional EST are needed. The are highly homologous (over 70% identity at pro- objective of this study was to generate EST to tein level) to humans and rodents. Our data al- better characterize the mammary gland lowed identification of growth factors (TGF-β, transcriptome. Construction of cDNA libraries HGF, FGF-receptor), transcription factors (Hox was performed using the open reading frame EST

27 Section A: Genome technologies

(ORESTES) method to avoid the transcript bias SNPs were found for every 1000 bp of good qua- inherent to mammary and to maximize discovery lity, aligned sequence. BLAST analysis of the rate of novel EST. ORESTES relies on arbitrarily 9264 unique sequences against the UK chicken primed RT-PCR to generate a partial expression EST database (http://www.chick.umist.ac.uk/), using profile that can be shotgun cloned. In a a significance threshold of E = e30, revealed 7070 preliminary study, six libraries were constructed hits (76.3%) and 2191 no hits (23.7%). The 2191 using mRNA from pre-pubertal mammary and sequences that did not have a significant hit in the primers that amplify the estrogen receptors. UK database were BLASTed against NCBI nr and Sequencing of 576 clones generated 455 GenBank est databases, and it was found that 660 had a quality submissions. Sequence assembly was used significant hit (E = e30), while 1531 sequences to assess rate of clonal redundancy relative to each (16.5% of the total unique sequences that were RT-PCR primer and provide tentative consensus originally BLASTed), remained unidentified. (TC) sequences for BLAST analysis. A total of 64 TC sequences were assembled from three libraries A008 leaving 178 singletons and an average redundancy Determination of RNaseH accessible sites along rate of 47%. BLAST analysis of the 242 unique the Sus scrofa FUT1 and FUT2 RNA sequences sequence elements revealed 81 sequences (17%) for design of efficient antisense oligonucleotides that had no match to GenBank nt and 147 (32%) were novel relative to cattle. Because ORESTES ANGELIKA GABLER, STEFAN KREBS, produced a high rate of novel sequences, this MARTIN FÖRSTER method will be exploited to generate mammary- University of Munich, Institute of Animal Bree- derived EST from Holstein- and Gir-derived ding, Munich, Germany libraries. The resultant breed specific Diarrhea in suckling piglets is supposed to be in polymorphisms and novel sequences will be a part due to adhesion of Escherichia coli F18 to valuable resource for understanding gene antigenic structures on the surface of small intes- expression differences between breeds. tine mucosa cells engineered by the products of the FUT genes. Animals lacking specific FUT A007 alleles are thought to be resistant to E.coli F18 Sequencing and comparison of total brain colonization. Temporary blockade of the FUT cDNA Libraries of White Leghorn chicken and gene product synthesis should therefore circum- Red Jungle fowl vent E.coli F18-related intestinal troubles. Thus we looked for a strategy to find targets for an- CAROLYN J. FITZSIMMONS1, PETER tisense oligonucleotide annealing along the FUT SAVOLAINEN2, GÖRAN HJÄLM1, RNA sequences. To do so, RNA was transcribed JOAKIM LUNDEBERG2, LEIF ANDERSSON1 in vitro and incubated with short, denatured DNA 1Swedish University of Agricultural Sciences, fragments obtained after DNase-digest of PCR- Department of Animal Breeding and Genetics, generated fragments corresponding to the RNA Uppsala Sweden; 2Royal Institute of Technology, sequences. Then RNase H was added in order to Department of Biotechnology, Stockholm, Sweden cleave the RNA at sites where DNA-RNA-hybrids We report the sequencing and comparison of two have formed. The resulting RNA fragment pattern total brain and two testis cDNA libraries from was analysed by MALDI-MS. The masses of all White Leghorn chicken (WL) and Red Jungle fowl possible FUT sequence segments have been cal- (RJ). The aim of the project has been to generate a culated in theory and compared to the measured large number of sequenced cDNA clones that can peak masses from MALDI-MS. Because multiple be used for gene expression analysis using cDNA fragments matched these conditions the analysis arrays. The detection of new SNPs supplements was further refined by subsequent RNase T1 di- our ongoing QTL mapping project which consists gest of the initially generated RNA fragments. As of a cross between the White Leghorn and Red RNase T1 cleaves RNA after each G the cleavage Jungle Fowl. A total of 25824 clones were sequen- pattern of each of the possible FUT sequence ced from all 4 libraries, and after removal of low segments could be calculated. Comparing these quality sequence, E. coli, vector, and mitochondri- data to the measured RNase T1 digest spectra al contamination, 16667 sequences were assem- allows for determination of the underlying se- bled in Phrap. 6209 singlets were identified, and quence segment. Thus by knowing the sequences 10458 sequences were assembled into 3055 con- of RNase H cleavage sites efficient antisense oli- tigs (9264 unique sequences). Of these contigs, gonucleotides can be constructed. 581 were single reads. 746 SNPs and 37 inserti- ons/deletions were tentatively identified in 479 contigs, and it was calculated that on average 1.4

28 Section A: Genome technologies

A009 Atlantic salmon is one of the most important Validation of sperm sexing in Bos taurus by aquacultural fish species with a yearly production dual color Fluorescence in situ Hybridization of more than 600,000 tons (year 2000) in the (FISH) European countries. The importance is reflected in the increased interest in the molecular evolution FELIX A. HABERMANN1, INGRID OLSA- and genetics of the species. Genetic maps of sev- KER2, PETER REICHERT3 , RUEDI FRIES1 eral salmonid species are currently being con- 1Technical University of Munich, Chair of Animal structed. However, the number of known gene Breeding, Freising-Weihenstephan, Germany; sequences in the public databases is highly lim- 2The Norwegian School of Veterinary Science, ited. The aim of the current project (SALGENE, Department of Morphology, Genetics and Aquatic EU FAIR-CT98-4314) was to increase the number Biology, Oslo, Norway and (3) Big-X AG, Seewen- of known Atlantic salmon ESTs considerably. Schwyz, Switzerland Non-subtractive and subtractive cDNA libraries Separation of X- and Y-bearing sperm cells and from nine different tissues (kidney, liver, gill, artificial insemination with sex-specific semen intestine, brain, muscle, testis, ovary, and spleen) makes it possible to pre-determine the sex of were constructed. A total of more than 15,000 calves. This has the potential to considerably im- EST sequences has been obtained, adding sub- prove cattle breeding, genetic resource manage- stantially to the number of known ESTs in Atlan- ment and particularly the efficiency of dairy and tic salmon. All obtained sequences were analysed meat production. The broad use of sexed semen, by the BLASTN/BLASTX programs against the however, will depend on availability, price, fer- public genomic and EST databases to identify the tilizability and the actual sorting purity of sperm corresponding genes. Also, more than 400 candi- doses. To validate the accuracy of sperm sexing in date SNPs were identified. The cDNA clones Bos taurus a simple, fast and reliable dual color comprise a valuable resource for the future con- FISH test has been developed: Y-bearing sper- struction of cDNA microarrays with the purpose matozoa are identified by hybridizing a DNA of gene expression analysis to enable identifica- fragment that recognizes a repetitive DNA block tion of differentially expressed genes from indi- on the bovine Y chromosome. Simultaneously a viduals exposed to different treatments. This will second DNA probe identifying a repeat block on facilitate the future identification of genes under- the bovine autosome 6 is used as indicator of suf- lying potential important QTLs in Atlantic ficient accessibility of the sperm DNA and ade- salmon. quate hybridization efficiency in each spermato- zoon. Both DNA probes can be amplified and A011 labeled effectively by PCR and provide specific Diagnoses of blood chimerism between twins in and clear signals even after short incubation with- cattle using quantiative PCR out the need of competitor DNA. The procedure was evaluated using the established Beltsville HANNES W. JOERG1, MIKA ASAI- sperm sexing technology that separates X- and Y- COAKWELL1, FREDI JANETT2, DARIA A. bearing sperm cells by flow-sorting according to GRAPHODATSKAYA1, DAGMAR STEIGER1, their DNA content. GERALD F. STRANZINGER1 1Swiss Federal Institute of Technology, Institute of A010 Animal Sciences, Zurich, Switzerland; 2University Development of a characterized cDNA resour- of Zurich, Faculty of Veterinary Medicine, Zurich, ce in Atlantic salmon (Salmo salar) Switzerland In cattle, genomic DNA for parentage control is LARS-ERIK HOLM1, FRANK PANITZ1, commonly extracted from blood samples. How- BJØRN HØYHEIM2, HEIDI HAGEN-LARSEN2, ever, in cases of blood chimerism, the detected JOHN TAGGART3, MARGARET CAIRNEY3, alleles may interfere with parentage control. GRACE C. DAVEY4, SARAH A. MARTIN4, Blood chimerism is also associated with freemar- NICOLE C. CAPLICE4, RICHARD POWELL4, tinism, where a female calf twinned to a male calf CHRISTIAN BENDIXEN1 is sterile. In cattle, 92% of females born as a twin 1Danish Institute of Agricultural Sciences, De- of a male calf are freemartins. Using a multiplex partment of Animal Breeding and Genetics, Tjele, TaqMan® assay, DNA from 6 pairs of chimeras Denmark; 2Norwegian College of Veterinary Me- of both sexes were analysed through quantitative dicine, MGA - Genetics, Oslo, Norway; PCR. The relationship between the amplification 3University of Stirling, Institute of Aquaculture, of a SRY fragment and an autosomal gene Stirling, Scotland; 4National University of Ireland, (MSHR) fragment was used to calculate the male Department of Microbiology, Galway, Ireland cell proportion. Our results showed amplification

29 Section A: Genome technologies of SRY with differing quantities in all 6 females. 20% of BAC-ends have significant BLASTN hits The efficiency of the MSHR and the SRY ampli- against non-repetitive segments of the human fication was different, so that a correction by dif- genome. These sequences are termed compara- ference of the mean values was performed. Nev- tively anchored sequence tagged sites (CASTS). ertheless the quantification of the proportion of The COMPASS III program is used to predict the male cells showed high variation and even pro- chromosome map location and position of CASTS portions above 100%. Seventeen samples of leu- in the cattle genome. For each CAST the pre- kocyte cultures of male twins were analysed dicted cattle genome position is determined com- through quantitative PCR and compared with their putationally in a look-up table that contains the corresponding male and female metaphase spread current cattle-human comparative map. This table counts. The correlation between the results of the relates all positions in the to all quantitative PCR and the chromosome typing was known positions in the cattle genome on the basis 0.3. To quantify the proportion of male to female of a previously published whole-genome radiation cells in different samples is not very accurate, hybrid-based cattle-human comparative map. Us- especially in samples with few female cells. ing this approach it is possible to scaffold up all of Therefore it is difficult to use quantitative PCR the contigs with multiple CASTS on the cattle systems to detect chimerism in random samples. chromosomes thus enabling the creation of a whole genome contig that reveals fine details of A012 chromosome rearrangements. Toward a comparatively anchored, sequence- ready whole genome physical map of the cattle A013 genome Production and Characterization of Chicken- Duck Chimeras DENIS M. LARKIN1, JACQUELINE SCHEIN2, CHERYL GREEN1, THOMAS R. DEKOJ1, ZANDONG LI, JIN SHA, SHENGHUA JIANG, SHARON BACHMANN1, PETER SCHWEIT- MENG JIANG, CHUNHAI LIU, JINSONG ZER1, MARK REBEIZ1, ANNELIE EVERTS- HUANG, NING WANG VAN DER WIND1, STEVEN JONES2, IAN Department of Biochemistry and Molecular Biol- BOSDET2, CARRIE MATHEWSON2, NATAS- ogy, College of Biology, China Agricultural Uni- JA WYE2, READMAN CHIU2, STEPHEN versity, Beijing 100094, P. R. China MOORE3, BERNHARD BENKEL4, JOHN W Avian blastodermal cells at stage X are pluripo- KEELE5, STEVEN M. KAPPES5, MARCO tent, they are capable of differentiating and devel- MARRA2, PIETER DE JONG6, JAMES E. WO- oping into various cells including germline cells. MACK7, HARRIS A. LEWIN1 In this study, blastodermal cells isolated from 1University of Illinois at Urbana-Champaign, fertilized White Leghorn eggs at stage X were Urbana, IL, US; 2British Columbia Cancer Agen- labeled or not with PKH26, and microinjected cy Genome Sciences Centre, Vancouver, BC, Ca- respectively into subgerminal cavities of Maya nada; 3University of Alberta, Edmonton Alberta, Duck eggs at the identical stage. In the PKH26- Canada; 4Agriculture and Agri-Food Canada, labeled group, gonads of 8-days-old embryos we- Lethbridge Research Center, Lethbridge, Alberta, re examined under a fluorescent microscope, and Canada; 5USDA-MARC, Clay Center, NE, USA; PKH26 positive cells could be observed in 30 out 6Children’s Hospital Oakland Research Institute, of 71 embryos (42.3 %). In the unlabeled group, Oakland CA, USA, 7Texas A & M University, brains and main visceral tissues isolated from the College Station, TX, USA treated embryos, were sectioned and detected by An international consortium has been formed in in situ hybridization using W chromosome- order to create a comparatively anchored bacterial specific repetitive sequences of chicken as DNA artificial chromosome (BAC) map of the cattle probe, the donor-derived cells could be found in genome. To date >130,000 clones have been fin- all these tissues, with the chimeric rate highest in gerprinted from the CHORI-240 and RPCI-42 liver at 40.9 %, lesser in muscular stomach, kid- cattle BAC libraries. A total of 14,844 contigs ney, heart and brain, and lowest in gonad at 11.4 have been identified. BAC-end sequencing has % (5/44). been performed on >10,000 ends. The finger- The results showed that the exogenous chicken printing and end-sequencing results are being blastodermal cells could incorporate into the de- integrated to produce a comparatively-anchored velopment of duck embryos, especially in the whole-genome physical map. The cattle BAC-end germinal system. It suggested it was possible to sequences are being anchored to the human ge- yield the germline chimera by the method of bla- nome by BLASTN similarity search against the stodermal cells transplantation. draft human genome sequence. Approximately

30 Section A: Genome technologies

A014 extensively characterized human syntenic region Construction of a 3.5X genome-coverage physi- on HSA 19q13.13 were used as starting points. By cal map of the porcine genome by BAC finger- a chromosome walking strategy gaps between printing clones could then be closed and a single contig of 59 clones was obtained. During the construction BRANDY M. MARRON AND JONATHAN E. of the contig 54 new sequence tagged site (STS) BEEVER markers were generated. Detailed physical map- University of Illinois, Department of Animal Sci- ping of this gene-rich region allowed the assign- ences, Urbana, Illinois, USA ment of 17 porcine genes orthologous to known The identification of genes that influence eco- human chromosome 19 genes to this contig. Ex- nomically important traits or ETL, has been the cept for the relatively well characterized porcine driving force for the development of genome RYR1 gene the other 16 genes represent novel maps in our agricultural species. To this end, the chromosomal assignments and 14 genes have genome map of the pig contains sufficient genetic been cloned for the first time in pig. Comparative markers to conduct genome scans for these ETL. analysis of the porcine BAC/PAC contig with the As a result of these studies, we are finding an human HSA 19q13.13 map revealed a completely increasing need to fine-map regions of the ge- conserved gene order of this segment between pig nome for the eventual identification of the under- and human. The compilation of a detailed porcine- lying gene or polymorphism causing the effect. human comparative map might help to resolve Restriction endonuclease fingerprinting of large- existing discrepancies between the currently insert clones, such as BACs, has been utilized in available human HSA 19 maps. the production of high-resolution genome-wide physical maps in organisms such as C. elegans, A016 humans and mice. The RPCI-44 BAC library, A putative gene therapy vector for prevention consisting of approximately 10.2X genome of transmisible spongiform encephalopathy equivalents, was used to construct a physical map (TSE) of the porcine genome by fingerprinting. DNA from approximately 70,000 BAC clones was iso- JAVIER MIANA-MENA1, JESÚS CIRIZA1, lated and digested with Hind III, followed by JULIO POZA1, INMACULADA MARTÍN- separation of the fragments by agarose gel elec- BURRIEL I.1,2, MARIA JESÚS MUÑOZ3, trophoresis and visualization by fluorescence im- CLEMENTINA RODELLAR1, PILAR ZARA- aging using SBYR Green I nucleic acid stain. GOZA1, ROSARIO OSTA1 Gel images were analyzed using IMAGE and 1Universidad de Zaragoza, Laboratorio de Gené- fingerprints were assembled into contigs using tica Bioquímica y Grupos Sanguíneos, Zaragoza, FPC. This high-resolution physical map will pro- España; 2Centro Nacional de Encefalopatías vide the foundation for fine-mapping of ETL and Espongiformes, Zaragoza, España; 3Universidad is the initial step towards a complete high density, de Zaragoza, Departamento de Farmacología, sequence ready map of the porcine genome. Zaragoza, España The Transmissible Spongiform Encephalopathies A015 (TSEs) are fatal, neurodegenerative diseases for A BAC/PAC contig of the porcine RYR1 gene which no effective treatments are available. Sev- region on SSC 6q1.2 and comparative analysis eral studies have suggested that gene therapy with with HSA 19q13.13 mutant PrP may be effective in preventing TSE diseases. Moreover certain antibodies anti-PrP FLÁVIA MARTINS-WEß1, RODJA VOß- have recently been shown to block in vitro prion NEMITZ1, CORD DRÖGEMÜLLER1, replication and disemination. We have considered BERTRAM BRENIG2, TOSSO LEEB1 that specific systems to deliver this therapeutic 1School of Veterinary Medicine Hannover, Insti- agents to the neurons should be developed. In vivo tute of Animal Breeding and Genetics, Hannover, gene transfer has been explored as an invasive German; 2University of Göttingen, Institute of method to deliver enzymatic activities to the brain Veterinary Medicine, Göttingen, Germany using viral vectors, which are efficient in trans- To generate a detailed physical map of the ryano- ducing cells. However safety concerns regarding dine receptor 1 gene (RYR1) region on SSC 6q1.2 the use of virus in humans make non viral deliv- a porcine bacterial artificial chromosome (BAC) ery systems an attractive alternative. Fragment C and a P1 derived artificial chromosome (PAC) of tetanus toxin (TTC) retains specific nerve cell library were screened resulting in a sequence- binding and transport properties of the holotoxin, ready ~1.2 Mb BAC/PAC contig. For the library lacking any toxicity. At this work we have used screenings several heterologous probes from the the naked DNA encoding ß-galactosidase-TTC

31 Section A: Genome technologies hybrid protein to transfect muscle cells in vivo. An School of Veterinary Science, Oslo, Norway, intramuscular injection of naked DNA resulted in 3GENO, Hamar, Norway a selective gene transfer of the enzymatic activity A group of in vivo produced preimplantation cat- to the motoneurons and their connections. Label- tle embryos frozen during 1992-1993 by GENO, ling in the motoneurons and motor cortex was the Norwegian cattle breeding and AI association, observed from 4 days postinjection whereas the β- was in 1999 released for research. Twenty of galactosidase expression was maintained for 60 these embryos, representing the stages from mo- days. We propose a gene therapy approach based rula to expanded blastocyst were chosen for con- on TTC hybrid proteins which appears to be a struction of a cDNA library. The pool of 20 em- feasible method of delivering a biological function bryos constituted about 5000 cells in total. This to the CNS for TSE therapy. yielded a fairly low amount of mRNA and the library was constructed by utilizing the „PCR A017 cDNA Library Construction Kit“ from Stratagen. Development of a novel homologous promoter Obtained average insert size is 800-900 basepairs. for expression of a recombinant glycoprotein in Initially 8000 clones were gridded in microtiter- Chinese Hamster Ovary cells. plates, and 2300 clones have at present been se- quenced. About 30% of the sequenced clones are CATHY NOONE, TERRY SMITH, MICHAEL unique and the rest (70%) are represented by on CAIRNS average, 4-5 copies. Preliminary homology National University of Ireland, Galway searches (BLAST) with a subset of the sequences Globally there is widespread use of CHO cell revealed that 20% contains SINE and / or LINE culture in the production of recombinant proteins. repeats and 30% gives match to known genes and The advantage of the CHO system is its ability to / or ESTs (mainly human and bovine). The rest achieve authentic glycosylation of products, un- seems to represent not yet described genes, but like bacterial, fungal or yeast systems. Without homology searches against the human genome this post-translational modification recombinant sequence has not yet been performed. This library products do not mimic the natural molecule. The should constitute a valuable resource for identifi- aim of this project is to improve upon current cation of genes involved in the early stages of high-level expression systems by the replacement cattle development. Updated results will be pre- of viral promoters with homologous CHO pro- sented. moters. A CHO genomic library was screened with a number of different probes derived from A019 cDNA’s of genes known to be highly expressed in Generation of dominant-negative murine CHO cells. One clone of 7Kb has been fully myostatin alleles sequenced and analysed by web-based promoter analysis programs (e.g. TESS). Promoter frag- DIMITRI PIROTTIN, LUC GROBET, ments have been cloned into the pGL3-Basic re- DOMINIQUE PONCELET, LUIS ROYO, porter vector and characterization of these, in BENOÎT BROUWERS, MICHEL GEORGES comparison to viral SV40 and CMV promoters, Department of Molecular Genetics, Faculty of has been carried out using a luciferase reporter Veterinary Medicine, University of Liege, B43, 20 assay. The promoter is currently being incorpo- Boulevard de Colonster, 4000, Liege, Belgium rated into a two-vector system expressing the bo- Loss of myostatin function in the mouse and in vine follicle stimulating hormone genes, with the several cattle breeds leads to a dramatic increase aim of producing and purifying recombinant FSH, in skeletal muscle mass. Myostatin is a member of which can be used for superovulation treatment of the TGF-beta superfamily of proteins which are cows. secreted as disulfide-bounded polypeptides and characterized by a proteolytic processing site A018 thought to mediate cleavage of the bioactive C- Construction of a cDNA library from preim- terminal domain from the N-terminal latency- plantation cattle embryos associated fragment. All loss of function mutations identified in cattle are recessive. Our INGRID OLSAKER1, KRISTINE G. aim is to produce potentially dominant-negative GAUSTAD1, WENCHE FARSTAD2, ELISA- variants of myostatin and to test them by a BETH KOMMISRUD3 transgenic approach using homologous 1Department of Morphology, Genetics and Aqua- recombination in ES cells. Four regions of the tic Biology, The Norwegian School of Veterinary protein were targeted : the proteolytic cleavage Science, Oslo, Norway, 2Department of Repro- site, the putative glycosylation site, a residue duction and Forensic Medicine, The Norwegian potentially involved in receptor binding and a

32 Section A: Genome technologies highly conserved sequence of 8 amino-acids in the did not find a homologous sequence in V28 the latency associated fragment. In order to increase most recent human genome assembly (Dec 24th, the efficiency of the recombination events, we 2001). This is indicative of assembly gaps in the have generated a murine ES cell line carrying a HGS. The comparative map covers 3,290MB, or floxed myostatin allele allowing repeated Cre- 98% of the presumed size of the human genome. recombinase mediated cassette exchange. Six porcine chromosomes, SSC2, 5, 6, 7, 12 and However, because of the relatively high efficiency 14 are syntenic with the three gene-richest human of homologous recombination at the myostatin chromosomes, HSA17, 19 and 22. Pig chromo- compared to a very inefficient recombinase somes 1, 8, 11 and X display a low DNA/marker mediated cassette exchange, all mutated ES cell ratio are syntenic with the ‘genome deserts’ clones were generated by classical homologous HSA18, 4, 13 and X. recombination. Germline chimeras have been produced from ES cell-diploid embryo A021 aggregation experiments for two of the four Construction of a canine BAC-library compa- variants until now and phenotypes of F2 mice are tible with PCR analysis being analysed. CLAUDE SCHELLING1, JÖRG SCHLÄPFER2, A020 ALAIN BILLAUT3, KARINA GUZIEWICZ2, A second generation EST radiation hybrid BENITA PINEROLI 1, ANDREAS GMÜR2, IRIS comparative map of the Porcine genome KATMANN2, OLIVER RICKLI2, CHRISTINE WITTWER2, ANGNIESZKA PIASECKA1, ANETTE RINK1, ELISABETH M. SANTSCHI2, BRIGITTA COLOMB2, GAUDENZ DOLF2 KIM A. VONNAHME3, KATIE E. EYER1, BEN- 1Federal Institute of Technology and Faculty of JAMIN M. ROELOFS1, KATHLEEN J. SHAR- Veterinary Medicine, Department of Animal Sci- KEY1, SUDKAMON LEKHONG1, ERIN F. ences, Zurich, Switzerland, 2University of Berne, MURPHY1, STEPHEN P. FORD3, MARTINE Institute of Animal Genetics, Nutrition and Hou- YERLE4, DENIS MILAN4, CRAIG W. BEAT- sing, Berne, Switzerland; 3Molecular Engines TIE1 Laboratories, Paris, France 1Department of Animal Biotechnology, University A canine Bacterial Artificial Chromosome (BAC) of Nevada, Reno, NV 89557, USA, 2Department of library was constructed from a male white shep- Surgical Sciences, School of Veterinary Medicine, herd. Approximately 96'768 clones were stored in University of Wisconsin, Madison, WI 53706, 96-well microtiter plates and aliquots were or- USA; 3Department of Animal Science, University ganized in a three-dimensional pooling system to of Wyoming, Laramie, WY 82071, USA; 4Institut prepare DNA ready for screening by PCR. The 42 National de la Recherche Agronomique, Labora- superpools each contained 24 plates of 96 clones, toire de Genetique Cellulaire, Castanet-Tolosan, that is, a total of 2'304 clones. Each superpool is France represented by 8 row pools, 12 column pools and Expressed Sequence Tag (EST) based radiation 24 plate pools. The screening of the library by hybrid maps are powerful tools for QTL identifi- PCR requires a total of 86 reactions plus appropri- cation or positional candidate approaches. We ate controls. This two-step procedure involves an have constructed a second-generation EST radia- initial screening of 42 superpool DNAs, followed tion hybrid comparative map of the porcine ge- by screening of 24 plate pool DNAs, 8 row pool nome by ordering >2,000 ESTs from immune DNAs and 12 column pool DNAs of the initially tissues and placentae on the IMpRH7000 panel and identified superpool. The library was screened identified the respective location for each human with 111 microsatellites representing all canine orthologue. Chromosomal maps were constructed chromosomes. Eighty-six primer sets (85 %) with a 2pt LOD of 6 or 8. The number of ob- yielded between 1 and 7 positive clones. In order served/expected markers per chromosome ranged to increase the genomic coverage the library will from 0.34 for SSCX to 2.06 for SSC12, respec- be expanded by adding another 30 superpools tively. Average marker retention frequency was representing different canine genomic insert sizes. 30.3%. Annotated ESTs represent 47% of the Interested researchers can access the library fol- markers, 30% ESTs showed homology to ESTs lowing the rules published at from a variety of species, 20% represent novel http://www.dogmap.ch. sequences (ns) with no significant match in any nucleotide or protein database, 3% of the ESTs only identify open reading frames (ORFs) in hu- man genomic sequence (HGS). More than 20% of the ESTs assigned to the physical map of the pig

33 Section A: Genome technologies

A022 nondai, Tsukuba, Ibaraki, 305-8602, Japan Recombineering of Porcine BACs for Somatic We are carrying out EST analysis produced from Cell Genomics porcine back fat cDNA. Assignment of those ESTs to the porcine map will facilitate compre- MARGARITA B. ROGATCHEVA1, LAURIE A. hensive mapping and construction of a compara- RUND1, JONATHAN E. BEEVER1, CHRISTO- tive map based on the human information. Radia- PHER M. COUNTER2, LAWRENCE B. tion hybrid (RH) panel is the most convenient and SCHOOK1 efficient tool for mapping non-polymorphic mark- 1University of Illinois, Department of Animal Sci- ers, such as EST. By fusing normal porcine aortic ences, Urbana, Illinois; 2DukeUniversity Medical endothelial cells (Cell Systems Corp. WA, USA), Center, Department of Pharmacology and Cancer which were irradiated with 7000 to 8000 rads, Biology, Durham, North Carolina, USA with recipient mouse cells, L-M (TK-)(ATCC; In current model species, functional genomics is CCL1.3), we established 110 hybrid cell lines. supported by the ability to develop individuals Among 1091 microsatellite (MS) markers investi- possessing specific modifications either by dele- gated for construction of framework map, we tion or substitution of genes. In pigs, the tools could type 842 markers (77 %) based on the 110 required to create such animals are lacking. Thus, clones. According to the typing data, we built a there is a need to develop in vitro correlates that framework map including 390 MS markers with will allow the validation of hypotheses with re- RHMAPPER-1.22 Z-extension program (Stein, spect to allelic variation and gene-gene interac- Kruglyak, Slonim, Lander, 1995, Whitehead In- tions associated with multigenic traits. It also stitute/MIT Center for Genome Research). After would be ideal that such an experimental system integrating additional 240 MS markers to the allowed the introgression of “alleles” into germ- framework map (placement map), this RH maß plasm. Using the recombigenic E. coli strain was comprised of 630 MS markers. The estimated DY380, we have rapidly generated multiple ge- average retention frequency of the RH panel was netic modifications in BAC clones containing the 30.6 %. We are presently mapping porcine back porcine myostatin gene. Single-stranded oligonu- fat ESTs on the framework map. cleotides and PCR-derived DNA fragments were used for targeting and generation of a point muta- A025 tion, a 68-bp deletion, and a 24-bp in-frame inser- Construction and characterization of a 15 fold tion of the sequence encoding the FLAG octapep- redundant BAC library of Salmo salar (Atlan- tide into exon 3 of pig myostatin. Modified BACs tic salmon) are identified by allele-specific PCR amplification from pooled bacterial cells. Individual targeted JIM THORSEN1,4, WILLIAM S. DAVIDSON2, clones were identified within positive pools and BEN KOOP3, BJ∅RN H∅YHEIM1 & PIETER J. sequenced to confirm the mutations. The ob- DE JONG4 served rate of modification is one per 170 to 400 1Norwegian School of Veterinary Sciences, Oslo, surviving cells. Constructs generated by this ap- Norway, 2 Simon Fraser University, Burnaby, proach can be used to develop specifically modi- Canada 3University of Victoria, Victoria, Canada fied somatic cells for gain-of-function [knock-in 4BACPAC Resources, Children’s Hospital of including either new functions or allelic substitu- Oakland Research Institute, Oakland, USA tion] or loss-of-function [knock-out] studies. The BAC (Bacterial Artificial Chromosome) libraries promise of such an approach in cell culture [with- have been an important and essential resource for out the complexity of whole animal models] is the genome studies, such as sequencing of whole ability to link genetic variation with rigorous ge- genomes, physical mapping and in reconstruction netic analyses [e.g., microarray expression profil- and positional cloning of genomic regions. The ing] to assess gene function. salmon BAC library was constructed using high molecular weight DNA prepared from sperm of a A023 single anonymous male. The genomic DNA pre- Establishment of the procine radiation bybrid pared was partially digested with EcoRI in the panel and construction of the framework map presence of EcoRI Methylase and separated by pseudo-double size fractionation using PFGE HIDEAKI SUZUKI1; AYUMI MIKAWA1, TA- (pulsed-field gel electrophoresis). Size selected KEYA MOROZUMI1, TADAYOSHI MITSU- DNA was then electro-eluted and ligated to the HASHI2, NORIYUKI HAMASIMA1 EcoRI sites in the pTARBAC2.1 vector. Ligation 1STAFF-institute, 446-1 Ippaizuka, Kamiyokoba, products were transformed into electro-competent Tsukuba, Ibaraki, 305-0854, Japan, 2National T1-resistant DH10B cells. A total of 300.000 clo- Institute of Agrobiological Sciences, 2-1-2 Kan- nes, representing 15 fold genome coverage, have

34 Section A: Genome technologies been generated and arrayed into 384-well micro- A027 titer dishes, and spotted onto 22x22cm nylon ChickRH6: a chicken radiation hybrid panel, high-density colony filters. Each hybridization and its use for improving the resolution of membrane represents over 18,000 distinct salmon GGA7 and GGA14 comparative maps BAC clones, stamped in duplicate. The clones in the library have an average insert size ranging MIREILLE MORISSON, CARINE JIGUET- from 170 kb to 197 kb with a none-insert rate of JIGLAIRE, THOMAS FARAUT, SUZANNE 2-3%. More than 20 oligo probes, including BARDES, KATIA FÈVE, MARTINE YERLE, micro-satellites and ESTs (expressed sequence ALAIN VIGNAL tags), have been chosen for screening of the high- INRA, Laboratoire de Génétique Cellulaire, density filters to obtain BACs for use in further Castanet-Tolosan, France characterization, e.g. finger-printing analysis and Previous reports on the possibilities of generating contig assembly. The BAC library will be publicly a chicken whole genome radiation hybrid map- available from BACPAC Resources. ping panel by fusion with rodent recipient cells, were rather pessimistic due to low retention fre- A026 quencies of chicken chromosome fragments in Strategy for efficient isolation of microsatellites most clones. However, we were successful in developing a panel of 90 clones, by applying a TERUAKI TOZAKI1, SUGURU MASHIMA1, drastic selection based on marker retention tested SHUN-ICHI NAGATA1, KEI-ICHI HIROTA1, with 46 markers chosen genome-wide, over 452 MASAHIKO KUROSAWA1, TELHISA HASE- hybrids produced by fusing female chicken fibro- GAWA2 blasts irradiated at 6,000 rads to HPRT-deficient 1Laboratory of Racing Chemistry, Utsunomiya, hamster cells. The average retention frequencies Japan; 2Equine Research Institute, Japan Racing in this 90 clones panel, are of 22.0% for the whole Association, Utsunomiya, Japan genome, 20.1% for macrochromosomes and The linkage maps will serve as very useful tools 25.7% for microchromosomes. To estimate the for tracing genes governing economically signifi- validity of the panel for mapping, we are cur- cant traits. It is necessary to isolate many micro- rently constructing radiation maps of macrochro- satellites for the purpose. In this study, we draw mosome 7 (GGA7) and microchromosome 14 the strategy for efficient isolation of microsatel- (GGA14). According to the state of the art of lites. The strategy contains (I) construction of an comparative maps, human genes located in the enrichment library, (II) high speed sequencing, corresponding human regions (HSA2q, and (III) genotyping using so-called “pig tail HSA16q13-3) are used to search, on a sequence PCR” with an adaptor sequence designed newly. similarity basis, for candidate orthologous chicken The library was constructed by following; capture ESTs. These ESTs, likely to map to GGA7 and microsatellites from genomic DNA by hybridizing GGA14, are used to design primers for the map- to biotin-conjugated probes, subsequent extraction ping experiment on the radiation hybrid panel. with magnetic beads coated with streptavidin, This computer assisted primer design is part of a nucleotide substrate-biased polymerase reaction, computer tool, ICCARE (Interspecific Compara- and amplification by PCR followed by cloning. tive Clustering and Annotation foR ESTs), devel- Isolated microsatellites were treated from culture oped in our laboratory. to sequencing with 96-well format. The genotyp- ing method used three primers of a sequence- A028 specific forward primer with the adaptor sequence BAC map of the chicken genome at its 5’ end, a sequence-specific reverse primer, and a fluorescent-labeled adaptor primer, and was JAN AERTS, RICHARD CROOIJMANS, SAN- performed with one reaction. In this study, we DRA CORNELISSEN, adapted the strategy to equine genome, resulting KAVEH HEMMATIAN, JAN VAN DER POEL, in isolation of 624 novel microsatellites. About MARTIEN GROENEN 20% of the microsatellites showed some homolo- Wageningen University, Animal Breeding and gous groups, such as ERE-1 and ERE-2. MLRE Genetics Group, Wageningen, The Netherlands (microsatellite-linked repetitive elements) was The Wageningen chicken BAC library contains characterized as a novel homologous group. The approximately 50,000 BACs, representing a 5.57- results proved the efficiency of our strategy in this fold redundant coverage of the chicken genome. A study. Once applied, the strategy will accelerate physical map of BAC contigs is created using the construction of linkage maps in many species. fingerprinting technique. The BAC DNAs are digested with the HindIII restriction enzyme. The band patterns are scanned and loaded into the

35 Section A: Genome technologies

Image program (Sanger Institute) to perform the RICHARD CROOIJMANS1, LAURIE band calling. To build contigs of these BACs, the GORDON2, SANDRA CORNELISSEN1, TARIK resulting band data are imported into the FPC RABIE1, JAN VAN DER POEL1, MIREILLE program (FingerPrinted Contigs, Sanger Institute). MORISSON3, ALAIN VIGNAL3, LISA As in the Human Genome project, the resulting STUBBS2, MARTIEN GROENEN1 number of contigs is high. Therefore, the finger- 1Wageningen University, Animal Breeding and printing results are combined with the results of a Genetics Group, Wageningen, The Netherlands; marker screening of the BAC library. A large 2D.O.E. Joint Genome Institute, Walnut Creek and repository of microsatellite markers is available, Genomics Division, Biology and Biotechnology with a known location on the linkage, cytogenetic Research Program, Lawrence Livermore National or radiation hybrid map. Following a two dimen- Laboratory, Livermore, California, USA; 3Institut sional PCR screening, BACs are identified that National de la Recherche Agronomique, Labora- contain these specific markers. The contigs cre- toire de Génétique Cellulaire INRA, Castanet- ated by the fingerprinting can be positioned rela- Tolosan, France tive to each other by incorporating this new in- Extensive conservation of synteny is observed formation. The 5' and 3' ends of the contigs will between the chicken and human genomes, which be sequenced and new markers will be con- is particularly apparent for many of the small mi- structed, followed by additional 2D PCR screen- cro-chromosomes in chicken such as GGA28. ing. This will allow for the anchoring of addi- Mapping of over 40 genes to chicken chromo- tional contigs on the existing maps of the chicken some 28 indicates that this chicken chromosome genome. over almost its entire length is syntenic to human chromosome 19p. A029 Thirteen BAC contigs covering over 7 Mbp of this Characterization of 10,000 canine ESTs chromosome have been constructed and aligned with the linkage and radiation hybrid maps. BAC H. MURUA ESCOBAR1, L. BORRMANN1, R. contig construction was initiated from markers NIMZYK1, J. BULLERDIEK1, known to be located on chromosome 28 and ex- I. NOLTE2 tended by chromosome walking. Additional BACs 1 Center for Human Genetics, University of Bre- and BAC contigs were identified using chicken men, Bremen, Germany; 2Clinic for Small Ani- EST sequences homologous to genes located on mals, Veterinary School, Hanover, Germany human chromosome 19p and by fingerprinting all During the past years the dog has become a more BACs from the Wageningen chicken BAC library and more interesting model organism for human (see abstract of Aerts et al.). Verification of the diseases including cancer. Nevertheless, molecu- chromosomal location of these additional BACs lar genetic tools allowing a more advanced was done by two-colour FISH or by radiation knowledge of canine molecular genetics are far hybrid mapping. This enabled a detailed align- behind of what is known for humans. ment of GGA28 with human chromosome 19p The representation of canine expressed sequence and resulted in the identification of multiple rear- tags (ESTs) at the NCBI database reflects the rangements on this chromosome relative to human current situation. The NCBI EST database con- and mouse. Representative BACs that constitute a tains currently (March 2002) roughly 11,000,000 minimal tiling path have been selected for se- entries covering all organisms. Compared to this quencing by the Joint Genome Institute in Walnut number, the dog stays clearly underrepresented Creek, California, USA. with just 7301 entries. Herein we present first results of our program A031 aimed at large scale sequencing of canine cDNAs. A BAC map covering 90% of chicken chromo- So far 10,000 cDNA clones have been sequenced some 10 thus almost doubling the number of available ca- nine ESTs. Furthermore, the results reveal that RICHARD CROOIJMANS1, ROSILDE based on the gene level the homology between DIJKHOF1, TINEKE VEENENDAAL1, men and dogs is much higher than estimated so MIREILLE MORISSON2, JAN VAN DER far. POEL1, ALAIN VIGNAL2; MARTIEN GROE- NEN1 A030 1Wageningen University, Animal Breeding and Comparative mapping and sequencing between Genetics Group, Wageningen, The Netherlands; chicken chromosome 28 and human chromo- 2INRA, Laboratoire de génétique Cellulaire, some 19p Castanet-Tolosan, France Chicken chromosome 10 (GGA10), one of the

36 Section A: Genome technologies larger microchromosomes and approximately 30 and Y at a LOD > 4.0. Assignment for each lin- Mb in size, is one of the best studied chromo- kage group was based on the USDA bovine linka- somes in chicken. The genetic markers known to ge map. Our initial map forms a frame for map- be located to this chromosome were used for BAC ping bovine genes and ESTs. contig mapping. Additional BACs were identified by chromosome walking and using STS markers A034 derived from chicken EST sequences. The A cost effective method for the production of chicken EST sequences were selected based on small gene number canine macroarrays known GGA10-HSA15 conserved synteny. Map locations of these BACs were confirmed either by A. RICHTER1, H. MURUA ESCOBAR1, B. FISH, SNP typing in a reference population or by MEYER1, K. BECKER1, I. NOLTE2, mapping on the chicken RH-panel. This resulted J. BULLERDIEK1 in the development of over 450 STS markers and 1Center for Human Genetics, University of Bre- the isolation of more than 900 BAC clones. To men, Bremen, Germany; 2Clinic for Small Ani- further increase the BAC coverage of this chro- mals, Veterinary School, Hanover, Germany mosome, BAC fingerprint information from Im- DNA hybridisation arrays are one of the major age and FPC of the Wageningen BAC library was trends in molecular biology. Covering a wide used (see abstract Aerts et al.). These combina- range of topics including cancer research, arrays tions of approaches resulted in the identification are being used in gene expression profiling and of BACs for GGA10 that cover 25 Mb of se- genotyping. While the domestic dog (Canis fa- quences, which is close to 90% of this chromo- miliaris) is supposed to join the company of some. model organisms for human disease, canine DNA arrays are still less common, often making the in- A033 house production of custom arrays necessary. Construction of a framework map of the Shi- High-density DNA arrays allow the investigation rakawa/University of Nevada Reno bovine Ra- of a large number of genes per experiment, but are diation Hybrid (SUN-bRH7) panel relatively costly due to the technical equipment needed for their production and analysis of ex- TOMOHITO ITOH1, TOSHIO WATANABE1, perimental data, thus limiting their availability for NAOYA IHARA1, CRAIG W. BAETTIE2; many laboratories. In this study, we evaluated the YOSHIKAZU SUGIMOTO1 production of low cost, small gene number canine 1Shirakawa Inst. Animal Genet., Fukushima, Ja- nylon arrays that can be made and analysed with pan; 2University of Nevada, Reno, USA common laboratory equipment. DNA of randomly Whole-genome radiation hybrid (WG-RH) panels chosen clones from a canine testis tissue cDNA have widely been used for localizing genes and library was amplified using different approaches, anchoring physical maps. In order to map bovine the resulting products (being whole plasmids, genes and ESTs, we have prepared a 7000 rad inserts isolated from plasmids and PCR products) bovine-hamster WG-RH panel (Mariani et al, 27th hand-spotted on nylon membranes. Hybridisation ISAG). Here we report the characterization of the conditions were optimised using 32P-labelled Shirakawa/University of Nevada Reno bovine cDNA from a canine mammary tumour cell line. Radiation Hybrid (SUN-bRH7) panel and provide Array analysis was conducted using standard an initial RH map of the bovine genome. laboratory techniques and PC imaging software. Retention frequencies of bovine chromosomes While whole plasmids were prone to unspecific were estimated by Genescan analysis of microsa- hybridisation, inserts and PCR products showed tellite loci with an ABI 3700 DNA sequencer. specific signals. PCR products amplified from The framework map was created using the bacterial clones appeared most suited due to re- RH2PT, RHMAXLIK program of RHMAP 3.0 duced production steps and cost. with a LOD threshold of four. Finally, this order was used the create placement map option of A035 RHMAPPER. Genetic and functional characterization of a To construct an RH map providing comprehensive candidate mutation for callipyge in sheep coverage of the bovine genome, we selected more than 1400 microsatellites from the USDA bovine MARIA A. SMIT(1), CAROLE CHARLIER(2), linkage map and used to amplify the SUN-bRH7 KARIN SEGERS(2), FRANCESCA BARALDI(2), panel comprising 92 hybrid clones. An average TRACY L. SHAY(1), FRÉDÉRIQUE CER- retention frequency of 19% was calculated with QUEIRA(3), ROBERT FEIL(3), GARY D. 1000 scorable markers. The RH map programs SNOWDER(4), CHRISTOPHER A. BIDWELL(5), established linkage groups covering BTA1-29, X

37 Section A: Genome technologies

MICHEL GEORGES(2) & NOELLE E. COK- AGENA - devoted to farm animals (cattle, pig, KETT(1) trout and chicken) dealing with genome structure, (1)Utah State University, Department of Animal, functional genomics and bio-informatics. We Dairy, and Veterinary Sciences, Logan, UT, USA, present here the construction of multi-tissue (2)University of Liège, Department of Genetics, cDNA libraries for chickens and pigs as a first Liège, Belgium, (3)Institut de Génétique Molécu- step in functional genomics. Total RNA from a laire, Montpellier, France, (4)USDA/ARS, U.S. large set of tissues (up to 42) from animals at dif- Meat Animal Research Center, Clay Center, NE, ferent stages of development or in different USA and (5)Purdue University, Department of physiological conditions were collected. Standard Animal and Poultry Sciences, West Lafayette, IN, multi-tissue libraries were made from 5 to 9 dif- USA ferent tissues. These libraries were mixed and the In an attempt to identify the causative callipyge pool was normalized: 46 x 106 clones (average (CLPG) mutation, we and others have recently insert length 900 bp, empty clones < 2%) were sequenced 180 kb spanning an imprinted cluster obtained for chickens and 6.4 x106 clones (aver- of genes whose expression pattern is altered by age insert length 1000 pb, 2% empty clones) for the CLPG mutation. The only CLPG-specific pigs. First data obtained by hybridization or spe- mutation identified was a single nucleotide poly- cific amplification of either actin or exogenous morphism (SNP) that lies within a conserved in- sequences indicated that the normalization process tergenic region of the imprinted domain. In the was successful. Sequencing (3’ and 5’ ends) of the present study, we tested the reported callipyge libraries is now underway. In order to reduce the founder ram (“Solid Gold”) for the presence of the residual redundancy, the sequencing strategy will SNP. Interestingly, Solid Gold was found to be be iterative: sequencing of a set of 5 to 10,000 heterozygous at the SNP site, but with a greater clones will be followed by subtraction of these proportion of the wild-type allele than the CLPG- clones from the starting library. 200,000 ESTs are specific mutation. Solid Gold was then genotyped planned. The clones will be spotted onto mem- for microsatellite markers known to be in com- branes and microarrays for gene expression stud- plete linkage disequilibrium with the CLPG allele, ies. All EST sequence data management, cluster- and he carried the marker haplotype correspond- ing, submission to public databases and annota- ing to that of his callipyge descendants. These tion will be achieved using the specially in-house results lead us to suggest that the mutation event designed bioinformatics software package SI- creating the SNP occurred in Solid Gold during GENA. early embryonic development, resulting in germ- line and somatic tissue mosaicism at the SNP A037 position. This finding further supports the hy- Generation of a 12,000 rads radiation hybrid pothesis that the newly identified SNP is the panel for fine mapping in pig. First compari- causative CLPG mutation. We predicted that if sons between IMpRH (7,000 rads) and the SNP is causative and functions as part of a IMNpRH2 (12,000 rads) regulatory element, this would result in a pertur- bation of its epigenetic conformation. Preliminary MARTINE YERLE, PHILIPPE PINTON, results from bisulfite sequencing and chromatin CHANTAL DELCROS, NADÈGE ARNAL, immunoprecipitation experiments indicate that DENIS MILAN, ANNIE ROBIE there is an allele-specific epigenetic mark at the Institut National de la Recherche Agronomique, SNP site. Laboratoire de Génétique Cellulaire, Castanet- Tolosan, France A036 We have constructed a 12,000 rads porcine Chicken and Pig Normalised Multi-Tissue whole-genome radiation hybrid panel to com- cDNA Libraries plement thefirst generation 7,000 rads panel (IMpRH) and allow higher resolution mapping A. BONNET1, F. HÉRAULT2, G. TOSSER- studies both in specific areas of interest and on KLOPP1, V. LE MEUTH-METZINGER2, F. the whole genome. We have analyzed 243 hy- BENNE1, C. DÉSERT2, C. CABAU1, S. VILLE- brid clones produced, on the basis of their ge- GER1, M.B. SOARES3, F. BONALDO3, M. nome retention frequency to constitute a final DOUAIRE2, panel of 90 hybrid clones with an average reten- F. HATEY1 tion frequency of 35.4%. The resolution of this 1INRA Laboratoire de Génétique Cellulaire 12,000 rads panel (IMNpRH2) was compared to Castanet-Tolosan France; 2INRA-ENSA Rennes the resolution of 7,000 rads panel (IMpRH) by France; 3The University of Iowa Iowa City USA constructing framework maps in the 2.4 Mb re- INRA is carrying on a genomic programme - gion of porcine chromosome 15 containing the

38 Section A: Genome technologies acid meat RN gene. In this region, two-point S. WINKLER2, H. MURUA ESCOBAR1, J.T. analysis was used to estimate RH distances and SOLLER1, K.CULMSEE2, demonstrated their reliability with the estimation J. BULLERDIEK1, I. NOLTE2 of physical distances. This study demonstrates 1Center for Human Genetics, University of Bre- that the 12,000 rads panel constitutes a powerful men, Bremen, Germany; 2Clinic for Small Ani- tool to construct high-resolution maps. mals, Veterinary School, Hanover, Germany IMNpRH2 (12 to 14 kb/cR12,000) is two to three The limiting factor for various histological and times more resolvent than IMpRH (35 to 37 genetic studies is the absence of the adequate tis- kb/cR7,000). As expected, the increase in the ra- sues. The establishment of a “Canine/Feline Tis- diation dose allows an increase of the mapping sue Bank” covering different races and tissue resolution in terms of kb/cR compared to types builds the base for various future studies. IMpRH, with the same suppleness of use for Samples were taken from normal and neoplastic mapping experiments. In addition the RH map tissues of dogs (~87.5 %) and cats (~12.4 %). In constructed in the region investigated proved to the past few years the dog has become an in- be more homogeneous on IMNpRH2 than on creasingly important model for genetic diseases IMpRH. affecting both species dogs and humans, e.g. hae- mophilia, narcolepsy, and various types of cancer. A040 For these diseases genetic factors are assumed to Comparative map between GGA15 and HSA12 be involved in both species. Factors like age, and HSA22 breed, sex, and hormonal reasons show involve- ment in different tumours. For example DANYEL JENNEN, BRAM KAMPS, JAN VAN Rottweilers show elevated incidence of osteosar- DER POEL, RICHARD CROOIJMANS, MAR- comas compared to Dachshunds showing reduced TIEN GROENEN appearance. Other tumours rely on sex and age Wageningen University, Animal Breeding and such as canine tumours of the mammary gland. In Genetics Group, Wageningen, The Netherlands female dogs showing adenocarcinomas of the To improve the physical and comparative map of mammary gland the age of 5 – 6 years is affected chicken chromosome 15 (GGA15, former linkage with 3.1% compared to the age of 11–12 years group E18W15) BAC contigs were constructed with 29.7%. The tissue bank currently consists of around loci previously mapped on this chromo- more than 10,000 samples taken from different some by linkage analysis. The BAC clones were tissues of 452 patients with several tumours for used for both sample sequencing and BAC end molecular examination and of tissues from 453 sequencing. STS markers derived from the BAC “healthy” patients for reference. To draw up our end sequences were used for chromosome wal- own empirical data, in addition to information king. In total 224 BAC clones were isolated, co- about type and localisation of the tumour, infor- vering almost 30 % of GGA15, and 114 STS were mation about breed, age and sex were recorded for developed (102 STS derived from BAC end se- every dog/cat that was treated because of a neo- quences and 12 STS derived from sequences plastic diseases. within genes). The partial sequences of the chik- ken BAC clones were compared to sequences A042 present in the EMBL/GenBank databases, and Development of an integrated large scale sam- revealed matches to genes, ESTs and genomic ple collection and DNA preparation system and clones located on human chromosome 12q24 and its application to paternity in sheep 22q11-q12. Furthermore 12 chicken orthologues of human genes located on HSA22q11-q12 were UWE LUKSCH1, MONIKA GUTSCHER1, directly mapped within BAC contigs of GGA15. JÖRDIS SEMMER2, GOTTFRIED BREM1, In addition 3 more genes (in total 15 genes) from JOHANNES BUITKAMP2 HSA 22 and 8 genes from HSA 12 were mapped 1Agrobiogen GmbH Larezhausen, Germany; to GGA15. These results provide a better align- 2Bayerische Landesanstalt für Tierzucht, Abt. Bio- ment of GGA15 with the corresponding regions in und Gentechnik, Poing, Germany human and mouse and identify several intra- With the development of techniques for the ge- chromosomal rearrangements between chicken notyping of individuals, there exists an increased and mammals. demand for the collection of large numbers of samples to be processed in the course of scientific A041 projects such as QTL association studies and Tumour and tissue bank for dogs and cats - practical applications, marker assisted selection base for various genetical and molecular- using bottom-up designs, or genetic diagnosis of biological studies inherited diseases. Currently the samples are

39 Section A: Genome technologies mainly collected as blood, milk, sperm, or tissue. associated with the CLPG allele and might have Each of these methods has its drawbacks, and they gone unnoticed by direct sequencing, we are are usually to cost and time consuming. To over- studying the physical integrity of the CLPG locus come these drawbacks, a system has been develo- by using dynamic molecular combing associated ped that allows the sampling of a small tissue with FISH. In this process, DNA molecules, at- probe using established -tag technology. The tached at one end, are combed on a silanized sur- TypiFix® system provides a simple, inexpensive by a receding air-water interface. Combed and reliable method (failure rate for the most pre- DNA is then hybridized to DNA probes that are cious samples is under 0,1 %) for the collection labelled with biotine or digoxigenin, and revealed and long-term storage of samples at room tempe- with fluorescent antibodies (respectively Texas rature. In combination with a new „One Step“ red or FITC). DNA isolation process (the passage of the DNA In our study, we have prepared combed DNA from through a column of newly applied absorption homozygous +/+ and CLPG/CLPG individuals. Six materials which hold back the undesired side BACs corresponding to a minimum tiling path span- components such as protein and low molecular ning the 400 Kb interval known to contain the CLPG substances and provides an average of 20 µg DNA locus were used as probes. Detailed results will be presented. at a quality level equivalent to existing systems), the preparation of a large number of samples can be archieved in a short time. Approximately 600 DNA samples have success- fully been used for the amplification of a micro- satellite set for paternity control among lambs at the test station of the Bavarian Institute of Animal Production.

A043 Monitoring the physical integrity of the cal- lipyge locus by molecular combing

Fanny Di Silvestro(1), Carole Charlier(1), Sandrine Caburet(2), Aaron Bensimon(2), Noelle Cockett(3) & Michel Georges(1)

(1)Department of Genetics, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium, (2)Laboratoire de Biophysique de l’ADN, Institut Pasteur, Paris, France and (3)Department of Animal, Dairy and Veterinary Sciences, College of Agriculture, Utah State University, Logan,USA The ovine callipyge phenotype is an inherited muscular hypertrophy subject to an unusual par- ent-of-origin effect referred to as "polar overdo- minance". The CLPG locus has been mapped to a 400 Kb interval on distal OAR18q. This interval has been shown to contain an imprinted domain comprising at least four genes: the paternally ex- pressed DLK1 and PEG11, and the maternally expressed GTL2 and MEG8 genes. We have pre- viously shown that the CLPG mutation enhances the expression of these four genes in cis without affecting their imprinting status. By resequencing 180 Kb within this interval we and others have identified an SNP that is perfectly associated with the callipyge phenotype. It is not yet known how- ever whether this SNP is the causal CLPG muta- tion. To exclude possible chromosomal rearrangements (insertions/deletions or inversions) that might be

40 Section B: MHC and Immunogenetics

Section B: P.D. Mammalian Immunogenetics Research Group, MHC and Immunogenetics Veterinary Science Faculty, University of Liver- pool, Crown Street, Liverpool L69 7ZJ, United B001 Kingdom Sequence analysis of a segment between the Normal immune surveillance depends on the abi- two swine leukocyte antigen (SLA) class I gene lity of Major Histocompatibility Complex class II clusters in the SLA class I region molecules (MHC II) to bind peptide antigens and carry them to the cell surface for presentation to ASAKO ANDO1, ATSUKE SHIGENARI1, TA- T-cells. Polymorphisms in DNA coding for the KASHI SHIINA1, CLAIRE ROGEL- MHC class II antigen binding sites can signifi- GAILLARD2, PATRICK CHARDON2, HIROSHI cantly affect antigen binding and hence disease YASUE3, HIDETOSHI INOKO1 susceptibility. The MHC class II DRA locus is 1Department of Molecular Life Science, Tokai distinct from most other class II loci, for little or University School of Medicine, Isehara, Kanaga- no sequence variation is seen in most species. wa, Japan; 2Laboratoire de Radiobiologie et This, however, is not the case with domestic equi- d’Etude du Génome, INRA-CEA, Jouy-en-Josas, dae. To date, five equine DRA alleles have been France; 3Genome Research Department, National detected by single strand conformation polymor- Institute of Agrobiological Sciences, Tsukuba, phism (SSCP) studies of DRA exon 2 in all equid Ibaraki, Japan species. Here, we report the discovery of new A contig map of the swine leukocyte antigen DRA alleles after screening a random panel of (SLA) class I region spanning about 1 Mb was equids using sequenced based typing (SBT) and constructed by alignment of YAC and BAC clo- RSCA. The future objective of our research is to nes. In this contig, 158 kb and 307 kb segments characterize Equine MHC class II genes with re- corresponding to the non-classical and classical spect to the extent of polymorphism, haplotypic SLA class I genes clusters have been recently association between alleles of different loci esta- sequenced, respectively. To obtain the entire se- blishing linkage disequilibrium, determining the quence of the SLA complex and compare the ge- distribution of alleles & haplotypes in different nomic structure between the pig and hu- breeds/equids, and to determine the functional man MHCs, we determined genomic sequences consequences and clinical relevance of ELA class of four BAC clones carrying the segment between II polymorphisms in relation to disease suscepti- the non-classical and classical SLA class I gene bility/resistance (Sarcoids, Sweet itch, Strangles, clusters by the shotgun method. These clones Grass sickness, Colic). included a total of 433 kb genome segments from the HCR gene (most centromere-side) to the FB19 B003 gene (most telomere-side) in the SLA class I regi- Structure and polymorphism of the upstream on. The genomic sequences thus obtained in swine regulatory region of bovine and equine DRB were compared to those of the corresponding hu- genes man class I segments. Nine homologues with re- 1 2 spect to the nine human genes, HCR, SPR1, SILVIANA DÍAZ , MARÍA V. RIPOLI , PILAR SEEK1, S, STG, TFIIH, DDR, FLOTLLIN, and PERAL-GARCÍA, GUILLERMO FB19 were identified in this segment. We also GIOVAMBATTISTA identified a pig homologue of the new human Universidad Nacional de La Plata, Facultad de gene represented by the cDNA clone Ciencias Veterinarias, Centro de Investigaciones (KIAA1885) which is expressed in brain. en Genética Básica y Aplicada). La Plata, Argentina [email protected] Furthermore, the length of this segment in the 1 SLA class I region was only 433 kb, as contrasted Fellow from Consejo Nacional de Investigacio- nes Científicas y Técnicas (CONICET) with as long as 582kb in the HLA class I region. 2 This remarkable difference in size between them Fellow from Universidad Nacional de La Plata could be explained by a higher density of repetiti- (UNLP) ve sequences in the human MHC. In the present work we communicate the sequence and polimorphism of the proximal upstream B002 regulatory region (URR) of bovine and equine Reference Strand Mediated Conformation DRB genes, since up to now there are not se- Analysis (RSCA) of Equine MHC Class II Loci quence information about this region of MHC class II genes in cattle and . The genomic BROWN, J.J., OLLIER, W.E.R., THOMSON, DNA was extracted from whole blood samples W., CARTER, S.D., MATTHEWS, J.B., CLEGG, corresponding to 49 bovine from 11 cattle breeds 41 Section B: MHC and Immunogenetics and 73 horses from 4 equine breeds. The URR chickens, respectively. A subtracted cDNA library was amplified by using the reverse primer de- from OS and CB thyroids was generated and used signed from the consensus among DRB sequences for differential screening. DIG labeled subtracted from different species, and as a forward primer we mixtures from OS and CB thyroids, respectively, used the oligonucleotide proposed by Turco et al., were used as hybridisation probes. All clones 1990. Six and four URR variants were detected in showing negative signals after differential scree- bovine and equine respectively through SSCP ning probably represent differentially expressed method. The PCR products were cloned and se- (2-5 times) transcripts between OS (SAT affected) quenced. The obtained DNA sequences are com- and CB thyroids. More than two hundred clones posed of highly conserved sequence motifs that were selected after this screening, sequenced and include from 5´ to 3´ direction the W, X, Y, database-searched using BLAST. Forty OS origin CCAAT and TATA-like boxes, showing the same clones and 21 CB origin clones have no match in organisation of the conserved regulatory elements the public DNA databases. No cDNA of OS ori- than previously reported DRB genes. In addition, gin was found in CB origin clones. We have iden- we observed the following: (i) the polymorphic tified several clones which are expressed only in sites were found within and between conserved the OS strain, and not in the healthy control CB sequence motifs, (ii) each DNA sequence corre- strain. Some clones showed quantitative differen- spond to a different SSCP variant, and (iii) our tial expression between the two strains. Several nucleotide sequences exhibit more identity with OS clones matched with transcripts expressed in HLA-DRB sequences than HLA-DQB sequences. macrophages (A1/OS, B15/OS), activated T lym- These evidences suggest that the sequence would phocytes (B2/OS), or during the inflammatory correspond to the bovine and equine DRB pro- process. moter. However, we are still not able to assign This work was supported by FWF grant Nr.: this sequence to a specific DRB gene neither in P12732-MED, and the BBSRC. horses nor in cattle. B005 B004 Characterisation of Major Histocompatibility Analysis of expressed sequence tags (ESTs) in Complex (MHC) FLA-DRB genes the thyroid glands of chickens suffering from in the domestic cat spontaneous autoimmune thyroiditis, an ani- mal model of human Hashimoto thyroiditis LORNA J. KENNEDY1, RUTH RYVAR1, RO- SALIND M. GASKELL1, DIANE D. ADDIE2, DUSAN VASICEK1, PETE KAISER2 KATARI- KIM WILLOUGHBY1, STUARD D. CARTER1, NA VASICKOVA1, MICHAL KOPECNY1; K. WENDY THOMSON1, WILLIAM E.R. OL- HALA1 LIER1, ALAN D. RADFORD1 1Institute for Pathophysiology, University of Inns- 1Mammalian Immunogenetics Research Group, bruck, Innsbruck, Austria; 2Institute for Animal Veterinary Science, University of Liverpool, UK; 2 Health, Compton, Berkshire RG20 7NN, UK Department of Veterinary Pathology, Bearsden The obese strain (OS) of chickens suffer from Road, Bearsden, Glasgow, UK spontaneous autoimmune thyroiditis (SAT) and To date, 71 feline leukocyte antigen (FLA) DRB provide an animal model for human Hashimoto's alleles have been identified in studies of cats in thyroiditis. SAT is regulated by approximately the USA and Japan. The number of DRB genes three autosomal genes, one of which, regulating present in the cat is not yet clear. PCR amplifica- susceptibility of the thyroid gland to autoimmune tion and DNA cloning were used to identify the attack, is recessive. So far, markers for this cha- FLA-DRB alleles in a group of 33 British domes- racteristic have not been identified. The mecha- tic cats of varying breeds from three locations nism of initiation of SAT is not yet known. It within the UK. Some cat family pedigrees were could be due either to the action of specific gene/s also analysed. Applying the strict criteria for as- products, or to differential gene/s expression in signing new alleles as used by established mam- the thyroid of disease-prone individuals. The aim malian MHC nomenclature committees, we iden- of this study was to understand the molecular tified 13 FLA-DRB alleles, including four previ- regulation of these processes. Healthy CB inbred ously unreported alleles. We found many se- strain birds were used as controls. We used quences that were 1-2 base pairs different from suppression subtractive hybridisation (SSH) to these alleles, and have shown that these are obtain clones of genes that are different or diffe- probably artifacts of PCR amplification. When the rentially expressed between affected (OS) and same criteria for allele acceptance were applied to healthy (CB) chickens. For SSH, RNA was prepa- other previously reported sequences, 11 further red from thyroid lobes of 3-day-old OS and CB alleles were confirmed. Thus there is good evi-

42 Section B: MHC and Immunogenetics dence for 24 FLA-DRB alleles fulfilling nomen- nal Medical Center, Duarte, California USA; clature criteria. Preliminary results of Reference 2Northern Illinois University, DeKalb, Illinois, Strand Conformational Analysis on the same cat USA samples suggest individual cats have between one Major histocompatibility complex (Mhc) B and three DRB loci. Within families, haplotypes haplotypes in the chicken differentially control carrying different numbers of DRB alleles can be immune responses to infectious disease. Particu- identified. This study suggests that there may be a lar B haplotypes are known to confer genetic resi- different distribution of DRB alleles in the UK stance to diseases caused by infection with Ma- compared to the USA, and that different FLA rek’s virus, Rous sarcoma virus, and Eimeria haplotypes may carry different numbers of DRB tenella. The differential responses to infectious genes, as is the situation in many other mammals disease in the chicken may be related to the com- including man, apes and cattle. pact nature of the B class I (B-F) and class II (B- L) gene regions and to the levels of expression of B006 the loci within these two regions. As yet however Sequence-based genotyping at eight major hi- the exact basis of B system conferred disease resi- stocompatibility complex (SLA) loci stance remains unknown. B recombinant haploty- in Westran pigs pes are powerful means for more closely defining Mhc genes conferring disease resistance. Recom- JUN-HEON LEE1, STACY WALTERS2, TINA binant haplotypes have already narrowed the B PATEL2, DENBIGH SIMOND1, RICHARD AL- gene region controlling Marek’s disease respon- LEN2, WAYNE HAWTHORNE2, PHIL ses. If available, animals bearing additional, more O’CONNELL2, JEREMY CHAPMAN2, finely defined recombinant haplotypes might be DOUGLAS SMITH3, CHRIS MORAN1 tested along side parental haplotypes to further 1Centre for Advanced Technologies in Animal map disease responses to particular B subregions Genetics and Reproduction, University of Sydney, or loci. Identification of B recombinant haploty- NSW 2006, Australia; 2National Pancreas Trans- pes has been hampered by lack of means by which plantation Unit, Westmead Hospital, NSW 2146, to easily identify alleles at the B-F and B-L loci. Australia; 3Transplant Immunology Laboratory, We have developed methods for B-G, B-F, and B- Baylor University Medical Center, Dallas, TX L typing to rapidly characterize B haplotypes. We 75246, USA demonstrate the capacity of these methods to de- Since pigs may be donors for xenotransplantation fine genotypic differences among a series of re- and SLA molecules elicit a xenoreactive response, combinant haplotypes developed in a common we examined the SLA1, SLA6, SLA7, SLA14, lineage and to reveal potential recombinant DQA, DQB, DRA and DRB loci in inbred Westran haplotypes within closed breeding populations. (Westmead Hospital transplantation) pigs, bred for transplantation research. Three Westran gene- B008 ration-six inbred animals and a Large White con- Structure of the Bovine MHC DRA and DRB3 trol were used to assess SLA variants. Three clo- genes nes from each locus in each animal were sequen- ced to assess within line diversity and similarity to GEORGE C. RUSSELL(1), JUDITH A. SMITH(2), known SLA sequences. Westran pigs have novel JOHN L. WILLIAMS(1) & ROBERT A. OLI- alleles at the SLA7, SLA14 and DQA loci. Only VER(1) SLA1 appeared to be segregating, consistent with (1) Roslin Institute, Roslin, Midlothian UK and (2) low microsatellite marker heterozygosity from Department of Veterinary Parasitology, Univer- recent deliberate inbreeding but also their deriva- sity of Glasgow, Glasgow UK. tion from a feral stock from Kangaroo Island, The cattle MHC class II DR gene product is a South Australia, established by the release of a heterodimer encoded by the BoLA-DRA and - single pair in 1803. The SLA genotypes will be DRB3 genes. Several groups have isolated cDNA useful for attempting modulation of immune re- and genomic clones for these genes, but the ge- sponses in xenotransplantation. nomic organization around the first exon of both genes has yet to be described due to the length of B007 the first intron. We have used a combination of Molecular genetic dissection of the major hi- long-range PCR, cloning and sequencing to define stocompatibility B complex in the chicken the organization of the DR genes on existing ge- nomic clones and in genomic DNA. We estimate RONALD M. GOTO1, W. ElWOOD BRILES2, the size of the coding region of the DRA gene to MARCIA M. MILLER1 be 4.7 kilo-base pairs (kbp), while that of the 1Beckman Research Institute, City of Hope Natio- DRB3 gene is about 11.4 kbp. Sequencing of these

43 Section B: MHC and Immunogenetics full-length genomic PCR products confirmed that sequences from the SLA β cluster displayed lim- they carried the complete DRA and DRB3 genes ited similarity between them and no relationship respectively and allowed the design of probes and with motifs of the SLA κ cluster. Remarkably, primers to isolate and characterise the class II only significant alignments exist between the pig promoter regions and the DRB3 3’-end. MIC2 containing segment and several human Fragments carrying the 5’-end of the DRB3 gene MIC segments from the α and β HLA clusters. and its promoter were identified in both the Thus the history of SLA and HLA gene duplica- lambda clone A1 and on a BAC clone carrying the tion differs and, within each species, the β and κ BoLA-DR genes. Interestingly, hybridization of a clusters were subject to independent evolutionary DRB3 exon 1 probe to BamHI digests of these pattern. clones showed a 10 kbp promoter fragment on the BAC clone and a 5 kbp fragment on clone A1. B010 The larger fragment was sub-cloned and a 1.7kbp Characterisation of duck (Anas Sp.) MHC fragment including exon1 and the promoter was region sequenced. A 3 kbp fragment encoding exons 4-6 and the entire 3’-untranslated region of the DRB3 SILVANA ROTUNDO1, GIULIANA SALTINI2, gene was sub-cloned from A1 and sequenced. LAURA SIRONI3, SILVIA CEROLINI3, PAOLA This now provides us with improved characteri- MARIANI1 zation of the existing lambda clones carrying the 1FPTP-CERSA, Segrate (MI), ; 2CNR-ITB, DRA*0101 and DRB3*0101 alleles, and also Segrate (MI), Italy; 3University of Milan, VSA cloned 5’- and 3’- flanking fragments for gener- Department, Milan, Italy ating DRB3 allele-specific constructs. In contrast to the well studied human and murine This work not only improves our understanding of MHCs, very little is known about the genomic bovine class II gene structure, but also provides organization of non-mammalian MHCs. Although the material to allow functional DR gene pairs some information is already available for a num- encoding selected alleles to be assembled for ber of avian MHC systems, chicken MHC, also analysis by transfection or transgenesis. referred to as B complex, is the most studied and better known. Within the B locus there are two B009 loci containing the classical class I (B-F) and class Evolution of the SLA class I region II (B-L) β genes homologous to human. Chicken MHC also contains the B-G (class IV) gene, so far CHRISTINE RENARD, MARCEL VAIMAN, identified only in this species. Within the B locus PATRICK CHARDON there is only one gene homologous to the human Laboratoire de Radiobiologie et d'Etude du Gé- class III, the complement component C4. We nome INRA CEA Jouy en Josas, France started characterising the duck MHC region by Chromosomal distribution of the MHC class Ia, Ib isolating MHC gene specific sequences. We de- and MIC sequences vary considerably in mam- rived primers specific for MHC classical genes mals. In the pig, class I genes are distributed in 2 from chicken, pheasant, and quail sequences de- clusters denoted β and κ whereas in humans a posited in GenBank. When possible, conserved third cluster α exists. Close to TNF, the β cluster regions were determined by aligning the sequen- contains MIC genes in both species and either ces. We amplified duck specific MHC gene se- class Ib in the pig or class Ia in humans. Further quences using genomic DNA as template. The along the chromosome, the κ cluster encompasses PCR products were sequenced and database sear- the whole set of SLA class Ia loci but no func- ches (EMBL, Genbank) were carried out using tional class Ia gene in HLA. Lastly at the telo- BLAST to ascertain the homology of the duck meric end of HLA, the α cluster presents a large sequences with MHC gene sequences in other set of sequences from the 3 series. We analyse the avian species. Thus, duck specific primers were sequences available in pigs and humans. We designed, further rounds of PCR were run and the identified an elementary duplication unit in the products sequenced. Polymorphisms within the SLA κ cluster comprising a mosaic of repeats MHC region were investigated by sequence and ending with a specific motif. Dot matrix analysis SSCP analyses. underlined similarity between human and pig orthologous κ fragments and unexpectedly be- B012 tween fragments from the pig κ cluster and frag- Bovine MHC class II DRB3 genes diversity in ments from human α cluster. Presumably, SLA11 Japanese black, Japanese shorthorn, Jersey and SLA4 were the oldest loci and the remaining and Holstein cattle class Ia loci arose from 5 additional subsequent rounds of gene duplication. By contrast intergenic YOKO AIDA1, MITSUO MORITA2, SHIN-

44 Section B: MHC and Immunogenetics

NOSUKE TAKESHIMA1 chains. First, we compared the average number of 1 RIKEN, Retrovirus Research Unit, Wako, Saita- nonsynonymous substitution per site with that of 2 ma; Livestock improvement association of Japan synonymous substitutions per site in pairwise of Inc., Maebashi, Gunma, Japan 103 BoLA-DRB3 and 236 human MHC (HLA)- We sequenced exon 2 of the bovine MHC (BoLA) DRB1 alleles. The rate of nonsynomymous sub- class II DRB3 gene from 200 Japanese black, 102 Holstein, 100 Japanese Shorthorn and 69 Jersey stitutions in BoLA alleles was higher than that of cattle using new PCR-Sequence-based typing HLA alleles. Next, we detected the selective force method. The 35 different previously published at single amino acid site in bovine and human alleles and the three novel alleles were identified. DR 1 domains by adaptsite program package In 38 alleles, 19 breed specific alleles were identi- which reported by Suzuki and Gojobori (1999). fied. These alleles were 80.0% to 100% identical Of the 22 ARSs, 6 were inferred as positively at the nucleotide level to BoLA-DRB3 cDNA clo- selected but none were inferred as negatively se- ne NR1. These specific alleles did not have a spe- lected in BoLA. By contrast, 8 were inferred as cific cluster in Neighbor-joining tree, indicating positively selected and 3 were inferred as nega- that these breeds have a common variation in point of the value of the average of nucleotide, tively selected in HLA. Collectively, these result amino acid, synonymous and non-synonymous shows that, several amino acids might have differ- substitution. To differentiate the allelic variations ent biological functions between human and cat- between four distinct breeds, we determined the tle, and, in addition, excess number of non- gene frequencies of the BoLA-DRB3 alleles in synomymous was might be the result of small each breed and compared with those of other po- negatively selected amino acid sites in cattle. pulations. All breeds examined showed extremely high DRB3 diversities, with heterozygosity rates B014 of between 90.5% and 94.1%, which were near Molecular cloning of cDNA encoding porcine the heterozygosity rates expected of between interleukin-16 88.7% to 91.4%. The DRB3*1101 was detected as a most frequent allele in Holstein (16.7%), the TOMOKO EGUCHI1, TOMOHIKO EBATA2, DRB3*4501 was in Jersey (18.1%), DRB3*1201 YOHTAROH TAKAGAKI3, was in Japanese Shorthorn (16.0%) and JUNICHIRO FUJIMOTO4, HIROSHI YASUE 1 DRB3*1001 was in Japanese Black (17.5%). 1National Institute of Agrobiological Sciences, 2 Furthermore, we constructed the UPGMA tree Ikenodai, kukizaki, Inashiki, Ibaraki 305-0901, based on distances of allele frequency in each Japan, 2Juntendo University School of Medicine, breeds. Holstein and Japanese Black were the 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan, closest to each other, and Jersey was farther from 3Kitasato University school of medicine, 1-15-1 these both breeds than Japanese shorthorn. Kitasato, Sagamihara, Kanagawa 228-8555, Ja- pan, 4National Research Institute for Child Health B013 and Development, 3-35-31, Taishido, Setagaya, Comparing of antigen binding groove of MHC Tokyo 154-8509, Japan class II DR molecule of cattle and human Interleukin-16 (IL-16) is a chemotactic cytokine that binds to the CD4 receptor, and considered to SHIN-NOSUKE TAKESHIMA, YOKO AIDA be involved in T cell functions. Analysis of swine RIKEN, Retrovirus Research Unit, Wako, Saita- IL-16 would contribute to understanding of T cell- ma, Japan related immune-response system which is relevant The major histocompatibility complex (MHC) to animal health as well as xenotransplantation. In polymorphism occurs predominantly at residues the present study, in order to characterize swine involved in peptide binding, and there is compel- IL-16, cDNA clones coding IL-16 were isolated from “full-length” cDNA library of swine ling evidence that the polymorphism is maintained “gamma/delta” T cell populations (described by some form of balancing selection. The essen- elsewhere). The cDNA inserts of the clones were tial role of the MHC molecules for immunological sequenced to demonstrate that swine IL-16 recognition of foreign peptide antigens implies mRNA was 2,296 base pairs (bp). The hypotheti- that the cause for this selection is related to the cal ORF for IL-16 cDNA is 1,905 bp, which en- influence of MHC polymorphism on host defense codes a 635 amino-acids sequence (pro-IL-16) against pathogens. To study the function of bo- (MW=67,177). When the amino-acid sequence vine MHC (BoLA)-DR molecule, we analyzed the was compared with those of human, crab-eating selective force of bovine and human DR 1 monkey and mouse, the homologies were calcu- lated to be 94.3%, 94.2% and 92.8%, respectively. 45 Section B: MHC and Immunogenetics

The comparison also predicted that the swine pro- B014 IL-16 is excised to yield a matured IL-16 consi- Protection of recombinant mouse single chain sting of 121 amino-acids (MW=12,523). antibodies against orthopox virus envelope proteins correlates with antigen affinity B016 Michaela Alex1, Katharina Hoelzle2, Claus-Peter Analysis of genomic structure and repertoire 1 of swine T cell receptor α chain gene Czerny , 1University of Goettingen, Institute of Veterinary 2 HIROHIDE UENISHI1, RYUJI YAMA- Medicine, Goettingen, Germany; University of MOTO1,2, TOMOKO EGUCHI1, HIROSHI YA- Zurich, Institute of Veterinary Bacteriology, Zu- SUE1, YOHTAROH TAKAGAKI2, TAKASHI rich, Switzerland AWATA1 The 14 kDa fusion protein (ORF A27L), the 32 1National Institute of Agrobiological Sciences, 2- kDa adsorption protein (ORF D8L) and a 35 kDa 1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Ja- membrane protein (ORF H3L) localized in the pan; 2Kitasato University School of Medicine, 1- envelope of intracellular mature orthopox virus 15-1 Kitasato, Sagamihara, Kanagawa 228- particles are key determinants in early and late 8555, Japan virus/host interactions and mainly responsible for T cells express heterodimeric receptors (TCR), the induction of B- and T-cell specific immune recognize antigens using these receptors and exhi- responses. High-affinity neutralizing antibodies various functions such as cytotoxicity or im- may be of therapeutic value in human and veteri- mune regulation. TCR genes consist of four mole- nary medicine for alleviating the symptoms in cules, α, β, γ and δ. T cells are classified into two infected individuals and for prophylaxis to infec- tion. We have engineered a panel of neutralizing types by their expressing receptor pairs, αβ and single-chain variable fragments (scFvs) from γδ. γδ Τ cells account for only several percents of BALB/c-mouse hybridoma cell clones producing all T cells in peripheral blood of human and monoclonal antibodies (MAb) that bind to se- γδ mouse, however, amount of T cells of swine quential and conformation-dependent protective are almost half of the whole T cells in periphery. antigenic sites of the fusion protein (aa 32-39), γδ Swine T cells are notable according to their adsorption protein (aa 290-304), and 35 kDa pro- unique character, such as MHC class II expression tein (aa 210-324). The DNA of the constructs of and antigen-presenting ability to αβ T cells. TCR about 750 bp was sequenced and analyzed com- α and δ chain genes (TRA/TRD) are located on paratively. Framework (FRW) and complemen- the same locus, and the locus spans about 1Mbp tary determing regions (CDR) of the recombinant of the germline genomic DNA. We focus antibodies were defined on the deduced amino TRA/TRD locus because its analysis may be a acid sequences. After cloning into the pQE30 clue to elucidation of a unique fashion of lineage vector (Qiagen, Hilden) the scFvs were expressed of αβ/γδ T cells in artiodactyls. We revealed ge- in E. coli and purified by an N2+-terminal 6xHis- nomic structure of the region including the TRA Tag. Binding to their epitopes was determined in constant (TRAC) region and joining (TRAJ) seg- standardized Michaelis-Menten kinetic studies ments. We newly identified one pseudo-TRAJ together with the corresponding full-size MAbs. segment in the germline sequence, however, The kinetic studies indicated, that binding capaci- whole structure of this locus is strikingly conser- ties of the scFvs were 11-27-times lower than ved among swine, human and mouse. We also binding capacities of the corresponding MAbs. identified 91 clones of swine TRA cDNA and Neutralizing potencies of purified scFvs against confirmed that 44 TRAJ segments identified in vaccinia virus were tested in vitro in plaque re- the swine germline sequence are expressed in duction assays and in vivo in a mouse challenge peripheral blood. Length of CDR3 of swine TCR model with the neurovirulent vaccinia virus strain α chain molecules is longer than those of human Munich 1. Protection correlated with antigen af- and mouse, and this suggests a different manner finity. for recognition of antigens in swine.

Protection of recombinant mouse single chain antibodies against orthopox virus envelope pro- teins correlates with antigen affinity

46 Section C: Functional Genomics

Section C: and transgenic crops as dietary components in animal agriculture. Functional Genomics C002 C001 Analysis of tissue-specific gene expression Diet and gene expression - Bridging the gap patterns using a 7653 gene cattle microarray with small intestine specific expressed se- MARK R. BAND1, JONATHAN U. PELED2, quence tags (ESTs) and cDNA microarrays ROBIN E. EVERTS2, ZONGLIN L. LIU2, in the chicken HARRIS A. LEWIN1,2 1 1 The W.M. Keck Center for Comparative and CHRIS M. ASHWELL , HYUN S. LILLE- 2 2 3 Functional Genomics; Department of Animal HOJ , ROSELINA ANGEL 1 Sciences, University of Illinois at Urbana- Growth Biology Laboratory, USDA-ARS; 2 Champaign, Urbana, IL, USA Parasite Biology, Epidemiology, and Syste- cDNA microarrays have been shown to be matics Laboratory-USDA-ARS, Beltsville, MD; 3 useful for monitoring global gene expression Department of Animal and Avian Sciences, patterns in normal and diseases states and in UMD, College Park, MD response to various environmental stimuli. We The predominant factor influencing growth in have constructed cattle cDNA microarrays broiler chickens is nutrient absorption across containing 7653 elements, of which ~80% the small intestine. Animal agriculture expends have putative human orthologs. The cattle its greatest resources on animal feeds and must cDNA clones used for creation of the array develop novel strategies to maximize nutrient were selected from more than 17,000 ex- absorption and retention, due to new environ- pressed sequence tags (ESTs) derived prima- mental regulations regarding animal wastes. rily from a normalized and subtracted placenta Additionally, the advent and use of novel feed cDNA library. Clones were annotated by se- additives and transgenic crops in animal agri- quential BLASTN and TBLASTX searches culture has increased the value of ascertaining against multiple public domain databases (see the exact impact, if any, dietary components accompanying abstract by Everts et al) inclu- have on animals at the molecular level. These ding 2917 genes that have one or more Gene studies were undertaken to both expand the Ontology functional annotations. Fifteen diffe- knowledge of expressed genes for chicken, as rent tissues were analyzed in order to identify well as to identify specific genes involved in gene expression patterns associated with speci- small intestinal function. A normalized cDNA fic organs and tissue types and to establish a library was constructed from small intestines functional baseline for future studies. Hybri- of both normal broiler chickens and those in- dizations were performed with Cy3 and Cy5- fected with Eimeria acervulina, a pathogen labelled cDNA derived from RNA collected known to limit nutrient absorption. Of the total from the tissue samples. All samples were number of unique ESTs sequenced, approxi- compared to a universal control sample com- mately 4,000 were randomly selected, repre- prised of a mix of cattle RNAs. Three exoge- senting both novel genes and GenBank homo- nous plant genes were used as spiking controls logs, to be amplified for spotting onto glass for data normalization. Results of various clu- slides for subsequent hybridization studies. stering methods to correlate gene expression With a goal of reducing the phosphorus con- profiles and define sequences lacking annotati- tent of manures, the first experiments using on will be presented. This research illustrates these microarrays are to determine the effects the potential of microarrays for understanding of limiting dietary phosphorus on gene expres- tissue-specific gene regulation in response to sion, specifically identifying those genes in- different environmental stressors. volved in phosphorus absorption and retention. Utilizing these resources, these and future stu- dies will identify the effects of dietary mani- C003 pulation on the expression of genes in the Affects of the brown locus (TYRP1) on coat small intestine. Monitoring intestinal gene color in cattle expression may also be a valuable tool for TOM G. BERRYERE1, SHEILA M. evaluating the impacts of novel feed additives SCHMUTZ1, C. MICHAEL COWAN2, JOHN

47 Section C: Functional Genomics

POTTER3 The region of ovine chromosome 18 that in- 1University of Saskatchewan, Department of cludes the callipyge mutation contains an im- Animal and Poultry Science, Saskatoon, Cana- printed gene cluster of at least six transcripts. da; 2Genetic Visions, Inc., Middleton, Paternal expressed gene 11 (PEG11) has a Wisconsin, USA; 3John Potter, Spruce Grove putative protein-coding sequence that produces Farm, Galien, MI, USA a paternally derived sense strand transcript and Skin biopsies from several cattle of different maternally derived antisense strand transcripts coat colors were used to prepare mRNA. The (antiPEG11). Expression of PEG11 and an- entire coding sequence of cattle tyrosinase tiPEG11 transcripts was analyzed using strand related protein 1 (TYRP1) was obtained using specific probes and northern blot analysis. cDNA prepared from mRNA. The cattle ami- Expression of a 6.5 kb PEG11 transcript was no acid sequence was found to be 97-86% detected in muscles that become hypertrophied similar to goat, , human and mouse se- including the longissimus dorsi (LD), semi- quence. A microsatellite detected in intron 5 of membranosus (SM) and gluteus medius (GM) TYRP1 from genomic DNA was used to linka- in 14-day, 56-day, and 84-day-old paternal ge map this gene to cattle chromosome 8 bet- heterozygous callipyge lambs (+mat/Cpat). The ween microsatellites BL1080 and BM4006. PEG11 transcript was not detected in the LD, Although 2 nonconservative amino acid chan- SM or GM from the other three possible ge- ges were detected, no association was found notypes (+/+, Cmat/+pat or CC). The PEG11 with diluted shades of black or red in Sim- transcript also was not detected in the su- mental nor Charolais cattle. Nor was an asso- praspinatus (SP), which does not undergo hy- ciation found with the dun coat color, inherited pertrophy, regardless of the lambs’ genotype. as a dominant trait, which occurs in Galloway Two antiPEG11 transcripts (1.7 kb and 0.8 kb) cattle. Therefore this gene does not appear to were readily detected in the LD, SM, GM and be one of the dominant or co-dominant diluter SP muscles of 14-day, 56-day and 84-day-old genes. However, in Dexter cattle dun coat Cmat/+pat and CC lambs but were weakly de- color is inherited as an autosomal recessive tected in +mat/Cpat callipyge lambs and not de- trait. An amino acid change was found in the tected in +/+ lambs. The callipyge mutation homozygous state in all 26 dun Dexter cattle has altered the expression of both PEG11 and examined, irrelevant of shade of dun which antiPEG11 transcripts. Expression of the ranges from a pale golden to dark brown. We PEG11 transcript only in the LD and pelvic suggest the dun Dexter is more correctly called limb muscles of the +mat/Cpat genotype was a "brown cow". Black Dexters had either one consistent with polar overdominant inheritance (28) or no (16) copy of this allele. We ex- of muscle hypertrophy in callipyge lambs. amined over 88 cattle of other breeds including Brown Swiss, Canadienne, Flamande, Guern- C005 sey, Jersey, Angus, Charolais, Hereford, Li- Physical mapping of genes expressed in the mousin, and Simmental and did not find this Corpus luteum of Cattle TYRP1 mutation in any of them. TINA B. BØNSDORFF1, ANDRÉ EGGEN2, C004 MATHIEU GAUTIER2, FRODE LINGAAS1, Expression of PEG11 transcripts in the mu- HANS-CHRISTIAN AASHEIM3, KNUT scles of normal and callipyge lambs (Ovis RØNNINGEN1, INGRID OLSAKER1 aries) 1Department of Morphology, Genetics and Aquatic Biology, The Norwegian School of CHRISTOPHER A. BIDWELL1, TRACY L. Veterinary Science, Oslo, Norway; SHAY2, CAROLE CHARLIER3, KARIN SE- 2Laboratoire de genetique biochimique et de GERS3, MICHAEL GEORGES3, NOELLE E. cytogenetique, Institut national de la recherche COCKETT2 agronomique, Jouy-en-Josas, France; 1Purdue University, Department of Animal 3Department of Immunology, The Norwegian Sciences, West Lafayette, Indiana USA; 2Utah Radium Hospital, Oslo, Norway State University, Department of Animal, Dairy Fertility, measured as % non-return, is highly and Veterinary Sciences, Logan, Utah USA; ranked in the merit index by the Norwegian 3University of Liège, Department of Genetics, cattle-breeding organisation because of the Liège Belgium considerable economical importance of this

48 Section C: Functional Genomics trait. The estimates of heritability for fertility found with the transcript including exons 1, 2, traits are small (0.01-0.05). However, there are 3, 7, 8 and 9 being predominant. This tran- indications that the genetic variation is signifi- script can further be subdivided in two iso- cant for these traits, which means that there is a forms due to alternative poly(A) site selection. potential to make long-term improvements in Promoter P1 usage was also observed in fetal fertility. A deeper understanding of the mo- liver and fetal ham. In these tissues there is an lecular mechanisms controlling fertility, i.e. the additional variant with exons 1, 2, 3, 7, 8 and 9 regulation of ovarian function, will contribute due to alternative splicing of exon 2. Promoter to the improvement of methods and strategies 2 (P2) usage results in transcripts encompas- to increase fertility and reproductive perform- sing exon 4, 7, 8 and 9 and exon 4, 4b, 7, 8 and ance in cattle as well as other mammals in- 9, respectively and is found in all tissues. Pro- cluding man. The corpus luteum of the ovary motor 3 (P3) and 4 (P4) are predominantly secretes progesterone and plays a central role used in various fetal tissues, in adult kidney, in the regulation of cyclicity and maintaince of adult muscle and to a low extent in adult liver. pregnancy. In order to identify bovine genes The transcript isoforms include exons 5, 7, 8 expressed in the corpus luteum at the most and 9 and 6, 7, 8 and 9 for promoter P3 and P4 active state, i.e. 10 days after ovulation, a rep- usage, respectively. For both transcripts iso- resentational differential analysis (RDA) was forms resulting from different polyA signal performed, with skeletal muscle as the sub- usage have been observed. tractive agent. Eleven of eighty initially ana- lysed clones were selected for further studies C007 based on their sequence and expression pro- The hairless (hr) gene in the ovine species files. A corpus luteum cDNA library was con- structed and cDNA clones for the RDA frag- RAFFAELLA FINOCCHIARO1, ANNA CA- ments were identified, sequenced, and mapped ROLI2, ELENA BUDELLI3, BALDASSARE by using a bovine radiation hybrid cell panel. PORTOLANO1, PATRIZIA BOLLA3, PIE- The mapping results as well as expression TRO GIACCONE1, GIUSEPPE DAMIANI4 analysis of the clones will be presented. 1University of Palermo, Department S.En.Fi.Mi.Zo., Palermo, Italy; 2University of C006 Bari, Department of Animal Health and Wel- Porcine IGF2 promoter usage and isoforms fare, Valenzano, Italy; 3University of Milan, in fetal and adult porcine tissues Department VSA, Milano, Italy; 4IBBA-CNR, Milano, Italy MARTIN H. BRAUNSCHWEIG1, MINH The hairless (hr) gene is often responsible for NGUYEN2, MICHEL GEORGES2, LEIF congenital hypotrichosis in mammalian spe- ANDERSSON1 cies. The protein codified by this gene is a 1 University of Agricultural Sciences, Depart- transcriptional corepressor for thyroid hormone ment of Animal Breeding and Genetics, receptors. So far no molecular study has been Uppsala, Sweden; 2University of Liege, De- carried out on hr gene in the ovine species. By partment of Genetics, Liege, Belgium using the primers designed on the conserved IGF2 is a candidate gene for a paternally ex- human, rat, and mouse sequences and the long pressed QTL on pig chromosome 2p affecting PCR technique, the entire ovine hr gene was skeletal and cardiac muscle mass. The aim of successfully amplified. Sequencing allowed this study was to characterize the forms of the continuous alignment of 62% the ovine hr IGF2 transcripts expressed in different tissues coding sequences, from exon 2 to 9. The high- and developmental stages. The porcine IGF2 est homology (80% identity) was found with gene encompasses 10 exons of which exons 1, human hr gene. The homology scores (ranging 2, 3, 4, 4b, 5 and 6 represent 5’ UTRs. The from 76% identity of ovine-mouse comparison exons 7, 8, and part of exon 9 encode the pre- to 95% of rat-mouse comparison) indicate that cursor protein of the 67-amino-acid IGF2 pep- hr gene is strongly conserved. The phyloge- tide. The IGF2 gene is imprinted and primarily netic tree agrees with the well known evolu- expressed from the paternal allele. The four tionary pathway of mammals, indicating that promoters show tissue- and development- the divergences of hr gene in Ruminants, Ro- specific activity. Promotor 1 (P1) is mainly dents, and Primates stemmed from a common used in adult liver. Four isoforms have been ancestor. Functional constraints may be re-

49 Section C: Functional Genomics sponsible for the low mutation rate, and for the ERICA E. DAVIS1, CHARLOTTE HARKEN- reduced phylogenetic divergence suggesting an JENSEN2, CAROLE CHARLIER1, ANETTE important physiological role of hr gene. This KLEIM2, TRACY L. SHAY3, BØRGE TEIS- hypothesis agrees with recent experimental NER2, NOELLE E. COCKETT3, MICHEL evidences indicating that the gene product GEORGES1 plays a wider role than previously suspected in 1University of Liège, Department of Genetics, development. Several pleiotropic effects were Liège, Belgiu;, 2University of Southern Den- detected in different mouse tissues. Therefore mark, Department of Immunology and Micro- the investigation of hr gene in domestic ani- biology, Odense, Denmark; 3Utah State Uni- mals could reveal interesting influences on versity, Department of Animal, Dairy, and productive traits in addition to the well known Veterinary Sciences, Logan, UT, USA effect on growth. Delta-like (DLK1) is a paternally expressed gene located in the imprinted domain encom- C008 passing the ovine callipyge (clpg) locus. Sequencing and mapping of the porcine Comparative analysis of DLK1 across the four Pyruvate Carboxylase (PC) gene possible genotypes has revealed that the cal- lipyge mutation induces overexpression of GUILLERMO DÁVALOS1, MARCEL mRNA in both CLPGM/CLPGP and +M/CLPGP AMILLS1, JOSÉ LUÍS NOGUERA2, animals, while at the same time, only the latter ARMAND SÁNCHEZ1 individuals exhibit the callipyge phenotype. 1Departament de Ciència Animal i dels Ali- Addressing the need for more extensive cha- ments, Facultat de Veterinària, Universitat racterization of DLK1 relative to callipyge, a Autònoma de Barcelona, Bellaterra 08193, multi-faceted expression study is underway in Spain; 2Àrea de Producció Animal, Centre which DLK1 expression is being examined on UdL-IRTA, Lleida 25198, Spain a panel of tissues collected from animals repre- The pyruvate carboxylase (PC) gene is a mem- sentative of the four callipyge genotypes span- ber of the biotin-dependent enzyme family and ning three stages of development. Despite the catalyzes the ATP-dependent carboxylation of overexpression of DLK1 previously demon- pyruvate to oxalacetate. In mammals, PC plays strated on the mRNA level in two distinct ge- an important role in gluconeogenesis and lipo- notypes, preliminary results reveal that only genesis. We have amplified three different callipyge +M/CLPGP individuals display DLK1 regions (exon 1 to 10, exon 9 to exon 15 and protein in muscle fibers. These results provide exon 15 to the 3’UTR) of the pyruvate car- evidence justifying the proposed model for boxylase cDNA in six Iberian, Pietrain and callipyge polar overdominance implicating a Large White pigs. Amplified products were trans effect of coregulated imprinted genes in sequenced forward and reverse and a silent producing the callipyge phenotype. Based on mutation C → T was identified at exon 6. This the perfect association of DLK1 protein with polymorphism can be typed by primer- the callipyge phenotype combined with the extension analysis. Furthermore, physical known role of DLK1 in cell proliferation and mapping of the pig PC gene was performed by differentiation, an in vivo approach is being using the INRA-Minnesota porcine radiation used to elucidate the direct involvement of hybrid panel (IMpRH). The porcine PC gene DLK1 in producing the muscular hypertrophy. mapped to the 2p14-17 interval, near the A transgenic construct consisting of the ovine SW2623 microsatellite (LOD score = 13.91). DLK1 ORF placed under the control of the Overall sequence identity between pig PC and Myosin Light Chain-3F promoter has been its human and murine counterparts ranged introduced into the mouse by pronuclear from 86-91 %. microinjection. Seven transgenic lines are cur- rently being established and will be subjected C009 to analysis of both DLK1 transgenic expression Extensive DLK1 expression profiling sub- and phenotype. stantiates the callipyge polar overdominance model in sheep implicating a trans effect of C010 coregulated imprinted genes in producing Prion protein (PRNP) genotype frequencies the callipyge phenotype in German breeding sheep suggest feasibili- ty of breeding for scrapie resistance

50 Section C: Functional Genomics

Sensorineurale deafness is a common con- CORD DRÖGEMÜLLER, TOSSO LEEB, genital disorder in Dalmatians with a genetic OTTMAR DISTL background for which different modes of in- Institute of Animal Breeding and Genetics, heritance have been proposed. The objective of School of Veterinary Medicine Hannover, our study was to investigate the mode of in- Hannover, Germany heritance of this disease by segregation analy- Genetic susceptibility to scrapie is associated ses using maximum likelihood procedures. with polymorphisms in three different codons These analyses were performed under four of the ovine prion protein (PRNP) gene (136, models, namely a major gene, a mixed inheri- 154, 171). In a number of countries, studies of tance, a polygenic and an environmental PRNP genotypes linked to scrapie revealed the model. The data of a large population from the resistance of homozygous PRNPARR / USA and a smaller one from Switzerland were PRNPARR animals and the high risk of pooled as the diagnoses were established based PRNPVRQ /PRNPVRQ and PRNPVRQ /PRNPARQ on the same protocol (brainstem auditory animals in scrapie affected flocks. Therefore, evoked responses) and the incidences were the selection of PRNPARR /PRNPARR genotypes quite similar. A total of 1697 dogs with 1441 may be a strategy for controlling clinical scra- known phenotypes entered the study. The pie at the population level. We genotyped 1674 prevalence of deafness was 19.2% (14.3% German breeding sheep from 15 different unilaterally deaf, 4.9% bilaterally deaf). Fe- breeds in Germany. Apart from the wildtype males showed significantly (p=0.030) more allele PRNPARQ at least four mutually exclu- affected animals (21.6%) than males (16.3%). sive allelic variants were found. The greatest The data were analyzed in two sets, the first variability within the PRNP gene was encoun- including normal, uni- and bilateral deaf dogs, tered in Texel sheep, where 14 different PRNP and the second normal and deaf dogs. With the genotypes were found. In the important meat first set (3 liability classes) the differences breeds Suffolk, German Whiteheaded, and between the genetic models was not signifi- Blackheaded Mutton the PRNPARR allele was cant. With the second set (2 liability classes) predominant. In these breeds simulation stud- the mixed inheritance model was significantly ies showed that four generations are necessary better than all other models. This model to breed scrapie resistant pedigree flocks. For showed the presence of a major gene with a the Texel sheep, the German Merino, the Ger- completely recessive deleterious allele man milk and land sheep breeds examined here (q=0.05) and important polygenic effect the frequency of the PRNPARR allele was much (h2=0.62). lower and for several breeds no homozygous rams were available for breeding purposes. C012 The breeding strategy in those breeds depends Characterization of bovine ectodysplasin 1 on the number of heterozygous animals. Nev- (ED1) mutations in cattle with hypotrichosis ertheless, resistant pedigree flocks can be and oligodontia achieved in nine generations at the most with- out loosing genetic heterogeneity of the breed. CORD DRÖGEMÜLLER, OTTMAR DISTL, TOSSO LEEB C011 Institute of Animal Breeding and Genetics, Segregation analyses of deafness in a large School of Veterinary Medicine Hannover, pedigree of Dalmatians Hannover, Germany The ectodysplasin 1 gene (ED1) encodes a CLAUDE GAILLARD1, VILMA YUZBASI- signalling molecule of the tumor necrosis fac- YAN-GURKAN2, EMILY ROBINSON tor (TNF) family that is involved in fetal de- NOLAN2, TAKESHI MIYAKE1, ANDRÉ velopment of ectodermal appendages. Muta- JAGGY3, GAUDENZ DOLF1 tions in the ED1 gene are responsible for X- 1University of Bern, Institute of Animal Ge- linked anhidrotic ectodermal dysplasia (ED1) netics, Bern, Switzerland; 2Michigan State characterized by impaired development of hair, University, Small Animal Clinical Sciences, teeth, and eccrine sweat glands in human and East Lansing, MI, USA; 3University of Bern, mice. We report the construction of a 480 kb Dept. of Clinical Veterinary Medicine, Bern, BAC contig harboring the complete bovine Switzerland ED1 gene on BTA Xq22-q24. A large genomic

51 Section C: Functional Genomics region including exon 3 of the ED1 gene is better than e-5 with at least one sequence in a deleted in cattle with hypotrichosis and oligo- UniGene cluster. GeneOntology (GO) terms dontia in a family of black and white German were parsed from LocusLink and putative Holstein cattle with four affected maternal half functions were assigned to a significant frac- sibs. An RT-PCR assay demonstrated the X- tion of the genes on the array. Approximately linked recessive segregation of the ED1 dele- 170 genes were upregulated and 100 genes tion and established the grand-maternal origin were downregulated in BL3* as compared to of the mutation. A second case of congential BL30. BLV infection was associated with de- X-chromosomal hypotrichosis and oligodontia creased transcript levels of 23/46 genes enco- in a family of red and white German Holstein ding ribosomal proteins suggesting a negative cattle with three affected males is caused by a effect on protein synthesis. By contrast, 10 point mutation within the 5’ splice site fol- transcription factors and many genes associa- lowing ED1 exon 8b. Interestingly, cDNA ted with cell cycle and cell proliferation were sequencing analyses revealed that this muta- upregulated in BL3*. These results demonstrate tion alters the splicing of both alternative ED1 the power of microarray analysis for studying transcripts thus indicating the presence of an the effects of retrovirus infection on host gene important regulatory element for the correct expression. splicing of the ED1 transcripts at the exon 8 / intron 8 junction. As the clinical, pathological C014 and genetic findings in human ED1 show A second generation ordered comparative similarities to the described phenotype in cat- map of the cattle and human genomes tle, this bovine disorder may serve as an ani- 1 mal model for human ED1. ANNELIE EVERTS-VAN DER WIND , SRINIVAS KATA3, SHREEDHAR NATA- 2 2 C013 RAJAN , MARK R. BAND , MARK RE- 1 2 3 Transcript profiling of bovine leukemia BEIZ , LEI LIU , TOM GOLDAMMER , 1 virus (BLV) infected and uninfected cell CHERYL A. GREEN , STEPHANIE 3 1 lines using a cDNA microarray containing McKAY , ROBIN E. EVERTS , JIM E. WO- 3 1,2 7653 cattle genes MACK , HARRIS A. LEWIN 1Department of Animal Sciences; 2W. M. Keck ROBIN E. EVERTS(1), MARK R. BAND(2), Center for Comparative and Functional Ge- ZONGLIN L. LIU(1), JONATHAN U. PE- nomics, University of Illinois at Urbana- LED(1), CHARU G. KUMAR(1), AL BARI(2), Champaign, Urbana, IL LEI LIU(2), & HARRIS A. LEWIN(1,2) 3Department of Vete-rinary Pathobiology, (1)Department of Animal Sciences and (2)The Texas A & M University, College Station, TX W.M. Keck Center for Comparative and Func- A second generation ordered comparative map tional Genomics, University of Illinois at Ur- of the cattle and human genomes was produced bana-Champaign, Urbana, IL 61801, USA using a 5000 rad whole genome radiation hy- A 7653-element cDNA microarray was used to brid panel and in silico predictive mapping investigate the effects of bovine leukemia virus tools. A total of 720 new markers were added (BLV) infection on host gene expression. to the published map, bringing the total num- Transcription profiling was performed on the ber of mapped markers to 2034 with LOD >9. uninfected B-lymphocyte cell line BL30 and its Of these, 1423 are cattle genes or ESTs with infected, genetically identical daughter cell line significant (E

52 Section C: Functional Genomics for prediction of cattle chromosome and RH association analyses were performed. The re- bin location. Comparative coverage is 94%, sults indicate significant associations of PKM2 with average marker spacing on the human with GP and glycogen content and of GPI with GB4 map of 9.2 cR. The large number of ge- lactate value at 1 hr post mortem (P < 0.05). nes and ESTs mapped in this study allows for a comprehensive comparison of gene order bet- C016 ween the cattle and human genomes. The Characterization of the equine AEG1 gene number of conserved segments and genomic and its role in fertility rearrangements is considerably large, but still smaller than between human and mouse. This ALEXANDER GIESE1, RONY JUDE1, new comparative map will provide a resource HEIDI KUIPER1, FRANCOIS PIUMI2, AL- for phylogenomic analysis of mammalian EXANDRA SCHAMBONY3, HARALD chromosomes and will greatly facilitate the SIEME4, GÉRARD GUÉRIN2, OTTMAR identification of candidate genes for economi- DISTL1, EDDA TÖPFER-PETERSEN3, cally important traits. TOSSO LEEB1 1Institute of Animal Breeding and Genetics, C015 School of Veterinary Medicine Hannover, Study of candidate genes for glycolytic po- Hannover, Germany; 2INRA, Laboratoire de tential in pig muscle Génétique biochimique et de Cytogénétique, Jouy-en-Josas, France; 3Institute of Repro- LUCA FONTANESI, ROBERTA DAVOLI, ductive Medicine, School of Veterinary Medi- LEONARDO NANNI COSTA, EMILIO cine Hannover, Hannover, Germany; SCOTTI, VINCENZO RUSSO 4Hanoverian State Stud Celle, Celle, Germany DIPROVAL - Sezione di Allevamenti Acidic epididymal glycoprotein 1 gene Zootecnici, Faculty of Agriculture, University (AEG1), also known as cysteine-rich secretory of Bologna, Reggio Emilia, Italy protein 1 (CRISP1), is a member of the CRISP Glycolytic potential (GP) of porcine skeletal protein family. The CRISP proteins are char- muscles has been shown to be a predictive acterized by 16 conserved cysteine residues at measure of meat quality. A high value of GP the C-terminus and expressed in the male may be caused by alterations of the glucose genital tract. It has recently been shown that metabolism in this tissue. Thus, for this study, the amount of CRISPs that are tightly bound we selected as candidate genes those that code on sperm surfaces correlates with the fertility for enzymes that play an essential role in the of . Therefore, their genes are of inter- glycolysis and glycogen metabolism of the est as candidate genes for inherited male fertil- skeletal muscle. Physical mapping was ob- ity dysfunctions and as putative quantitative tained for PYGM (Sscr 2), ALDOA (Sscr 3), trait loci for male fertility traits. Here, we re- AGL (Sscr 4), PFKM (Sscr 5), GYS1 (Sscr 6), port the cloning and DNA sequence of 90 kb PKM2 (Sscr 7), ENO3 (Sscr 12), GAA (Sscr of horse genomic DNA from equine chromo- 12), PRKAB1 (Sscr 14) and PGAM2 (Sscr 18). some 20q22 containing the complete equine SNPs were identified for PYGM, LDHA, AEG1 gene, which consists of eight exons PKM2, GAA, PRKAB1 and PGAM2 and linka- spanning 31 kb. The transcription start site was ge mapping was obtained for the latter four mapped by the RLM-RACE technique and genes. Allele frequencies for these loci were three closely spaced transcription start sites studied in seven pig breeds (Large White, were annotated. Further analysis of equine Landrace, Duroc, Belgian Landrace, Piétrain, AEG1 transcripts did not reveal any evidence Hampshire and Meishan). GP of m. biceps for alternative splicing. RT-PCR analysis dem- femoris at 1 and 24 hrs post mortem and other onstrated that AEG1 is expressed in different meat quality traits were measured in 507 parts of the epididymis, whereas it is hardly commercial pigs. Among these animals, 60 detectable in the testis. The naturally occurring pigs were selected according to the GP value diversity of the equine AEG1 gene in different and typed to exclude the presence of the RYR1 horse breeds was investigated and several n allele and of the PRKAG3 RN– allele. Mo- polymorphisms are reported, including one that reover these animals were analysed for the affects the amino acid sequence. markers identified and for a polymorphism already presented in literature for GPI and

53 Section C: Functional Genomics

C017 Switzerland Identification and chromosome assignment Coat colour in mammals is determined by the of genes potentially different expressed in relative amounts of eumelanin (black) and meat and dairy cattle pheomelanin (red) produced in melanocytes. The mealanocortin 1 receptor (MC1R) is acti- TOM GOLDAMMER1, UTE DORROCH1, vated by α-melanocyte stimulating hormone RONALD M. BRUNNER1, SRINIVAS R. (α-MSH) which increases the amount of cAMP KATA2, JAMES E. WOMACK2, MANFRED in the cell. This activates tyrosinase resulting SCHWERIN1 in eumelanin synthesis. The Agouti signalling 1Research Unit for Molecular Biology, Rese- protein (ASP) acts as an antagonist to MC1R arch Institute for the Biology of Farm Animals, by binding to the receptor and preventing the Dummerstorf, Germany; 2Department of Vete- MC1R-MSH interaction. The cAMP concen- rinary Pathobiology, Texas A&M University, trations were measured in cells lines express- College Station, TX, USA ing 5 MC1R alleles and stimulated with α- We investigate in the identification and loca- MSH. The recessive red allele e and the domi- lization of genes potentially different ex- nant black allele ED were unresponsive to a pressed in liver and intestine of lactating Ger- wide range of αMSH concentrations. Two man Holstein and Charolais cows to analyze alleles from brown cattle Ed1, Ed2 and one al- active metabolic pathways and chromosome lele found in red cattle ef responded to an α- regions that could cause the different phento- MSH stimulation in a dose-dependent manner. types of these milk and meat type cows. The Cells transfected with the ef allele reached the homology search for twenty-four bovine ex- same cAMP concentrations as those with the pressed sequence tags (ESTs) in human geno- Ed1 and Ed2 alleles at 10 times a higher con- me data bases resulted in hits with high se- centration of α-MSH. These results indicate quence similary with 18 genes and 6 unknown that the e MC1R allele is a non-functional re- coding sequences in genomic DNA clones. ceptor, ED is a constitutively activated recep- The human DNA sequences were comparative tor, and Ed1 and Ed2 are hormone activated assigned in the bovine genome using a cattle- receptors. The delay in the ef allele response hamster somatic hybrid cell panel, a cattle- may explain the similarity of e and ef pheno- hamster 5000 rad whole genome radiation type. No differences were found in the coding hybrid panel, and fluorescence in situ hybri- sequences of ASP between red, black and dization (FISH). Synteny could be declard for brown cattle. Incubation of the cells trans- 20 ESTs. Twenty-three ESTs were RH- fected with ASP together with the cells trans- mapped and assigned in the established cattle fected with Ed1 and Ed2 resulted in decreased WGRH5000 map. The software RHMAPPER cAMP production. was used for calculation of RH-mapping data. Two genes were assigned by FISH on cattle C019 chromosomes. The identified genes represent The bovine gastrointestinal tract: A gene potentially candidates for the description of expression profile metabolic differences in meat and dairy cows at the gene transcription level and generally for CHRISTIANE HANSEN1, ANNA FU1, YAN economical important traits. The new loci MENG1, CHANGXI LI1, ERASMUS contribute to the completion of the bovine gene OKINE1, CHRISTOPH W. SENSEN2, PAUL map and the comparative assignments will GORDON2, STEPHEN S. MOORE1 increase our knowledge about genome evoluti- 1Dept. of Agricultural, Food and Nutritional on between cattle and human. Science, University of Alberta, Edmonton, AB Canada; 2Dept. of Biochemistry and Molecular C018 Biology, University of Calgary, Calgary, AB Functional analysis of bovine MC1R and Canada ASP using in vitro cell systems Understanding the genetic mechanisms at work in the bovine gastrointestinal (GI) tract would DARIA A. GRAPHODATSKAYA, HANNES aid considerably in our understanding of rumi- W. JOERG, GERALD F. STRANZINGER nant digestion. However, while the gross Swiss Federal Institute of Technology, Institute functions of most regions of the bovine GI of Animal Sciences, ETH-Zentrum, Zurich, tract are known, a lack of information concer-

54 Section C: Functional Genomics ning the specific processes occurring in each IL), a recently discovered member of proteins region exists. In addition, little is known about produced in tendon tissue. Tenomodulin has these regions from a genetic point of view. We been mined from human and murine EST da- have used gene expression profiling of the tabases by similarity with chondromodulin-I bovine rumen, reticulum, omasum, abomasum, (ChM-I) mRNA, an anti-angiogenic extra- duodenum, jejunum and ileum to examine cellular matrix (ECM) protein secreted by car- differences between them. Directionally cloned tilage cells. The reconstructed 1214 bases nu- cDNA libraries of each of the tract’s regions cleotide sequence of equine tenomodulin en- were constructed using the Stratagene ZAP codes 371 amino acid sequence, and showed cDNA synthesis kit and approximately 2000 high similarity and common motifs with hu- clones were sequenced for each region. Se- man and murine tenomodulin sequences. The quences were submitted to the MAGPIE pro- expression of this molecule in the regenerating gram, a system for the automated analysis of part of injured SDFT tissue had been visual- biological sequences, functional assignments ized by in situ hybridization. Therefore, the were made for the various ESTs, gene ontolo- role and function of this molecule are sub- gy assignments were then made and sequences jected to study as well as its possibility as a were assembled into contigs where possible. It molecular marker of functional tenocytes. was found that significant differences in gene expression exist as one moves from one com- C022 partment to the next. While lysozymes are very African Swine Fever virus (ASFV): abundant in the abomasum, for example, they microrarray analysis of differential gene are virtually absent from the other regions. expression in porcine macrophages Ribosomal sequences too showed differences in expression between regions. Finally, a num- PAUL A. HOPWOOD(1), FUQUAN ber of the GI regions did not appear to express ZHANG(2), CHARLES ABRAMS(2), LINDA a particular type of sequence preferentially. A DIXON(2), ALAN ARCHIBALD(3), RI- comprehensive overview of the differences CHARD TALBOT(3), DAVE BURT(3) &. found will be presented. STEWART LOWDEN(1) (1) Preclinical Veterinary Sciences, Royal C020 (Dick) School of Veterinary Studies, University Molecular cloning and characterization of of Edinburgh, Scotland, UK. (2) Institute of mRNA for Equus caballus tenomodulin gene Animal Health, Pirbright, Surrey, UK (3) UK (TNMD) Centre for Functional Genomics in Farm Ani- mals, Roslin Institute, Edinburgh, Scotland, TELHIAS HASEGAWA1, MUTSUMI UK MATSUTA1, FUMIO SATO1, ATSUTOSHI A porcine cDNA microarray has been con- KUWANO2 structed to examine gene expression profiles in 1Japan Racing Association, Equine Research macrophages infected with African swine fever Institute, Laboratory of Molecular and Cellu- virus (ASFV). ASFV is a serious threat to the lar Biology, Utsunomiya, Japan; 2Japan pork industry worldwide, causing rapidly fatal Racing Association, Equine Research Institute, haemorrhagic fever in domestic pigs. Howe- Clinical Sciences and Pathobiology Division, ver, although the virus infects African species Utsunomiya, Japan (e.g warthog and bushpig), they are resistant to Although the superficial digital flexor tendon disease through unknown mechanisms. Using a (SDFT) is frequently injured in equine athletes microarray to investigate global gene expressi- and presenting a significant problem in equine on in infected cells has provided insights into sports medicine, there are not enough informa- the virus-host interaction & immune-evasion tion regard to mechanisms on development and strategy and to improved understanding of regeneration of tendon tissues. Because tendon viral pathogenesis. The parallels with other cells, so called ‘tenocytes’, should contribute haemorrhagic fevers such as Ebola and Mar- to both processes, characterization of tenocytes burg suggest ASFV as a valuable model for is essential in these studies. Thus, we at- these diseases. tempted to clone and characterize an mRNA The microarray consists of 2500 encodes equine tenomodulin (synonyms: ten- cDNAs comprising selectively cloned target din, chondromodulin-I like protein, and ChM- transcripts including cytokines, cell surface

55 Section C: Functional Genomics markers and signal transduction molecules, and C024 clones derived from subtracted macrophage Characterisation of Gene Expression in RN- libraries. To establish the differential gene Pigs Carrying a Mutation in PRKAG3 expression pattern associated with ASFV in- fection, the microarray has been interrogated PER HORN1, JAKOB HEDEGAARD2, RENE with probes from Sus scrofa macrophages; LAMETCH2, EMØKE BENDIXEN2, CHRI- both uninfected and infected in vitro with a STIAN BENDIXEN1 virulent strain of ASFV. We will present this Departments of 1Animal Breeding and Gene- data and also describe ongoing experiments tics; 2Animal Product Quality; Danish Institute which aim to investigate gene expression in of Agricultural Sciences, P.O. Box 50, DK- macrophages from ASFV-resistant species and 8830 Tjele, Denmark cells infected with mutant virus. These studies The porcine RN-genotype has large effects on will further elucidate the mechanisms of viral meat quality and processing yields. This ge- pathogenesis and host-resistance. notype possess a dominant mutation resulting in a ~ 70 % increase in muscle glycogen con- C023 tent in glycolytic muscle cells, which is con- Polymorphic markers for three genes ex- verted into lactate postmortem resulting in a pressed in the horse macrophage lowered ultimate pH. Furthermore, a number of other physiological changes are observed, PETR KRÁLÍK, LUDMILA LUKESZOVÁ, including enlarged sarcoplasmatic compart- JÁN MATIAŠOVIC, PETR HOŘIN ments and higher density and altered morpho- University of Veterinary and Pharmaceutical logically of mitochondria. The RN- gene has Sciences, Institute of Animal Breeding and been mapped to chromosome 15 and was re- Genetics, Brno, Czech Republic cently identified as the PRKAG3 gene enco- Detection of polymorphisms of genes ex- ding a muscle specific isoform of the regulato- pressed in macrophages is a prerequisite for ry γ3 subunit of adenosine monophosphate application of functional genomics in studying activated protein kinase (AMPK). The RN- mechanisms of innate immunity and of genetic genotype is a single nucleotide substitution in resistance to intracellular pathogens. Here, PRKAG3 causing a change from arginine to polymorphisms of three horse genes crucial for glutamine. macrophage function were studied. A part of We have examined the gene expression pat- the horse interferon-gamma gene from 5' UTR terns in the RN-pigs by restriction fragment down to exon 4 was amplified and screened differential display (RFDD), 2D-gels and using a set of restriction endonucleases. Two Northern blot analysis. RFDD showed the polymorphic sites were found by AfaI, one majority of differentially expressed genes to be localized within intron 1 and the second within of mitochondrial origin. Further analysis intron 3. Homozygotes as well as heterozy- showed a general increase in mitochondrial gotes in both polymorphic sites were found in gene expression probably accounting for the Old Kladruber horses. were higher respiration seen in RN-pigs and this found to be polymorphic only in the intron 3 phenomenon is likely to be explained by the polymorphic site. A PCR-RFLP method for higher density of mitochondria. The 2D-gels detecting previously reported allelic variants of showed an elevated expression of UDP- the interleukin-12 p40 gene was developed. A glucose pyrophosphorylase 2 (UDPG2). This 1587 bp long PCR product containing poly- observation was confirmed by Northern blot- morphic nucleotide position 714 in exon 6 was ting and can, at least in part, explain the higher cleaved with the Aci I restriction endonuclease glycogen content seen in the RN-pigs. detecting the A/G substitution involved. All three possible genotypes were identified in Old C025 Kladruber horses. A GT microsatellite was Characterization of the porcine peroxisome found within the 3´UTR of the TNF-alpha proliferator activated receptor gamma coac- receptor 1 gene. Preliminary results suggest tivator 1 (PPARGC1) existence of at least three alleles whose length varied from 42 to 58 GT repeats. Kathleen Jacobs1, Gary Rohrer2, Heinz Bart- henschlager3, Antonin Stratil4, Mario Van Poucke1, François Piumi5, Martine Yerle6,

56 Section C: Functional Genomics

Marc Mattheeuws1, Alex Van Zeveren1 & Luc C026 Peelman1 Partial cloning and chromosomal mapping 1Department of Animal Nutrition, Genetics, of the porcine ADRP gene Breeding and Ethology, Ghent University, Merelbeke, Belgium, 2USDA ARS US Meat TAE-HUN KIM1, NAM-SUN KIM1, BONG- Animal Research Center, Clay Center, NE, HWAN CHOI1, JUN-HEON LEE2, CHRIS USA, 3Institute of Animal Husbandry and MORAN2, IL-CHEONG CHEONG1 Breeding, Department of Animal Breeding and 1Animal Genomics & Bioinformatics Division, Biotechnology, University of Hohenheim, National Livestock Research Institute, RDA, Stuttgart, Germany, 4Institute of Animal Physi- Suwon 441-350, Republic of Korea; 2Centre ology and Genetics, Academy of Sciences of for Advanced Technologies in Animal Genetics Czech Rpublic, 277 21 Libechov, Czech Re- and Reproduction, University of Sydney, NSW public, 5Laboratoire de Radiobiologie et 2006, Australia d’étude du génome, INRA CEA jouy-en-Josas, Adipose differentiation related protein (ADRP) France & 6Laboratoire de Génétique Cellu- expression increases rapidly from a very low laire, INRA, Castanet-Tolosan Cedex, France. level as undifferentiated adipocytes mature. Peroxisome proliferative activated receptor We report here the molecular cloning, charac- gamma coactivator 1 (PPARGC1) is a coacti- terization, and chromosomal localization of the vator of nuclear receptors with an important porcine ADRP gene. Partial cDNA was ampli- function in adaptive thermogenesis. It influ- fied from total RNA of porcine adipose tissues ences genes involved in regulation of body by RT-PCR using consensus primers designed weight and composition. Therefore, PPARGC1 from human and cattle sequences. PCR pro- can be considered as a candidate gene for car- ducts of 700 bp and 580 bp were cloned and cass and meat quality traits. A BAC clone, sequenced. 1213 bp of porcine sequence was isolated using a PPARGC1 PCR fragment has compared with human, cattle, and mouse sho- been mapped by FISH to Sscr8p21. A (CA)n- wing amino acid sequence identities of 89%, microsattelite (SGU0001) isolated from the 89% and 82%. Analysis of the amplification BAC has been mapped to porcine chromosome pattern in the 27 clones of the French porcine- 8 by RH mapping. The most significantly rodent somatic cell hybrid panel allowed re- linked marker (2pt analysis) is SWR1101 gional assignment to SSC1q2.3–q2.7. The (57cR; LOD=5.47). SGU001 was also mapped human location at HSA9p21.3 and porcine at the same position as marker KS195 (32.5 location are consistent with the known syntenic cM) by linkage mapping on the MARC refer- relationship between SSC1 and HSA9. The ence family. PPARGC1 was located between characterization of this gene provides a useful the most proximal marker SW905 and the fol- starting point for evaluation of a positional lowing SW933 with the respective supports candidate gene for frequently reported fatness 8.03 and 11.92 in sex specific linkage analysis QTL in the long arm of SSC1. on the Hohenheim reference family by using an AseI polymorphism located in exon 8 of the C027 gene. The coding exons of the porcine gene Candidate genes for progressive external were sequenced and compared to human, ophthalmoplegia (PEO) in cattle mouse and rat sequences. The coding regions HEIDI KUIPER1, GABI HAUKE1, CORD and surrounding sequences harbouring known 1 1 splice sites of the gene were scanned for poly- DRÖGEMÜLLER , TOSSO LEEB , JOHN L. WILLIAMS2, OTTMAR DISTL1 morphisms. Five intronic and four exonic 1 SNP’s were found. No SNP’s were found in Institute of Animal Breeding and Genetics, School of Veterinary Medicine Hannover, known regulatory sequences. Some exonic 2 SNP’s have effect on the translation product of Hannover, Germany; Roslin Institute (Edin- the gene. Transcription of the gene was de- burgh), Rosling, Midlothian, Scotland tected by RT-PCR in porcine adipose, muscle, Bilaterial convergent strabismus with kidney, liver, brain and heart tissues and in exophthalmus (BCSE) is a widely spread here- lower amounts in duodenum and adrenal tis- ditary disease in many cattle populations. It is sues. known that autosomal dominant progressive external ophthalmoplegia with various mito- chondrial DNA deletions in man can be caused

57 Section C: Functional Genomics by mutations in the genes C10orf2 (chromo- ducing milk with a fishy off-flavour and nor- some 10 open reading frame 2), POLG, (poly- mal milk, respectively. The affected cow was merase (DNA directed) gamma) and SLC25A4 homozygous for a R238X nonsense mutation (solute carrier family 25, member 4). In our that prevents >50% of the peptide synthesis. A study we mapped the three genes C10orf2, pyrosequencing test showed a strong associa- POLG, and SLC25A4 by flourescences in situ tion between this mutation and off-flavour hybridization on bovine metaphase spreads. milk in a case/control material. The R238X Using a comparative approach human cDNA frequency among 100 SRB individuals was clones from these genes were obtained to 0.155 whereas we did not find the mutation in screen the bovine RPCI-42 BAC library. Posi- samples from a few other breeds. Sequence tive bovine BAC clones were labelled with analysis of RT-PCR products from cattle liver digoxigenin and then used for FISH on GTG mRNA revealed only barely detectable levels banded chromosomes. The cattle ortholog of of the mutant FMO3 transcript in two het- the gene POLG was mapped to BTA21q17- erozygous carriers of R238X. This could q22 and the orthologs of C10orf2 and SLC25A probably be explained by nonsense-mediated were mapped to BTA26q13-q21 and mRNA decay (NMD). BTA27q14-q15, respectively. The results of our FISH experiments were corroborated by C029 radiation hybrid mapping of STS markers de- Characterization of perixome profilator- rived from the BAC end sequences using the activated receptor gamma (PPARγ) iso- 3,000 rad Roslin/Cambridge bovine whole- forms in the pig genome radiation hybrid panel. RH mapping results confirm the known synteny conservati- TOSHINORI OMI1, ŠPELA ŠPILAR1, BER- on between parts of HSA15 with BTA21, parts TRAM BRENIG2, GERALD STRANZIN- of HSA10 with BTA26, and HSA 4q with GER1, STEFAN NEUENSCHWANDER1 BTA27. The development of microsatellites of 1Swiss Federal Institute of Techology, Institute the candidate gene containing BAC clones of Animal Sciences, Zürich, Switzlerland; provides the basis for future linkage studies 2University of Göttingen, Insitute of Veterinary that might clarify the role of these genes for Medicine, Göttingen, Germany BCSE in German Brown cattle. Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor which C028 controls genes that are involved in the regula- A non-sense mutation in the FMO3 gene tion of energy metabolism, cell differentiation, causes fishy off-flavour in cow’s milk apoptosis and inflammation. So far, two PPARγ isoforms (γ-1b, γ-1c and γ-1d) which STEFAN MARKLUND, LEIF ANDERSSON, are isolated from a porcine fat tissue cDNA VICTORIA GUSTAFSSON, ANNE library or by RT-PCR using total RNA from LUNDÉN fat tissue. The cDNA structure and genomic Department of Animal Breeding and Genetics, organization of the PPARγ showed that free Swedish University of Agricultural Sciences, exons are associated with the γ-1 cDNAs (A1, Uppsala, Sweden A‘, A2) and one exon (B) with the γ-2 cDNA. Fishy off-flavour sometimes occurs in milk PPARγ-1b is a splice variant of the known γ-1a from cows of the Swedish Red and White starting with exon (B) with the γ-2 cDNA. breed (SRB). It was recently shown that the PPARγ is a splice variant of the knwon γ-1a off-flavour comes from high levels of trimeth- starting with exon A1, whereas PPARγ-1c and ylamine (TMA) in the milk. ‘Fish-odour syn- γ-1d start from a seperate leader exon (A‘) drome’ is a similar human phenotype charac- which is located approximetaly 1kb down- terized by high levels of TMA in the body stream of exon A1. Based on sequence com- fluids caused by recessive loss-of-function parisons we found 6 SNPs in the PPARγ and γ- mutations in the FMO3 gene encoding flavin- 2 sequence. The relevance of the allelic vari- containing monooxygenase 3. This liver en- ants will be investigated. Furthermore, expres- zyme converts TMA to the odourless TMA sion fat γ-2 and γ-1 showed the same expres- oxide. We used cross-species PCR on genomic sion level whereas in the subcutaneous fat DNA to amplify and determine ~50% of the more γ-1 expressed than γ-2. Among PPARγ-1 coding FMO3 sequence from two cows pro- transcripts the γ-1a isoform is most abundant

58 Section C: Functional Genomics and the transcripts which do not contain exon ferent breeds of dogs. The incidence of this A2 are much less expressed. inherited congenital anomaly is highest in Dalmatian dogs with 20-30%. Mutations in C030 various genes are causative for congenital non- Analysis of nucleosome – Stat5 relationship syndromic hearing impairment in humans or in βLG transgene expression mouse and 26 of these genes were chosen as candidates for mapping in dogs. In an effort to RAMONA N. PENA, BRUCE WHITELAW identify these canine genes, human 32P–labeled Division of Gene Expression and Development, inserts of IMAGE cDNA clones were used to Roslin Institute, Roslin, Scotland, UK screen the high density filters of the RPCI-81 The proximal promoter of sheep β- canine BAC library. Positive BAC clones were lactoglobulin (βLG) gene is well characterised isolated and the BAC end sequence data were and includes three Stat5 binding sites, 190-bp used to design canine-specific primers for away, which promote prolactin induction of mapping on the canine RHDF5000 radiation this gene. Using the monomer extension tech- hybrid panel. The BAC clones were labeled by nique we have previously identified a strong nick translation and used for fluorescence in sequence dependent nucleosome positioning situ hybridization (FISH) on GTG-banded over the proximal promoter of this gene which canine metaphase chromosomes. Identification coincides with the region spanning the three of the chromosomes was done according to the Stat5 sites. In order to further analyse the role established GTG- and DAPI-banded karyotype of this nucleosome-directing DNA sequence of the domestic dog. So far, 17 candidate genes we generated six constructs where the region have been mapped on the canine genome. The comprising the three Stat5 sites was inserted obtained results confirm the established syn- (both in forward and reverse orientations) at teny of the latest integrated canine RH map. several distances from the original site. The The development of microsatellite markers of insertion of these short DNA sequence en- the candidate gene containing BAC clones hanced gene expression in stably transfected provides the basis for future linkage studies HC11 cells in all forward-inserted constructs. that might clarify the role of these genes for In a second set of constructs a LacZ-based congenital nonsyndromic sensorineural deaf- reporter gene replaced the βLG transcription ness in Dalmatian dogs. unit. For each construct, the total level of LacZ expression was correlated to the percentage of C032 expressing cells as assessed by a FACS/Scan Statistical Analysis of Gene Expression Data protocol. We are currently analysing the in from Hypertrophying and Normal Muscle vivo effect of this sequence repetition in trans- Tissue genic mice. Expression studies and nu- JAMES M. REECY1, DAN NETTLON2 cleosome-position analysis is currently under- 1 2 way in these animals. Department of Animal Science; Department of Statistics, Iowa State University, Ames, IA C031 50011 Mapping of candidate genes for nonsyn- Knowledge of the molecular mechanisms un- dromic sensorineural deafness in dogs derlying skeletal muscle growth could lead to new methods for enhancing lean tissue deposi- SIMONE G. RAK1, CORD DRÖGEMÜL- tion in livestock. Affymetrix GeneChip tech- LER1, HEIDI KUIPER1, TOSSO LEEB1, nology was used to measure the relative levels PASCALE QUIGNON2, CATHERINE messenger RNA transcripts in the soleus mus- ANDRÉ2, OTTMAR DISTL1 cle isolated from gastrocnemius ablated or 1Institute of Animal Breeding and Genetics, sham operated rats. We used this data on tran- School of Veterinary Medicine Hannover, script abundance to screen for evidence of Hannover, Germany; 2UMR 6061 CNRS, Gé- increased or decreased gene expression in hy- nétique et Développement, Faculté de Méde- pertrophying tissue relative to control tissue. cine, 35043 Rennes Cedex, France An ANOVA approach, coupled with boot- Congenital nonsyndromic sensorineural deaf- strapping of residuals, was used to determine a ness has been reported in several livestock p-value for each gene, where small p-values species and, particularly, in more than 60 dif- are indicative of differential expression be-

59 Section C: Functional Genomics tween treatment and control tissues. Resam- immunohistochemical detection of preferential pling-based multiple testing techniques were signals for C/EBPalpha and PPARgamma in used to adjust p-values to control the overall intramuscular fat cell nuclei over the muscle type I error rate and provide a list of genes that tissue. Thus, C/EBPalpha and PPARgamma may play a role in promoting skeletal muscle may be useful as indicators for marbling scores growth. We found 19 genes with step-down in finished cattle. At present, we are going to adjusted p-values less than 0.05. The small investigate age-dependent expression patterns number of animals used in this study (three rats of these factors in Musculus longissimus of per treatment) is a concern. To address this Japanese Black and Holstein breeds and to concern we computed adjusted p-values de- elucidate the molecular mechanism underlying signed to control the false discovery rate. intramuscular fat development during bovine These FDR-adjusted p-values are designed to fattening. produce a list of differentially expressed genes of which a certain percentage (5%) is believed C034 to be false positives. We found 125 genes with TYRP1 mutations in dogs affect coat and FDR-adjusted p-values less than 0.05. The use nose color of FDR analysis dramatically increases the number of differentially expressed genes. SHEILA M. SCHMUTZ, TOM G. BERRYERE C033 University of Saskatchewan, Department of Relationship between expression patterns of Animal and Poultry Science, Saskatoon, C/EBP family and PPARgamma genes and Canada marbling scores in the muscle of Japanese Skin biopsies of several dogs of various coat Black Cattle colors were used to prepare mRNA and cDNA which allowed us to obtain the first 1536 base REI SAKOYAMA, YUKIO TANIGUCHI, pairs of the 1612 bp of tyrosinase related pro- TAKAHISA YAMADA, YOSHIYUKI tein 1 (TYRP1). Three polymorphisms were SASAKI found to be associated with brown coat color in Graduate School of Agriculture, Kyoto Univer- dogs which had normal melanocortin 1 recep- sity, Kyoto, Japan tor (MC1R) sequence: a premature stop codon Intramuscular fat accumulation is an economi- in exon 5, a proline deletion in exon 5, and a cally important trait in beef cattle and an cysteine to serine change in exon 2. These unique but yet unidentified molecular mecha- mutations caused brown nose and pad color in nism is thought to be involved in the expressi- dogs which had at least one copy of wild type on of this trait. As a first step to elucidate this MC1R. Dogs which were homozyogous for a mechanism, we investigated expression pat- premature stop codon in MC1R, had yellow to terns of the transcription factors which play red coat color but the pigmentation of their important roles in adipocyte development, i.e., nose and pads was brown instead of black C/EBPalpha, beta and delta and PPARgamma, when they contained 2 copies of these TYRP1 in the muscle in accordance with marbling mutations. All 100 dogs from 29 breeds fit the scores. Competitive-PCR analysis revealed coat and nose colors predicted by these muta- that expression levels of C/EBPalpha and tions. No differences in shade of brown were PPARgamma but not C/EBPbeta and delta, noted amongst homozygotes of the 3 different increased in proportion to an increase of mar- TYRP1 mutations. All 3 mutations are propo- bling scores. In immunohistochemical study, sed to be relatively old since they all occur in we detected C/EBPalpha signals mainly in longhaired, shorthaired, and wirehaired breeds. intramuscular fat cell nuclei, C/EBPbeta and This study documented gene interactions at the delta signals mainly in cell nuclei in connecti- molecular level between MC1R and TYRP1 ve tissue and muscle bundle and rarely in in- which were proposed by Little (1957) and tramuscular fat cell nuclei, and PPARgamma Winge (1950), but not been previously proven. signals mainly in cell nuclei in connective tissue and in some degree in intramuscular fat cell nuclei. The result of association of C/EBPalpha and PPARgamma expression levels with marbling scores is consistent with

60 Section C: Functional Genomics

C035 BERTRAM BRENIG2, TOSSO LEEB1, Interacting phenotypic effects of co-existing OTTMAR DISTL1 variants within a single gene - cellular stress 1Institute of Animal Breeding and Genetics, response is significantly affected by interac- School of Veterinary Medicine Hannover, tions between promoter and 3´-UTR vari- Hannover, Germany; 2Institute of Veterinary ants of the porcine .2 gene Medicine, University of Göttingen, Göttingen, Germany MANFRED SCHWERIN1, STEFFEN Candidate genes being involved in the embryo MAAK2, RAINER FUERBASS1 implantation in the pig between day 10-20 1Department of Molecular Biology, Research were chosen in consideration of expression Institute for the Biology of Farm Animals, patterns of endometrial proteins during early Dummerstorf, Germany, 2Institute of Animal pregnancy. Additional selection criteria were Breeding and Husbandry with Veterinary the knowledge about the function of homolo- Clinic, Martin-Luther-University, D-06108 gous proteins in other species and QTL studies. Halle, Germany For the genes CTSL, ITIH4, EGF, LIF poly- Cellular tolerance to stress is mediated by a morphic DNA markers (microsatellites or family of proteins termed heat-shock proteins SNPs) were developed and they were physi- (HSP). As shown in this study of porcine pri- cally assigned by FISH and RH mapping. mary fibroblasts hsp70.2 is induced to produce Furthermore, LIF and CTSL were molecularly abundant amounts of transcript upon heat characterized. CTSL (cathepsin L) is a lyso- shock treatment. However, transcript levels somal cysteine protease. The complete DNA and heat shock response was found to be dif- sequence of this gene which spans about 5.6 kb ferent in various individuals. While previously and consists of 8 exons was determined. CTSL described functional promoter variants of this was assigned to SSC10q11→q12. The LIF gene can partly account for the high variability (leukemia inhibitory factor) gene which en- of heat-induced transcript levels, they are un- codes a pleiotropic cytokine, spans about 6.3 related to the observed highly variable absolute kb and consists of 5 exons including three al- amounts of hsp70.2 transcripts. Comparative ternative first exons (1D, 1M, 1T) spliced onto sequence analysis revealed an alteration of the common second and third exons. LIF was 3´-UTR sequences in these samples. The vari- mapped on SSC14q21→q22. ITIH4 belongs to ant 3´-UTR allele proved to extend the half life the inter-α-trypsin inhibitor family of serine of the hsp70.2 mRNA. It is suggested that the protease inhibitors. This gene was localized on cellular stress response is significantly affected SSC13q21→q22. The polypeptide EGF (epi- by the action and interaction of both, promoter dermal growth factor) stimulates growth and and 3´-UTR variants of the hsp70.2 gene oc- proliferation of skin epithelia in the embryo. In curring naturally in different pig breeds. The the uterus it may induce endometrial growth mRNA stabilising 3´-UTR variant was signifi- and differentiation. EGF was mapped on cantly more frequently associated with the SSC8q23→q24. The developed markers will promoter-"down" variant in two pig popula- be used for upcoming association studies to tions analysed. This implies that the mutant 3´- show significant additive and dominant gene UTR may be advantageous for the carrier in effects on the embryonic survival and number the context of an impaired promoter. Our re- of piglets born alive. sults demonstrate that polymorphisms with opposite effects on gene expression occur even C037 within a single gene. This makes evident the Differences in Stearoyl-CoA Desaturase limits of candidate gene experiments where mRNA Levels in Muscle between Japanese one polymorphism in a gene is investigated on Black and Holstein cattle its relationship to phenotypes. MASAAKI TANIGUCHI1, HIDEYUKI C036 MANNEN2, YASUHIKO SHIMAKURA3, Candidate genes for embryonic survival in AKIO OKA4, HIROKO WANABE5, the Pig MASANORI KOMATSU5, GREGORY S. 6 2 1 HARPER ; SOICHI TSUJI ANDREAS SPÖTTER , CORD 1 1 1 Kobe University, Graduate school of Science DRÖGEMÜLLER , HEIDI KUIPER , and Technology, Kobe, Japan, 2Kobe Univer-

61 Section C: Functional Genomics sity, Faculty of Agriculture, Kobe, Japan, retrieval. We have amplified one fragment of 3Animal Inspection Office, Gifu, Japan, the porcine DBH cDNA encompassing exon 4 4Animal Science Division, Hyogo Central In- and 12 in eight pigs belonging to four different stitute of Agriculture, Kasai, Japan, breeds (Iberian, Landrace, Large White and 5Department of Livestock and Grassland Sci- Pietrain). Direct sequencing of the PCR ence, National Agricultural Research Center product revealed 87%, 82% and 88% for Western Region, Oda, Japan, nucleotide identity with the orthologous 6CSIRO Livestock Industries, Brisbane, human, mouse and bovine sequences, Australia respectively. The pig dopamine β-hydroxylase Japanese Black cattle have a fatty acid profile mRNA was expressed in the uterus, ovary, that differs from that of other breeds in terms testicle and hypophysis. Moreover, we have of unsaturated fatty acids. The enzyme stea- performed the physical mapping of the DBH royl-CoA desaturase (SCD, also known as ∆9- locus by using the INRA-University of desaturase) has been implicated in creating this Minnesota somatic cell radiation hybrid panel breed difference. In this study, we compare the (IMpRH). The DBH gene was significantly level of the SCD gene expression in muscle linked to the EST SSC10D08 and the between two cattle breeds, Holstein and Japa- microsatellite marker SW1301 on 1q2.13 with nese Black. First of all, we cloned SCD cDNA LOD scores of 12,99 and 6.09, respectively. and sequenced it so that the RT-PCR is appli- Comparative mapping between pig and human cable. In so doing, we identified five nucleoti- revealed that this result is consistent with the de substitutions in the cDNA sequence. As chromosomal location of the human DBH gene those substitutions are linked each other, two at Hsa9q34. haplotypes, A and G, were observed. The gene frequency of haplotype A is 55% in Japanese C039 Black and 39% in Holstein. The nucleotide Improving the human-pig comparative substitution at 878bp causes the substitution of map: sequencing of 16,000 ests and mapping amino acid alanine for valine in the protein. 175 pig genes with human orthologs There were significant differences of SCD mRNA content and unsaturated fatty acids CHRISTOPHER K. TUGGLE1, JON A. composition between two breeds; Japanese GREEN2, CARLOYN FITZSIMMONS1, Black showed higher SCD mRNA (3.4-fold) in RAMI WOODS2, RANDALL S. PRATHER2, muscle and higher unsaturated fatty acids (1.3- SERGEI MALCHENKO3, M. BENTO SOA- fold) in fat tissue than Holstein. However, RES3, CHAD A.ROBERTS4, KEVIN PE- there were no differences of SCD mRNA con- DRETTI4, THOMAS CASAVANT4, DANIEL tent and unsaturated fatty acid composition POMP5, J. BRAD EDEAL5, YUANDAN between two haplotypes, and no correlation ZHANG1, MAX F. ROTHSCHILD1, KEVIN between SCD mRNA content and unsaturated GARWOOD6; WILLIAM BEAVIS6 fatty acid content in fat tissue within a breed. 1Department of Animal Science, Iowa State University, Ames, IA USA; 2Department of C038 Animal Science, University of Missouri- 3 Sequencing and physical mapping of the Columbia, Columbia, MO USA; Department porcine dopamine β-hydroxylase gene of Pediatrics, University of Iowa, Iowa City, IA USA; 4Department of Electrical and Computer ANNA TOMÁS, MARCEL AMILLS, Engineering, University of Iowa, Iowa City, IA 5 ARMAND SÁNCHEZ USA; Department of Animal Science, Univer- Universitat Autònoma de Barcelona. Depar- sity of Nebraska, Lincoln, NE USA; 6 tament de Ciència Animal i dels Aliments, National Center for Genomic Resources, Barcelona, Spain Sante Fe, NM USA Dopamine β-hydroxylase (DBH) is the enzyme We are developing extensive sequence and that catalyzes the conversion of dopamine to mapping data for cDNAs expressed in female norepinephrine. The transfer of catecholamines reproductive tissues. We have produced a total across the placenta is essential for embryo of 22 libraries from different stages of estrus or survival. Female mice with targeted disruption gestation for embryo, anterior pituitary, hy- of the DBH gene showed an impaired maternal pothalamus, ovary, uterus, and term placenta. behavior caracterized by a lack of pup More than 16,000 sequences from random

62 Section C: Functional Genomics clones have been produced and 90% submitted and search for associations with fat deposition to Genbank. The average read length across and fatty acid composition. this dataset is >400 base pairs. As assessed by clustering analysis, these data represent nearly C041 10,000 different genes. A BLAST analysis Targeted gene identification using exon using 9,336 clusters, as of February 2002, indi- trapping on bovine chromosome 6 cates that 4,099 of these clusters are unique relative to porcine Genbank genes/ESTs ROSEMARIE WEIKARD, CHRISTA KÜHN, (BLAST score <50). To facilitate selection of TOM GOLDAMMER, MANFRED genes for comparative mapping, we have de- SCHWERIN veloped software to predict the cytogenetic Research Institute for the Biology of Farm location of pig ESTs. We identified human loci Animals, Research Unit Molecular Biology, with a BLAST score >200 to our EST dataset, Dummerstorf, Germany and then predicted the pig location of high- Linkage analyses strongly suggest several QTL scoring ESTs based on human cytogenetic and for production, health and conformation traits RH mapping data, along with human:pig in the middle region of bovine chromosome 6 chromosome painting information. Pig EST (BTA6). For identification of genes located in matches to human loci with consistent cytoge- this region we focused on both comparative netic and RH mapping locations total 1,486. mapping of genes from orthologous human Within the human genome, there is an average chromosome 4 onto BTA6 by high-resolution distance of 9.4±5.8 cR between loci with a pig radiation hybrid mapping and targeted identifi- EST match. To date, 175 loci have been map- cation of coding sequences in subchromosomal ped using both the SCHP and the RH panel, regions of BTA6 poorly covered with infor- with nearly complete agreement to prediction. mation from the human gene map. A bovine A website (http://pigest.genome.iastate.edu) BAC library was screened with 60 BTA6 has been established for access to these se- markers comprising the region of BTA6 quences and the analysis data. flanked by markers BMS2508-ILSTS87. As an initial step towards systematic transcript analy- C040 sis in the critical BTA6 region we performed Identification of one single nucleotide poly- exon trapping on four selected region-specific morphism at exon 7 of the porcine cytosolic BACs. Twenty one unique putative exon traps malate dehydrogenase (MDH1) gene were detected. The chromosomal location of 19 putative exons was confirmed by remapping ORIOL VIDAL1, ARMAND SÁNCHEZ1, them onto their parent BACs by PCR. The LUÍS VARONA2, MARCEL AMILLS1 trapped sequences were used as templates to 1Departament de Ciència Animal i dels Ali- screen public gene databases for in silico gene ments, Facultat de Veterinària, Universitat identification. However, there was no identity Autònoma de Barcelona, Bellaterra 08193, to known genes and ESTs, although 6 exon Spain; 2Àrea de Producció Animal, Centre traps revealed substantial similarity to bovine UdL-IRTA, Lleida 25198, Spain repetitive sequences. To this point, 14 of the Cytosolic malate dehydrogenase (MDH1) is an trapped sequences are novel and could be pre- enzyme that catalyses the conversion of oxa- dicted to originate from unknown genes. The lacetate to malate, that afterwards is converted present exon sequence information provides to pyruvate by the malic enzyme. These two probes for screening cDNA libraries to isolate biochemical reactions are coupled and release full-length cDNA and contributes to establish a most of the NADPH required for fatty acid detailed transcription map for the specific synthesis. We have sequenced the pig MDH1 BTA6 region for positional cloning efforts. gene in six Landrace (LD), Large White (LW) and Pietrain (PI) pigs. Total RNA from liver C042 was reverse transcribed and amplified yielding Comparison of Pedigrees of BSE infected one fragment of 1 Kb (from exon 2 to 9). This Holstein-cows with the normal Holstein fragment was sequenced forward and reverse. population in Germany We found one silent C→ T mutation at exon 7. We have designed a primer-extension analysis MAIK WITTEMEIER1, WALTER SCHULZ- protocol in order to type this polymorphism SCHÄFFER2, BERTRAM BRENIG1, HER-

63 Section C: Functional Genomics

MANN H. SWALVE3, EKKEHARD Institute of Animal Breeding and Genetics, SCHÜTZ4, WILHELM WEMHEUER1 School of Veterinary Medicine Hannover, 1University of Göttingen, Institute of Veteri- Hannover, Germany nary Medicine, Göttingen, Germany; In cattle the polled phenotype shows tight link- 2University Clinics of Göttingen, Göttingen, age with genetic markers of the centromeric Germany; 3University of Halle-Wittenberg, region of BTA1, but several studies have not Institute of Animal Breeding and Animal Hus- been able to order the polled locus relative to bandry, Halle/S., Germany; (4) Chronix Bio- the markers used. Without knowledge of true medical, Benicia, CA, USA marker and gene order on proximal BTA1 The question whether there is a genetic dispo- maps, the selection of candidate genes using sition for susceptibility of BSE infection is still comparative mapping data may lead to failure. under debate. Furthermore it is unclear yet Current comparative maps between HSA21 whether the risk of BSE differs between or and the proximal part of BTA1 are insufficient within the cattle breeds. There is a large and to define chromosomal rearrangements due to convincing body of evidence that genetic dif- the low density of mapped genes in the bovine ferences of PrP in both, Scrapie in sheep as genome. Recently, the availability of the com- well as CJD in Humans having a major impact plete sequence and gene catalogue of the long on the disease risk. Therefore it seemed likely arm of HSA21 has provided valuable tools for to find such disease favouring variants in the a more detailed comparative analysis of corre- Bos Taurus PrP gene as well. But in contrast, sponding segments on BTA1. In this study we in cattle no variant of the PrP gene could been constructed a comprehensive RH map of the found to be associated with an increased risk of proximal part of BTA1 on the 3,000 rad Ros- acquiring BSE. However, that does not exclude lin/Cambridge bovine whole-genome radiation still undiscovered genetic dispositions for this hybrid panel. A set of known bovine markers, disease at other gene loci. The objective of the i.e. 10 microsatellites, 5 genes and 2 cattle study was therefore, to generate pedigrees of ESTs, was used to construct a RH map. Fur- BSE-positive cows in comparison to a control thermore, we mapped eight new STS markers group, with consideration of race, age and derived from bovine RPCI-42 BAC clones. region, to prove whether there are significant These BAC clones contain bovine orthologs of differences. We tried to achieve a 5-generation HSA21 genes, i.e. GRIK1, CLDN8, TIAM1, pedigree of all black and white and red and HUNK, SYNJ1, OLIG2, IL10RB, and KCNE2, white German Holstein-cows, which were and were physically assigned by FISH to tested BSE-positive. The inspection of 90 cows BTA1q12.1-q12.2. Concluding, the RH- found in 2000, 2001 and early 2002 resulted in mapping of 25 loci greatly improved the map 40 complete pedigrees. A randomly selected resolution of the proximal part of BTA1 and age, breed and region matched group of dis- revealed previously unknown details of the ease-free animals (n=xx) served as control. We cattle-human comparative map. found no differences between the BSE and the control groups of pedigrees. In Germany, even C044 the distribution of the breeds of all infected Characteristics of gene expression levels in cows is similar to the typical regional cattle somatic nuclear transfer-derived cloned population. The result of this study suggests bovine placenta based on array analysis of that there is no distinct genetic disposition for a 1,353 genes BSE- infection in the German Holstein popu- lation. In addition we could show that there is HIROSHI GOHMA1, YUKIO TANIGUCHI1, no strain difference in susceptibility of BSE HIROSHI YASUE2, KAZUYOSHI HASHI- between the German cattle breeds. ZUME2, TAKAHISA YAMADA1, YOSHI- YUKI SASAKI1 C043 1Graduate School of Agriculture, Kyoto Uni- A comparative RH map of the polled gene versity, Kyoto, Japan, 2National Institute of region in cattle Agrobiological Sciences, Ibaraki, Japan Somatic nuclear transfer-derived cloned bovine ANNE WÖHLKE, CORD DRÖGEMÜLLER, fetus and placenta are known to be susceptible ANKE BADER, HEIDI KUIPER, TOSSO to a developmental abnormality associated LEEB, OTTMAR DISTL with abortion or stillbirth, as compared to em-

64 Section C: Functional Genomics bryo transfer-derived ones. This susceptibility mone (bGH) gene and its relationship to back- may be partly attributable to an abnormal ex- fat thickness in Hanwoo cattle (Korean cattle). pression of genes at early developmental stage A 522 bp fragment from eight unrelated Han- in the former cloned bovine fetus and placenta. woo cattle was amplified by PCR and subse- Our present study was thus designed to char- quently cloned and sequenced. An MspI RFLP acterize gene expression levels in the somatic corresponding to a C to T transition was obser- nuclear transfer-derived cloned bovine pla- ved at position 2258. It is predicted to be a centa. We have firstly made a catalog of 1,521 missense mutation (Arg to Trp) at codon 166. clones isolated randomly from directionally Codominant Mendelian segregation of the two cloned cDNA library of 60 embryonic day-old alleles was confirmed in two full-sib F2 fami- placenta, yielding clone collection of 795 spe- lies (n = 32, African taurine B. taurus Afri- cies. We have next made a macroarray com- can zebu B. indicus). MspI allele frequencies prising of clones of 1,353 species obtained were calculated in 13 different breeds of cattle from placenta cDNA library as well as from (European and Asian taurines). An analysis of 60-day-old whole fetus cDNA library. Using variance (ANOVA) was conducted to investi- the macroarray, we have examined expression gate the effects of the three genotypes on live levels of 1,353 genes in 60 embryonic day-old weight, backfat thickness and carcass traits in abnormal placentas derived from the cloned 64 Hanwoo bulls. A significant association (P cattle, and in age-matched normal placentas < 0.05) was found with backfat thickness. This from the cloned and artificially inseminated result indicates that the bGH RFLP newly cattle. There was more marked difference in identified might be used as a marker for selec- gene expression level between abnormal and tion against backfat thickness in Hanwoo normal placentas than between normal pla- cattle. centas. This difference almost corresponded to an increase rather than a decrease in abnormal C046 against normal placentas. Three genes, HBA1, Cloning and mapping of cattle caspases HBA2 and SPTB, and one gene, IGF2, respec- tively, expression levels of which showed de- Y.Q.YUAN1,2, L. PEELMAN1, A. DE crease and increase in the abnormal placenta, KRUIF2, A. VAN ZEVEREN1, A. VAN have been suggested to be possible candidates SOOM2 for responsible genes for susceptibility to de- 1Department of Animal Nutrition, Genetics, velopmental abnormality in the cloned cattle. Breeding and Ethology, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium; C045 2Department of Reproduction, Obstetrics and A novel polymorphism in exon 5 of the Bo- Herd Health, Ghent University, Salisburylaan vine Growth Hormone gene and its relati- 133, B-9820, Belgium onship to backfat thickness in Hanwoo Sequential activation of cysteine-aspartic acid cattle proteases (caspases) plays a central role in the execution of cell apoptosis. Studies in caspase DU-HAK YOON1, HONGHE CAO2, YONG- inhibiting or inactivating could be helpful in MIN CHO1, OLIVER HANOTTE3, developing drugs. Currently 13 caspase family IL-CHEONG CHEONG1 members have been characterized in mouse 1Division of Animal Genomics and Bioinfor- while 11 caspases were discovered in human. matics, National Livestock Research Institute, However, only bovine caspase 13 has so far RDA, Suwon 441-350, Korea, 2institute of been described. The aim of this study was to Animal Science, Chinese Academy of Agricul- search for other bovine caspases and map tural Sciences, Beijing 100094, China; them. Fragments of the caspases were ampli- 3International Livestock Research Institute fied by RT-PCR followed by cloning and se- (ILRI), PO Box 30709 Nairobi, Kenya quencing. Mapping of genes was done by use The Growth Hormone (GH) is a member of the of radiation panel. Caspase 1, 4, 5, 6, 7 and 8 somatotropin/prolactin family of hormones. Its have been successfully detected, cloned and role in growth control has been extensively mapped at this moment. studied in human, mice and livestock. We re- port here a novel PCR-RFLP polymorphism within the exon 5 of the bovine Growth Hor-

65 Section C: Functional Genomics

C047 bruck, Austria; 3University of Milano, Veteri- Mapping of the canine HMGA1 gene - a nary faculty, Milan, Italy gene frequently involved in human mesen- The 2140 bp fragment of the bovine kappa chymal tumors casein (K-CN) promoter region has been se- quenced. DNA sequence analysis revealed a K. BECKER1, H. MURUA ESCOBAR1, A. number of potential binding sites for transcrip- RICHTER1, B. MEYER1, I. NOLTE2, tion factors as YY1, Stat5, NF1, AP2, CREBP, J. BULLERDIEK1 CREB and GRE. The consensus TATA box 1Center for Human Genetics, University of sequence was found proximal to the transcrip- Bremen, Bremen, Germany; 2Clinic for Small tion start site. Due to their important role in the Animals, Veterinary School, Hanover, regulation of lactoprotein gene expression we Germany focused on the functionality of the Stat5 bind- The dog has become a more and more inter- ing sites in the bovine K-CN promoter. DNA esting model organism for human diseases fragments containing Stat5 sequences present including cancer. Parallels between human and in the bovine K-CN promoter were used for canine tumors have often been described. Ac- electro mobility shift assay (EMSA) and cordingly, the results of canine gene mapping DNase1 footprinting. The cell lysate from studies will be of considerable significance. transfected COS7 cells, over-expressing the C Rearrangements of its human counterpart on terminally deleted forms of Stat5a and Stat5b 6p21 involving the HMGA1 gene have been proteins was used as a source of enriched tran- described in a variety of mesenchymal tumors scription factor. EMSA and DNase1 foot- as e.g. pulmonary chondroid hamartomas, printing revealed specific binding of Stat5 to uterine leiomyomas and lipomas. So far, it is the target sequences. In order to test the pro- not clear yet if comparable translocations occur moter activity in vitro we transfected bovine in the corresponding canine tumor as well. To mammary gland derived cells (BME UV1/2) further elucidate that question, we have with the luciferase reporter gene construct mapped the canine HMGA1 gene. By fluores- containing the short (925 bp) and the long cence in situ hybridization (FISH), we have (2064 bp) version of the K-CN promoter. In mapped the canine HMGA1 to CFA 23. The the luciferase assay almost no expression was assignment of the canine HMGA1 gene to CFA found after transfection with the shorter pro- 23 clearly shows that the chromosomal region moter variant, however, the longer promoter where the canine HMGA1 gene has been fragment induced weak but specific expres- mapped to is not a hotspot of chromosomal sion, suggesting that 2000 bp of the K-CN breakpoints seen in canine tumors. Therefore promoter are sufficient for induction of expres- in contrast to humans the activation of that sion under in vitro conditions. gene as a result of chromosomal translocations does not seem to play a considerable role in C049 canine tumors. This may be due to the fact that Characterization of a candidate gene for the corresponding changes are not able to in- performance in racehorses duce benign tumors in the dog or to stimulate their growth. Alternatively, there may be fac- N.A. ELLIS, I. TAMMEN, F.W. NICHOLAS, tors favouring the occurrence of the structural H.W. RAADSMA changes in humans which are lacking in dogs. ReproGen, Centre for Advanced Technologies in Animal Genetics and Reproduction, The C048 University of Sydney, Camden and Sydney, Functional analysis of the bovine kappa Australia. casein promoter The control of arterial blood pressure is largely regulated by angiotensin-converting enzyme MARUŠA DEBELJAK1, WOLFGANG DOP- (ACE), a component of the renin-angiotensin PLER2, ANTONELLE BALDI3, PETER system. In humans, a polymorphism in the DOVC1 ACE gene is associated with elite endurance 1University of Ljubljana, Biotechnical Faculty, performance. This gene is therefore one of Department of Animal Science, Domzale, Slo- interest when investigating the effect of gene- venia; 2University of Innsbruck, Department of tics on racing performance in horses. The aim Medical Chemistry and Biochemistry, Inns- of this study is to fully characterise the equine

66 Section C: Functional Genomics

ACE gene. A BAC clone containing this gene Dexter Cattle Australia (DCA) chose to initiate has been obtained through the International and support research to develop a DNA-test to Equine Gene Mapping Workshop (IEGMW). identify carrier animals, prevent carrier x car- Using comparative information, approximately rier matings, and hence reduce the incidence of half of the gene has been sequenced from the chondrodysplastic foetuses. Potential candidate BAC. Collaboration with the IEGMW has also genes were identified, and homozygosity map- led to the mapping of the gene on the somatic ping in regions predicted to contain these cell and radiation hybrid panels, with the re- genes identified a region of interest. A candi- sults confirmed by FISH mapping. ACE was date gene predicted to be in this region was found to be on ECA 11p13, which agreed with mapped on the bovine linkage and physical predictions from comparative maps. Two pools maps, confirming location in the region of of horse DNA are being created for screening interest. One disease causing mutation was for polymorphisms based on sequencing. Fol- identified in the candidate gene, a 4 bp inser- lowing the detection of polymorphisms, asso- tion causing a frame shift which leads to a ciation studies investigating possible links premature termination codon. The resulting between identified ACE haplotypes and racing protein is predicted to contain less than one ability will be performed. Phenotypic data is third of the original amino acid sequence. A being collected in collaboration with the Syd- DNA test has been developed to identify ani- ney University Equine Performance Labora- mals heterozygous for the mutation. tory, which has facilities for the measurement of physiological parameters of racing perform- C052 ance. Other performance data such as racing PorDictor – predictors of pork quality de- records will also be included in the analysis. rived from gene expression profiles of pre- Future work will include more in-depth analy- natal muscle tissue sis of associations with performance, with the aim of identifying DNA markers for athletic BARBARA HARLIZIUS1, MARINUS F.W. ability in race horses. TE PAS2, KIN-CHOW CHANG3, ROBERTA DAVOLI4, JAN W.M. MERKS1, ROLAND C050 WÖRNER5, HUBERT EPING6, EDUARD Molecular genetic characterisation of the MURANI7, SIRILICK PONSUKSILI7, KARL gene causing chondrodysplasia in Dexter SCHELLANDER7, JAN M. PRIEM2, NUNO cattle DA COSTA3, MASSIMO CAGNAZZO4, LUCA FONTANESI4, BARBARA LAMA4, JULIE A.L. CAVANAGH1, IMKE TAM- HUBERT HENNE5, KLAUS WIMMERS7 MEN1, PETER A. WINDSOR1, FRANK W. 1Institute for Pig Genetics BV, 6641 SZ Be- NICHOLS2, HERMAN W. RAADSMA1 uningen, The Netherlands; 2ID-Lelystad, In- 1Reprogen, Centre for Advanced Technologies stitute for Animal Science and Health, 8200 AB in Animal Genetics and Reproduction, The Lelystad, The Netherlands; 3University of University of Sydney, Camden, Australia and Glasgow Veterinary School, Department of 2Reprogen, The University of Sydney, Sydney, Veterinary Pathology, Glasgow G61 1QH, Australia UK; 4University of Bologna, Dipartimento di Dexter cattle are a small breed of cattle origi- Protezione e valorizzazione Agroalimentare nating in Ireland that have been bred in Aus- (DIPROVAL) Sezione di Allevamenti Zootecni- tralia for many decades. There have been re- ci, 42100 Reggio Emilia, Italy; ports of mutant, aborted foetuses in this breed 5Züchtungszentrale Deutsches Hybridschwein of cattle, described as chondrodysplastic foe- GmbH, 21337 Lüneburg, Germany; tuses (or “bulldog” calves) occuring world- 6Landesverband Rheinischer Schweinezüchter, wide. The affected foetuses display dispro- 53115 Bonn, Germany; 7University of Bonn, portionate dwarfism, a short vertebral column, Institute of Animal Breeding Science, 53115 marked micromelia, a relatively large head Bonn, Germany with a retruded muzzle, cleft palate, protruding Meat quality traits are of major economic tongue and a large abdominal hernia. Dexter importance in livestock production. The chondrodysplasia is inherited in an incom- genetic background of meat quality is to a pletely dominant manner. As part of an ap- large extend determined by factors active proach to controlling the disease in Australia, during prenatal muscle development. Recently

67 Section C: Functional Genomics an EU funded project, PorDictor (QLK5-2000- ween Wild Boar and Yorkshire pigs. In the 01363), has been started aiming at the present study a total of 16 litters, crosses bet- identification of new predictors for pork ween H and Yorkshire/Landrace (Y/L) or pure quality derived from gene expression profiles Y, were analysed regarding KIT genotypes and of embryonic/fetal skeletal muscle. The project various blood cells measures. Blood samples, 3 comprises (1) identification of candidate genes to 7 per piglet, were collected from the age of for meat quality traits by comparing breeds that 2 days and until 2 months after birth. Several are extremes in meat quality and muscularity blood parameters were recorded, including and (2) confirmation of gene effects on meat numbers of erythrocytes and leukocytes, hae- quality in commercial crossbreeds. In total moglobin and hematocrit levels. The Kit ge- about 2000 embryos/fetuses of seven key notypes were determined using pyrose- developmental stages of the breeds Duroc and quencing. Several genotypes were found Pietrain have been collected for analysis of amongst the Y/L sows confirming a high alle- differential gene regulation in (presumptive) lic diversity at the KIT locus. A good agree- M. longissimus dorsi. Several techniques of ment was found between coat colour and ge- expression profiling, i.e. cDNA-microarrays, notypes, as all piglets carrying the IBe allele differential display-RT-PCR, in situ had black spots, except one. A segregation hybridisation, subtractive hybridisation and distortion was indicated in the inheritance of quantitative real time RT-PCR are applied to the IBe allele and a preliminary analysis indi- detect breed-specific, stage-specific and/or cated that pigs carrying the IBe allele had higher phenotype-associated transcripts. These will be numbers of leukocytes and hematocrit levels. screened for polymorphism and their association with meat quality traits will be C054 evaluated in performance tested porkers of the Detection of genes differentially expressed commercial crosses Pietrain x Landrace and in porcine leukocytes due to transport stress Duroc x Large White. The project will provide by using cDNA–AFLP and Differential Dis- new insights in muscle development and play largely contribute to improve meat production and to overcome the existing antagonisms PATCHARIN KRUTMUANG, SIRILUCK between leanness and meat quality traits. PONSUKSILI, KARL SCHELLANDER, KLAUS WIMMERS C053 University of Bonn, Institute of Animal Breed- Variation at the porcine KIT locus influence ing Science, 53115 Bonn, Germany peripheral blood cell measures Transport stress is one of the problems in pig production. The process of pig management A. JOHANSSON1, G. PIELBERG2, I. often includes the transport of piglets from VAARA3, L. ANDERSSON2, I. EDFORS- several producers to fattening units and then to LILJA1 slaughterhouses. Stress is the reaction of the 1Department of Biosciences and Process Tech- body to stimulus, which disturbs the normal nology, Växjö University, Växjö, Sweden; homeostasis, physiology and biochemical ac- 2Department of Animal Breeding and Genetics, tivities of an animal. Stress response likely SLU, Uppsala, Sweden and 3Central Hospital, leads to adverse effects on growth, behavior, Växjö, Sweden reproductive performance, meat quality and The KIT gene encodes the mast/stem cell disease resistance. The objective of this inves- growth factor receptor essential for normal tigation is to identify candidate genes which development of melanocytes, hematopoietic are differentially expressed under transport stem cells and germ cells. Dominant white coat stress. cDNA-AFLPs were used to compare the colour in pig is due to a gene duplication and a expression pattern of leukocytes before and single-nucleotide splice mutation, whereas the after transport stress. Differential display Belt phenotype (IBe/IBe) in the Hampshire (H) analysis was conducted for expression profil- breed is assumed to be caused by a regulatory ing of leukocytes of animals with high and low KIT mutation. A reduced number of peripheral cortisol response after transport stress. Five blood leukocytes in pigs homozygous for the and six potential candidate genes/ESTs were dominant white allele have earlier been obser- detected by cDNA-AFLP and differential dis- ved in the F2-generation of an intercross bet- play, respectively. The chromosomal localiza-

68 Section C: Functional Genomics tion of these ESTs was done using the IMpRH- maternal transcript level. Further study to panel. Quantitative real-time PCR was used to evaluate their contribution to embryo quality confirm differential expression of the ESTs. and developmental competence will comple- Two ESTs detected by cDNA-AFLP were ment this study. found to be differentially expressed (P<0.05) before and after transport. One EST from dif- C056 ferential display was differentially expressed Expression analysis of HMGA1 in normal (P<0.001) between the high and low cortisol and neoplastic canine mammary cell lines group. These ESTs represent candidate genes using macroarrays for understanding and for dissecting genes which may have major effect on stress resis- B. MEYER1, H. MURURA ESCOBAR1, A. tance. RICHTER1, K. BECKER1, I. NOLTE2, J. BULLERDIEK1 C055 1Center for Human Genetics, University of Mapping of some maternal transcript ESTs Bremen, Bremen, Germany; 2Clinic for Small in cattle pre-implantation embryos Animals, Veterinary School, Hannover, FRG Mammary neoplasms are twice as frequent in SOLOMON G. MAMO1, KLAUS WIM- dogs (Canis familiaris) as in humans and ac- MERS1, MARCUS GILLES1, KATA SRINI- count for more than 50% of the tumours ob- VAS2, KARL SCHELLANDER1, SIRILUCK served in the female dog. Due to their similar PONSUKSILI1 histological and biological characteristics ca- 1University of Bonn, Institute of Animal nine mammary carcinomas are considered to Breeding Science, 53115 Bonn, Germany; be valid models for studying the molecular 2Texas A&M University, Department of mechanisms underlying tumour development Veterinary Pathology, Texas, USA in humans. A gene frequently involved in neo- Gene mapping is an important tool towards plastic transformation and metastatic tumour identification of candidate genes, applications progression is HMGA1, an architectural tran- in comparative genomics and evolutionary scription factor, which is expressed in high studies. The objectives were to identify and levels during embryogenesis and in dividing allocate genes expressed in cattle embryo pre- cells, whereas it is only detectable at very low implantation stage. Nine ESTs (Expressed levels or even absent in non-dividing fully Sequence Tags) derived from matured oocytes differentiated cells. Overexpression of HMGA1 and 4- cell embryo cDNA libraries were recently has been observed in primary human mapped using radiation hybrid panel. A primer breast cancer. Increased expression was also pair was designed per EST to amplify products correlated to increased metastatic potential of and their identity was confirmed by sequenc- both mouse and human mammary epithelial ing. RH mapping revealed average retention cancer. In order to investigate mRNA expres- frequencies, calculated as the proportion of sion levels of HMGA1 in canine mammary clones retained a given marker, of 16.5 %. Six tumours, macroarray and northern blot analy- markers (ESTBb003, ESTBb007, ESTBb008, ses of normal, benign, and malignant mam- ESTBb011, ESTBb4c1 & ESTBb4c6) have mary cell lines were carried out. For the benign strong similarity with genes; Sterol C5 desatu- mammary tumour cell line these experiments rase, Normal keratinocyte mRNA, KIAA0197 revealed a strong expression of HMGA1 as protein, Host cell homolog, Catenin associated well as of the tumour-relevant genes c-MYC delta 1 & Capping protein. They are assigned and HER2. Furthermore the northern blot to chromosomes 15, 14, 15, 10, 15 and 3, re- analysis showed signals for all three examined spectively. These localizations fit the current cell lines, indicating expression of the HMGA1 human bovine comparative map as shown by mRNA not only in the benign but also in the using the COMPASS software. Some of the normal and malignant mammary cell lines functions of these genes include cholesterol verifying the results obtained by the arrays. biosynthesis, calcium binding, intracellular protein transport, cell-cell adhesion, protein complex assembly and role in cell motility. This study ordered some of the developmen- tally important genes that are expressed at

69 Section C: Functional Genomics

C057 sucklings. We are aiming to address candidate Coat color and mutations of MC1-R locus genes for these processes of reproduction. infour sheep breeds There is experimental evidence that trans- forming growth factor beta 1 and relaxin affect LUIS V. MONTEAGUDO1, M. TERESA the prenatal development and the rearing abil- TEJEDOR1, ISIDRO SIERRA2, ALICIA PO- ity, especially the mammary gland phenotype. STIGLIONI3, ROSA GAGLIARDI3, MI- In order to identify SNP within the TGFB1 and GUEL D‘ANGELO3, RAUL BARRAO1, RI- RLN gene porcine cDNA-amplicons were CARDO PONZ1, M. VICTORIA ARRUGA1 comparatively sequenced in animals of five pig 1University of Zaragoza, Laboratory of Cyto- breeds. A transition (A>G) was detected in genetics and Molecular Genetics, Zaragoza , exon 5 (position 797 of cds) of TGFB1. A Spain; 2University of Zaragoza, Department SSCP procedure was established for genotyp- of Animal Production and FoodScience, Zara- ing. Two SNPs were identified in the RLN goza, Spain; 3Universidad de laRepública, gene, a transversion from T to G in intron 1 at Laboratory of Cytogenetics , Montevideo, position 9, and a transversion from A to C in Uruguay exon 1 at position 22. RFLP and SSCP are MC1-R locus was investigated by PCR-RFLP used to genotyping these SNPs. The SNP methodology inMerino (black variety, an an- within exon 1 at position 22 of RLN leads to an cient breed), and in several black individu- amino acid exchange (isoleucine to leucine). alsfrom Merino Corriedale, Rasa Aragonesa The segregation of all alleles was observed in and Salz sheep breeds. M73K andD121N 21 families of DUMI F2 resource population mutations ofMC1-R were found in the black and Mendelian inheritance of the alleles could Merino and Corriedale individuals. In fact,both be demonstrated. Linkage mapping of the mutations cosegregated with dominant black genes is in agreement with previous physical color coat in the Blackvariety of Merino and in mapping data. The SNPs identified here are Merino Corriedale. However, in Rasa Ara- valuable tools for ongoing linkage and asso- gonesa andSalz breeds, black color appeared ciation analyses with traits related to reproduc- as a recessive character and was not associated tion. to these mutations.Genotype coincidence was always found for both mutations; in every C059 studiedbreed, individuals were homozygous Mapping and identification of the gene dominant, heterozygous or homozygousreces- causing hereditary zinc deficiency in Angus sive for both M73Kand D121N mutations. cattle These findings suggest the existence of only two alleles, one of themcarrying simultane- IMKE TAMMEN1 , ROGER W. COOK2, ously both mutations and the other one free of JANE GITSCHIER3, FRANK W. NICHO- them.Dominant mutations associated to black LAS1, HERMAN W. RAADSMA1 1 color should be considered as anancestral char- University of Sydney, Centre for Advanced acter, with a commun and remote origin in Technologies in Animal Genetics and Repro- Black Merino and Merino Corriedale sheep duction – ReproGen, Camden & Sydney, Aus- breeds. tralia; 2 NSW Agriculture, Wollongbar, Aus- tralia and University of California, Howard C058 Hughes Medical Institute and Departments of SNP detection and linkage mapping of the Medicine and Pediatrics,San Francisco,USA porcine TGFB1 and RLN genes as candi- Bovine hereditary zinc deficiency (lethal trait dates for reproductive traits A46) was first reported in Friesian cattle in 1964. It is inherited as an autosomal recessive SIRIWADEE CHOMDEJ, SIRILUCK PON- disorder and is considered to be homologous to SUKSILI, KARL SCHELLANDER, KLAUS Acrodermatitis enteropathica (AE) in humans. WIMMERS In 1993 hereditary zinc deficiency was diag- University of Bonn, Institute of Animal nosed in a herd of Angus cattle in Australia. Breeding Science, 53115 Bonn, Germany Although the disease appeared to be confined Reproductive performance comprises of gene- to a single herd it was further investigated due sis of conceptuses, their prenatal development to its potential to provide a model for gene and growth as well as postnatal rearing of therapy as affected animals survive with treat-

70 Section C: Functional Genomics ment. We applied a breeding program to en- short chain monounsaturates to Lc-HUFA. The large the Angus pedigree and used retrospec- elongase and desaturase genes have now been tive sampling to obtain DNA from affected cloned from an anadromous (Atlantic salmon) Friesian cattle. Due to the limited material we and several marine species. Predicted protein initiated a homozygosity mapping approach to sequences show that the gene products have map the defective gene in cattle. Before com- high homology among fish species. Functional pletion of the genome screen, the human gene characterisation is ongoing. for AE was mapped to HSA 8q24.3 and re- cently identified. Applying comparative map- C061 ping information, we mapped hereditary zinc Quantification of differentially expressed deficiency in Angus cattle to a region on transcripts in in-vitro produced cattle pre- chromosome 14, which is homologous to hu- implantation stage embryos using fluores- man HSA8q24.3. The data for Friesian cattle cence monitored real time PCR technique was inconclusive. After identification of the gene causing AE, we started sequencing the DAWIT TESFAYE, KLAUS WIMMERS, orthologous bovine gene and identified a non- MARKUS GILLES, KARL SCHELLANDER, sense mutation in Angus cattle, which is not SIRILUCK PONSUKSILI present in affected Friesian cattle. Further re- University of Bonn, Institute of Animal search will be required to determine if the dis- Breeding Science, 53115 Bonn, Germany ease in Friesian cattle is caused by a different Determining the physiological timetable of mutation in the same gene. differential gene expression in pre- implantation stage embryos requires the ability C060 of accurately quantifying stage specific mRNA Fatty acyl desaturase and elongase genes of transcripts. The aim of this study was quantifi- marine and freshwater teleosts cation of differentially expressed transcripts in in-vitro produced cattle 8-cell, 16-cell, morulae MORRIS AGABA, NICOLA HASTINGS, and blastocyst embryos. Of 16 clones identi- DOUGLAS R. TOCHER, CATHERINE A. fied and sequenced, clones 2C14 and 1C9 DICKSON, JOHN R. SARGENT, ALAN J. which were identified in 8-cell stage and share TEALE high homology with human Pleckstrin homol- Institute of Aquaculture, University of Stirling, ogy, Sec 7 coiled domain 2 (PSCD2) mRNA Stirling, Scotland FK9 4LA, UK and Nucleosome assembly protein 1 (NAP1L1), Enzymes that lengthen and desaturate the car- respectively, were quantified using ABI Prism bon chain are required for biosynthesis of the 7000 instrument. cDNA from the four stages of long chain highly unsaturated fatty acids (Lc- embryos were subjected to real time PCR HUFA) arachidonic and docosahexaenoic quantification utilising specific primers de- acids. The Lc-HUFA are essential for normal signed using the Primer express software and health and development of all vertebrates. Ma- SYBR green PCR master mix (Applied Bio- rine teleosts, unlike their freshwater counter- systems). ß-actin was used for internal nor- parts, appear to have a dietary requirement for malization. The relative expression level of Lc-HUFA that may be due to defective fatty 1C9 clone to 8-cell stage amounted 1.05, 0.82 acid elongation and/or desaturation. In exami- and 0.75 fold difference in 16-cell, morulae ning this possibility we are comparing the and blastocyst stages, respectively. Similarly, structures of the desaturase and elongase genes the relative expression level of clone 2C14 to of representative marine and freshwater spe- 8-cell stage amounted to 0.97, 0.97, 0.96 fold cies, and the function of their products. The difference in 16-cell, morulae and blastocyst zebrafish (Danio rerio) elongase and desatura- stage respectively. These results indicated that se genes were the first that we characterised. while clone 1C9 showed a significant reduc- Interestingly, the products of both genes ap- tion in expression up to blastocyst stage, a pear to be less functionally constrained than weak difference was observed in the expres- the desaturases and elongases of higher verte- sion of clone 2C14. Further quantification of brates. The zebrafish desaturase gene encodes these clones in other developmental stages and a novel bifunctional enzyme having both delta different embryo qualities will supplement 5 and delta 6 desaturase activities, while the these findings. elongase substrate specificity ranges from

71 Section C: Functional Genomics

C062 through linkage disequilibrium studies. To this Mapping and examination of ion channel end, we made an effort to determine the gene genes as candidate genes for the hereditary content of the chromosomal region harbouring disease 'Congenital progressive ataxia and DGAT1 (BTA 14q12). Sequences of human spastic paresis' in swine genes within a range of 640 kb around DGAT1 (HSA8q24.3) listed in the NCBI MapView A. KRATZSCH, B.KORCZAK, S. NEUEN- were used to obtain bovine sequence informa- SCHWANDER, G. STRANZINGER, tion for the corresponding genes using BLAST P. VÖGELI analysis of the EST division of GenBank. Bo- Institute of Animal Science, ETH-Zurich, vine EST information of five neighbouring Zurich, Switzerland genes and sequence information of five BAC The congenital progressive ataxia (CPA) and ends of clones containing DGAT1 were used spastic paresis in pigs, recently identified in for generating PCR probes. Two compound Switzerland, is a disease with unknown etiol- probes were compiled, one consisting of gene- ogy. It manifests itself shortly after birth as a specific probes and one consisting of BAC- severe neuropathy. The disease seems to be end-specific probes, and were used to screen a controlled by a single autosomal recessive bovine BAC library (RPCI-42). A total of 19 allele designated cpa. In a previous study we overlapping clones encompassing about 500 kb demonstrated close linkage of cpa to the mi- was isolated. The order of BACs and the posi- crosatellite Sw902 on porcine tion relative to DGAT1 of 25 genes were de- (SSC3). Sw902 is mapped in close proximity to termined by BAC-end and gene-specific PCR the physically and genetically mapped IL1 amplification from the 19 BACs. Genes and locus on SSC3q13-21. Available human-swine BAC ends are now comparatively sequenced to comparative maps predict correspondence of find SNPs, which will be the basis of linkage this region to human 2q1-2 region, where ion disequilibrium studies. channel genes (Ca2+, Na+) and a cholinergic receptor gene are mapped. Epilepsy and ataxia C065 in humans seem to be caused by mutations in Intra- and inter-subspecific variation in these genes. The calcium channel beta 4 gene complete Bos taurus and Bos indicus mito- (CACNB4) was mapped to SSC3q14-q21, chondrial genomes - a molecular basis for while the sodium channel gene (SCN2A) and maternal lineage effects the cholinergic receptor gene (CHRNA1) were located on SSC15. So far mutation screening, STEFAN HIENDLEDER1, AXEL JANKE2, expression studies and drug treatment of the ECKARD WOLF1 porcine CACNB4 gene did not reveal any 1Department of Molecular Animal Breeding modification in affected piglets. and Biotechnology, Gene Center, Ludwig- Maximilians-University Munich, Germany; C063 2Division of Evolutionary Molecular Sys- Physical mapping of a bovine chromosome tematics, Department of Genetics, University region with an effect on milk fat content of Lund, Sweden Maternal lineage effects have been reported to ANDREAS WINTER, ARIANE ALZINGER, influence milk production traits, fertility and, RUEDI FRIES more recently, nuclear transfer efficiency in Technische Universität München, Lehrstuhl für cattle. However, information on the molecular Tierzucht, Freising-Weihenstephan, Germany basis of such effects is very limited. We have A QTL affecting milk fat content on bovine analyzed maternal lineages of 5 B. taurus chromosome (BTA) 14 has been independently breeds and one B. indicus breed for mitochon- confirmed by several studies. A mutation drial DNA (mtDNA) control region (CR) se- within DGAT1 (diacylglycerol acyltransferase quence polymorphism and used the CR data- 1), a functional and positional candidate gene, base to infer phylogenetic relationships. All is associated with the QTL effect. Directly analyzed sequences fell into the two major proving the causality of this mutation by gene- clusters of Bos primigenius mtDNAs, B. taurus tics means is not feasible. Therefore we at- and B. indicus. One B. taurus mtDNA (Sim- tempt to provide indirect proof by excluding mental) and the B. indicus mtDNA other positional genes in the QTL region (Zwergzebu) were chosen for complete se-

72 Section C: Functional Genomics quence analysis, cloned into plasmid vectors analysis to successfully prosecute cases. Deer and sequenced by standard procedures. Se- STRs can link meat to bloodstains or remains quence comparison of the new 16338 nt B. in poaching cases. Additionally, sex of the taurus sequence with the bovine reference deer can be determined so that laws relating to sequence revealed 10 polymorphisms in total. the harvesting of antlerless deer can be en- Six polymorphic sites were located in coding forced. regions, and one of four substitutions in protein With multi-species DNA analysis capabilities, coding genes was non-synonymous. Compari- more crimes will be solved in the future. If a sons between the 16339 nt B. indicus and the suspect owns an animal or works around ani- B. taurus mitochondrial genomes showed 237 mals, chances are that animal hair will be pre- polymorphic sites in total. Non-synonymous sent on his person. Even with gloves or other substitutions were present in subunits of respi- protective measures that a suspect might take ratory chain enzymes NADH-dehydrogenase, to prevent transfer of his or her own DNA, bc1-complex, Cytochrome c-oxidase and ATP- animal hair has been left behind at crime sce- synthase. We conclude that the molecular basis nes time after time. Increased utilization of for mtDNA effects in B. taurus cattle is lim- available animal DNA analysis methods will ited, but it is extensive in cattle populations insure that more cases will not remain unsol- where B. taurus and B. indicus haplotypes ved. occur simultaneously, e.g. New Zealand and Australia. C067 Comparative sequence analysis of the TH- C066 IGF2 region for Q and q alleles at an im- Forensics and Animal Genetics: An Emer- printed QTL mapping to proximal SSC2 ging Science MINH NGYUEN, CATHERINE COLLETTE, M. KETCHUM, M. ALVAREZ, J. ALVA- CARINE NEZER, NADINE BUYS, MICHEL REZ, M. KONIECZNY, et al. GEORGES DNA Diagnostics, Inc. d.b.a. Shelterwood Department of Genetics, Faculty of Veterinary Laboratories, Carthage, TX Medicine, University of Liège In the past, various methods of human DNA While performing a whole genome scan to analysis have been employed to link suspects identify QTL influencing growth and carcass to crime scenes. Now, crimes are also being traits in a Piétrain x Large White intercross, we solved with the use of animal DNA testing. identified a QTL with major effect on muscu- Both mitochondrial DNA as well as STRs are larity and fat deposition towards the centro- now commonly used in the animal industries. meric end of SSC2 (Nezer et al., 2001). In recent years, these resources are also being Comparative mapping information pointed used to solve crimes. Cases have ranged from towards HSA11 as the human orthologue, and fraud disclosed using equine mitochondrial thereby to IGF2 and MyoD as potential DNA to linking a suspect to the crime scene of positional candidates. Mapping these genes a homicide with canine mitochondrial DNA. with respect to the porcine marker map showed Additionally, multi-species STRs yield results that IGF2 co-localized with the QTL. As- in just as wide a variety of forensic cases. suming that IGF2 was indeed responsible for Equine STRs coupled with human STRs were the QTL effect and that the IGF2 gene would utilized to not only identify a racehorse injec- be imprinted in the pig as it is known to be in ted with cocaine, but also to identify the hu- the human and mice, we made the prediction man profile of the suspect that injected the that in our F2 population a significant substi- horse. Canine STRs amplified from DNA tution effect would be found between the pa- extracted from saliva stains on a victim’s ternally inherited Piétrain versus Large White clothing after a brutal dog attack resulted in alleles, but not between the corresponding linking a neighbor’s dogs to the attack and maternally inherited QTL alleles. We demon- resulting criminal negligence charges. Bovine strated that IGF2 is indeed imprinted in the and equine STRs have been utilized on nu- pig, and that only the QTL alleles inherited merous occasions on various cattle rustling from the boar influenced the phenotype, cases as well as horse theft with great success. thereby supporting our hypothesis (Nezer et Wildlife enforcement also utilizes animal DNA al., 2001). Similar results were simultaneously

73 Section C: Functional Genomics obtained in a Wild Boar x Large White inter- be the nucleotide sequence of the pig RH cross by Jeon et al. (2001). cDNA, the chromosomal mapping and two Despite the strong candidacy of IGF2 (given allelic variants of the pig RH gene. The pig RH its known role in myogenesis), our results did cDNA has an open reading frame (ORF) of not formally prove that this gene caused the 1365 nucleotides (GenBank; AB067771), observed QTL effect. IGF2 indeed maps to an which encodes for a protein of 423 amino imprinted domain containing other putative acids. On the amino acid level, the pig RH candidates. To refine the map position of the shares 60.8% and 61.0% identity with the hu- QTL, we (i) constructed a BAC contig span- man RHD and RHCE, respectively. The pig ning the TSSC5-H19 interval containing the RH protein appears as a composite encoding KVLQT1 and IGF2 imprinted domains and amino acids from the human RHD and RHCE. from it developed a high density microsatellite However, the pig RH protein contains 6 amino and SNP-based marker map of proximal SSC2, acid residues more than the human RH pro- and (ii) identified six distinct ”Q” and “q” teins. Hydrophobic analysis suggests that the chromosomes by marker assisted segregation pig RH protein also has a 12-transmembrane analysis performed in 32 large (> 100 off- domain, which is conserved among all mem- spring) boar families. Comparison of the “Q” bers of the RH family. RH transcripts could be bearing (muscle increasing) boar chromosomes detected by RT-PCR in the spleen and bone revealed a shared chromosome segment in the marrow but not in the heart, the kidney, or the interval bounded by p57KIP2 and the 3’ UTR of lung. The pig RH gene was mapped to chro- IGF2, thereby mapping the QTL to this inter- mosome 6q22-23 and porcine RHBG gene to val. As INS and IGF2 are the only known chromosome 4q21-22 by somatic cell hybrid paternally expressed genes in this well- panel. A SINE sequence and a short tandem characterized interval, both remained good repeat (STR) of (CAAA)n were detected in the causative candidates. We therefore rese- pig RH (GenBank; AB039288), corresponding quenced 28 Kb corresponding to the 3’ TH- 3’ to the human RH intron 4. So far, two alleles, IGF2 interval for the shared “Q” haplotype as (CAAA)1 and (CAAA)4, have been found in well as the six “q” chromosomes. We identi- the pig. fied > 140 different SNPs in this segment of which all but 30 showed a perfect allelic seg- C069 regation between the Q and q chromosomes. Evolution of the bovine Y-chromosomal Further analysis is in progress to identify the multicopy TSPY gene family putative causal mutation responsible for the QTL effect. Corresponding results will be E.L.C. VERKAAR, J.A. LENSTRA presented. Department of Infections diseases and Immu- nology and Department of Equine Sciences, C068 Faculty of Veterinary Medicine, Utrecht Uni- cDNA cloning and characterization of the versity, The Netherlands porcine RH gene The non-recombining region (NRY) is impor- tant for maintaining integrity of the Y- TOSHINORI OMI1, STEFAN NEUEN- chromosome. A typical feature of the NRY is SCHWANDER1, ŠPELA ŠPILAR1, the rapid evolution and amplification of male PETER VÖGELI1, EIJI KAJII2, GERALD specific sequences in order to prevent recom- STRANZINGER1 bination with the X-chromosome. The repeti- 1Swiss Federal Institute of Technology, Insti- tive TSPY (testis specific protein Y-encoded) tute of Animal Sciences, Zurich, Switzerland, gene encodes a nucleosome binding protein 2Jichi Medical School, Department of Legal and has been found in several species. TSPY Medicine & Human Genetics, Tochigi, Japan genes have been shown to be dispersed over The Rhesus (RH) homologous gene family the whole bovine NRY. We have studied the includes RH (D /CE), RHAG, RHBG, and organization of the TSPY loci by isolation and RHCG in the human. RHD and RHCE, origi- analysis of BAC clones. Southern blotting nally identified in human red cells, encode for showed two types of restriction patterns. Se- the antigens of the human RH blood group quence analysis of representative clones re- system and were of central importance in vealed that one pattern corresponds to a cluster transfusion medicine. In this paper, we descri- of four closely linked and heterogeneous TSPY

74 Section C: Functional Genomics pseudogenes. The BAC clones with the less human TECTA sequence, we amplified and complex patterns contain a single TSPY gene sequenced products from genomic dog DNA with an intact open reading frame, which is isolated from EDTA blood. BLAST results for highly similar to published expressed sequence Exon 10 indicated an average of 88 % homol- tags. Comparison of the sixth intron sequences ogy between the dog and the human sequence. from several BAC clones and from related Exon 10 sequences of affected dogs, normal bovine species indicated sequence heterogene- animals and control animals from different ity as well als concerted evolution. The ampli- breeds were compared and analyzed for poten- fied TSPY sequence from taurine cattle resem- tial mutation sites. The analysis for Connexin bled the sequence of zebu, gayal and gaur, 26 is in progress. while the TSPY sequences of bison, wisent and yak form a seperate cluster. Our data suggests C071 that repetitive elements as well as repetitive cDNA cloning, molecular analysis and ex- genes on the NRY are comparable to centro- pression of the ovine uroporphyrinogen meric satellite repeats by being subject to a decarboxylase gene dynamic concerted evolution, presumably via successive amplification events. REZA NEZAMZADEH, ANDREA KREM- PLER, BERTRAM BRENIG C070 Georg-August University of Göttingen, De- SWMolecular analysis of candidate genes partment of Veterinary Medicine, Göttingen, for congenital deafness in dalmatians Germany Uroporphyrinogen decarboxylase (Uro-D) is a T. KUTZER, I. PFEIFFER, B. BRENIG cytosolic enzyme which catalyzes the fifth step Institute of Veterinary Medicine, University of of the heme biosynthetic pathway, the sequen- Goettingen, Germany tial decarboxylation of uroporphyrinogen III Congenital deafness has been reported for ap- yields coproporphyrinogen III. Defects at the proximately 60 dog breeds. The disorder is Uro-D locus cause the genetic disease Por- usually associated with pigmentation patterns. phyria cutanea tarda (PCT). It was first de- A large amount of white in the hair coat in- scribed in domestic animals ahead with light creases the likelihood of deafness. Two pig- sensitivity and photodermatosis. In females the mentation genes are often associated with defect also leads to reduced fertility by inacti- deafness in dogs: the merle gene and the pie- vation of the ovaries and reduced estrus. bald gene (Dalmatian, English Setter). But not The genomic structure of the Uro-D gene has all breeds with these genes have been reported been characterized in man and cDNA se- to be affected. The deafness usually develops quences are knwon from human, mouse and in the first few weeks after birth. A degenera- rat. According to the human genomic organi- tion of a part of the blood supply to the cochlea sation we found 10 exons spanning over 3 Kb. is discussed as a reason. In most cases the inci- On screening animals with the disease we dence of congenital deafness in different found a point mutation in the coding region of breeds is unknown because of the limited Uro-D gene resulting in a Leu → Pro substitu- number of studies. In the Dalmatian breed the tion at amino acid position 392. The ovine occurrence is up to 30%. Deafness in Dalma- Uro-D cDNA was isolated from sheep liver. tians appears to be an autosomal recessive The open reading frame with a length of 1104 disorder, possibly caused by the presence of bp encodes a 367-amino acid polypeptide with two different autosomal recessive deafness a predicted molecular mass of 45 kDa. genes, or a syndrome with incomplete pene- trance. Further studies will be required to de- C072 termine the mechanisms. At present no genetic Isolation and characterization of the porcine tests exist to identify affected or carrier ani- ß-Glucuronidase gene (GUSB) as candidate mals. We investigated the alpha-tectorin for congenital hernia (TECTA) and Connexin 26 (Cx26) genes as candidates for deafness in dalmatians. The J. BECK1, CHRISTOPH KNORR1, E. study included 119 dalmatian dogs, 92 healthy SCHUETZ2 & BERTRAM BRENIG1 dogs, 10 unilaterally and 17 bilaterally deaf 1Institute of Veterinary Medicine, Georg- dogs. Using PCR primers based on published August-University of Göttingen, Göttingen

75 Section C: Functional Genomics

(Germany) autochthonal domestication of the wild aurochs 2Chronix Biomedical, Benicia, CA, USA (Bos primigenius) in Middle Europe. Ancient The gubernaculum is a loose connective tissue DNA analyses on Neolithic bones were per- organ that plays a major role during testicular formed in order to collect genetic data of dif- descent. In the pig, the first phase of transab- ferent populations. Samples from all over dominal migration is brought about by growth Middle Europe, the Balkan and the Near East of the gubernaculum through the inguinal will be compared to find out where centers of canal into the scrotum. This dilates the domestication were and if crossbreeding with inguinal canal preparing the pass of the testis wild aurochs took place. The first results from through this bottleneck. Development of the mitochondrial markers (d-loop) are presented gubernaculum during this phase is character- from both Neolithic domestic cattle and au- ized by rapid cell proliferation and accumula- rochs. tion of intercellular water. This process is mainly mediated by synthesis of hyaluronic C074 acid (HA), since this poly-anionic macromole- Molecular characterization of a 95kDa zona cule is known to have a large hydrodynamic receptor kinase fragment volume. Our assumption is that swelling of the gubernaculum exceeds through diminished LEONARD BULL, STEPHAN JANSEN, degradation of HA and causes that the inguinal BERTRAM BRENIG canal remains open, which predisposes male Institute of Veterinary Medicine, Georg-August pigs for the development of hernia. As ß- University of Goettingen, Germany Glucuronidase is involved in the biodegrada- Gamete binding involves complex recognition tion of HA, it is a candidate gene for this con- mechanisms which are directed by specialised genital disorder. Here we report the isolation, molecules. The male adhesion molecules are characterization, and localization of the por- divided in primary and secondary ligands. cine GUSB gene. Our first step was to screen a Primary ligands and their receptors are respon- porcine genomic PAC-library by PCR, with sible for the first recognition of the gamete and primers derived from exon 6 & 7 of the feline the initiation of metabolic pathways as can be GUSB gene. A clone of approximately 80 kbp seen in the acrosome reaction, while secondary was isolated, containing the whole GUSB receptors and ligands become important gene. We assigned GUSB to porcine chromo- shortly before the sperm’s penetration through some SSC3p14-p16 by FISH and confirmed the zona pellucida. In our experiments, our findings by hybrid panel analysis. Animals we aim to find out whether the initial binding phenotypically affected with hernia inguinalis of the gametes can be modulated. We hypothe- and non-affected animals were used to detect sis that binding affinity to the complementary SNPs in the procine GUSB gene by CEL I gamete can be modified by the state of phos- digestion of heteroduplex DNA and compara- phorylation in certain proteins. Under the as- tive sequencing. sumption that the degree of phosphorylation in binding proteins is affected by environmental C073 factors, lack of fertility and the inability to The origin of domestic cattle (BOS TAU- bind to the complementary gamete -either RUS) in Middle Europe: ancient DNA permanently or temporarily- would then beco- Analysis on Neolithic bones me explainable. We have cloned a porcine fragment of the 95 kDa receptor kinase. Se- RUTH BOLLONGINO, JOACHIM BUR- quence analysis reveals that the porcine re- GER, KURT W. ALT ceptor fragment shows a homology of 88.5% University of Mainz, Molecular Archaeology and 74% to the murine and human receptor Group, Institute of Anthropology, Mainz, kinase. The fragment has been also success- Germany fully expressed in a bacterial system. This is an We present the first results of a project on the important prerequisite for further experiments origin of Middle European domestic cattle in which the binding capacity in a phosphory- (Bos taurus). From archaeological and ar- lated and dephosphorylated state can be mea- chaeozoological record it is not quite clear sured. whether the first domestic cattle were im- ported from the Near East or if there was an

76 Section C: Functional Genomics

C075 mited success and development of antibiotic The porcine CALC-A/α-CGRP gene: Isola- resistance is also a concern. tion, characterization, physical mapping, PathoCHIP is an EC funded Framework 5 and detection of SNPs (single nucleotide project, aiming to use microarrays to identify polymorphisms) both host and pathogen genes that are diffe- rentially expressed during Haemophilus para- CHRISTOPH KNORR, BERTRAM BRENIG suis infection. It is hoped that identification of Georg-August-University of Goettingen, bacterial virulence genes will lead to develop- Institute of Veterinary Medicine, Goettingen, ment of novel treatments for the disease whilst Germany identification of host candidate genes respon- Calcitonin and α-CGRP are products of the sible for disease resistance may lead to the alternatively spliced gene CALC-A/α-CGRP. production of animals that are resistant or less Calcitonin mRNA is predominantly found in susceptible to the disease via marker assisted thyrodial C cells and mRNA encoding the selection. neuropeptide calcitonin gene-related peptide An animal challenge model will be utilized and (CGRP) in neural tissues. In vivo, calcitonin optimized to reproduce the disease in vivo. reduces serum calcium and acts antagonistic to Two lines of pigs will be used in the challen- the parathyroid hormone. Primers derived from ges to increase the amount of genetic differen- the canine mRNA were designed to screen ces in host susceptibility to infection. Differen- porcine genomic libraries. A positive PAC ces in host responses to the experimental chal- clone has been isolated and the CALC-A/α- lenge will be determined by assessment of CGRP gene was completely sequenced. Cal- clinical signs, bacteriology, pathology as well citonin is translated after alternative splicing of as PCR testing. Samples will be taken from all exons 1 to 4 and α-CGRP is generated by al- points of infection (lung, pericardium, menin- ternative splicing of exons 1, 2, 3, 5, and 6. ges, joints, tonsils and lymph node) to allow Ten SNPs (single nucleotide polymorphism) preparation of both host and bacterial RNA. have been detected in the gene after com- This material will be used to identify bacterial perative sequencing of animals of different and host genes that are differentially expressed α during the infection via the use of bacterial and origins. The porcine CALC-A/ -CGRP gene host microarrays. A Haemophilus parasuis → was localized to chromosome 2p11 p13 con- genomic DNA library containing 18,000 clo- firming synteny between the p arms of chro- nes (> 6x genome coverage) will be used to mosomes HSA11 and SSC2. analyse the bacterial genes expressed during the pathogenesis. A host pig normalized cDNA C076 library from infected tissues containing about PathoCHIP: Identification of genes ex- 15,000 clones and a pig immune tissue cDNA pressed during the pathogenesis and control library containing about 9,000 clones will be of Haemophilus parasuis infections in pigs used to construct the host pig microarrays which will be used to analyse gene expression 1 SAFFRON DORNAN , LUCINA GALI- differences. Differentially expressed genes will 1 1 NA ,CAROLE SARGENT , LUSHENG be sequenced to identify candidate genes asso- 1 2 HUANG , ISABEL BLANCO , JOACHIM ciated with disease resistance or susceptibility. 3 3 ULLRICH , VOLKER SPEHE , PAUL Host genes will also be identified using SSH 3 1 SELTZER , MARNIE MELLENCAMP , cDNA libraries. 2 1* ANA CANALS & GARY EVANS It is hoped that the PathoCHIP project may www.pathochipproject.com generate additional tools such as diagnostics to 1 Sygen Laboratory, Department of Pathology, identify infected pigs, identification of bacteri- 2 University of Cambridge, UK, INIA-CISA, al genes that can be used to develop vaccines 3 Madrid, Spain and Intervet, Frankfurt, and creation of genetic tools and resources to Germany study the interaction between other pathogens Haemophilus parasuis is a gram-negative and the host. bacterium causing polyserositis primarily in * To whom correspondence should be young pigs. Current treatments include anti- addressed biotics and vaccination. Vaccination is of li-

77 Section D: Marker, Polymorphism and Biodiversity

Section D: Vitamin D binding protein (GC) polymor- Marker, Polymorphism and phism has been reported in many mammalian Biodiversity species. In addition, GC has been shown to be linked to albumin and α-fetoprotein and these constitute a gene family. In cattle, three GC D001 variants have been described and marked breed Mitochondrial D-loop and cytochrome B differences have been observed. Utilizing 12% gene sequences as a tool to preserve the ge- PAGE, pH 7.9, and Identification of two new netic diversity in an Iberian pig population variants of vitamin D binding protein (GC) in cattle immunoblotting with antiserum to the ESTEFÂNIA ALVES, CRISTINA ÓVILO, human GC protein, two new GC variants, CARMEN RODRÍGUEZ, LUÍS SILIÓ designated GC M and GC D, have been found INIA, Departamento Mejora Genética Animal, in Piedmontese and Angus cattle respectively. Madrid, Spain The Piedmontese variant migrates between the A conservation programme of Iberian pig is F and S variants and occurred only at low fre- based on the Torbiscal line, produced by quency (n=316; F = 0.30, S = 0.67, M = 0.03). blending four old strains of Iberian pigs: two An examination of samples from Angus cattle Portuguese (Ervideira and Caldeira) and two revealed a much slower migrating variant Spanish (Puebla and Campanario) strains. The which was also relatively rare (n=1144; F = complete genealogy of all animals is available 0.11, S = 0.88, D = 0.01). Whilst GC F and S back to 1945, with 21 generations from the homozygotes are commonly found, in this founders to the present animals. These genea- study the GC M and GC D forms and also GC logical records allow the identification of eight C occurred only as heterozygotes. Neuramini- maternal lineages: five corresponding to Cam- dase treatment of plasma samples followed by panario strain and the others to Puebla, Cal- two-dimensional electrophoresis (IEF 4.5-5.4, deira and Ervideira strains, respectively. The 10% PAGE, pH 7.9) and immunoblotting indi- complete sequence of the D-loop region (1246 cated that the microheterogeneity observed in bp) and cytochrome B (CytB) gene (1140 bp) the five known GC variants was due in part to of mtDNA were determined by direct se- differences in sialylation. quencing of PCR-product from eight animals representing the eight quoted maternal linea- D003 ges. We found the existence of six haplotypes and Trotter Blood Group defined by 14 single nucleotide polymor- and DNA Allele Frequency in Hungary phisms, 10 at the D-loop region (positions 15544, 15558, 15578, 15714, 15715, 15741, BEÁTA BÁN1, ALICE GYURMAN1, 15758, 16127, 16139 and 16141) and four at CSILLA JÓZSA2 the CytB gene (positions 14309, 14717, 14740 1National Institute for Agriculture Quality and 15264). One out of the six haplotypes was Control; 2University of Veszprém shared by three maternal lineages belonging to Two thoroughbred and two trotter stocks were Campanario strain and the other five were typed (N=88) with 5 biochemical systems (Tf, specific to each one of the remaining lineages. Al, Es, Gc, A1B), blood groupwithin the D Our results show that mitochondrial DNA se- system by 13 serums and a set of 12 microsat- quence analysis is useful for the assessment of ellites by ABI 310 automatic prism analyzer. non-autosomal genetic variability in a conser- The results indicated that the allele frequencies vation programme and to improve new conser- characteristic for the species in the basis of by vation strategies. blood group and polymorphical tests can be detected by the microsatellite tests as well. D002 Those group which can be seperated with spe- Identification of two new variants of vitamin cies on the basis of alleles by tradtional meth- D binding protein (GC) in cattle ods, can be seperated by microsatellite tests, too. HELEN ARTHUR, T. KEVIN BELL Australian Equine Genetics Research Centre, University of Queensland, St. Lucia, Australia

78 Section D: Marker, Polymorphism and Biodiversity

D004 DODD, BOB G. MORRIS Evaluation of certain genetic and phenoty- Stormont Laboratories Inc., Woodland, CA pic parameters on growth performance in 95776, USA crosses of Hampshire x Indigenous Pigs in Using the restriction fragment length polymor- Assam phism test of Meyer et al. (1995), 447 North American Bison (Bison bison) were tested for TEJENDRA BARDOLOI, KABERI DEKA variation in the mitochondrial cytochrome b Assam Agricultural University, Department of gene. The amplicons (359 bp in length) were Animal Genetics and Breeding, digested with the restriction enzyme Inf I and College of Veterinary Science, Khanapara , resolved by polyacrylamide gel electrophore- Guwahati-781022,Assam ,India sis. Two restriction profiles were identified: Data on growth records of 621 half-bred and one characterized by two DNA fragments of 697 graded (crosses of Hampshire and Indige- 198 and 161 bp, generated by the presence of a nous) progenies born to 122 dams mated to 70 single Inf I restriction site in the amplified sires were utilized to analyse the effects of region, and a second one characterized by three various genetic and non-genetic factors, to fragments of 198, 117 and 44 bp generated by estimate the heritability, genetic and phenoty- an additional Inf I restriction site in the smaller pic correlation coefficients on body weights at fragment. The first restriction profile was ex- birth, weaning,16th week, maturity and at pected in agreement with the published DNA slaughter age respectively. Least squares ana- sequence for bison (Hassanin, A. and Douzery, lysis of variance revealed highly significant E.J.P., 1999), and was called B for bison spe- effect of genetic groups on body weights at cific. The unexpected second profile was birth,8th, and 24th week, highly significant found to be in accordance with the published influence of sex on body weights at 8th, 24th DNA sequence for cattle (Anderson, S. et al., and 32nd week. Period of birth and season of 1982), and therefore called C for cattle spe- birth had highly significant effects on body cific. If the presence of the C restriction pro- weights at all these stages of growth. Faster file among bison is used as an indication of growth in half-bred and graded pigs born du- past cattle-bison hybridization, then its occur- ring winter and pre-monsoon seasons is a fa- rence among the tested populations was esti- vourable information to the breeders to maxi- mated at 0.028 ± 0.016 (n = 107) in commer- mize profit by restricting the farrowings to cial herds, at 0.018 ± 0.012 (n = 114) and at these seasons. Heritability estimates by pater- 0.066 ± 0.017 (n = 226) in two protected pub- nal half-sib correlation method indicated better lic park herds for an average of 0.045 ± 0.010. performance on overall population, followed Due to the low occurrence of the C restriction by graded and then in half-breds. The genetic profile, the differences between herds’ fre- correlation coefficients in graded pigs were quencies were not statistically significant. better than overall estimates, followed by half- 2 breds. The coefficient of determinant (R ) va- D007 lues indicated that efficiency in prediction of th Co-amplification of ten microsatellites for body weight at 24 week is more, as compa- identity and parentage testing in Alpaca red to body weight at 32nd week, when the linear regression coefficient on body weight at 1 1 th MICHELE BLASI , ADELE LANZA , 16 week, partial regression coefficients on 1 th th CINZIA VARLOTTA , MARYAM body weights at 8 and 16 weeks and mul- MOTAVALIAN1, ANDREA ROSATI1, tiple regression coefficients on body weights at 2 th th GABRIELLA BOZZINI birth, 8 and 16 weeks of ages are considered 1Laboratorio Gruppi Sanguigni, Italy; to be the independent factors. 2Associazione Italpaca, Italy Alpaca breeding in Italy is in its initial stages D006 of growth but promises to grow fairly quickly, Restriction fragment length polymorphism just as it is happening in other European coun- analysis of a conserved region in the mito- tries. As the alpaca breeding population in- chondrial cytochrome b gene in the North creases, it inevitably requires that breeders American Bison (Bison bison) have a means for identifying single animals and their exact maternal and parental links. For DOMENICO BERNOCO, JONATHAN N. this purposes, the Laboratorio Gruppi San-

79 Section D: Marker, Polymorphism and Biodiversity guini, a longstanding center dedicated to the That’s why in future it is necessary to work out genetic improvement of livestock in Italy, has new methodical approaches to evaluation and defined a protocol based on the amplification forecasting possibility of appearance of ani- of 10 microsatellites. The ten microsatellites mals of given genotype. that have been selected by our laboratory for the identification and exclusion of parentage in D009 alpacas were extracted from published scien- Polymorphism evaluation of three bovine tific literature. The microsatellites selected are microsatellite markers in different Italian the following: LCA192, LCA23, LCA56, cattle breeds LCA71, LCA65, LCA5, LCA68, LCA66, LCA8, LCA77. To demonstrate the validity of this G. BONGIONI, A. POZZI, K. PARATI, A. protocol we analysed 50 non-sib Alpacas. Par- GALLI entage exclusion probability has been calcu- Istituto Sperimentale Italiano “Lazzaro lated for each locus and for the all 10 loci to- Spallanzani” Milano, Italy gether and is equal to 0.999. If one of the par- In Italy the official control of quality semen is ents is sure, 999 out of 1000 mistakes in the regulated by law and executed by the Lazzaro attribution of the other parent can be identified. Spallanzani Institute on random samples of frozen semen (at least 10% of all batches cir- Last researches based on fruit flies (Droso- culating in Italy). The official control includes phila) (4 pairs of chromosomes) that were the so called “identity test”, which is done by conducted by us, show that on the basis of comparing the pattern of DNA extracted from traditional calculation of genotype frequency reference sample (blood or semen) versus of any breed in general genotype of hybrid it is DNA extracted from semen of each sampled impossible to identify actual genotype of every batch. For this test we used at least three mi- separate animal. It is generally known that crosatellites belonging to the ISAG recom- genotype of hybrids ABC, received from mended set : TGLA 122, TGLA 126, TGLA ABxC, is ¼ A, ¼ B and ½ C. But the analysis 227. of possible versions of segregation shows that The aim of this work was to compare seven in practice far not all the animals have such Italian cattle breeds: Italian Brown (01), Italian ratio of A,B,C genotypes in themselves, and Frisian (02), (03), even double-breed individuals may be met Italian Simmenthal (04), Piedmontese (05), (table 1). (10) and Grey Alpine (11) concerning ♂АВ х С♀ the following genetic parameters: effective 12341234 number of alleles (A. Ns.), Heterozygosity 1А АВВВСССС (He) and Polymorphism information content ВАВВСССС ВВАВСССС (PIC) obtained from these three microsatellites ВВВАСССС in 1204 sires. 2А ААВВСССС The DNA extracted was amplified by PCR and ВААВСССС ВВААСССС alleles were visualized by 373 DNA automatic АВАВСССС sequencer (ABI Prism–Applied Biosystems) . АВВАСССС The following table showes the obtained re- ВАВАСССС 3А ВАААСССС sults. АВААСССС ААВАСССС TGLA 122 TGLA 126 TGLA 227 Breed A. He PIC A. He PIC A. He PIC АААВСССС Ns. Ns. Ns. 4 ААААСССС 01 13 0.78 0.75 6 0.63 0.55 10 0.80 0.77 4 ВВВВСССС 02 17 0.82 0.78 7 0.73 0.69 12 0.83 0.81 03 14 0.82 0.79 7 0.76 0.70 13 0.86 0.84 04 13 0.79 0.76 8 0.65 0.59 9 0.81 0.78 05 19 0.86 0.83 6 0.77 0.74 12 0.88 0.87 Besides this, even animals that correspond to 10 9 0.68 0.64 6 0.65 0.60 8 0.79 0.75 the counted parts are not the same as for 11 7 0.76 0.71 4 0.63 0.57 11 0.82 0.79 genotypes. The division of chromosomes lasts in more complicated manner in population as The most variable breed was represented by well as in individuals during hybridization of Piedmontese in all three locus either for Het- received three-hybrids descendants with indi- erozygosity or for PIC. viduals of D-breed.

80 Section D: Marker, Polymorphism and Biodiversity

D010 sponded to the Mountain subpopulation, 51 to Development, comparison and application the Valley and 33 from the Regional Park of of different PRNP typing systems Manzanares. All birds were genotyped using 7 microsatellites (NVHfp13, NVHfp31, JOHANNES BUITKAMP1, CHRISTA NVHfp79-4, NVHfp89, NVHfp92-1, KÜHN2, MARTINA HAIDUCH1, URSULA UCMfp517, UCMfp347) and AFLPs (Ampli- IRPS3, JÖRDIS SEMMER1, JÖRN MOSNER3 fied Fragment Length Polymorphisms) through 1 Bayerische Landesanstalt für Tierzucht, Abt. the combination of TaqI and EcoRI and prim- Bio- und Gentechnik, Poing, Germany; ers E45 and T32. Genotypes were used by 2Forschungs-institut für die Biologie landwirt- scoring absence or presence of an allele with schaftlicher Nutztiere, Dummerstorf, Ger- "0" or "1", and genetic distances estimated many; 3 GAG BioScience, Bremen, Germany according to Dice. The graphic representation Scrapie is a transmissible spongiform en- shows the grouping of Mountain and Regional cephalopathy (TSEs) of sheep. Sheep that are Park sub-populations with the clear segrega- experimentally infected with BSE agent via the tion of Valley sub-population. A low percent- oral route develop almost the same clinical age (3%) of the total genetic variability, esti- signs as scrapie. Natural scrapie causes eco- mated using F statistics, is due to the sub- nomic losses that could be reduced by the use populations for which the inbreeding level was of scrapie resistant sheep. It is well known that relatively high (17%). Analysis of diversity variants of the prion protein coding gene shows that if extinction of Regional Park and (PRNP) are associated with resistance or sus- Mountain sub-populations is considered, 52% ceptibility to scrapie and several labs provide of the total genetic diversity would be lost. genetic tests. Depending on the application PRNP test systems have to meet different re- D012 quirements. When used in scientific projects a Chromosomal localization, and genetic precise definition of the alleles may be re- variation of the Ovine heart fatty acid- quired including detection of novel alleles binding protein gene (H-FABP) whereas mass-screening systems will need robust and cheap single-pass typing of a large JORGE H. CALVO1, NADIRA SAÏDI- number of sheep. We have implemented three MEHTAR2, DANIEL VAIMAN3, JUAN J. different methods accomplishing these differ- JURADOL, MALENA SERRANOL ent needs. For complete analysis of the geno- 1Departamento de Mejora genética, INIA, type we used direct PCR sequencing, for me- Madrid, Spain; 2Laboratoire de Biologie dium-throughput applications we developed a Moléculaire et Génétique, Université Oran Es- fluorescent multiplex 'amplification refractory Sénia, Oran, Algeria; 3Laboratoire de mutation system', and for the mass-screening a Génétique Biochimique et de Cytogénétique, fully automated MALDI-TOF based method Département de Génétique Animale, INRA, was established. The pros and cons of these Jouy-en Josas, France methods will be discussed. Fatty acid-binding proteins (FABPs) are small intracellular proteins involved in fatty acid D011 transport from the plasma membrane to the Genetic Diversity of the Falco peregrinus sites of β oxidation and triacylglycerol or populations of central Iberian Peninsula phospholipid synthesis. The heart type FABP (H-FABP) protein is present in several tissues E. CALONGE, Y. MÍNGUEZ, S. DUNNER, with a high demand for fatty acids such as J. CAÑÓN cardiac and skeletal muscle and lactating Dpto. Producción Animal, Facultad de Veteri- mammary gland. By the other hand, this pro- naria, Madrid, Spain tein has been associated with mammary gland A total number of 124 Falco peregrinus be- development. Thus, the ovine H-FABP is a longing to three different ecological niches of candidate gene for milk and meat quality, and the central Iberian Peninsula were studied with for uddertraits. the purpose of analysing the existence of a Exons two, three and four and flanking intronic subjacent genetic structure, and also studying sequences were determined as well as a small the inbreeding levels associated to these DNA fragment of the 3’ untranslated region, niches. From the 124 birds analysed, 35 corre- from an autochthonous breed specialized in

81 Section D: Marker, Polymorphism and Biodiversity milk production (Manchega breed). Eleven D014 SNPs were detected up to now, showing simi- Gene linkage mapping of the porcine lar haplotypes. Besides, a variable CTC inser- chromosome X region harbouring QTL for tion and a variable poli A tract were also de- fat deposition tected in intron 3. These polymorphisms have been characterized in Manchega breed. STANISLAV CEPICA1, GARY A. RO- The sheep H-FABP gene was located on HRER2, MARTIN MASOPUST1 chromosome 2 by sheep sequence-specific 1Institute of Animal Physiology and Genetics, PCR on DNA from a sheep/rodent cell hybrid Academy of Sciences of the Czech Republic, panel. Libechov, Czech Republic; 2USDA, ARS, U. S. H-FABP pseudogene-like sequences have been Meat Animal Research Center, PO Box 166, isolated, showing a 7-bp deletion in putative Spur 18D, Clay Center, Nebraska, USA exon 3, and various nucleotide substitutions, as The QTL for backfat thickness and intramu- well as codon stops. scular fat content on SSCX is well documented In conclusion, this genetic variation will enable in Meishan x Western breed pedigrees. The us to investigate the role of the H-FABP gene QTL has been mapped to the chromosome in milk and meat quality. region between microsatellites SW2456 and SW1943. In the French pedigree with more D013 than 1100 F2 animals the QTL mapped at posi- An IRS-PCR (Interspersed Repeated Se- tion 73 – 74 Kosambi cM with 95% confidence quence-PCR) method for the determination interval ranging from 65 to 85 Kosambi cM. of sex and other traits in bovine embryos For fine mapping and comparative positional candidate cloning, alignment of human and IRENE CAPPUCCIO, ALESSIO swine gene maps is essential. Using PCR we VALENTINI have partially cloned and sequenced porcine Dipartimento di Produzioni Animali, orthologs of genes from HSAX and subse- Università della Tuscia, Viterbo, Italy quently detected SNPs (PCR-RFLPs) within Control of the sex ratio of domestic species is seven genes. These markers have been typed potentially of great commercial importance for on the USDA-MARC backcross pedigree. animal production. Several methods for bovine Gene location on the current USDA-MARC embryos sexing have been developed. The linkage map of chromosome X follows: optimal one must be accurate, non invasive, AMELX - 8.7 cM, RPS4X – 74 cM, POU3F4 inexpensive, rapid. Methods based on PCR fit – 74 cM, FACL4 – 80 cM, CAPN6 – 81 cM, all these requirements, moreover they can al- PAK3 – 82.5 cM, and BGN – 128,0 cM. Final low the characterisation of bovine embryos for order of genes mapped to the USDA-MARC 2 multiple traits and in this respect they are supe- linkage map of the chromosome X region with rior to the use of sexed sperm for the artificial 95% confidence interval of the QTL contains insemination. The reduced amount of DNA genes in the following order: AR – 74 cM, obtained from few biopsed cells represents a PGK1 – 74 cM, ZNF261 – 74 cM, RPS4X – major limitation to perform multiple analyses 74 cM, PLP1 – 74 cM, POU3F4 – 74 cM, or for repeating the analysis to improve its SERPINA7 – 75.5 cM, FACL4 – 80 cM, accuracy. CAPN6 – 81 cM, and PAK3 – 82.5 cM. From Several technologies based on PCR amplifica- comparison of cytogenetic and linkage maps it tion of unknown DNA sequences are available is apparent that the chromosome region be- to increase the overall amount of DNA and longs among those with a low recombination some of them have been successfully used. rate. Now with more than 20 genes on the In this study an IRS-PCR (Interspersed Re- porcine linkage map there is no evidence of peated Sequence-PCR) was performed to in- rearrangements in gene order between porcine crease the amount of DNA present in 2-4 days and human X chromosomes. bovine embryos and its amplified products were used for a successful and accurate sex D015 typing. The possibility of multiple loci embryo Milk protein variability in African Bos Ge- characterisation is investigated. nus breeds

GABRIELLA CERIOTTI1, GIULIANA

82 Section D: Marker, Polymorphism and Biodiversity

TIRELLA1, STEFANIA CHESSA1, ANNA biochimique et Cytogénétique, INRA, Jouy-en- CAROLI2, CASIMIRO CRIMELLA3 Josas, France; 3Station d’Amélioration Géné- 1University of Milan, Department VSA, Mi- tique des Animaux, INRA, Castanet-Tolosan, lano, Italy; 2University of Bari, Department of France Animal Health and Welfare, Valenzano, Italy; Rabbit (Oryctolagus cuniculus) is a small but 3University of Milan, Institute of Zootechnics, active animal sector for meat and fur in South- Milano, Italy ern Europe agriculture and is a good model for The importance of milk protein loci is well human diseases and for studies on physiology, known due to the relationships with productive immunology and transgenesis. However, the traits. The polymorphism of αs1-casein rabbit genome has been poorly studied until (CSN1S1), β-casein (CSN2), k-casein (CSN3) now. Since there is an increasing need of effi- and β-lactoglobulin (LGB) was investigated in cient mapping tools to identify genes of inter- African Bos taurus (Somba, Lagunaire), Bos est in this species, the construction of an inte- indicus (Zebu Peul Soudanais) and Bos taurus grated cytogenetic and genetic map has started. x Bos indicus (Borgou) populations at DNA The aim of the work is to build a first genera- level. The employed techniques were: ASP- tion microsatellite map with a resolution of 10 PCR (CSN1S1), PCR-RFLP (BLG), and PCR- cM that means the isolation of about 300 SSCP (CSN2 and CSN3). Milk protein loci polymorphic microsatellites. Microsatellite were all polymorphic, except CSN1S1 in typing will be performed on informative three Lagunaire. CSN2 presented four alleles in Bor- generation families to establish genetic dis- 1 2 1 gou and Zebu (A , A , B, I), while only A and tances. The availability of both a rabbit BAC A2 variants were found in Somba (A1 = 75%) library and data on reciprocal human-rabbit and Lagunaire (A1 = 69%). The CSN2*I vari- chromosomal painting allowed the develop- ant, recently described in European breeds, ment of an integrated approach. The BAC li- showed a rather high frequency (10%) in Zebu, brary has been screened by PCR or by high and was detected at a lower frequency (1%) in density filter hybridization for genes chosen Borgou. At CSN3 locus, B allele was predomi- for their hypothetical position on rabbit chro- nant in pure taurine breeds (Somba: 76%; mosomes. BAC clones for 74 distinct genes Lagunaire: 75%), while H was predominant in have already been isolated and 55 clones were Zebu and Borgou (35% both). CSN*H allele mapped by FISH to all but the X chromo- can only be detected at DNA level, being somes. Thirtytwo microsatellite sequences identical to CSN*A at protein level by electro- with more than 10 repeats were identified from phoretic techniques. In addition CSN3*A1, 22 BAC clones and unexpectedly half of them which is a silent mutation based on CSN3*A, corresponded to (TC)n repeats. PCR primers was observed in Zebu (17%) and Borgou (8%). were designed for 20 microsatellites. Isolation At LGB locus, the B allele was predominant, if of other microsatellites and analysis of the compared to LGB*A, with a decreasing trend allelic polymorphism is in progress. from Zebu (90%) to Borgou (81%), Somba (78%) and Lagunaire (51%). The discriminat- D017 ing power of milk protein genes in differenti- Radiation hybrid mapping of 22 loci con- ating between indicine and taurine breeds was taining endogenous retroviral sequences in verified by statistical analyses. the pig and characterization of the retrovi- ral genomic flanking sequences D016 1 2 Towards the construction of an integrated Z. LIU , C. ROGEL-GAILLARD , N. 2 2 3 cytogenetic and genetic map of the rabbit BOURGEAUX , J. C. SAVE , M. YERLE , D. MILAN3, P. CHARDON2 1 C. CHANTRY-DARMON1,2, D. VAIMAN2, Laboratory of Agrobiotechnology, China Ag- H. HAYES2, H. DE ROCHAMBEAU3, D. ricultural University, Beijing, China; 2 ALLAIN3, C. URIEN1, N. BOURGEAUX1, Laboratoire de Radiobiologie et Etude du M. BERTAUD2, P. CHARDON1, C. ROGEL- Génome, UMR INRA-CEA 13.314, Jouy-en- 3 GAILLARD1 Josas, France; Laboratoire de Génétique 1Laboratoire de Radiobiologie et Etude du Cellulaire, INRA, Castanet-Tolosan, France Génome, UMR INRA-CEA 13.314, Jouy-en- Genomic integration of mobile elements such Josas, France; 2Laboratoire de Génétique as transposons and retroviruses is known to

83 Section D: Marker, Polymorphism and Biodiversity contribute to the evolution and plasticity of using computer program „Primer 3”, while genomes. Sixtytwo BAC clones containing primers for the 3’gene end originated from type C porcine retroviruses (PERVS) were literature (Ernst et al., 1993). previously isolated from a porcine BAC library Exon 1 showed low polymorphism with two and mapped by fluorescent in situ hybridiza- SSCP patterns: A (two bands) in 98% of ani- tion to all chromosomes except 6, 12, 15, 16 mals from group I and 94% in group II, and E and 18, allowing us to identify 24 distinct inte- (three bands) in 2 fatteners from group I and 6 gration loci. We report here the more precise in group II. Exon 2 in all studied animals was mapping of 22 loci using the ImpRH radiation monomorphic. Exon 3 showed high polymor- hybrid (RH) panel and sequence analysis of phism with four patterns: A, B, C and D alleles PERV genomic flanks. BAC end sequencing with one to three bands. Pattern A was the was done for all BAC clones and 104 sequence most frequently observed (88% in group I, tagged sites were recovered. PCR was per- 82% in group II), the remaining patterns oc- formed on the RH panel with 31 primer pairs curred in 2-10% of fatteners. designed from the STS. Two loci at position Sequence analysis showed substitution T→A 17q12 were clearly distinguished due to the in exon 1; deletion T in exon 3 (B allele) and resolution of the RH mapping. Two PERV substitution G→T in exon 3 (C allele). Allele clusters had been described at positions 3p15 D had the same sequence as allele B. and 17q21 but the RH mapping results strongly In non-coding region of 3’ end of myogenin suggested a unique locus at each position. gene we found three RFLP genotypes: F - ho- Further fingerprinting analysis of the BAC mozygote non-digested with MspI (PCR prod- clones confirmed the single locus and the ex- uct equal to 353 bp), G - homozygote digested istence of two alleles at each chromosomal with MspI (two bands - 219 bp and 134 bp) position. A massive sub-cloning of all PERV and H - heterozygote including F and G bands. genomic flanks was launched. One BAC clone Genotype H was the most frequent in both per locus was digested by EcoR1 or HindIII groups of fatteners (0.85 and 0.65 in I and II and a long terminal repeat (LTR) probe spe- group, respectively), genotype G frequency cific for PERVs was used to recover the LTR was equal to 0.13 in group I and 0.32 in group flanking genomic sequences on both sides of II, while genotype F was the most rare one the PERV. Analysis of the sequencing results (0.02 and 0.03, respectively). will help to study genomic segments permis- sive for stable retroviral integration in the pig. D019 Microsatellite DNA polymorphism of Jeju D018 native horses in Korea RFLP and SSCP polymorphism in the myo- genin gene in crossbred pigs GIL JAE CHO1, YOUNG JAE LIM1, SUNG JEAN KIM1, BYUNG WOOK CHO2 KRYSTYNA M. CHARON, ZUZANNA 1Korea Racing Association, Equine Blood NOWAK, JÓZEF KULISIEWICZ & ALEK- Typing Laboratory, Gwachon, Korea; SANDRA HERMANOWICZ-ŚWIERCZEK 2Miryang National University, Department of Agricultural University, Faculty of Animal Animal Science, Miryang, Korea Science, Warsaw, Poland Jeju native horses (JNH) are one of the native Polymorphism in the chosen regions of myo- animals that were designated as a natural genin gene was analysed in two groups of monument in Korea. They have been isolated crossbred fatteners, born by crossbred sows in Jeju island for a long time and some of them (Polish Large White x Polish Landrace) and have been used as racing horses. Ramdom sired by (I) Duroc boars and (II) crossbred samples of 62 JNH were genotyped with 16 boars (Duroc x Pietrain) from the Warsaw microsatellites markers including nine interna- Agricultural University experimental farm. tional minimum standard panel. Multiplex The total DNA was isolated from a whole PCR was used for amplifying these microsat- blood using phenol/chloroform extraction. ellite markers and genotyping reproducibility SSCP polymorphism was analysed in exons 1, was observed based on size precision of the 2 and 3, while RFLP (MspI) polymorphism in PCR fragments. Allele frequencies, heterozy- non-coding region of 3’ end of the myogenin gosities, polymorphic information contents gene. Primers for exons 1 to 3 were designed (PIC) and exclusion probabilities (PE) were

84 Section D: Marker, Polymorphism and Biodiversity calculated. The number of alleles of the mark- sent unique horse genetic resources and pres- ers was varied between 5 and 13 with an aver- ervation efforts should be continued. age number of alleles of 8.38. The heterozy- gosities were ranged from 0.388 to 0.847 and D021 mean expected heterozygosity was 0.721. Ob- Two-point linkage mapping of the ovine served PIC was from 0.366 (HTG6) to 0.823 calpain II regulatory gene (ASB17) and mean PIC was 0.685. This result indicated that JNH are more polymorphic than H. Y. CHUNG1, C. D. KIM1, C. Y. CHO1, S. Thoroughbred horses. The PE was observed H. YEON1, H. J. JIN1, M. E. DAVIS2, H. C. from 0.222 (HTG6) to 0.691 (ASB17) and the HINES2, A. M. CRAWFORD3 total exclusionary power of all markers was 1National Livestock Research Institute, Dept. 0.9999. These results can give basic informa- of Performance Testing, Chenan, Korea; 2The tion for developing parentage verification and Ohio State University, Dept. of Animal Sci- individual identification system in JNH in Ko- ences, Columbus, USA; 3University of Otago, rea. Dept. of Biochemistry, Dunedin, New Zealand The ovine calpain II regulatory gene, which is D020 calpain 4, was screened with primers for 8 Genetic diversity of two rare horse breeds sheep reference families containing 122 cross- from Poland bred animals from New Zealand. The primer sequences were selected based on the bovine GRZEGORZ CHOLEWIŃSKI1, BARBARA cDNA sequence (GenBank accession No. GRALAK2, E. GUS COTHRAN3, EWA J05065), and focused on the exon regions (ex- IWAŃCZYK1 ons 3 and 4, 5 and 6, and 7 and 8). Genetic 1Agricultural University of Poznan, Poland; variants were observed using polymerase chain 2Institute of Genetics and Animal Breeding of reaction single strand conformational poly- Polish Academy of Sciences, Jastrzebiec, Po- morphism (PCR-SSCP) analysis of the three land; 3University of Kentucky, Lexington, KY, segments, and heterozygosities for each seg- USA ment were estimated to be 0.45 (CAPN4L34), In Poland, as in many other countries, a pro- 0.41 (CAPN456), and 0.48 (CAPN478). Two- gram for the preservation of the genetic re- point linkage analysis was performed to iden- sources represented by different farm animals tify the location of the calpain 4 segments species has been developed. In this study 27 among microsatellite loci with CRI-MAP ver- genetic markers (10 microsatellites – AHT5, sion 2.4. The results strongly suggested that HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, the calpain regulatory gene is located on ovine HTG7, HTG10, VHL20; 7 blood group loci – chromosome 14. A, C, D, K, P, Q, U and 10 biochemical poly- morphisms – ALB, A1B, ES, GC, HBA, PGD, D022 GPI, PGM, PI, TF) were use to assess genetic Cytochrome B sequence variation as a tool diversity of two horse breeds (the Polish for tracing the origin of three local Spanish Primitive Horse and Hutsul) covered by the pig breeds. National Farm Animal Genetic Resources Conservation Program. Blood group and ALEX CLOP1, ANA FERNÁNDEZ2, MAR- biochemical diversity was relatively low in the CEL AMILLS1, JUAN CAPOTE3, CORI Hutsul compared to the average for domestic RAMON4, LEIF ANDERSSON5, ARMAND horse breeds but was well above average for SÁNCHEZ1 the Polish Primitive Horse. In contrast, the 1Departament de Ciència Animal i dels Hutsul had higher heterozygosity at microsat- Aliments, Facultat de Veterinària, Universitat ellite loci than did the Polish Primitive (both Autònoma de Barcelona, Bellaterra 08193, breeds had values near the mean of microsat- Spain; 2Departamento de Mejora Genética y ellite variability for horse breeds). This differ- Biotecnología, INIA, Madrid 28040, Spain; ence can be accounted for by monomorphism 3Unidad de Producción Animal, Pastos y For- of the HTG6 locus in the Polish Primitive rajes, Instituto Canario de Investigaciones Horse. Based upon genetic distance values, Agrarias, La Laguna 60, Spain; 4Departament neither breed shows close relationship to any de Biologia, Universitat de les Illes Balears, breed that we have tested. These breeds repre- Palma 07071, Spain; 5Department of Animal

85 Section D: Marker, Polymorphism and Biodiversity

Breeding and Genetics, Swedish University of Bank database. Microsatellite sequences were agricultural Sciences, Uppsala S-75124, Swe. downloaded from GenBank and queried The pig has been domesticated both from a against the databases using Blastn and Blastx European and Asian subspecies of the Wild searches. Out of 312 microsatellites compared Boar. The origin of mitochondrial DNA can be to the databases, 17 loci had a significant traced in the maternal line by analysing Cyto- match to known genes. We were able to map chrome B sequence polymorphism. We have nine of these genes in the tilapia linkage map, typed four SNPs located at positions 15036, providing anchors for comparative mapping 15038, 15041 and 15045 of the porcine Cyto- between tilapia and other vertebrates. The chrome B gene in three Spanish breeds (n = rapid in silico approach utilized in this study, 83) by direct sequencing of the PCR product. previously used in mice and livestock, in- Different combinations of these SNPs yield creased the number of genes in the tilapia link- four different haplotypes: EI (TGCG), EII age map from 14 to 23, and identified 7 more (TGTG), AI (CATA) and AII (CATG). Three genes which match unmapped microsatellies. different Spanish breeds, Iberian (Torbiscal and Guadyerbas) (IB), Porc Negre from Mal- D024 lorca (PN) and Cerdo Negro Canario (CAN), Mapping of mammary gland-derived ex- were studied. The analysis of 50 pigs from the pressed sequence tags (ESTs) in cattle IB breed showed that all of them had the EI european haplotype. This observation could be ERIN E. CONNOR1, TAD S. SON- explained by the purity of both the Torbiscal STEGARD1, JOHN W. KEELE2, GARY L. and the Guadyerbas experimental lines. The BENNETT2, JOHN WILLIAMS3, RICHARD PN pigs also had the EI haplotype (n = 22) PAPWORTH3, MELISSA S. ASHWELL1 except one individual that showed a new hap- 1U.S. Department of Agriculture, Agricultural lotype (TGCA). This new haplotype might Research Service, Beltsville, MD, USA; 2U.S. have been produced by a single mutation on Department of Agriculture, Agricultural Re- the first SNP of EI. Finally, CAN pigs (n = 11) search Service, Clay Center, NE, USA; 3Roslin displayed three different haplotypes: EI (n = Institute, Roslin, Midlothian, Scotland 3), AI (n = 3) and AII (n = 5). This result indi- To improve the comparative map between the cates that this pig breed has a mixed European cattle and human genomes, bovine expressed and Asian origin. Possibly, the AI and AII sequence tags (ESTs) were assembled into haplotypes were introduced by the ancient tentative consensus (TC) sequences and those multiple settlers that colonized the Canary with an expression bias for the mammary Islands. gland (≥75% of ESTs in TC) were selected for radiation hybrid (RH) and linkage mapping. To D023 initiate mapping, selected TC sequences were Fishing in silico: searching for tilapia genes aligned with a human genome draft sequence using sequences of microsatellites DNA to predict gene identity, map position, and markers genomic sequence structure information. Of more than 400 TC sequences analyzed, ap- AVNER CNAANI, MICHA RON, GIDEON proximately 100 met criteria as candidates for HULATA, EYAL SEROUSSI mapping. These sequence alignments were Institute of Animal Science, Agricultural Re- used to design primer pairs for PCR that ampli- search Organization, P.O. Box 6, Bet-Dagan fied a predicted intron, which limited this 50250, Israel number to 58 primer pairs. PCR products am- Genetic linkage maps of some edible fish spe- plified from bovine genomic DNA from sires cies, constructed in recent years, consist of of the USDA Meat Animal Research Center hundreds of DNA markers but only few genes. (MARC) reference population were directly Microsatellites DNA markers are short tandem sequenced to verify amplification of the tar- repeats, with unique flanking sequences. They geted gene region and to identify single nu- are highly abundant throughout the genome cleotide polymorphisms to be used as markers and appear in coding and non-coding regions. for linkage mapping. Currently, 22 bovine Therefore, it is likely that the flanking se- genes are being placed on the USDA-MARC quences can be part of a gene, which can be Linkage Map and/or the Roslin RH Map. identified by similarity searches against Gen- Mapping of bovine ESTs improves the bovine

86 Section D: Marker, Polymorphism and Biodiversity comparative map with the human genome se- gene. From the analysis of published se- quence and assists in the integration of the quences in pig, human and cattle we have de- bovine linkage and physical maps. signed consensus primers to amplify a frag- ment of the promoter region (1270bp).The D025 sequence analysis was carried out on two indi- Genetic diversity in and burro viduals each of the following breeds: Mar- populations chigiana, , Piedmontese, , Holstein Friesan, Belgian Blue, Limousine, E. GUS COTHRAN Pezzata Rossa, Brown and Charolaise, The Department of Veterinary Science, University promoter region contains several binding sites of Kentucky, Lexington, KY, 40546, USA for transcriptional factors which may play an Genetic diversity in 78 populations of feral important role for the regulation of the protein horses has been measured using blood group, and consequently of muscular development. biochemical, and microsatellite polymor- From sequence data analysis we found a T/A phisms. There is a tremendous amount of ge- polymorphism at –371 (relative to ATG start netic diversity if feral horses, approaching that codon) in Belgian Blue breed. Then we carried observed across domestic horse breeds. This is out a PCR-RFLP test on fifthteen to twenty largely due to the diverse origins of the feral individuals of each breed. The T/A variant is herds. Heterozygosities range from 24.6% to present both in heterozygous and homozygous 43.2%. There was a general trend for variabil- condition with different allele frequencies in ity to be correlated to population size but the all breeds. relationship was not statistically significant. Eight populations have been tested multiple D027 times over several years. In only one case was Polymorphism of κ-casein in Italian goat there a significant change in variation and that breeds: a new ACRS-PCR designed DNA was a loss of variation. Twelve feral burro test for discrimination of A and B alleles populations have been tested for microsatellite variation. Genetic variation levels in burros MARIA FELIGINI1, VLATKA CUBRIC- was similar to that of horses although consis- CURIK2, PIETRO PARMA1, JASMINA tently lower (Ho=21.7% to 55.1%). Feral LUKAC-HAVRANEK2, GIAN FRANCO populations of burrows showed lower variation GREPPI1, INO CURIK3, GIUSEPPE ENNE1 than domestic Standard donkeys. There were 1Istituto Sperimentale Italiano "Lazzaro Spal- some differences in patterns of variation within lanzani", Milan, Italy; 2Faculty of Agriculture feral burro populations compared to feral horse University of Zagreb, Dairy Science Depart- populations. These differences may be due to ment, Zagreb, Croatia; 3Faculty of Agriculture differences in social structure. University of Zagreb, Animal Science Depart- ment, Zagreb, Croatia D026 Genetic polymorphism of κ-CN gene was Sequence and analysis of the myostatin analysed in Italian goat breeds by a newly de- promoter region in cattle signed ACRS-PCR DNA test for rapid char- acterisation of goat kappa-casein (κ-CN) A and A. CRISÀ, C. MARCHITELLI, B variants. Thus, the 167-bp PCR product A. VALENTINI surrounding the nucleotide mutation was am- Dipartimento di Produzioni Animali, plified from genomic DNA and the PCR prod- Università della Tuscia, Viterbo, Italy uct was digested with MaeIII. After digestion Myostatin or GDF8 acts as a negative regulator the A allele showed two fragments of 77 and of muscle growth; mutations in this gene are 65 bp in comparison to the B allele which has responsible for the double muscling phenotype given two fragments of 90 and 77 bp. The found in several European cattle breeds. In analysis of allele frequency distribution at κ- some breeds double muscling is not associated CN locus, based on 401 individual samples, with any disruptive mutation in the gene and a revealed significant differences between three possible explanation could be attributed to the goat breeds from the north of Italy (Nera di effect of transcriptional gene regulation. For Verzasca, Frontalasca and Alpine) with fre- this reason we have identified and character- quency of κ-CN B allele around 0.3, versus ized the upstream 5’ region of the myostatin two goat breeds from the south of Italy (Mal-

87 Section D: Marker, Polymorphism and Biodiversity tese and Sarda) with frequency of κ-CN B different partners that could be implicated in allele around 0.5. While two goat breeds the interaction with this threonine, specially (Maltese and Nera di Verzasca) did not show spinophilin. significant deviations from the Hardy- Weinberg equilibrium, a highly significant D030 excess of heterozygote genotype (AB) was Analysis of new variants within the ovine observed in Alpine, Frontalasca and Sarda and bovine PrP-gene goats. Here developed ACRS-PCR method for discrimination of A and B alleles as well as JULIA ECKERT, SIEGFRIED PREUSS, YI- high frequency of κ-CN B allele give a prereq- HUA HAN, HERMANN GELDERMANN uisite for simultaneous estimation of casein University of Hohenheim, Institute of Animal haplotype effects on milk production and Husbandry and Breeding, Department of Ani- cheese-making properties. mal Breeding and Biotechnology, Stuttgart, Germany D029 Strong effects of DNA variants within the PrP The ink4a/arf locus evolution in primates (Prion Protein) gene on the incidence and pathogenesis of a number of TSE (Transmissi- ANNE DI TOMMASO1, CEDRIC SOLER1, ble Spongiform Encephalopathy) syndromes CHRISTIAN ROOS2, ALAIN KITZIS1, VE- have been reported. Three allelic amino acid RONIQUE LADEVEZE1 positions in the PrP of sheep are associated 1Laboratoire de Génétique Cellulaire et Molé- with Scrapie incidence. But this association culaire, UPRES EA2622, Université de Poi- does not sufficiently explain Scrapie distribu- tiers and CHU de Poitiers, France; 2German tion and breed specific differences. In cattle no Primate Center, Göttingen, Germany amino acid substitutions have been identified The human ink4a/arf locus encodes two cell so far and only one silent mutation and a vari- cycle regulatory proteins, the cyclin dependent able repeat motifs coding for an octapeptide kinase inhibitor (p16ink4a) and the p53 activator are known for the bovine PrP gene. The few (p14arf), through use of alternative first exons. DNA markers available do not allow efficient This locus genomic organization is unique in genotyping. Aim of this study was to identify eukaryotes, with two different proteins ob- new polymorphic positions within the PrP gene tained using different reading frames. The aim that will allow rapid screening and may reveal of this study is to characterize the ink4a/arf possible associations with TSE-resistance. locus origin and to determine the molecular Potentially polymorphic loci were identified by process which allows this locus organization. “etandem” (sequence analysis package EM- The divergence between mouse or opossum BOSS was kindly provided by the UK HGMP p19ARF and human p14ARF is very important Resouce Centre) software-assisted screening of whereas proteins have the same nucleolar lo- PrP sequences published in genomic databases calization and function. We studied the exon (i.e. Genbank). Three animals for each of 1β of p14arf in 12 different species of primates. eleven genetically diverse sheep breeds and We didn't find any polymorphism in studied four animals for each of eight genetically dis- species (monkeys, apes and humans). These tinct cattle breeds were submitted to PCR- sequences are very similar with only one to based screening and analysed for polymor- four amino acids substitutions as compared to phisms. Divers alleles were cloned, sequenced the human sequence, while mouse and opos- and compared to database information. Of the sum sequences show many differences. 24 analysed positions in cattle nine displayed Moreover we show three amino acid substitu- polymorphisms. The number of alleles ranged tions in new world monkeys and lemurs which between two and seven. In eight of the 23 po- are not present in old world monkeys, apes and sitions analysed in sheep polymorphisms were humans. More surprisingly we observe a found with the number of alleles variing be- threonine at position 31 in all human se- tween two and five. Seven of the positions quences whereas an alanine is always present amplified in sheep have not previously been in all pre-monkey, monkey and ape sequences. described. The allele structures displayed com- Our results indicate that this substitution could plex DNA differences. These results will help play an essential role in the control of cell- to refine and simplify present day genotyping cycle arrest and apoptosis. We will discuss the of the PrP gene and to analyse the distribution

88 Section D: Marker, Polymorphism and Biodiversity of variants among species and breeds. Laboratoire de Génétique Cellulaire, INRA, Toulouse, France D031 Our aim is to define syntenic breakpoints be- Blood protein polymorphism in some native tween the human and porcine genome maps horse types in Turkey through RH mapping of porcine ESTs in order to permit a rapid and efficient exploitation of OKAN ERTUĞRUL1, BILAL AKYÜZ2, genetic information between species. Indeed, AHMET KOPAR3 the construction of high-resolution compara- 1Ankara University, Veterinary Faculty, De- tive maps that define gene order within syn- partment of Genetics, Ankara, Turkey; tenic regions will provide valuable molecular 2Erciyes University, Veterinary Faculty, De- genetic information for mapping agriculturally partment of Genetics, Kayseri, Turkey; important traits. The previous comparative 3Veterinary Control and Research Centre, map studies based on non dense localisation of Laboratory of Blood Groups and Genetics, genes distributed over all chromosomes and on Etlik, Ankara, Turkey bi-directional painting between human and Different breeds and types of horses raised in swine chromosomes showed conservation of different areas in Turkey. The numbers and synteny without allowing the precise determi- types of native horses are in steady decrease in nation of syntenic breakpoints. To construct time because of expanding agricultural mecha- high-resolution maps, we proceed as follow. nization. The bloods of 87 native Turkish According to the state of the art comparative horses of different types were analysed for maps, human genes located near evolution- blood protein polymorphism. The horses were nary breakpoints are used to search, on a se- from different geographical areas of Turkey quence similarity basis, for candidate ortholo- (Kars; Erzurum, Cukurova, Adapazarõ). Starch gous pig ESTs. These ESTs are further on gel electrophoresis, alkaline polyacrylamide processed to design primers for the mapping gel electrophoresis and polyacrylamide isoe- experiment on a swine 7000 rad radiation hy- lectric focusing electrophoresis were used to brid panel (IMpRH). This computer assisted identify genotypic variants of AIB glycopro- strategy for comparative mapping is integrated tein (AIB), Albumin (ALB), Vitamin D bind- in a computer tool, Iccare (Interspecific Com- ing protein (GC), Hemoglobin-α (HBA), parative Clustering and Annotation for ESTs), Transferrin (TF), Carboxylesterase (ES), 6- developped in our laboratory. An example will phosphogluconate dehyrogenase (PGD) and be presented, the determination of the precise Phosphoglucomutase (PGM) Loci. Direct breakpoints between porcine chr3 and human Counting Method was used for the calculation chr 7, 2 and 18. of gene frequencies of blood proteins systems. Homozygosity degrees, effective allele num- D033 bers, and efficiencies of genes were also cal- Validation of milk origin in goat dairy culated. In albumin system A and B allele fre- products by species-specific PCR quencies were found equal. The gene frequen- cies of I allele in ES system; K allele in AIB MARIA FELIGINI1, PIETRO PARMA1, system, F allele in GC, PGD, PGM systems VLATKA CUBRIC-CURIK2, GIAN were determined quite high ranging from 0,782 FRANCO GREPPI1, GIUSEPPE ENNE1 to 0,994. The highest frequency in Transferrin 1Istituto Sperimentale Italiano ‘Lazzaro Spal- system was found 0,431 in F2 allele. BI allele lanzani’, Milan, Italy; 2Faculty of Agriculture, frequency (0,534) was determined highest University of Zagreb, Departement of Dairy frequency than other allele frequencies in Hb Science, Zagreb, Croatia System. In order to verify the quality and authenticity of dairy products and protect consumers from D032 fraud, adequate control methods are required. RH mapping of porcine ESTs to improve Identification of animal species using DNA Human-Pig comparative map at evolution- analysis has become more important to detect ary breakpoints adulterations in food products. In this study an adapted DNA extraction in combination with Y. LAHBIB-MANSAIS, T. FARAUT, F. specific polymerase chain reaction-restriction MOMPART, S. LEROUX, M. YERLE fragment length polymorphism analysis were

89 Section D: Marker, Polymorphism and Biodiversity applied to validate milk origin in goat dairy analysis revealed a new allele/haplotype, products. MC1R*7, that corresponds to the combination The DNA from somatic milk cells was ex- MC1R*1 and MC1R*6 alleles. This new hap- tracted from pasteurised milk, powdered milk, lotype results from a mutation in the form of a fresh and mature cheeses by phenol and chlo- 2bp insertion at 23 codon. We have genotyped roform. The cleared lysate was purified by a MC1R alleles by PCR-RFLP test and fragment commercial kit. The target for PCR amplifica- analysis of PCR products on an ABI 3100 in tion and restriction enzyme analysis was a black (n= 31), red (n= 109) and (n= selected partial sequence of the Capra hircus 79) Iberian pigs, MC1R*6 and MC1R*7 alleles kappa casein region (exon 4). The 167-bp am- were found in red and chestnut pigs, and black plified sequence contained the MAEIII restric- pigs were homozygous for the MC1R*3 allele. tion sites for k-CN A and B alleles. The diges- tion results were analysed by continuous poly- D035 acrylamide gel. Specific restriction enzyme Expression and Characterization of the Bo- recognition sites, exclusively located within vine Prion-Doppel and Analysis of PRND the goat PCR product, permitted the accurate Gene Polymorphisms validation after digestion. This k-CN polymor- phism-based method is applicable to validate LAURENT R. CHIARELLI2, SERGIO the origin of dairy products from goat milk and COMINCINI1, MARIA GABRIELLA FOTI3, to characterise k-CN alleles from cheeses in UMBERTO AGRIMI5, GIOVANNI DI research related to the estimation of polymor- GUARDO4, GABRIELE VACCARI5, DO- phism effects on technological properties and NATA VERDINELLI1, MICHAEL TRANU- cheese making yield. LIS6, INGRID HARBITZ6, JOERG SCHLAEPFER7, GAUDENZ DOLF7, DAVID D034 HILLS8, JOHN L. WILLIAMS8, GIOVANNA Identification of a new haplotype for the VALENTINI2, LUCA FERRETTIl MC1R gene in Iberian pig 1Dipartimento di Genetica e Microbiologia, Università di Pavia, Italy; 2Dipartimento di ANA FERNÁNDEZ, CRISTINA ÓVILO, Biochimica, Università di Pavia, Italy; CARMINA CASTELLANOS, CARMEN 3CERSA, FPTP, Lodi, Italy; 4Instituto RODRÍGUEZ, MIGUEL ANGEL TORO, Zooprofilattico Sperimentale delle regioni LUIS SILIÓ Lazio e Toscana, Roma, Italy; 5ISS, Roma, INIA, Department of Mejora Genética Animal, Italy; 6Norweagian School of Veterinary Madrid, Spain Science, Oslo, Norway; 7Institute ofAnimal Molecular interaction between the G-protein Breeding, University of Berne, Switzerland; coupled melanocortin receptor 1 (MC1R) and 8Roslin Institute, Roslin, Scotland the agouti protein is the main regulatory sys- The bovine recombinant Dpl(26-155) was tem know to control the synthesis of eumelanin purified from E.coli inclusion bodies with and phaeomelanin. Extension(MC1R) and yields of 2/3 mg/liter. Edman degradation and Agouti loci make up a regulatory system that is Carboxypeptidase Y treatment revealed a sin- known to control this process. In previous gle species shortened by 12 aa albeit with the studies, the sequence analysis of MC1R gene expected Alanine 155. Titration of free thiol has revealed six MC1R alleles, MC1R*1 groups with Edman's reagent in the presence needed for the expression of the wild-type of urea gave negative results suggesting that color, MC1R*2 associated with the black phe- cysteines were blocked in disulfide bonds. The notype in Large Black and Meishan pigs, comparison of tryptic maps obtained by HPLC MC1R*3 associated with the black color in of the peptides highlighted two prominent Hampshire, MC1R*4 associated with the red peaks that disappeared after treatment with phenotype in Duroc, MC1R*5 presents in DTT. Two N-terminal sequences were ob- Japanese wild boar, and MC1R*6 associated tained for each peak, matching the portions of with white and red phenotype in Landrace, Dpl that are involved in the formation of the Yorkshire and Linderöd. We have analyzed two predicted disulfide bonds (Y77-K96/H146- DNA sequence of MC1R gene by direct se- R153 for C94-C151 and F103-R122/E140-K145 for quencing of the PCR product in Iberian pigs C108-C142). Our data suggest that the (black, red, and chestnut animals), sequence rDpl(39-155) is properly folded. The entire

90 Section D: Marker, Polymorphism and Biodiversity

PRND ORF was sequenced in 60 swiss cattle genetic variation in wild Japanese Sika deer in (27 affected, 33 controls), 56 Italian Sarda the areas of Nikko, Gunma, Hokkaido and sheep (32/24), 52 Italian Comisana sheep Tsushima was investigated using sequence (12/40), 50 Norwegian Rygja sheep (16/34), polymorphism in the mtDNA D-loop regions, and 37 Italian Ionica goat (17/20). Eleven and mother-child relationships between popu- polymorphisms were detected in cattle (I15V; lations were assessed. C22S; R50H; D69N; K96E; I105V; A114T; DNA samples of 110 Japanese Sika deer were R132Q; T144I; R158Q; R158K). Variation collected from Nikko (Nikko National Park), was higher in affected cattle compared to Gunma, Hokkaido and Tsushima areas be- controls, with codons 50, 69 and 158 signifi- tween January and March in every year from cantly represented in the diseased animals 1996 to 2001. DNA was extracted from hepa- (P<0.003). On the contrary, PRND was rinized whole blood or a tooth. The mtDNA largely monomorphic in the sheep breeds in- D-loop region was amplified using PCR meth- vestigated, except for two synonimous muta- ods with primers constructed by Nagata et al. tions (I12I; A26A). Eleven polymorphisms (1998):LD5-AGCCATAGCCCCACTATCAA were detected in goats (C8W; W9G; L10V; and HD8-TTGACTTAATGCGCTATGTA. K34T; T47R; T51A; Y77D; L129V; E140D, PCR was performed using a PCR reagent kit G156R; Q163H), but variation was apparently (TAKARA) according to the instructions pro- unrelated to the diseased animals. vided by the manufacturer. The PCR reaction Acknowledgments: This work was supported mixture was heated at 93°C for 2 min, and by EU MASSES (FAIR5 CT97 3311) amplified under 35 sequential cycles at three different temperatures : 1 min at 94°C, 1 min D036 at 55°C and 1 min at 72°C. Nucleotide se- Genetic variation of mitochondrial DNA D- quences of PCR products were determined loop regions in Japanese Sika deer using an automatic sequencer. Alignment of sequences was achieved using the CLUS- 1 EMIKO FUKUI , MASAAKI KO- TALW software package. Haplotypes in the 2 1 GANEZAWA , MIDORI YOSHIZAWA mtDNA D-loop region in Japanese Sika deer 1 Department of Animal Breeding and Repro- were defined by comparison with mtDNA D- duction, Utsunomiya University, Utsunomiya, loop sequences of Japanese Sika deer from 2 Japan; University Forest, Faculty of Agricul- Hokkaido (accession number D50128, a-type) ture, Utsunomiya University, Utsunomiya, published by Nagata et al. (1998). Eight hap- Japan lotypes for the D-loop region were identified in Wild Japanese Sika deer (Cervus nippon) are Sika deer from the Nikko area, and homogene- distributed throughout the Japanese Archipel- ity among these haplotypes was 82.4~99.7%. ago, including Honshu, Hokkaido, Kyushu, Six haplotypes were identified in the popula- Shikoku and other islands. Some populations tion in the Gunma area, with homogeneity of of these deer were domesticated and used for 95.0~98.0%. The phylogenetic tree con- the production of venison and velvet. Se- structed from these results demonstrated that quence polymorphism in mitochondrial DNA the Tsushima population is the oldest of the displacement loop (mtDNA D-loop) regions four populations examined. has been widely used to study phylogenetic relationships at specific and subspecific levels D037 and between various populations. In cattle, Coat colour and polymorphism in the nucleotide sequences of mtDNA D-loop re- MC1R gene of the Hérens cattle breed gions have been examined by many research- ers since the complete sequence of bovine HANNES W. JÖRG1, STEPHAN J. RIEDER1, mtDNA was reported. However, the complete ELLIE FELLAY2, JEAN-JACQUES LAU- sequence of mtDNA in Japanese Sika deer has VERGNE3, CLAUDE GAILLARD4 not previously been reported, although several 1Swiss Federal Institute of Technology, Insti- studies have reported population genetics. The tute of Animal Sciences, Zürich, Switzerland; comparison of genetic variation among various 2Fédération d’èlevage de la race d’Hérens, populations of Japanese Sika deer is vital for Sion, Switzerland; 3INRA CRJ, Laboratoire de the development of useful management and Génétique Factorielle, Jouy-en-Josas, France; conservation strategies. In the present study, 4University of Bern, Faculty of Veterinary

91 Section D: Marker, Polymorphism and Biodiversity

Medicine, Bern, Switzerland microsatellites loci, were analyzed by estimat- The Hérens cattle breed has three different coat ing the useful parameters to evaluate the ge- colour phenotypes: red, black with a red dorsal netic profile of the populations. The obtained strip, and black. In mammals, dominant alleles frequencies were used to estimate the expected of the Extension (E) locus are associated with heterozygote percentage following Hardy- black pigment, while the recessive alleles lead Weinberg equilibrium. Frequencies of hetero- to production of the red pigment. However, in zygote animals, actually observed, were esti- the Hérens breed, the red coat colour is not mated by using the software ARLEQUIN 2.0. completely recessively inherited. Molecular Genetic distances were estimated following the studies have revealed that the E locus encodes model proposed by Bowcock about the pro- a melanocyte stimulating hormone receptor portion of shared alleles. The obtained dis- (MC1R). In this study, 11 animals of various tances matrix was used to build a Neighbour- coat colours were selected. Analysis of the Joining diagram using the software PHYLIP. MC1R gene showed three polymorphic sites. From the obtained results there is no evidence A deletion of a G nucleotide at position 311 of significant difference among the two groups resulting in a frameshift was identified in two therefore Barrà animals are not a separated animals. This frameshift is associated with the group from the Pustertaler animals. red coat colour in Holstein cattle. However, the low frequency of the allele in our sample con- D039 firms that red is not only due to this recessive Reliability of DNA certificates in parentage allele in the Hérens breed. A nucleotide sub- verification for cattle stitution of a T to an A at position 296 was identified only and in each black animal. This MARIE-LOUISE GLOWATZKI-MULLIS, allele encodes a constitutively active receptor JEANNETTE MUNTWYLER and results in the black phenotype. A duplica- University of Berne, Institute of Animal Ge- tion of 12 nucleotides starting at position 650 netics, Nutrition and Housing, Berne, Switzer- was found in two of the four black animals land with a red dorsal stripe and is not informative Since April 1999, STR profiling is routinely for the described phenotypes. Our results show used for parentage verification and identity that the black phenotype is due to an allele of testing in our lab. In some cases the genotype the MC1R gene. The red and the black with a of a progeny has to be verified with data from red dorsal stripe phenotypes are controlled by DNA certificates. 9 STR markers were se- another gene, which likely interacts with the lected as international ISAG standard: function of MC1R. BM1824, BM2113, ETH10, ETH225, INRA023, SPS115, TGLA122, TGLA126, D038 TGLA227. For DNA certificates, the results Genetic characterization of two alpine au- are adjusted to a reference sample. Projects tochthon cattle breeds exist to establish DNA databases which would serve as central platforms for storage and com- MICHELE BLASI1, ADELE LANZA1, parison of data. Thus, the reliability of DNA ELENA GENZINI1, ANDREA ROSATI1, typing results is of crucial interest. However DANIELA IAMARTINO2, FABIO PILLA2 interlab concordance is not always attained. 1) 1Laboratorio Gruppi Sanguigni,Potenza- Reports of ISAG Comparison Tests. For none Cremona, Italy; 2Università degli Studi del of the 9 ISAG markers the participating labs Molise, Campobasso, Italy reported absolutely identical results in The autochthon cattle breed named Barrà is 1999/2000. Best marker was BM1824: 2 labs reared in Piedmont, a northern Italian region. out of 33 (40 samples tested) differed in more This is a breed producing both milk and meat. than one allele, 2 labs failed in typing at least The goal of this research is to verify the ge- 39 samples, 5 labs made false adjustments to netic origin of Barrà cattle and particularly to the reference. 2) Casework experience. 90 control the possibility to distinguish this breed DNA certificates were requested: in 21 of them from the Pustertaler Sprinzer cattle breed, from the results were either not adjusted to the refer- which, most probably, the Barrà originated. 64 ence or allele designation was erroneous. 3) samples of Barrà and 29 samples of Pustertaler Allelic ladders are not usual in cattle casework. Sprinzer were used. Genotypes, obtained by 16 This requires caution when comparing results

92 Section D: Marker, Polymorphism and Biodiversity obtained from different types of DNA Se- JAIME GONGORA, CHRIS MORAN quencers. ETH225: difference between allele 1 Centre for Advanced Technologies in Animal and 2 (same PCR product) is 12bp (sample 1 Genetics and Reproduction, University of Syd- ISAG CT 2001/2002) when the fragments are ney, NSW 2006, Australia run on the 373A slab gel system and 16bp There are many examples of microsatellite when run on the capillary 3100 system from primers designed from one species being used ABI. to amplify products from another. Interspecific amplification has been demonstrated among D040 cattle, sheep, goats and deer, among chickens, Characterization of the prion protein gene quail and turkeys and among domestic pigs region in Swiss sheep breeds based on ge- and wild Suiformes. Here 61 porcine microsat- netic polymorphisms ellite primer pairs were tested for amplification of microsatellite products from seven Colom- ANDREAS GMÜR, CLAUDE GAILLARD, bian Collared peccaries (Tayassu tajacu), with GAUDENZ DOLF 47 (74%) yielding products. Fluorescent University of Berne, Institute of Animal Ge- genotyping of the PCR products revealed that netics, Nutrition and Housing, Berne, CH 10 (16%) were polymorphic. All observed Polymorphisms at PRNP in sheep seem to be peccary alleles fall within the range of allele associated with the incubation time of both sizes found in Australian commercial pigs. The scrapie and BSE. In the light that sheep can be high success rate with porcine primers on pec- experimentally infected with BSE, the question caries suggests low nucleotide divergence whether Swiss sheep potentially are suscepti- between Suidae and Tayassuidae. The amplifi- ble to the disease becomes extremly important cation efficiency agrees reasonably well with in the context with public health. The aim of our previous study (16/18; 89%), although the this study was to characterize 50 animals of polymorphism level here is lower than in the each of the four major Swiss sheep breeds, previous study (11/16; 69%) By contrast, the namely Swiss Oxford Down (BFS), Swiss only other reported study on peccaries (Low- Black-Brown Mountain (SBS), Valais Blac- den, pers. comm) found an amplification suc- knose (SN) and Swiss White Alpine (WAS) cess rate of only 29% (9/31) using porcine with respect to polymorphisms (codons 112, primers. Perhaps surprisingly, many porcine 136, 137, 138, 141, 151, 154, 171 and 211) in microsatellites primers promise to be useful the exon 3 of the PRNP. First we amplified tools for population and phylogenetic studies part of exon 3 using Goldmann's primers (up- of peccaries. per 5’-CCGCTATCCACCTCAGGGA-3’ and lower5’-TCTCATAGTAGGATAGGGGCAA- D042 3’) and then we sequenced the PCR products Polymorphism of pig breeds for the pres- from both ends using the primers (5’- ence of an active full-length endogenous AAGGTGGTAGCCACAGTC-3’ and 5’- retrovirus (PERV) at position 1q24. CAGGAGGGGAAGAAAAGAGGAT-3’) which allowed us to double check the se- M. GORBOVITSKAIA1, N. BOURGEAUX1, quences. At codon 143 a novel polymorphism Z. LIAN2, N. LI2, P. CHARDON1, C. ROGEL- was found. Based on the genotypes allele fre- GAILLARD1 quencies were estimated at the polymorphic 1Laboratoire de Radiobiologie et d’Etude du codons. Substantial polymorphism was found Génome, UMR INRA-CEA 13.314, Jouy-en- only at codon 171 in all breeds and to a lesser Josas, France; 2Laboratory of extent at codon 112 and 154 in SBS, and at Agrobiotechnology, China Agricultural codon 136 in WAS. At other codons or in other University, Beijing, China breeds polymorphism is rather low or was not Pig (Sus scrofa) is a potential organ donor for observed. If codon 171 indeed plays a major man. However, the porcine endogenous retro- role in susceptibility to BSE, we expect all four viruses (PERVs) represent a major concern for breeds to be potentially susceptible for BSE. the patients and it has not yet been clearly es- tablished whether these sequences are safe or D041 not. A major goal would be to select poten- Further studies on amplification of peccary tially active PERV-free pigs. We have previ- microsatellites using porcine primers ously identified 24 integration loci for type C

93 Section D: Marker, Polymorphism and Biodiversity

PERVs using porcine BAC clones. It was fur- man diversity values for populations were ther shown that at least three loci contained positively correlated with divergence compo- PERVs, that were replication competent upon nents and negatively with within population transfection of the corresponding BAC clones components of Petit et al. (1998). The com- into susceptible human cells (J Virol, 76:2714- parison of Weitzman marginal loss of diversity 2720). We have thus started to study the poly- and the contribution to total diversity defined morphism of distinct pig breeds for the pres- by Petit et al. (1998) were only weakly posi- ence of a PERV at one of these loci, at position tively correlated. The results suggest that set- 1q24. The PERV genomic flanks were sub- ting conservation priorities for the studied cloned from the BAC clone using a viral probe sheep breeds only with the Weitzman ap- specific for the Long Terminal Repeats proach can be misleading, if intrabreed genetic (LTRs). Two DNA fragments of 2021 and variation is ignored. 2445 base pairs were recovered for the LTR 5 and 3 prime junctions, respectively. The se- D044 quencing results indicated that the PERV inter- Eurofins-TAGTM for meat DNA traceability rupted a tandem of 8 repetitions of a 196 bp and a cattle rustling case element within the second repeat at position 181, leading to the duplication of a small motif HÉLÈNE PFITZINGER, NATHALIE AR- AGAC. PCR primers were designed to amplify CIDIACONO, STÉPHANIE GUILLET both PERV junctions. Fourteen pig breeds Eurofins Scientific, 44323 Nantes, France were tested, including 11 national Chinese Since the BSE crisis, professionals have to breeds. Amplification products were obtained restore consumer confidence in beef products for some but not all the breeds, clearly sug- and to guarantee maximum food safety and gesting a polymorphism for the presence of the transparency. In this context, the DNA tech- active PERV at position 1q24 and the possibil- nology using genetic markers such as micro- ity of genetic selection. satellites provides a very efficient mean for the identification of the animal of origin. D043 Based on this molecular technology but also on Weitzman approach and components of an important logistic and dedicated sampling diversity in Northern European sheep tools, the Eurofins-TAGTM system assures the breeds traceability of cattle from farm to fork using a combination of microsatellite markers recom- ILMA GRIGALIUNAITE1, MIIKA TAPIO2, mended by ISAG. This system is based on the LARS-ERIK HOLM3, SVEN JEPPSSON4, comparison of DNA profiles of reference and JUHA KANTANEN2, ILONA check samples (muscle, hair, cartilage…) that MICEIKIENE1, INGRID OLSAKER5, can be taken at any stage during animal’s life HALDJA VIINALASS6, EMMA or along the production chain. EYTHORSDOTTIR7 A large scale pilote project involving 14000 1Lithuanian Veterinary Academy, Kaunas, cows was conducted with the first beef pro- Lithuania; 2MTT Agrifood Research Finland, ducing french region “Pays de Loire”. System- Jokioinen, Finland; 3Danish Institute of Agri- atic sampling and DNA fingerprinting were cultural Science, Tjele, Denmark; 4Swedish realized throughout the beef production chain. Board of Agriculture, Jönköping, Sweden; The DNA profiling results allowed to show a 5The Norwegian School of Veterinary Science, good correlation with the paper-based trace- Oslo, Norway; 6Institute of Animal Science of ability. Estonian Agricultural University, Tartu, In addition to the validation of beef traceability Estonia; 7Agricultural Research Institute, schemes, Eurofins-TAGTM also permits to re- Reykjavik, Iceland alize parentage testing or to certify a breed Weitzman marginal loss of diversity and the origin, and has been used to elucidate a cattle contribution to total diversity described by rustling case. Petit et al. (1998) were applied to a set of 32 sheep breeds from Northern Europe that were D045 genotyped for 22 microsatellite markers. Con- Evaluation of 26 microsatellites for pater- servation values given by the methods for in- nity testing in dog dividual populations were compared. Weitz-

94 Section D: Marker, Polymorphism and Biodiversity

GAUDENZ DOLF1, KARINA GUZIEWICZ1, (Reprogen), The University of Sydney, NSW, BRIGITTA COLOMB1, CLAUDE Australia SCHELLING2 Proline-rich proteins (PRPs) account for 70% 1University of Berne, Institute of Animal Ge- of the protein in human saliva and have been netics, Nutrition and Housing, Berne, Switzer- attributed with a variety of functions including land; 2Federal Institute of Technology and the ability to bind fimbriae of various patho- Faculty of Veterinary Medicine, Department of genic organisms (e.g. Porphyromonas gingi- Animal Sciences, Zürich, Switzerland valis, Candida albicans), to precipitate tannins The microsatellites ZuBeCa1 to ZuBeCa23, and to act as a masticatory lubricant. We have ZuBeCa25, ZuBeCa26 and CanBern6 have previously reported the identification of a pro- been typed in 37 Cairn Terriers (FCI 3), 50 tein homologous with the human salivary PRPs Dachshunds (FCI 4), 31 Flat-Coated Retrievers (HsPRPs), isolated from porcine milk. The (FCI 8), 30 Papillons (FCI 9), 69 Siberian porcine milk PRP (PmPRP) gene is at least 5kb Huskys (FCI 5), 52 Vizslas (FCI 7) and 42 and, like the human genes, consists of four Whippets (FCI 10). Average PIC values across exons with the third exon comprised almost the 7 breeds (designated PIC-B) ranged from entirely of tandemly repetitive sequence. The 0.000 (ZuBeCa8) to 0.845 (ZuBeCa2). Micro- tandem repeat unit in the PmPRP gene se- satellites with a PIC-B < 0.4 tend to be mono- quence is 33 nucleotides in length and is re- morphic in single breeds. As an exception Zu- peated between 41 and 45 times, as determined BeCa21 with a PIC-B of 0.512 was monomor- by PCR and nucleotide sequencing. HsPRPs phic in Flat-Coated Retrievers. Microsatellites are the products of a six-gene cluster that spans with PIC-Bs > 0.7 (ZuBeCa2, ZuBeCa4, Zu- approximately 700kb of HSA12p13.2, a chro- BeCa6, ZuBeCa12 and ZuBeCa16) were diffi- mosomal region that has been shown to have cult to type due to the high numbers of alleles conserved synteny with the proximal part of (26 to 40). The average PIC value over all the SSC5 q arm. Physical mapping of the microsatellites, without ZuBeCa8, (designated PmPRP gene with a pig/rodent somatic cell PIC-M) was lowest in Flat-Coated Retrievers hybrid panel is currently underway. with 0.400 where 4 microsatellites (ZuBeCa5, ZuBeCa10, ZuBeCa21 and ZuBeCa22) were D047 monomorphic, and highest in Siberian Huskys Genetic analysis of Korean native horses with 0.591 where only ZuBeCa3 was mono- using microsatellite markers morphic. PIC-Ms may rather reflect population size and breeding effects than sample size. S. K. HAN1, H. D. BYUN1, E. R. CHUNG2 Based on these findings CanBern6 (CFA33, 11 1Laboratory of Molecular Genetics, Depart- alleles, PIC-B 0.596), ZuBeCa1 (CFA10, 9 ment of Dairy Science, College of Animal and alleles, PIC-B 0.586), ZuBeCa14 (CFA24, 5 Life Science, Konkuk University, Seoul, Korea; alleles, PIC-B 0.491), ZuBeCa18 (CFA9, 8 2Laboratory of Animal Molecular Genetics, alleles, PIC-B 0.417), ZuBeCa25 (CFA17, 9 Division of Applied Animal Science, College of alleles, PIC-B 0.610) and ZuBeCa26 (CFA27, Life Science and Natural Resources, Sangji 7 alleles, PIC-B 0.642) are proposed to be University, Seoul, Korea evaluated for inclusion in an international Korean native horse (KNH) is the oldest breed panel for paternity testing. of domestic animals at Jeju island in Korea. This breed classified two population as pure D046 breed (KNH I) and crossbreed (KNH II). KNH Physical mapping, sequencing and detection I has been conserved as natural monument. of VNTR of the porcine milk proline-rich The genetic variation of KNH was measured protein gene using data from 9 microsatellite loci (AHT5, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, ANDREW J. HALL1, HELEN ARTHUR1, T. HTG10 and VHL20) and compared with those KEVIN BELL1, RYAN MIDDLETON2, of Przewalski’s horse (EPR) and the Tho- CHRIS MORAN2 roughbred horse (TH). Allele frequencies and 1Australian Equine Genetics Research Centre, heterozygosities were calculated using CER- The University of Queensland, St. Lucia, QLD, VUS Ver. 2.0 (Marshall, 1998). The number Australia; 2Centre for Advanced Technologies of alleles of those varied from 5 to 11 with an in Animal Genetics and Reproduction average number of 7.22. When the results were

95 Section D: Marker, Polymorphism and Biodiversity compared with those of TH and ERP by 6 loci, individuals from 13 yak populations had a HMS3, HMS6, HMS7, HTG6, HTG10 and cattle mtDNA with the frequencies of cattle VHL20 (Breen et al., 1994), the number of introgression ranging from 1.5% (Tianzhu alleles of KNH I (8.00) and KNH II (7.666) White) to 10.6% (Tianzhu Black). More par- were more than those of TH (5.66) and EPR ticularly, cattle mtDNA were present in seven (4.33). The average heterozygosities are 0.697 yak populations from China (Tianzhu White and 0.773 in KNH I and II, respectively. And (1/68), Tianzhu Black (5/47), Sunan (2/35), the average heterozygosity of KNH I (0.699) at Luqu (1/32), Datong (2/84), Jiulong (1/24) and the 6 loci was higher than that of EPR (0.681) Maiwa (2/38)), three yak populations from but lower than those of KNH II (0.786) and TH Mongolia (Gobi Altai (1/40), Arkhangai (2/60) (0.715). The most frequent alleles of KNH I and South Gobi (1/30)), two yak populations were different from those of KNH II at 5 loci, from Bhutan (west (1/35) and central (1/36)) AHT5, HMS6, HTG6, HTG7 and VHL20. In and one yak population from Nepal (1/25). No HMS6 and HTG6 loci, the most frequent alle- cattle mtDNA introgression were detected in les of KNH II were the same as those of TH. Maqu (0/65), Xiahe (0/17), Jianzha (0/33), Jiali 12 alleles were presented at 4 loci (HMS3, (0/50), Pali (0/48) and Sibu (0/50) yak popula- HMS6, HTG10 and VHL20), which are unique tions from China; in Hovsgol (0/20) and Ubs- to KNH I and II, and especially, the new allele Khovd (0/19) yak populations from Mongolia; of 108bp was identified in VHL20 locus of in the east Bhutanese yak population (0/40); in KNH I. The genetic distance was estimated the Indian (0/23) and the Pakistani (0/44) yak among the 4 breeds, with the result that KNH I populations. In general, the introgression of was closer to EPR than KNH II, and KNH II cattle mtDNA in yak is rare and it is mostly was closer to TH. This result was similar to limited to marginal and agro-pastoral areas that of Han et al. (1998) using data from pro- where yak and cattle coexists. tein markers and we found the gene introgres- sion of TH to KNH II by crossbreeding. These D049 results suggest that KNH represent unique Evaluation of Linkage Disequilibrium in native breed as a horse genetic resource and commercial PIGS’s populations preservation efforts should be continued, al- though the genetic variation of KNH populati- NATACHA HARMEGNIES, FRÉDÉRIC on has been deduced. FARNIR, NADINE BUYS, WOUTER COP- PIETERS, MYRIAM MNI, PATRICIA D048 SIMON, MICHEL GEORGES Cattle mitochondrial DNA introgression in University of Liège, Department of Genetics, yak (Poephagus or Bos grunniens) Liège, Belgium Extensive linkage disequilibrium (LD) has QI XUEBIN1,2, HAN JIANLIN2,4, EDWARD been demonstrated in cattle, offering possibili- O. REGE3, OLIVIER HANOTTE2 ties to exploit this information for mapping and 1State Key Laboratory of Arid Agroecology, fine-mapping purposes using the presently Lanzhou University, Lanzhou, China; available medium density maps. To verify 2International Livestock Research Institute, whether the same possibilities might exist in Nairobi, Kenya; 3International Livestock Re- pigs, we have measured pair-wise LD between search Institute, Addis Ababa, Ethiopia; 13 microsatellite markers (S0369, SW1989, 4International Yak Information Centre, Gansu SW1401, S0284, S1263, SWR1002, SW1683, Agricultural University, Lanzhou, China SW1309, S1004, S1006, SW2608, SW1510 and To assess the level of cattle mtDNA introgres- SW1262) mapping on SSC15. The distance sion in yak, a 357 bp cattle specific D-loop and between adjacent markers ranged from 0.9 to a 590 bp cattle-yak conserved 16S rDNA 9.6 cM. Fourthly and 33 unrelated individuals fragments were PCR amplified in a single were respectively sampled in a Large White multiplex reaction. The amplification of the derived line (Line A) and in a synthetic line 16S rDNA was used as an internal control (T. (Line B). Multiplex microsatellite genotyping Ward et al. (1999) Animal Conservation 2, 51- was performed using automatic capillary se- 57). PCR products were separated on a 2% quencers. Haplotype frequencies maximising agarose gel. A total of 963 samples from 24 the likelihood (L) of the genotype data were yak populations were analysed. Twenty-one estimated using an EM algorithm. LD was

96 Section D: Marker, Polymorphism and Biodiversity measured using (i) the statistical significance we found, indicate mapping of equine APAF1 of the L ratio (Ldata|LD/Ldata|linkage equilib- proximal to IGF1 on ECA28q. This result per- rium (LE)) under the null hypothesis of LE as fectly fits to the gene order known from the determined by genotype permutation, (ii) the human gene map and is helpful for the orienta- normalised D’ measure of LD computed from tion of the group of microsatellite markers and the maximum L haplotype frequencies, and genes on ECA28q. (iii) the square of the correlation coefficient between loci (r2) measured from the same D051 haplotype frequencies. Preliminary results Temporal changes of gene frequencies in suggest that sufficient LD will be present in three breeds of horses these pig populations to devise mapping ap- proaches (association studies on DNA pools or UWE HERTNER1, IVICA MEDUGORAC2, individual DNAs as well as TDT approaches MARTIN FÖRSTER1,2 using individual DNAs) based on LD only. 1Tierzuchtforschung e.V. München, Grub, Germany; 2Institut für Tierzucht der Ludwig- D050 Maximilians-Universität, München, Germany Two polymorphisms detected in equine In order to develop a suitable , APAF1 and possible linkage to ECA28q original breeds of German horses have been modified by incrossing of breeds like Arabian, JULIA HENNER1,2, CAROLA MAU1,2, Thoroughbred and East Prussian Trakehner GERALD STRANZINGER1,2, STEFAN RIE- since the middle of the 20th century. In con- DER1 trast, Thoroughbreds have had a closed stud- 1Institute of Animal Science, Breeding Biology, book for about two centuries. In this study we Swiss Federal Institute of Technology, Zürich, compare two age groups of Hanoverian, Hol- Switzerland; 2Faculty of Veterinary Medicine, steinian and Thoroughbred horses. Animals are University of Zürich, Switzerland grouped in these classes by their year of birth: Apoptotic protease activating factor 1 plays an Hanoverian I, 1949 – 1970 (N = 134); important role in the apoptosis pathway and is Hanoverian II, 1993 – 1999 (N=1851); Hol- of particular interest in human melanoma re- steinian I, 1955 – 1971, (N = 123); Holsteinian search. APAF1 maps to HSA12q23. This re- II, 1993 – 1998 (N = 464); Thoroughbred I, gion also contains IGF1, TMPO and KITLG. 1948 – 1966 (N = 111); Thoroughbred II, 1999 The equine homologs of those genes belong to (N = 1221). Changes in gene frequencies are ECA28q. Two fragments of equine APAF1 evaluated using 5 polymorphic protein loci were amplified from horse genomic DNA. (A1B, ALB, ES, GC, TF). Laboratory results Sequence analysis and polymorphism screen- for age group I have been obtained by retyping ing revealed two single nucleotide polymor- of stored plasma samples sent to our laboratory phisms (APAF1 SNP I3 and APAF1 SNP I6). before the year 1978. Results for age group II In order to test whether equine APAF1 would are from routine paternity testing. As expected, map to ECA28q, a linkage analysis was carried lowest differences are observed between the out using known microsatellite markers from two classes of Thoroughbreds, whereas the ECA28q (IGF1, HTG30, UM003, TKY333) differences of allele frequencies between the and the two detected APAF1 SNP’s. A family two classes of Holsteinians show the highest of 66 Franches-Montagnes horses, a native significancy. breed from Switzerland, was genotyped. Link- age between microsatellites on ECA28q was D052 confirmed and a tendency for linkage between An MspI polymorphism in the GHRH- APAF1 SNP’s and marker IGF1 was found Receptor gene and its association with (APAF1 SNP I3: Z=1.50 θ=0.0; APAF1 SNP growth traits in Angus beef cattle I6: Z=0.72 θ=0.14). Microsatellite IGF1 was only poorly informative (PIC=0.4) in our fam- QUN ZHAO, M. E. DAVIS, H. C. HINES ily. We could not show linkage between IGF1 Department of Animal Sciences, The Ohio and HTG30, even linkage between these two State University, Columbus, OH 43210, USA markers is known. However, the available Growth hormone releasing hormone (GHRH) comparative mapping data and the tendency plays a major role in stimulation of both syn- for linkage between APAF1 SNP’s and IGF1 thesis and release of GH in the anterior pitui-

97 Section D: Marker, Polymorphism and Biodiversity tary through its receptor. Therefore, the mans, was examined in 860 dogs from 31 GHRH-receptor gene is a candidate gene for breeds and 9 alleles were identified by DNA growth traits in domestic animals. Genetic sequencing. We then investigated the relation- marker information on this gene could be used ship between behavioral profiles and DRD4 to facilitate selection and breeding through exon 3 allele frequency in each dog breed. marker assisted selection. An MspI polymor- Significant correlation was recognized between phism was detected in exon 6 of the GHRH- the allele 498 and score of aggression-related receptor gene. Sequencing result showed a behaviors such as ‘aggression to dogs’, ‘domi- point mutation of A in allele M to G in allele nance over owner’ and ‘territorial defense’. N. The polymorphism was examined in 194 Furthermore, we examined the association of Angus beef cattle, which were divergently DRD4 genotype and behavioral traits in the selected for high or low blood serum IGF-I Labrador Retriever trained as drug dogs. concentration. The genotypic frequencies were Seven behavioral traits were evaluated by .79 for MM, .19 for MN, and .02 for NN. The trainers and exon 3 polymorphism was found associations of the polymorphism with growth to be related to ‘aggression to dogs’ and ‘af- traits and IGF-I concentration were analyzed fection demand’. Another polymorphism was using the GLM procedure in SAS. A linear also found in exon 1 of DRD4 gene and this model was fitted for birth weight, weaning polymorphism was related to ‘excitability’. weight, weight at d 28 and 56 of the 140-d This information might be of use in the effec- postweaning test, off-test weight, weight gain tive training program for working dogs such as during the 20-d period between weaning and police dogs and guide dogs. the beginning of the postweaning test, post- weaning gain, serum IGF-I concentration on d D054 28, 42, and 56, and mean serum IGF-I concen- Diversity of mitochondrial DNA in Asian tration. No significant associations were found native goats in these animals. More genotyping is needed to further study this polymorphism. MISAKO ISHIDA1, HIDEYUKI MANNEN1, KEN NOZAWA2, SOICHI TSUJI1 D053 1Kobe University, Faculty of Agriculture, Allelic variation of the dog dopamine recep- Kobe, Japan; 2Koto University, Primate Re- tor D4 gene polymorphic region and its rela- search Institute, Aichi, Japan tion to behavioral traits Domestic goats (Capra hircus) were the first herbivores to be domesticated. Despite their MIHO INOUE-MURAYAMA1, NAOTO importance, the origins of domestic goats re- MATSUURA1, HIDEYUKI ITO1, YUKO main uncertain and controversial. Recent mito- UEDA1, KEIICHI TONOSAKI2, YUICHI chondrial DNA study revealed three highly MURAYAMA3, MITSUO MORITA4, HITO- divergent mitochondrial lineages in goat SHI KITAGAWA1, TOSHIROH IWASAKI5, (C.hircus A-C). In this study, we determined SHIN’ICHI ITO1 complete sequences of mitochondrial D-loop 1Gifu University, Gifu, Japan; 2Meikai Univer- region in 12 Myanmar, 36 Mongolian and 33 sity, Sakato, Japan; 3National Institute of Ani- Chinese native goats. Phylogenetic analysis mal Health, Tsukuba, Japan; 4Livestock Im- was performed with these sequences and pub- prov. Assoc. of Japan, Maebashi, Japan; lished complete D-loop sequences of 16 do- 5Tokyo University of Agricul-ture and mestic goats and three wild goats (two mark- Technology, Fuchu, Japan hor, and one bezoar). The phylogenetic tree The dog (Canis familiaris) has the longest revealed four distinct major clusters, three history among domestic animals and more than were consistent with C.hircus A-C and one 400 breeds have so far been established all consisted of markhor sequences was located as around the world. Purebred dogs are signifi- outgroup. Subsequently we assessed the fre- cantly different from each other in their be- quency of the lineages by PCR-RFLP and havioral traits, suggesting that some behavioral mismatch PCR methods in 180 Myanmar, 33 traits are under genetic control. The dopamine Chinese, 96 Mongolian, 25 Laotian and 60 receptor D4 (DRD4) gene polymorphic region Northern Vietnam natives. The results were in exon 3, which is possibly related to the per- follows; China (A:0.88, B:0.03, C:0.09), Mon- sonality trait known as novelty seeking in hu- gol (A:0.91, B:0.02, C:0.07), Myanmar

98 Section D: Marker, Polymorphism and Biodiversity

(A:0.55, B:0.45), Laos (A:0.36, B:0.64) and ANDRZEJ JANIK1, MARIAN KAMYCZEK2, Northern Vietnam (A:0.35, B:0.65). The ANNA KWACZYŃSKA2, MARIAN RÓŻY- C.hircus B lineage was detected at high fre- CKI1, MONIKA SIKORA2, TOMASZ ZĄ- quencies in Southeast Asia. The C.hircus C BEK1, BARBARA BOJCZUK3, ANNA lineage was observed only in Chinese and RADKO1 Mongolian native goats at low frequencies. 1National Research Institute of Animal Pro- duction, Kraków/Balice, Poland; 2National D056 Research Institute of Animal Production, Usefulness of a set of six microsatellites for Experimental Station, Pawłowice, Poland; parentage control in horses in Slovakia 3Świętokrzyska Academy, Institute of Biology, Kielce, Poland DANIELA JAKABOVÁ1, JOZEF Synthetic line 990 was formed from 6 breeds: TRANDŽÍK1, JOZEF CHRASTINA1, LUD- Polish Large White, Belgian Landrace, Ger- MILA HUDECOVÁ1, ERIKA ZETO- man Landrace, Welsh Landrace, Duroc and CHOVÁ1, JOZEF BULLA2, PETER KO- Hampshire. Total of 548 pigs line 990 was ZLÍK1, FRANTIŠEK JAKAB3 genotyped for 11 blood group systems (A, B, 1State Breeding Institute, Nitra, Slovak Repub- D, E, F, G, H, K, L, M, N), 2 lipoprotein sys- lic; 2Research Institute of Animal Production, tems (LPB, LPR) and 2 erythrocyte enzyme Nitra, Slovak Republic; 3State Breeding systems (GPI, PGD) and plasma protein (TF). Inspection, Nitra, Slovak Republic It was proved that blood groups frequencies in Parentage control in horses has been performed synthetic line 990 was different from results in using microsatellites tests (DNA typing). The the earlier study in the increased frequency DNA typing panel consisted of 6 dinucleotide particulary in alleles: Eedgh (0,344→0,393), Fbd repeat microsatellites (ASB2, HMS3, HMS6, (0,836→0,914), Gb (0,605→0,681), Ha HMS7, HTG4, VHL20). In this study, the (0,526→0,650), Kbf (0,403→0,450) and Na polymorphism of six microsatellites was in- (0,389→0.566). The frequency of the Lpb5 vestigated in population of Thoroughbred (0,945) was found to be the highest in com- (n=303 ), Slovak (n= 16) and parison to the all the remaining Lpb alleles. In Trotter (n= 26). We applied a combination of GPI system the higher frequency was for GPIB microsatellite PCR and semiautomatic fluores- (0,761) than for GPIA allele (0,239). The fre- cence - based detection. Amplified PCR prod- quency of PGDA allele (0,641) was higher than ucts were separated and visualised by an for PGDB allele (0,359). Three alleles contro- Automated Laser Fluorescent DNA sequencer ling the heterogeneity of transferrin (TfA, TfB, (A.L.F. DNA sequencer, Pharmacia). Allele TfC) were found in the pigs of line 990 with frequencies, observed and expected heterozy- respective frequencies (0,193, 0,713, 0,094). gosity and probability of exclusion are pre- sented for each STR locus and for all com- D058 bined loci in these three populations. The re- Detection of polymorphism in the pig heat sults showed high polymorphic information shock protein 70 gene by PCR-SSCP content PIC (0,63 - 0,86). The combined ex- clusion probabilities (CPE) estimated using six HYUN J. JIN1, IN C. KIM1, JUN C. PARK1, microsatellites were in Thoroughbred DONG S. SON1, CHOONG H. YOO1, HO Y. CPE=0,988, Slovak Warmblood CPE= 0,996 CHUNG2, IL C. CHEONG2, SHU T. FENG3, and Trotter CPE= 0,995. These results demon- ZAN D. LI4, CHOUNG I. KIM5 strate that the present DNA typing is useful 1National Livestock Research Institute, RDA and sufficient for individual identification and Cheonan 330-800, Korea; 2National Livestock parentage verification of Thoroughbred, Slo- Research Institute, RDA Suwon 441-350, Ko- vak Warmblood and Trotter. rea; 3Institute of Animal Science, CAAS Beijing 100094, China; 4College of Biological Science, D057 China Agricultural University, Beijing 100094, Genetic structure of synthetic line 990 pigs China; 5College of Animal Resource Science, defined on the basis of polymorphism of the Kangwon National University, Chunchon 200- blood groups, lipoprotein allotypes and 701, Korea erythrocyte enzymes Polymorphism of expressed pig major histo- compatibility complex (MHC) class Heat

99 Section D: Marker, Polymorphism and Biodiversity shock protein 70 (HSP 70) gene was investi- of 56 pigs. All Hal+ animals had increased gated in seven different breed groups (Duroc, serum CPK concentration. All tested pigs were Yorkshire, Landrace, Korean native pig, homozygous at 6-PGD and only A allele was Minzhu, Xiangzhu, Wuzhishanzhu). The established. Two allelic genes A and B, in primer sequences were selected based on the frequencies 0.29 and 0.71, respectively, were cDNA sequence (GenBank accession No. segregated within PHI locus. All animals had X68213), and segments for the primer were normal C/C genotype at RYR1 locus. The re- conducted in 290~512, 830~1424 and sults obtained show that halothane sensitivity 1363~2041 of HSP 70 gene. The polymor- and PSS are present in Yugoslav wild pig even phism of HSP 70 was found by polymerase in absence of C/T mutation at RYR1 locus. chain reaction–single strand conformation Possible multifactorial influence as the base of polymorphism (PCR-SSCP) analysis in ampli- PSS (MH) phenotype in pigs has not been con- fied fragment of 223bp, 595bp and 679bp. firmed yet. HSP 70 genotypes were detected as AA, AB, Acknowledgement: The invetigation was pre- and BB for HSP 70 (290~512), HSP 70 pared thanks to the support of NORAGRIC, in (830~1424) and HSP 70 (1363~2041). The the scope of Norwegian program for updating frequency of alleles in seven breed groups was academic education and research in agriculture HSP 70 (290~512) A = 0.367, HSP 70 in South Eastern Europe. (830~1424) A = 0.414 and HSP 70 (1363~2041) A = 0.109. The allele frequencies D060 were used to estimate the genetic distances and Genetic analysis of three Lithuanian native to construct both a neighbor joining tree and a horse breeds unweighted pair group method with arithmatic mean (UPGMA) tree by the Phylip programs. R. JURAS1, E. G. COTHRAN2 The two closer breeds were Wuzhishanzhu and 1Lithuanian Institute of Animal Science, Xiangzhu, while the two more different were Baisogala, Lithuania; 2University of Kentucky, Xiangzhu and Korean native pig. HSP 70 Lexington, KY, USA (1363~2041) genotypes were associated with Genetic variation in three Lithuanian native significant effects (P<0.05) on litter size (total horse breeds (Zemaitukai, large-type born) from Artificial Insemination (AI) of fro- Zemaitukai and Lithuanian Heavy Drought) zen boar semen, but failed to provide evidence was investigated using serological and for significant effects of pork quality electrophoretic procedures to detect genetic traits(color, firmness, pH and cooking loss) in variation in seven blood groups (A, C, D, K, P, Duroc, Landrace and Yorkshire breed. Q, U) and 10 protein systems (A1B, ALB, ES, GC, HBA, PGD, GPI, PGM, PI, TF) and DNA D059 typing of 16 microsatellites (ASB17, VHL20, Some genetic markers and Halothane sus- HTG10, HTG4, AHT5, AHT4, HMS3, HMS6, ceptibility in Yugoslav Wild pigs (SUS HMS7, ASB23, LEX3, LEX33, ASB2, HTG6, SCROFA FERUS) KTG7, HMS2). A total of 83 animals were tested. No significant deviations from Hardy- SLOBODAN J. JOVANOVIC, MIROSLAV Weinberg equilibrium were observed. SARAC, MILA SAVIC, RUZICA Rodger’s genetic similarity and Nei’s genetic TRAILOVIC, VLADIMIR DIMITRIJEVIC distance were calculated from the gene Faculty of Veterinary Medicine University of frequencies between Lithuanian native horses Belgrade, Department of Animal Breeding and and other horse breeds. Populational Genetics, Belgrade, Yugoslavia inbreeding level was estimated by Wright’s A total of 56, 3 moths, old wild pigs, were Fis. Restricted maximum likelihood (RML) tested for PSS by 5 minute exposure to 3% analysis was used to construct the dendograms. Halothane inhalant solution. Blood samples Genetic variability of the Zemaitukai breeds with K3EDTA were collected from all tested based upon blood groups and biochemical loci animals and genetic variations at PHI and 6- was higher than average for domestic horse PGD loci were tested by starch gel electropho- breeds. For microsatellite loci, the variability resis, while C-T mutation at nucleotide 1843 of of the large-type Zemaitukai and the Heavy RYR1 gene was tested by PCR-RFLP geno- Draught breeds also were high compared to the typing. Halothane test revealed 6 positives out mean variation for domestic breeds, however,

100 Section D: Marker, Polymorphism and Biodiversity

for the Zemaitukai breed, variation levels were surprisingly, that the extracts from the OA(Po) very near the average for horses. Zemaitukai cultivated synoviocytes reveal MMP-2 and horses showed greatest resemblance to the their secretion pattern contains MMP-2/MMP- American Appaloosa breed and the large-type 9 only. MMP patterns of the OA synovial flu- Zemaitukai to Morgan horses. However, ids contain whole set of the normal ones, but, phylogenic analysis paired Lithuanian horses firstly, the relative contents of MMP-9 are 1.5– with the Skyros pony from Greece. 2.5 times more as compared to N ones, and secondly, the summarysing MMP patterns may D061 increase until 8 - 12 MMP fractions. We con- Equine osteoarthrite (OA) chondrocytes clude that the OA chondrocyte cells in situ contribute to the OA matrix may contribute to the OA MMP pattern metalloproteinase pattern of the synovial through the secretion into the OA synovial fluids more than OA synoviocytes fluids more than the OA synoviocytes.

EVGUENI KARAKINE, IGOR PONO- D062 MAREV, DIRK BARNEWITZ, INGO GUJ0040: a Z chromosome-specific micro- WILKE satellite marker in Japanese quail Research Center for Medical Technique and Biotechnology, Department of Tissue Engi- BONIFACE B. KAYANG1, MIHO INOUE- neering and Department of Veterinary Chirur- MURAYAMA1, TAKUYA HOSHI1, KOJI gie, Bad Langensalza, Germany MATSUO1, HIDEAKI TAKAHASHI2, MI- The predisposing genetic factors have been TSURU MINEZAWA2, MAKOTO MIZU- shown to play an important role in the osteo- TANI3, SHIN'ICHI ITO1 arthritic (OA) diseases (Vikkula et al., 1994), 1Faculty of Agriculture, Gifu University, Gifu, although the etiology of the OA remains ob- Japan; 2National Institute Of Agrobiological scure. For instance, it was demonstrated that Sciences, Tsukuba, Japan; 3Laboratory Animal the matrix metalloproteinases (MMPs), namely Research Station, Nippon Institute for Biologi- MMP-9 of the OA synovial fluids, is a prera- cal Science, Kobuchizawa, Japan diological molecular marker of the OA disease Japanese quail (Coturnix japonica) is one of in horse (Clegg et al., 1997), as in a man the economically important members of the (Koolvijk et al., 1995; Ahrens et al., 1996). But family Phasianidae used widely as a labora- both the causes and the sources of the increas- tory research animal and a pilot animal for ing of the content of MMP-9 are unclear. poultry because of its advantages of small Normal (N) and OA (inflammation, trauma, body size, rapid generation turnover, high egg chips) equine chondrocytes from the different production and inexpensive rearing require- joints were cultivated, their MMP patterns ments. In order to construct a molecular ge- including the secretion ones, were screened by netic linkage map for Japanese quail we iso- the reverse zymography technique and were lated and characterized 100 microsatellite compared with the MMP patterns of the culti- markers. Genotyping revealed that one vated synoviocytes and with those of the same marker, GUJ0040, was heterozygous in males synovial fluids. The extracts from both the N only. In a random sample of 26 males and 58 (Po) cultivated chondrocytes and N (Po) syno- females, heterozygosity was observed in 8 viocytes contain the only MMP-2, their secre- males, while all females were hemizygous. tion patterns reveal usually MMP-2 and MMP- These results suggest that GUJ0040 is on the 9. The MMP patterns of the N synovial fluids Z chromosome. Furthermore, cross-species contain MMP-2, MMP-9, MMP-85 kDa, amplification of this marker was observed in MMP-220 kDa and also three additional minor chicken (Gallus gallus), guinea fowl (Numida MMP fractions, namely MMP-105 kDa, meleagris), ring-necked pheasant (Phasianus MMP-120 kDa and MMP-145 kDa. By the colchicus), and Asian blue quail (Coturnix contrast, the extracts from the OA (Po) culti- chinensis), and the genotyping results in these vated chondrocytes express MMP-2 or MMP- species were consistent with those in Japanese 2/MMP-9 patterns, but their secretion ones quail. This indicates that GUJ0040 would be contain 7– 9 MMP fractions including a set of useful for mapping the Z chromosome in the the synovial MMPs and also MMP-53 kDa, family Phasianidae. Recently, two plumage MMP-68 kDa and MMP-74 kDa. It was also colour loci in Japanese quail, albino (AL) and

101 Section D: Marker, Polymorphism and Biodiversity brown (BR), have been reported to be linked guished by good disease resistance, adaptive to the Z chromosome with a recombination and exterior qualities. By the end the XXth frequency of 38.1 cM. Thus, to determine the century the number of these animals was relative position of GUJ0040 by linkage sharply reduced, and in 1996 Vyatka was given analysis, three-point cross experiments among the status of disappearing breed (WWL- AL, BR and GUJ0040 loci are now in prog- DAD:2). Now areas of Vyatka horses have two ress. nuclei - Udmurt and Kirov populations. Gene frequencies at 9 blood group and protein D063 polymorphism loci (A, D, C, K, Al, Tf, Es, 6- Characterisation of the bovine kappa casein PGD and Ca) are given for basic populations promoter of Vyatka horses. Investigated loci had a suffi- cient polymorphism level. 124 Vyatka horses AILEEN F. KEATING, MICHAEL CAIRNS1, were used in a study designed for blood group TERRY SMITH1, PAUL ROSS2 and protein polymorphism analysis. The poly- 1National Diagnostic Centre, National Univer- morphism analysis indicated that a distribution sity of Ireland, Galway, Ireland; 2Dairy Prod- allele frequency was characteristic for native ucts Re- search Centre, Teagasc, Moorepark, northern wood breeds in the whole. The dis- Fermoy, Cork, Ireland tinctions on the investigated systems in genetic Milk serves as a complete food source for the structures of the Vyatka horse populations neonate. It contains a rich source of vitamins, from different regions were revealed. So minerals, lipids, carbohydrates and amino ac- horses from Kirov area have no alleles TfD ids. Bovine milk proteins are extremely well and TfH in Tf locus and differ sufficiently by characterised, both genetically and chemically. distribution allele frequencies in loci 6-PGD The caseins account for 80% of milk protein, and Alb. There is a tendency to accumulation and expression of the kappa casein gene ac- of gomozygotes on a number of loci in some counts for approximately 10.4% of total milk subpopulations. The highest degree of genetic protein. In this study it is proposed that single heterogeneity was found in a large farm of nucleotide polymorphisms (SNP’s) in the Udmurtiya, where it is kept the basic popula- kappa casein promoter region account for tions of the breed. variations in the expression of kappa casein between different bovine breeds. Ten breeds D065 of lactating cows were chosen, and the kappa Diversity of African domestic sheep (Ovis casein promoter amplified and sequenced. aries) revealed by mitochondrial D-loop Mutations in three transcription factor binding sequence differences sites were identified in 28.5% of animals G. KIERSTEIN(1), J. HIRBO(1), P. WAFULA(1) screened. Both the wild type and mutant forms J.E.O. REGE(2) & O. HANOTTE(1) of the kappa casein promoter have been cloned (1)International Livestock Research Institute into the pGL3-basic reporter vector. Differ- (ILRI), Nairobi, Kenya, and (2)International ences in expression between full-length pro- Livestock Research Institute (ILRI), Addis moter constructs, and also between truncated Ababa, Ethiopia forms of the promoters were investigated in a To better understand the origin and diversity of number of transiently transfected mammary sub-Saharan African domestic sheep, we cha- cell lines, by measuring luciferase levels. racterized the molecular genetic diversity of the mitochondrial D-loop sequence of 37 indi- D064 viduals belonging to twelve different popula- The genetic structure of Vyatka horse tions from seven African countries (Ethiopia, populations Nigeria, Mali, Senegal, South Africa, Botswa- na). These sequences were compared with L. A. KHRABROVA, A. M. ZAITCEV European, New Zealand, Near/Middle East Laboratory of Immunogenetics, The All- domestic sheep sequences and wild sheep se- Russian Institute of , Divovo, quences obtained from the GenBank database. Russia As is the case with the European haplotypes, The Vyatka is ancient native the African D-loop consensus sequence is 1180 known since the XIVth century. The Vyatka nucleotides (nt) long, including four tandemly horse is typical northern wood breed distin-

102 Section D: Marker, Polymorphism and Biodiversity repeated motifs of 75 nt length. All African cestral mitochondrial sequences. The average sequences represent new unique haplotypes. pairwise sequence divergence (uncorrected We observed 15 indels and found 130 variable "p") between river and swamp buffalo was sites, including 122 transitions and 11 trans- calculated to be around 7 %. Similar genetic versions. The pairwise distance between the differences have been reported between Afro- African haplotypes under the HYK85 model, European and Indian cattle. The average diver- ranged from 0.0 to 0.05 with an average of gence within the two buffalo types was each 0.02. Interestingly, within the conserved cen- less than one percent. Assuming an evolutiona- tral domain of the D-loop, we observe an A → ry rate of 7.5 x 10-8 substitutions per site and T transversion separating all New Zealand year and using the Tamura-Nei model with sequences from all other sequences analysed. gamma correction, we calculated that swamp Network analysis of the central domain sug- and river buffaloes diverged at least 913,000 gests two ‘groups’ of haplotypes, previously years ago. This time interval clearly exceeds described as A and B (Wood and Phua (1996) the time since their domestication around Animal Genetics, 27: 25-33), with the majority 7,000 to 9,000 years ago. of African haplotypes closer to subgroup B. However, in Africa we found intermediate D067 haplotypes connecting both groups. Molecular cloning and chromosomal as- signment of three type I markers on porcine D066 chromsome 7 Network analysis demonstrates new insights into water buffalo (Bubalus bubalis) phylog- SONJA KIERSTEIN1, HIAM AL-BAYATI2, eny SONJA KOLLERS3, FELIX A. HABER- 3 2 (1) MANN ,BERTRAM BRENIG GEROLD KIERSTEIN , MARCELO VAL- 1 (2) (2) International Livestock Research Institute LINOTO , ARTUR SILVA , MARIA P. 2 (2) (3) (ILRI), Nairobi, Kenya; Institute of Vetrinary SCHNEIDER , LEOPOLDO IANNUZZI & 3 Medicine, Göttingen, Germany; Institute of BERTRAM BRENIG(4) Animal Breeding, Muenchen-Weihenstephan, (1)International Livestock Research Institute Germany (ILRI), Nairobi, Kenya, (2)Centro de Ciencias In course of a genome analysis project with the Biológicas, Laboratorio de Polimorfismo de aim to map and characterize type I markers on DNA, Universidade Federal do Pará, Belém, porcine chromosome 7 we analysed two ge- Brazil, (3)National Research Council (CNR), nomic PAC clones designated F11 and B5. IABBAM, Laboratory of Animal Cytogenetics Fluorescence in situ Hybridisation (FISH) and and Gene Mapping, Naples, Italy and PCR analysis of a somatic as well as a radiati- (4)Institute of Veterinary Medicine, on hybrid panel assigned both PAC clones to Georg-August University, Göttingen, Germany porcine chromosome 7q13-14 and 7q11-13, The phylogeny of water buffaloes (Bubalus respectively. On PAC F11 we sequenced the bubalis) is still a matter of discussion. In the gene coding for methylmalonyl-CoA mutase past the divergence time of river and swamp (mut), which we consider as a candidate gene buffaloes calculated by different authors varied for neuro-muscular disorders in pig. Se- between 10,000 and 1.7 million years ago, due quencing of PAC B5 revealed several regions to different analysis methods. To obtain more that are homologous to a human BAC se- insight, we analysed the complete mitochon- quence localized on HSA 6q21-21.3. By means drial D-loop region of 80 water buffaloes of of comparative genome analysis we were able four different breeds. We demonstrate here a to map the exon-intron boundaries of the comprehensive network analysis, which is of porcine orthologue of the recently in humans great interest when analysing genetic differen- described GPIM gene. The GPIM gene (GPI ces between individuals of one species, as e. g. and MAM protein) is highly conserved bet- the median-joining algorithm takes into ac- ween man and pig. The gene is coding for a count the different genetic separation within a MAM-, Ig- and fibronectin-domain containing species compared to the situation between protein with the capability to anchor to cell different species. Our results not only confir- membranes by the GPI motif. Similar structu- ming the clear divergence between swamp and ral features are found in different types of cell river buffalo, but also give evidence for an- adhesive molecules (CAM). Additional se-

103 Section D: Marker, Polymorphism and Biodiversity quence analysis led us to postulate a second, (This study was supported by the Foundation still unknown gene on the same PAC clone. for Polish Science, contract 13/2000) Although no corresponding cDNA could be obtained by database search, computational D069 analysis of the porcine and human genomic Comparison of two microsatellite panels for DNA sequences indicate at least six intron- parentage verification in pigs in Czech Re- separated exons. Several regions of high ho- public mology, which do not refer to known repetitive elements, support our hypothesis of the pre- ALEŠ KNOLL, LENKA PUTNOVÁ sence of a transcribed gene. Mendel University of Agriculture and Forestry Brno, Brno, Czech Republic D068 The genotype determination and parentage The first linkage groups in the red fox (Vul- verification in pigs in Czech is performed ac- pes fulvus) and arctic fox (Alopex lagopus) cording to Law No. 154 (2000). This prelimi- genomes nary study compares the utilization of two DNA microsatellite panels for parentage veri- JOLANTA KLUKOWSKA, MACIEJ fication in Czech Republic. The first panel (A) SZYDŁOWSKI, MAREK SWITONSKI is based on our assembled and at present for Department of Animal Genetics and Breeding, genotyping used set of 9 porcine microsatel- August Cieszkowski Agricultural University of lites (MS) and second from set of 10 MS de- Poznan, Poland scribed by Nechtelberger et al. (panel B). We The dog (Canis familiaris), the arctic fox (Alo- tested unrelated animals from different farm pex lagopus) and the red fox (Vulpes fulvus) and breeds Large White (LW, N=30), Lan- belong to the family Canidae but diverged drace (Lc, N=30) and Czech gene reserve from a common ancestor some 10 million Black Pied Prestice (BPP, N=20). Genotyping years ago. It is well known that majority of was done on ABI 310 genetic analyser. We canine primers can be successfully used for described the number of founded alleles (NA) PCR amplification of the microsatellites from and their heterozygosity (H): the red fox and arctic fox DNA. Polymorphism and linkage analysis of nineteen Panel A LW Lc BPP canine-derived microsatellite loci (CPH3, MS NA H NA H NA H S0068 7 0.83 5 0.63 5 0.75 CPH6, CPH8, CPH11, 2004, 2010, 2019, S0107 8 0.90 5 0.70 7 0.85 2140, 2168, 2206, 2281, 2319, 2320, 2541, SW24 5 0.70 5 0.73 7 0.90 2594, ZuBeCa4 i ZuBeCa6) in the silver fox SW840 3 0.13 3 0.10 4 0.60 SW353 5 0.57 5 0.73 4 0.35 and the arctic fox genomes were carried out. SW936 4 0.70 6 0.70 6 0.80 Altogether, 14 silver fox families (87 animals) S0070 6 0.80 8 0.67 8 0.80 and 17 arctic fox families (145 animals) were SW72 6 0.73 3 0.63 5 0.90 TNFB 8 0.90 9 0.97 6 0.90 included into this study. Ten markers appeared S0005 8 0.88 6 0.86 8 0.88 to be linked in the red fox genome, with a lod- S0090 4 0.67 5 0.88 5 0.93 score 4 ≥ Z ≥ 3. Two linkage groups were S0101 6 0.67 4 0.70 4 0.65 S0155 4 0.64 4 0.60 5 0.92 identified: LGV 1: CPH8-2004-2319-2019 and S0355 6 0.77 3 0.58 6 0.79 LGV 2: 2010-2168-2281-2541-ZuBeca4. Eight S0386 3 0.52 5 0.48 5 0.84 markers were linked in the arctic fox genome. SW24 5 0.70 5 0.73 7 0.90 Three linkage groups were identified with a SW240 6 0.57 5 0.77 7 0.80 SW857 7 0.63 9 0.79 7 0.85 lod score ≥ 5.0: LGA2: ZuBeCa6-2594-2140; SW951 4 0.63 3 0.36 2 0.50 LGA 3: ZuBeCa4-2320 and LGA 4: 2010- 2168-2281 and one group with a lod score 4 ≥ The results from testing of larger set of tested Z ≥ 3 : LGA 1: CPH3-CPH6. Using the physi- animals will be available in time of poster cal mapping data we could assigned one link- presentation. age group (LGV2) to the red fox chromosome 14 and two linkage groups to the arctic fox chromosomes: 10 (LGA2) and 4 (LGA3). The identified linked microsatellite loci are also linked in the dog genome.

104 Section D: Marker, Polymorphism and Biodiversity

D071 Blood group and sera protein polymorphisms Genetic diversity of cattle from Mongol, were investigated in 4905 Thoroughbred Korea and Japan using microsatellite analy- horses in Turkey. Blood groups systems (A, C, sis D, P, Q, U) were analyzed by Hemolytic and agglutination tests. Starch gel electrophoresis, MIKI KONO1, HIDEYUKI MANNEN2, KOU alkaline polyacrylamide gel electrophoresis NOMURA3, TAKASHI AMANO3, JUNG S. and polyacrylamide isoelectric focusing elec- YEO4, SOICHI TSUJI2 trophoresis were used to identify genotypic 1Kobe University, Graduate School of Science variants of AIB glycoprotein, Albumin, Car- and Technology, Kobe, Japan; 2Kobe Univer- boxylesterase, Vitamin D binding protein, sity, Faculty of Agriculture, Kobe, Japan; Hemoglobin-α, Transferrin loci. The gene 3Tokyo University of Agriculture, Tokyo, Ja- frequencies of the A, D, Q blood group sys- pan; 4Yeungnam University, College of Natu- tems were calculated by Maximum likelihood ral Resources, Kyeungbuk, Korea method. Direct Counting Method was used for Information on the genetic structure and dis- the calculation of other blood groups and tance among breed is necessary for meaningful plasma proteins systems gene frequencies. Five conservation of genetic diversity. The objec- alleles (D, F, H, O, R) were identified for Tf. tive of this study is to characterize the genetic The highest frequency in this system was variability of native cattle breeds in the North- found in F allele. Two alleles (F, S) were iden- eastern Asia using microsatellite markers. The tified in GC system. Three alleles (F, I, S) have 15 microsatellite markers (INRA063, been detected in Carboxylesterase system. INRA005, ETH225, ILSTS005, HEL5, HEL1, Frequency of I was the highest among all al- INRA035, BM1824, HEL13, INRA037, leles. Two alleles (K, S) were detected in the BM1818, HAUT24, TGLA122, TGLA53, AIB (Xk) system. F allele, which is present in SPS115) were analyzed for three cattle breeds some breeds of the horse, was not found in this in Mongol (n=50), Korea (n=50) and Japan study. The estimated frequency of K in the (n=50). The average numbers of allele were study material of the thoroughbred horses was 8.0 in Mongolian, 6.4 in Korean and 5.5 in the highest. In Hemoglobin-α system, the fre- Japanese native cattle. Mean heterozygosity quency of BII was the highest. However AII and PIC values were calculated at 0.71 and allele was not found. The gene frequencies in 0.67 in Mongolian, 0.63 and 0.59 in Korean, blood groups and protein polymorphism in and 0.68 and 0.63 in Japanese native cattle, thoroughbred horses in Turkey were similar to respectively, indicating the highest genetic those thoroughbred horses in Europe than the diversity in Mongolian cattle. In addition, once reported in other countries. some microsatellite markers revealed Mongol- specific alleles. Neighbor-joining tree using the D073 genetic distance showed close genetic relation- A full genome scan panel of horse (Equus ship between Mongolian and Korean natives, caballus) microsatellite markers applied to and Japanese native as outgroup. These results different equid species are useful information not only for conserva- tion of genetic diversity, but also for studies on KARIN KRÜGER1,2, GERALD STRANZIN- the origin and the history of the Northeastern GER1,2, STEFAN RIEDER1 Asian cattle. 1Institute of Animal Science, Breeding Biology, Swiss Federal Institute of Technology, Zürich, D072 Switzerland; 2Faculty of Veterinary Medicine, Gene frequencies of blood group and blood University of Zürich, Switzerland proteins in the Thoroughbred horses in Conservation and homology among mammal- Turkey ian genomes is widely recognized. Thus, mo- lecular approaches using sequence and marker 1 2 AHMET KOPAR , OKAN ERTUĞRUL information from one species to establish spe- 1Institute of Etlik, Veterinary Control and Re- cific sequence and marker information in a search Center, Laboratory of Blood Groups second or third species, belong to the "state of 2 and Genetics, Etlik, Ankara, Turkey; Ankara the art" techniques in animal genetic research. University, Veterinary Faculty, Department of We applied a panel of microsatellite markers Genetics, Ankara, Turkey covering all 31 horse autosomal chromosomes

105 Section D: Marker, Polymorphism and Biodiversity and the X chromosome to different members tibility and resistance to BSE. In order to carry of the equid family: Equus caballus, Equus out an automated screening for this polymor- asinus, Equus africanus somaliensis, Equus phism a MALDI-assay was established that hemionus, Equus kiang. Horse microsatellites allows genotyping of a larger sample number. were found polymorphic in all mentioned Furthermore, a case control study with BSE equids. PCR conditions for equine markers affected animals is under way to investigate needed adjustment in all species other than the association between the polymorphism and Equus caballus itself. Allele sharing statistics BSE. for population demarcation and a maximum likelihood approach to assess allocation suc- D076 cess of individual animals to a specific popu- Genetic polymorphism of GPI and PGD in lation cluster were proceeded. The results the pigs of Polish Large White, Polish Lan- demonstrate the application of these molecular drace and synthetic line 990 breeded in Po- tools in evolutionary biology and as an instru- land ment for conservation programs of endangered species. ANNA KWACZYŃSKA1, MARIAN RÓŻY- CKI2, MARIAN KAMYCZEK1 D074 1National Research Institute of Animal Pro- A new silent mutation in German Simmen- duction, Experimental Station, Pawłowice, tal and Brown cattle could be used as a Poland; 2National Research Institute of Animal marker for a BSE case control study Production, Kraków/Balice, Poland The studies on the genes located on chromo- OLIVER KUPPINGER, STEFAN KREBS, some 6 in pigs, which determine polymor- RENATE DAMIAN, MARTIN FÖRSTER phism of erythrocyte enzymes glucose phos- University of Munich, Department of Animal phate isomerase (GPI) and 6- Breeding, Munich, Germany phosphogluconate dehydro-genase (PGD) Bovine spongiform encephalopathy (BSE) is a were carried out on the material tested at Pig fatal neurodegenerative disease caused by ac- Performance Stations. A total of 709 Polish cumulation of abnormal prion protein (PrP) in Large White (PLW), 1341 Polish Landrace cattle. Mutations within the PrP coding region (PL) and 535 synthetic line 990 (SL990) pigs from amino acid 100 to 231 in mice, sheep and were used. The analysis of genotypes of the humans are associated to incubation period and GPI and PGD loci was conducted on blood resistance in experimental and natural TSE. samples using the agarose gel electrophoresis However, such an association is not known for according to method Gahne and Juneja. Only the bovine PrP. Our present work aims at two alleles, A and B, were identified within identifying a genetic marker in the bovine gene both systems. There were estimated genotypes in order to conduct a case control study about frequency of GPI and PGD and of GPI-PGD its association to disease. The genomic DNA linked loci. In GPI system frequencies of the encoding PrP 95-231 was analysed by nucleo- AA, AB, BB genotypes were 0,259, 0,504, tide sequencing. Initial screening of 100 Ger- 0,237 respectively in PLW pigs, 0,031, 0,300, man Simmental and 100 Brown Swiss cattle 0,669 in PL pigs and 0,069, 0,400, 0,531 in yielded a silent point mutation for asparagine SL990 pigs. As regards the PGD locus in at position 173. All three possible genotypes: PLW frequencies of the AA, AB and BB AAC/AAC (wildtype), AAC/AAT and genotypes were 0,536, 0,395 and 0,069 in PL AAT/AAT could be detected and verified by 0,310, 0,496 and 0,194 and in SL990 pigs either MALDI or RFLP. Further 294 German 0,387, 0,467 and 0,146. Analysis of GPI-PGD Simmental and 300 Brown Swiss cattle were linkage reveal that the highest frequencies genptyped by HincII-RFLP. Frequency of the were in PLW AB-AA (0,294), in PL BB-AB mutant was 12,93 % for German Simmental (0,327) and in SL990 pigs BB-AA (0,238). In and only 7,17 % for Brown Swiss cattle breed. each breed the lowest frequency was for AA- Despite the high sequence identity to sheep, BB genotype linkage. Taking into considera- mice and human no other mutations could be tion both loci, the PL gene frequencies were detected. However, PrP genotyping of the more similar to those calculated for SL990 identified mutation could give hints to a yet than the PLW pigs. unidentified factor that may influence suscep-

106 Section D: Marker, Polymorphism and Biodiversity

D077 D078 Genetic diversity of European cattle Genetic characterisation of Istrian sheep by microsatellites THE EUROPEAN GENETIC CATTLE DI- VERSITY CONSORTIUM (J. A. LENSTRA1, MARIA LONGERI1, MARIA GIUSEPPINA I. J. NIJ-MAN1, G. MOMMENS2, G. DOLF3, STRILLACCI1, ROBERTA P. WIENER4, D. BURTON4, J. L. WIL- LEONARDUZZI2, FABIO PILLA3, LIAMS4, K. MOAZAMI-GOUDARZI5, D. ROBERTO RASERO4, ANDREA FRAGHI5, LALOË5, S. DUNNER6, J. CANON6, P. MARTA ZANOTTI1 ZARAGOZA7, C. RODELLAR7, I. MARTIN- 1University of Milan, Faculty of Veterinary BURRIEL7, A. SANCHEZ8, G. ERHARDT9, Medicine, Istituto di Zootecnica, Milan, Italy; O. JANN9, C. WEIMANN9, P. AJMONE- 2University of Udine, Dep. Scienze della MARSAN10, R. NEGRINI10, A. VALEN- Produzione Animale, Udine, Italy; 3University TINI11, M. C. SAVARESE11, M. ZANOTTI12, of Molise, Dep. SAVA, Campobasso, Italy; F. PILLA13, A. BRUZZONE13, D. IAMAR- 4University of Turin, Faculty of Veterinary TINO13, I. OLSAKER14, I. MICEIKIENE15) Medicine, Dep. Produzioni Animali, Turin, 1Faculty of Veterinary Medicine, Utrecht Italy; 5Ist. Zootecnico Caseario per la Univerity, The Netherlands; 2PolyGenLab, Sardegna, Sassari, Italy Malle, Belgium; 3Institute of Animal Breeding, Two small Istrian sheep populations, Istrian Nutrition and Housing, University of Berne, Pramenka and Carsolina bred in Istria (Croatia) Switzerland; 4Roslin Institute (Edinburgh), and in Carso (Italy) respectively, have been Roslin, UK; 5INRA, Jouy-en-Josas, France; analysed in order to define their genetic iden- 6Facultad Veterinaria, Madrid, Spain; tity. The two groups, originally coming of the 7Facultad Veterinaria, Zaragoza, Spain; same stock, have been divided following to the 8Facultad Veterinaria, Barcelona, Spain; politic partition of the breeding area after the 9Department of Animal Breeding and Genetics, Second World War. Samples randomly chosen Justus Liebig Universität, Giessen, Germany; out of the two breeds have been compared each 10Institute of Zootechnics, Catholic University other and with three Italian sheep breeds, dif- of S. Cuore, Piacenza, Italy; 11Istituto di ferent both for origin and morphology: Sarda, Zootecnica, Universitá della Tuscia, Viterbo, Comisana and Delle Langhe. Microsatellites Italy; 12Instituto di Zootecnica, University of CSSM31, OARAR119, OARCP34, Milano, Italy; 13Facoltà di Agragria, Campo- OARFCB11, MILVET07-08, ILSTS005-11- basso, Italy; 14Division of Genetics, The Nor- 29, MAF214 have been analysed by automatic wegian School of Veterinary Science, Oslo, fluorescent methods. Allelic frequencies and Norway; 15Gyvunu Genetikos Laboratorija, Hardy-Weinberg equilibrium by GENEPOP Kaunas, Lithuania software and heterozygosity index according to The EU-sponsored project Towards a strategy Nei (1979) have been calculated. Genetic dis- for the conservation of the genetic diversity of tances within populations according to Nei by European Cattle (Resgen CT98-118, 1999- DISPAN software and Principal Component 2001) carried out a molecular assessment of Analysis (PCA) by SAS’ PRINCOMP proce- the genetic diversity of European Cattle and dure have been performed. High homozygosity formulated recommendations for conservation due to high level of inbreeding, particularly in policies. Estimates of relative genetic distances Carsolina has been recorded. Comparing allelic were based on two different types of genetic frequencies, significant differences among the marker, microsatellites and AFLP polymor- breeds and between Carsolina and Istrian have phisms. Microsatellite typing was completed been shown. Genetic distances and PCA con- for 68 breeds with 30 markers and 25 to 50 firmed wide differences among all the popula- animals per breed. AFLP typing has been done tions. Despite morphological homogeneity on 49 continental breeds, 20 animals per breed. between the two Istrians, genetic markers Factors that influence the relative estimates of showed strong differences due to the bottle- distance measures (typing methodology, his- neck and genetic drift occurred after the politi- tory of the species or of the breed, geographi- cal partition. cal origin of the breed) will be discussed, as will how these data are of relevance for guid- ing the conservation of genetic diversity.

107 Section D: Marker, Polymorphism and Biodiversity

D079 tion, USA; 4AgResearch , Dunedin, New Zea- Identification of SNPs in the interleukin-2 land, 5Livestock Systems - Molecular Biology, gene (IL2) of cattle and goat South Australian Research and Development Institute (SARDI), Glenside, Australia; and GESINE LÜHKEN, GEORG ERHARDT 6Department de Génétique Animale, Institut Justus-Liebig-University of Giessen, Depart- National de la Recherche Agronomique ment of Animal Breeding and Genetics, (INRA), Jouy-en-Josas, France. Giessen, Germany The most recent sheep linkage map, published Polymorphisms in cytokine genes and cytokine in 2001, comprises 1,091 markers representing receptor genes have shown to influence genetic 1,062 unique loci. One hundred and twenty- disease resistance and susceptibility. In a pre- one of these loci are genes, and as such repre- vious study, we characterized the gene of the sent potential links between the sheep and cytokine interleukin-2 in sheep and found 12 human maps, while the remaining 941 loci are SNPs comparing sequence data derived from anonymous. One way to increase the useful- seven breeds (GeneBank AF287479). Here we ness of the sheep linkage map, and take describe the sequence analysis of IL2 in one advantage of more of the information genera- sample each of Bos taurus breeds (German ted by the human genome project, is to increa- Angus, German Simmental, Holstein-Friesian, se the number of links between the sheep and Pinzgau, N’Dama, Anatolian Black), of the human maps. This can be achieved by positio- Bos indicus Nelore, of five Capra hircus ning more genes on the sheep linkage map. breeds (Saanen, Toggenburg, Boer, Norwe- We have developed a number of new ruminant gian, Orobica) and of wild goat (Capra aega- gene and expressed sequence tag (EST) asso- grus) and Markhor (Capra falconieri). After ciated microsatellite markers following physi- PCR amplification and cloning of the ap- cal screening of ovine EST and bacterial artifi- proximately 4.8 kb gene, sequencing was done cial chromosome (BAC) libraries, and in silico by primer walking using two vector primers screening of ruminant EST and gene GenBank and eight specific primers. Interestingly, all of entries. These markers have been characteri- the specific primers developed for sequencing sed, and their sheep linkage map locations of ovine IL2 were useful in goat, while in cat- determined by genotyping the International tle, half of the primers had to be replaced by Mapping Flock (IMF, AgResearch, NZ). This primers with cattle-specific sequences. We mapping information, together with mapping found more than six SNPs in the 5’flanking information for new anonymous loci, has been and intronic region of caprine IL2 and more integrated with data for the existing map to than three SNPs in the intronic region of bo- construct a new sheep linkage map. The new vine IL2, and more potential polymorphisms map, containing 240 genes and EST sequences, are in progress to be verified in both species. will be presented at ISAG 2002 and can be found at D080 http://rubens.its.unimelb.edu.au/~jillm/jill.htm. An enhanced sheep linkage map comprising 240 gene and EST associated markers D081

1 North European Cattle Diversity JILLIAN F. MADDOX, IAN FRANKLIN , (N-EURO-CAD) CYNTHIA D.K. BOTTEMA2, UDAYA DES- 1 3 ILVA , DAVID L. ADELSON , CRISTINA JOLANTA MALEVICIUTE1, ILONA MI- 4 5 DIEZ-TASCÓN , GREG NATTRASS , CEIKIENE1, INGRID OLSAKER2, SIRJE 3 2 CLARE GILL , GRAHAM WEBB , KEN VÄRV3, HALDJA VIINALASS3, ERLING 4 6 DODDS & DANIEL VAIMAN FIMLAND4, ZIEDONIS GRISLIS5, JUHA Centre for Animal Biotechnology, University of KANTANEN6 Melbourne, Victoria, Australia; 1Lithuanian Veterinary Academy, Kaunas, 1 Commonwealth Scientific & Industrial Rese- Lithuania; 2The Norwegian School of Veteri- arch Organisation (CSIRO) Livestock Indu- nary Science, Oslo, Norway; 3Institute of Ani- 2 stries, Blacktown, Australia; Department of mal Science of Estonian Agriculture Univer- Animal Science, University of Adelaide, Rose- sity, Tartu, Estonia; 4Nordic Genebank for 3 worthy, Australia; Department of Animal Farm Animals (NGH), Aas, Norway; 5Latvian Science, Texas A& M University, College Sta- University of Agriculture, Jelgava, Latvia;

108 Section D: Marker, Polymorphism and Biodiversity

6Agrifood Research Finland MTT, Jokioinen, reference families, which we are constructing. Finland These expressed functional genes including The aim of the N-EURO-CAD project is to microsatellite would be useful markers for analyse genetic diversity within and between studying comparative mapping when the link- North European and Baltic cattle breeds and to age map of Japanese quail is constructed in the promote conservation of cattle genetic re- near future. sources (http://www.neurocad.lva.lt/). At pres- ent 16 microsatellites have been typed in 31 D083 cattle breeds. Expected heterozygosities, pair- Age of myostatin alleles causing double wise DA genetic distances and GST were cal- muscling in some cattle breeds culated and correspondence analysis was per- formed. The heterozygosities varied from 0.57 CINZIA MARCHITELLI, MARIA to 0.71. About 11% of the total genetic vari- CARMELA SAVARESE, ALESSANDRA ability was due to differences between breeds, CRISÀ, ALESSIO VALENTINI indicating a moderate subdivision. A neigh- Dipartimento di Produzioni animali, bour-joining tree was constructed by using the Università della Tuscia, Viterbo, Italy DA genetic distances. The branching pattern of Myostatin (or GDF8), a negative regulator of the tree and correspondence analysis suggested muscle cell growth, is highly conserved across a grouping of the cattle breeds into four main species. The loss of functional myostatin groups. causes the “double muscled” phenotype in several cattle breeds. Targeted disruption of D082 the myostatin gene in mice and mutations in Development of microsatellite markers from the third exon in Belgian Blue, Piedmontese a heart cDNA library in Japanese quail and cattle breeds, results in skeletal muscle hyperplasia. For nearly 200 HIDEYUKI MANNEN1, KAYO MURATA1, years, double-muscled animals have captured MAKOTO MIZUTANI2, SOICHI TSUJI1 the attention of livestock breeders and re- 1Kobe University, Faculty of Agriculture, searchers The aim of this work has been to Kobe, Japan; 2Nippon Institute of Biological estimate the onset of mutations in myostatin Science, Kobuchizawa, Japan gene using linked genetic markers. The esti- Japanese quail (Coturnix japonica) has been mates, based on intra-allelic variation, follow used not only for meat and egg producer but from the exponential decay of linkage disequi- also as a model animal for poultry because of librium due to recombination and mutation. its small size, short generation intervals and To maximize the information about allele age high egg production. However, linkage map of of myostatin gene, here we choosed five linked Japanese quail has not been developed yet. microsatellites that map on chromosome 2 , at Nevertheless, the expressed sequence markers varying distance from GDF-8 locus. We ana- are becoming important for comparative map- lysed both “double muscled” and normal cattle ping studies. In this study we isolated micro- breeds. satellites from a Japanese quail heart cDNA In Marchigiana breed the mutation in myo- library using a (CA/GT)n repeat as probe. Af- statin gene appeared recently, while in Belgian ter screening from 200,000 cDNA clones, 37 Blue and in Piedmontese thousands of genera- of the clones gave strongly positive signals and tions seem to have elapsed from its appear- subsequently were isolated, purified and se- ance. quenced. From the 37 positive clones 26 clones showed a high percentage of sequence homol- D085 ogy with sequences from other species present PrP genotyping in autochthonous Spanish in the International DNA database. Of the 26 sheep: Genetic characterisation of healthy clones 18 occurred only once. Seventy-three and scrapie flocks percent of the clones were complete (CA/GT) repeats, and 27% were incomplete repeats. The CRISTINA ACÍN1, INMACULADA number of (CA/GT)n repeats in these clones MARTÍN-BURRIEL1,2, CLEMEN RODEL- varied from 11 to 31. The average number of LAR2, JUAN J. BADIOLA1, PILAR repeat units was 17.6. These markers will be ZARAGOZA2 tested for polymorphism to Japanese quail 1University of Zaragoza, National Reference

109 Section D: Marker, Polymorphism and Biodiversity

Centre for TSEs, Faculty of Veterinary, selection programme to recover an old and rare Zaragoza, Spain; 2University of Zaragoza, pig variety located near the extinction. When Bichemical Genetics and Blood Groups Labo- the effective number of reproducers in a ratory, Faculty of Veterinary, Zaragoza, Spain population is small, the allele frequencies will Scrapie is a neurodegenerative disease that be different in males and females, which affects small ruminants like sheep and goat. It causes an excess of heterozygotes in the prog- is a Transmissible Spongiform Encephalopathy eny with respect to Hardy-Weinberg equilib- (TSE). The sheep PrP gene encodes a protein rium expectations. It is important to detect of 256 amino acids. At least 10 different mutu- population bottlenecks in threatened and man- ally exclusive polymorphism are present in PrP aged species because bottlenecks can increase and an association between susceptibil- the risk of population extinction. Early detec- ity/resistant to natural scrapie and the poly- tion is critical and can be facilitated by statisti- morphism at amino acid codons 136, 154 and cally powerful monitoring programs. We 171 has been reported. Valine (V) at codon evaluate the accuracy and precision of the het- 136 and Glutamine (Q) at codon 171 are asso- erozygote-excess method using two data sets ciated with susceptibility and Alanine (A) at from a Chato Murciano pig. One of them is a codon 136, Histidine (H) at codon 154 and original population and the other is a Arginine (R) at codon 171 are associated with F3+F4+F5 generations of a line created from resistance. This association has been proved in the mating of one Chato Murciano stallion certain breeds, we report the study of these with one LargeWhite female, followed of an polymorphisms in autochthonous Spanish absorption programe based in backcrosses. sheep breeds from the region of Aragón (Rasa Analysis of highly polymorphic loci detects the Aragonesa, Ojinegra, Roya Bilbilitana and experimental bottleneck and is a very good Ansotana). Fifty animals, belonging to the reference to estimate the magnitude of the regional genotyping plan, from each breed bottleneck severity in the original population. were genotyped by PCR/RFLPs. Allelic and genotypic frequencies were calculated for each D088 breed. Moreover, a natural scrapie flock (250 The AMPK gene family in cattle: Mapping sheep and 3 goats) of Rasa Aragonesa has been and SNP detection genotyped. Three scrapie postitive animals were diagnosed in this flock showing all of STEPHANIE D. McKAY, JAMES E. WO- them the ARQ/ARQ genotype. Allelic and MACK genotypic frequencies of this scrapie flock Dept of Veterinary Pathobiology, Texas A&M were compared with the results obtained in the University, College Station, Texas Rasa Aragonesa population from the Regional The 5`-AMP-activated protein kinase (AMPK) Genotyping Plan, in order to stablish possible family is an ancient stress response system differences between both groups. whose primary function is regulation of cellu- lar ATP. Activation of AMPK, which is insti- D086 gated by environmental and nutritional The use of microsatellite markers to detect stresses, initiates energy conserving measures bottlenecks in a Chato Murciano Pig popu- that protect the cell by inhibition and phospho- lation rylation of key enzymes in energy consuming biochemical pathways. Initially the seven JOSE L. VEGA-PLA1, AMPARO M. genes that compose the bovine AMPK family MARTÍNEZ2, ANGEL POTO3, BEGOÑA were mapped in cattle using a radiation hybrid PEINADO3, JUAN V. DELGADO2 panel. Seven genes mapped to six different 1Laboratorio de Grupos Sanguíneos, Servicio chromosomes in cattle, each with a LOD score de Cría Caballar, Córdoba, España; 2Unidad greater than 10.0. PRKAA1 mapped to de Veterinaria del Departamento de Genética, BTA20, PRKAA2 and PRKAB2 to BTA3, Universidad de Córdoba, España; 3Centro de PRKAB1 to BTA 17, PRKAG1 to BTA5, Investigaciones Agroalimentarias, Consejería PRKAG2 to BTA 4 and PRKAG3 to BTA 2. de Agricultura, Murcia, España Five of the seven genes mapped to regions Genetic variability at 26 microsatellite loci was expected from human/cattle comparative maps. analysed in two populations of Chato Mur- PRKAB2 and PRKAG3, however, have not ciano pig. These populations were part of a been mapped in humans. We predict these

110 Section D: Marker, Polymorphism and Biodiversity genes to be located on HSA 1 and 2, respec- visible traits, was accompanied by αs1-casein tively. Additionally, one synonymous and one polymorphism reduction. non-synonymous single nucleotide polymor- phism (SNP) have been detected in PRKAG3. D090 Various herds of mixed breed cattle have been Variation of genetic markers in the Göttin- tested for these SNPs by use of high resolution gen Minipig after 40 years of selection on electrophoresis of allele specific restriction low body weight. enzyme digests. Due to the physiological im- portance of this gene family, we believe that its JAN- N. MEYER1 , HORST R. BRANDT2 , individual genes are candidate genes for con- ROSEMARIE CLEMENS1 , CLAUDIA ferring differential disease resistance in cattle. KALTWASSER1 , PETER LUDEWIG1 , PE- TER GLODEK1, HENNER SIMIANER1 D089 1University of Göttingen, Institute of Animal Evolution of the αs1-casein polymorphism Breeding and Genetics, Albrecht-Thaer-Weg 3, in Orobica goats undergoing the standardi- 37075 Göttingen, Germany; 2University of sation process Giessen, Institute of Animal Breeding and Genetics, Ludwigstr. 21b, 35390 Giessen, DANIELA MEGGIOLARO, PAOLA Germany CREPALDI, MARTA MARILLI, LUISA The breeding of the Göttingen Minipig has VERDOGLIA, LUCIA FRANCESCHI, started in 1960 by Fritz Haring and Ruth MARIO CICOGNA Gruhn with the Minnesota miniature pig and Istituto di Zootecnia Generale, Facoltà di the Vietnamese pot-bellied pig. Agraria, Università degli Studi di Milano, Italy The first identification was done 1967 by The Orobica is a goat breed raised on the Schahmirzadi with 41 defined blood group Alps, protected by the regional factors and 6 not determined antisera, which measures for the conservation of autochtho- were tested in more than 500 minipigs. nous breeds. The standard of this breed was In 1969 Peter Glodek established an SPF approved in 1992 and the flock book started in population in the new breeding center on the 1995. This population now consists of about experimental station Relliehausen. The white 4200 subjects, including 2000 animals from and the spotted line was tested in 1971 with 9 190 farms registered in the flock book. In order blood group systems and 35 factors. Additional to evaluate if with the breed standardisation the 7 biochemical systems were included. These polymorphism at the CSN1S1 locus remained preliminary results were compared with analy- unchanged, in the year 1999-2000 milk and sis of blood groups and biochemical systems of blood samples were taken on 193 registered 1980, 1990 and 2002. In the 1980 and 1990 goats from 31 farms. The polymorphism of analysis 6 new biochemical systems were αs1-casein was analysed, at protein level, by added. IEF, for the detection of the alleles A, B and C In addition to the blood group and biochemical and, at genomic level, by three PCR protocols systems, two sets of 15 microsatellites were for the detection of CSN1S1 F, E and 01. To tested for the remaining white line in 2002, to assess the evolution of the polymorphism at get first results of molecular genetic markers in this locus, the results were compared with the Göttingen minipig population. those of our previous study based on the typing of CSN1S1 variants by IEF on 198 Orobica’s D091 milk samples collected in 1993-1994. The Genetic variability of 5 honeybee popula- present investigation shows that, in about six tions from the Basque Country (Western years, a relevant reduction of the studied Pyrenees) inferred by mitochondrial and polymorphism occurred, as the number of ob- microsatellite markers served genotypes decreased from 11 to 6. Con- cerning the gene frequencies, a decrease was IRATI MIGUEL1, LIONEL GARNERY2, observed for all the alleles except for F that, on MIKEL IRIONDO1, ANDONE ESTONBA1 the contrary, increased significantly (P< 1University of the Basque Country, Dpt. of 0.001). The frequency of the null allele 01 did Animal Biology and Genetics, Bilbao, Spain; not show significant variation. It thus appears 2Laboratoire populations, génétique et evoluti- that the breed standardisation, mainly based on on, Centre national de recherche scientifique

111 Section D: Marker, Polymorphism and Biodiversity

(CNRS), Gif-sur-Yvette, France Evidence of a new taurine mitochondrial A total of 639 colonies from 5 populations of lineage the local honeybee (Apis mellifera) from the Basque Country (Western Pyrenees), including M. M. MIRETTI1,2, H. A. PEREIRA JR.1, M. 2 localities where conservatories are to be A. POLI3, E. P. B. CONTEL2, J. A. FERRO2 established have been analysed using mito- 1Depto. de Tecnologia, FCAV, UNESP, Jabo- chondrial (DraI RFLP of COI-COII region) ticabal, SP, Brazil; 2Depto. de Genética, and nuclear (10 microsatellite loci) markers. FMRP, USP, Ribeirão Preto, SP, Brazil; Our aim is to study the genetic variability of 3Instituto de Genética, CNIA-INTA, Castelar, these populations, to detect the introgression Argentina level from north Mediterranean lineage, and to We report the nucleotide diversity within the analyze the phylogenetic relationship of our control region of 42 mtDNA sequences from honeybees with the Iberian (A. m. iberica) and an Argentinean and four Brazilian (AB) native French populations (A. m. mellifera). The in- cattle breeds. The analysis in conjunction with trogression detected is quite low (0-4%). African (A), European (E), Near Eastern (NE), Nuclear and mitochondrial results are well Japanese B.taurus and Indian B.indicus correlated in the most of populations, except in (n=550), allowed the recognition of 8 new one population where the level of introgression haplotypes and their relative positions in a in mtDNA is zero while the nuclear level up to phylogenetic network. The structure of genetic 2’1%. In this population the practice of trans- variation among different hypothetical grou- humance is habitual, so genetic hybridation pings was tested through the molecular varian- could be due to foreign drones’ genetic contri- ce decomposition and pairwise Fst distance, bution. The genetic diversity detected is higher which were best explained by haplotype- than described in French populations, but si- groups components. We then classified milar to Iberian populations. The distribution B.taurus haplotypes into NE, A and E taurus- of mitochondrial haplotypes is similar in the 5 derived haplotypes (haplogroups) based on the populations with high percentage of M haplo- sequence state at five positions (16050-16057- types (80-100%), confirming that this popula- 16113-16189-16255). As AB, as well as Por- tions are at the extreme of the distribution area tuguese cattle, is in fact an admixture of highly of haplotype A. The haplotypes M31 and M32 divergent haplotypes, more confident informa- are described for the first time. Their sequen- tion could be obtained from a gene-tree instead ces indicate that they are closely phylogeneti- of a population-based analysis. Two haploty- cally related to haplotype M7 and their low pes within the A cluster were more divergent frequency suggest that they are rare. The pair- from the A consensus than the latter from the E wise FST values point to a gradient of diffe- consensus. A NJ tree confirms haplogrouping rentiation correlated with geographic distance, and shows the position of two haplotypes rela- indicating that these populations are a product tive to the E/A lineage splitting, which diver- of “natural” evolution with low influence of gence might have occurred subsequent to that modern apicultural management as the impor- between AA and A. This putatively ancestral tation of foreign queens by bee-keepers. Phy- mitochondrial lineage (AA) is supported by the logenetic analyses indicate that Basque Coun- calibration of sequence divergence based on try's populations are intermediate between the Bos-Bison separation. These data could Iberian (A.m.i) and French (A.m.m) popula- reflect the haplotype distribution of the Iberian tions, in agreement with their geographical cattle five centuries ago. location and are in concordance with the evo- lutionary history of the West European honey- D093 bee. These results show that these populations Characterization of a novel SspI-family re- are good candidates to preserve local populati- petitive sequence on the chicken W chromo- on of honeybee due to their low introgression some level and the relatively high genetic diversity detected. YUICHIRO ITOH1, SHIGEKI MIZUNO2 1Gene Research Center, Tohoku University, D092 Sendai, Japan; 2Department of Agricultural African derived mitochondria in South and Biological Chemistry, College of Biore- American native cattle breeds (Bos taurus). source Sciences, Nihon University, Fujisawa

112 Section D: Marker, Polymorphism and Biodiversity

252-8510, Japan The number of animals genotyped per full-sib A clone λWS44 containing a 223-bp insert family varied from 20 to 100. 350 polymorphic from the chicken W chromosome-specific AFLP markers were found, and in 90 % of genomic library was noticed because it showed these, the segregation of AFLP markers could repetitive sequence-like and female-specific be followed from offspring to parents in at signals in Southern blot hybridization, yet was least one of the full-sib families (i.e. one parent different from XhoI- and EcoRI-family se- was heterozygous, and the other homozygous quences. A major repeating unit was a 0.5-kb for the null allele). Linkage analysis was per- sequence produced by digestion with SspI. A formed using CRIMAP, disregarding the cases genomic clone pWPRS09 containing a 508-bp for which both parents of a family were het- SspI unit was subsequently obtained. The 0.5- erozygous for the marker. The proportion of kb units showed sequence diversity, as demon- the markers segregating in a non-Mendelian strated by occasional absence of a single EarI fashion was larger than what was expected by site in the unit. Slot blot hybridization demon- chance, which is consistent with previous strated that the 0.5-kb SspI unit repeated about studies showing that the salmonids have re- 11,300 times on the chicken W chromosome. mains of tetraploid inheritance. The 0.5-kb unit contains a number of tandemly repeated GGAGT and GGAGA elements but D095 does not consist of regular internal repeats and Allozyme variability in wild and domesti- is a non-curved DNA. The SspI-family is cated common carp (Cyprinus carpio L.) unique to the genus Gallus (chickens and jun- populations from Germany, Uzbekistan and gle fowls). FISH to the W lampbrush chromo- East Asia some demonstrated that the SspI family was located to the chromomere-6 near the terminal KLAUS KOHLMANN1, ASIYA MURA- non-heterochromatic region on the short arm, KAEVA1,2, PETRA KERSTEN1 and thus served as a good positional marker for 1Leibniz-Institute of Freshwater Ecology and functional genes. FISH to interphase nuclei Inland Fisheries, Department of Inland Fish- demonstrated that the SspI-family sequence eries, Berlin, Germany; 2Institute of Biochem- was much less heterochromatic comparing to istry, Tashkent, Uzbekistan XhoI- and EcoRI- family sequences. The pre- Common carp is among the most important sence of SspI-family (5.8 Mb) in addition to cultured fish species worldwide. The natural XhoI- (21 Mb) and EcoRI-(11Mb) families distribution of its wild ancestor is believed to narrows down non-repetitive DNA regions to range from Western Europe throughout Eura- at most 16 Mb on the W chromosome (about sia to China, Japan and South-East Asia. Rep- 54Mb). resentative wild and domesticated populations from these regions (five from Germany, six D094 from Uzbekistan and three from East Asia) An AFLP genetic map of Atlantic salmon were selected to describe the genetic variability within and differentiation between them by THOMAS MOEN1, KJERSTI T. FJAL- examining eight enzymatic systems (= 22 loci). ESTAD1, HEGE MUNCK1, BJÖRN HOY- The observed level of polymorphism was high: HEIM2, LUIS GOMEZ-RAYA3 1.4 to 1.9 alleles per locus, 26.3 to 47.4% po- 1AKVAFORSK, Aas, Norway; 2Norwegian lymorphic loci and expected heterozygosities School of Veterinary Science, Oslo, Norway; from 0.098 to 0.191. Highly significant differ- 3 Àrea de Producció Animal, Centre UdL-IRTA, entiation based on FST values from pairwise Spain comparisons was found between Uzbek and A genetic map of Atlantic salmon has not yet German, Uzbek and East Asian, German and been published, in spite of the economic im- East Asian populations, and between all Uzbek portance of the species. 33 microsatellites, wild and Uzbek domesticated and some of the distributed on 22 linkage groups, were used as German feral and domesticated carp. The den- a framework to construct an drogram based on 13 populations showed a AFLP/microsatellite map. Two full-sib fami- general grouping of populations according to lies sired by the same male were genotyped their geographic origin: Europe, Central Asia with respect to these microsatellites and 64 and East Asia. High bootstrap support was AFLP primer combinations. found for groups consisting of German domes-

113 Section D: Marker, Polymorphism and Biodiversity ticated carp (87.4%), German feral carp products were then purified, cloned and se- (89.5%), Uzbek wild carp (95.2%) and East quenced in both strands using a LICOR 4200 Asian wild carp (86.2%). About 30.5% of total sequencer to determine their exact length. So variation could be attributed to the variability far, we have sequenced 25 loci and calculated among the three geographic regions, about the correspondence of the sequence with the 11% could be explained by variation among mean allele length obtained by the collaborat- populations within regions and 58.5% repre- ing laboratories. The full sequencing of micro- sented the within population component of satellite loci represents an absolute standard for variation. the laboratories working on cattle. The se- quence can also provide information on the D096 structure and the type of repeats of the locus Length standardization of microsatellites and definition of allelic variation not revealed used in analysis of biodiversity in cattle by apparent mobility of PCR products.

M. C. SAVARESE1, L. PARISET1, J. A. D097 LENSTRA2, I. J. NIJMAN2, G. MOMMENS3, Study of polymorphism of leptin receptor P. WIENER4, D. BURT4, J. L. WILLIAMS4, gene in Iberian and Landrace pigs K. MOAZAMI-GOUDARZI5, S. DUNNER6, C. RODELLAR7, G. ERHARDT8, C. WEI- CRISTINA ÓVILO1, BÁRBARA MELÉN- MANN8, M. ZANOTTI9, F. PILLA10, A. DEZ2, JAVIER BENÍTEZ2 BRUZZONE10, A. VALENTINI1 1INIA, Dpto Mejora Genética Animal, Madrid, 1Department of Animal Production, Universitá Spain; 2CNIO, Dpto Genética Humana, della Tuscia, Viterbo, Italy; 2Faculty of Veteri- Madrid, Spain nary Medicine, Utrecht University, Utrecht, The The leptin receptor gene is candidate for fat- Netherlands; 3PolyGenLab, Malle, Belgium; ness and body composition traits, as it is in- 4Roslin Institute, Roslin, Midlothian, Scotland, volved in food consumption and it maps on pig UK; 5INRA, Jouy-en-Josas, France; 6Dpto. chromosome six, in a region where several Produccion Animal, Facultad Veterinaria, QTL have been found for these traits. The aim Madrid, Spain; 7Lab. Genetica Bioquimica Y of this work was to identify SNP on coding Grupos Sanguineos, Facultad Veterinaria, regions of this gene, which could be causal Zaragoza, Spain; 8Department of Animal mutations of phenotypic variations. In first Breeding and Genetics, Justus Liebig Univer- place genomic DNA was analyzed by se- sität, Giessen, Germany; 9Istituto di Zootecnia, quencing of the largest exons (4, 6, 9, 15 and Universitá di Milano, Milan, Italy; 10SAVA, 20, 1604bp) in Iberian and Landrace samples. Facoltà di Agraria, Campobasso, Italy This resulted in the identification of three SNP, Conservation of genetic variation is recognized located on exons 4 (C/T), 9 (C/A) and 20 as a crucial concern at international level to (G/A). Among them, the ones on exons 4 and 9 preserve a basis for selection. Microsatellite produce a change in the aminoacid composi- markers are valuable tools for assessing ge- tion of the protein (thr/met and ala/asp respec- netic diversity and phylogeny in many species. tively). Due to the complex structure of this This work is part of a project aimed to facili- gene (20 exons, some of them very small), tate the comparison of European breeds using a cDNA was also analyzed. RNA was obtained panel of 30 microsatellites originally selected from muscle samples of Iberian and Landrace through an EC funded programme and now origin and cDNA was amplified and sequenced adopted by the FAO. DNA from a set of refer- on five fragments covering exons 4 to 17 ence individuals has been distributed among (2376bp). Seven new SNP were identified the participating laboratories. To standardise from cDNA. Four of them, located on exons 4, the allele calling, the reference individuals 6, 15 and 16, have the new allele at low fre- were typed independently by the laboratories quency. The other three SNP, on exons 13, 14 and the local allele size of the microsatellites and 16, are more informative, with different reported. Subsequently, three different refer- frequencies on Iberian and Landrace samples. ence animals were used for the sequence Moreover, the one on exon 14 (C/T) produces analysis. Homozygous individuals were chosen a change in the aminoacid coded (leu/phe). to minimize problems due to stutter bands. Missense polymorphisms on exons 4, 9 and 14 Microsatellites loci were PCR amplified, the are being genotyped by pyrosequencing on pig

114 Section D: Marker, Polymorphism and Biodiversity populations with phenotypic records, for asso- naria, 28040 Madrid, Spain ciation studies. Dogs show the most diverse phenotypes among the mammalian species and in the spe- D098 cial case of the pointing dogs, most breeds are Use of AFLP markers for the evaluation of morphologically quite different, although their genetic diversity of Coregonus lavaretus (L. behaviour is alike. This study focuses on the 1758) after restocking in Bolsena Lake, Italy determination of the genetic relationship and genetic diversity between 5 pointing dogs LORRAINE PARISET1, MARIA CARMELA breeds: German Short-haired Pointing (GSP, SAVARESE1, PAOLA BONDANELLI2, n=30), Deutsch Drahthaar (n=8), Epagneul PAOLO COLOMBARI2, ALESSIO Breton (n=15), English Pointer (n=48) and VALENTINI1 English Setter (n=63) by typing 21 microsatel- 1Department of Animal Production, Università lites, and sequencing 652 bp from the mito- degli Studi della Tuscia, Viterbo, Italy; chondrial D-loop. Highest levels of genetic 2Department of Environmental Sciences, variability at the mitochondrial level are shown Università degli Studi della Tuscia, Viterbo, by Drahthaar with 7.14 variable sites over a Italy total of 30 contained in 17 haplotypes. In the Coregonus lavaretus (L. 1758) has been intro- other hand, GSP only shows 1.77 variable sites duced in Italian lakes since the second half of and also the lowest heterozygosity (56.7%) the XIX century and nowadays it represents towards Pointer (67.6%). For the genome loci, one of the main fishery resources of these deep Drahthaar had the lower number of effective lakes of the country. Programs finalized to the alleles (3), and Pointer the highest (3.8). De- evaluation of the genetic effects caused by spite their common origin, distance between restocking have not been proposed yet. The some of them, e.g. Drahthaar and Breton is current reproduction techniques can bring specially high either at the genome (FST=0.15) negative effects on natural populations due to a nor at the mitochondrial level where sequence drop in genetic variability that can result in diversity estimated by Kimura-2 distance was fitness reduction. With the aid of molecular 0.014. The overall Fst was 0.11, which shows a markers, the genetic diversity of the popula- high variability level compared to other do- tions in the Bolsena Lake, Italy, has been mestic species (e.g.0.08 in horses, 0.064-0.082 studied in order to evaluate if reared C. in bovine breeds) but lower than 0.23 shown in lavaretus are suitable to be employed for re- studies for other canine breeds. stocking in the wild. The amplified fragment length polymorphism (AFLP) technique has D100 been used to investigate the level of genetic Analysis of the variability at mtDNA in diversity within and between wild (adults) and three indigenous Spanish sheep reared (from Marta and Bolsena incubators) populations. AFLP markers, generated by SUSANA PEDROSA, JUAN J. ARRANZ, EcoRI/TaqI digestion and amplified with 8 YOLANDA BAYÓN, FERMÍN SAN primer combinations on 20 individuals per PRIMITIVO population, produced polymorphic bands that Departamento de Producción Animal, were analysed using a Licor 4200 sequencer. Universidad de León, León, Spain To evaluate genetic variability, the percentage Genetic variation at the mitochondrial DNA of homozygosity and the Jaccard index have level was investigated in three Spanish sheep been calculated and PCA analysis has been breeds: Churra, Castellana and Alcarreña. The performed. Results are useful for a manage- study was performed on 1209 pb of the ment of restocking finalized to conservation, mtDNA control region, which is known to be by maintaining the genetic diversity of the more variable than other sequences. On the populations. basis of published information about this re- gion in sheep, different primers were designed D099 which allowed for an analysis of the whole Genetic diversity in pointing Dogs selected sequence, divided into three overlap- ping segments. Total DNA was extracted from D. PARRA, S. DUNNER, J. CAÑON blood samples and each of the three segments Dpto. Producción Animal, Facultad de Veteri- amplified through PCR and subsequently cy-

115 Section D: Marker, Polymorphism and Biodiversity cle-sequenced using an ABIPRISM 377 DNA Breeding and Genetics, Tjele, Denmark sequencer. The alignment of the resulting se- Genomic maps are becoming more and more quences was performed to obtain the consensus detailed as new technologies for genetic re- sequence. Comparison of individual sequences search are developed. The numbers of known revealed a total of 219 single nucleotide sub- genes and genetic markers are increasing at a stitutions, among which transitions predomi- tremendous speed creating high density chro- nated over transversions, in accordance with mosome maps. A speciel attention is paid to the general patterns known in mammalian mi- single nucleotide polymorphisms (SNPs) tochondrial evolution. Moreover, two of the which are predicted to replace traditional positions revealed insertion/deletion mutations. markers (microsatellites etc.) in most future Phylogenetic analysis of mtDNA haplotypes genetic assays. A lot of time and effort is in- was performed in order to derive genetic rela- vested in detection and localisation of SNPs tionships within and between groups of ani- and the resulting high-density chromsome mals. maps will ease detection of genes and QTLs. In this project we detect and evaluate SNPs in D101 the Pig genome. The SNPs are detected in Characterisation of bovine dopamine recep- overlapping EST-sequences which are proc- tors 1, 4 and 5 essed through a pipeline including Phred, Phrap, PolyBayes, and Consed (see also ab- ANDY HAEGEMAN, ALEX VAN stract by Frank Panitz et al.). Consensus se- ZEVEREN, LUC PEELMAN quences of clusters containing SNPs are Department of Animal Nutrition, Genetics, BLASTed against the human RefSeq to get an Breeding and Ethology, Ghent University, idea of the identity of the gene, the chromoso- 9820 Merelbeke, Belgium mal location, the exon-intron boundaries and if The balance of calorie uptake and expenditure the SNPs are synonomous or non-synonomous. is of vitable importance for each living organ- ism. Chronic excessive calorific uptake or D103 fasting will lead to tipping of that balance and Quantitative analysis of KIT copy numbers result in starvation or obesity with the resulting in domestic pigs (SUS SCROFA) using Py- severe health risks. Neurotransmitters and their rosequencing technology receptors play a major role in determining and modelling feeding behaviour, feeding rewards GERLI PIELBERG1, ANDERS ALDER- and satiating effects. A prime candidate is the BORN2, MONICA PETTERSSON2, LEIF dopaminergic system. The prominent role of ANDERSSON1 dopamine receptor (DRD) 2 in the several 1Swedish University of Agricultural Sciences aspects of feeding behaviour has been exten- (SLU), Department of Animal Breeding and sively documented in human and rat models. Genetics, Uppsala, Sweden; 2Pyrosequencing Although a lot is known about DRD2, much AB, Uppsala, Sweden less information about the potential role of Mutations in the KIT gene, encoding the DRD1, DRD4 and DRD5 is available. We mast/stem cell growth factor receptor (MGF), report here for bovine, the isolation and se- are responsible for coat color variation in do- quencing of DRD1, the localisation of DRD1 mestic pigs. The dominant white phenotype is and DRD4 by RH mapping, mutation screen- caused by at least two mutations, a gene dupli- ing and identification (using SSCP and/or se- cation and a splice mutation in one of the co- quence comparison) in DRD1, DRD4 and pies leading to skipping of exon 17. We have DRD5. recently shown an unexpectedly high allelic diversity at the KIT locus in the domestic pig D102 (Pielberg et al. 2002). By applying minise- Evaluation of SNPs in the Pig genome quencing and pyrosequencing for quantitative analysis of the number of copies with the spli- ANETTE H. PETERSEN, LONE B. ce form, we found evidence for at least two MADSEN, FRANK PANITZ, HENRIK new KIT alleles in pigs, both with a triplication STENGAARD, CHRISTIAN BENDIXEN of the gene. With such a high allelic diversity Danish Institute of Agricultural Sciences, Re- at the KIT locus, the determination of genoty- search Centre Foulum, Dept. of Animal pes at the KIT locus in domestic pigs is com-

116 Section D: Marker, Polymorphism and Biodiversity plicated. Here we present a new approach for MICHELE POLLI1, PAOLA MAGNETTI2, the analysis of KIT copy numbers. The recently MARIA LONGERI1, STEFANO MARELLI1, identified unique duplication breakpoint (Giuf- MARTA ZANOTTI1, LUIGI G. CAVAL- fra et al. submitted) is amplified by PCR and CHINI1 gene copy number estimated using Pyrose- 1University of Milan, Istituto di Zootecnica, quencing technology. Our new method con- Faculty of Veterinary Medicine, Milan, Italy; firms the existence of KIT haplotypes with 2University of Milan, Departement of Biology, triplication. The results imply that KIT alleles Faculty of Science, Milan, Italy with the duplication are genetically unstable Genetic variation at 10 canine microsatellite and new alleles are most likely generated by loci (AHT121, CXX20-123-263-403, FH2159- unequal crossing-over. This study provides an 2137-2138-2001-2132) was estimated to de- excellent method for genotyping the compli- termine the genetic diversity and phylogenetic cated Dominant white/KIT locus in pigs and a relationship in 15 different dog breeds (Ber- general approach for determining gene copy gamasco, Bolognese, Bracco Italiano, Cirneco numbers. dell'Etna, Corso, Fonnese Italiano, Volpino Italiano, Akita-inu, Czech Wolfdog, German D104 shepherds, Dobermann), in Italian wolf (Canis A microdissected whole canine X chromo- lupus italicus) and in red fox (Vulpes vulpes). some paint applied for 3 related canid spe- All the samples have been collected from ani- cies mals unrelated in two generations and were analysed by automatic fluorescent methods. ALDONA PIEŃKOWSKA1, MARIOLA Microsatellite allele frequencies were exam- ZAWADA2, GERALD STRANZINGER3, ined and evaluated by GENEPOP statistic CLAUDE SCHELLING3 package. Our data showed heterozygosities 1Agricultural University, Department of Ani- ranging from 0,47 (Dobermann) to 0,80 mal Genetics and Breeding, Poznań, Poland; (Maremma sheepdog) and PIC (polymorphism 2Institute for Human Genetics, Polish Academy information content) from 0,36 (Dobermann) of Science, Poznań, Poland; 3Federal Institute to 0,92 (Volpino Italiano). Mean number of of Technology and Faculty of Veterinary Me- alleles per breed ranged from 3 in Dobermann dicine, Department of Animal Sciences, Zurich, to 8 in Maremma sheepdog. Genetic distances Switzerland between populations were measured according A whole chromosome painting probe was de- to Nei's standard genetic distances by DISPAN veloped after microdissection of canine X statistic package. Both UPGMA dendrogram, chromosomes. This canine X chromosome based on Nei's genetic distances and Principal paint was applied by FISH to the chromosomes Component Analysis showed all dog breeds of three other canid species: red fox (Vulpes and Italian wolf closely related, and clearly vulpes), arctic fox (Alopex lagopus) and Chi- distant from wild fox. Data confirm the exis- nese raccoon dog (Nyctereutes procyonides p.). tence of a lower degree of genetic differentia- In the raccoon dog and the red fox the FISH- tion among the breeds of dog considered with staining was restricted to the entire X chromo- the Italian wolf and high genetic distance with some. In the arctic fox, beside the staining of other canids. the entire X chromosome, chromosome arms carrying constitutive heterochromatin were D106 also stained. Comparison among three methods to study This work has been supported by the Polish the double-muscling locus (mh) in Piedmon- State committee for Scientific Research (6PO tese cattle 6D 028 21). A. POZZI, G. BONGIONI, A. GALLI D105 Istituto Sperimentale Italiano “Lazzaro Spal- Genetic variation and phylogenetic relati- lanzani”, Milano, Italy onships in some dog breeds, Italian wolf The Piedmontese bovine is one of the most (Canis lupus italicus) and in red fox (Vulpes important and finest Italian beef breed. Its in- vulpes) based on microsatellite DNA poly- terest is determined by the exceptional devel- morphisms opment of muscle mass, known as “double- muscled”, given by a point mutation (G938A)

117 Section D: Marker, Polymorphism and Biodiversity of the myostatin gene (GDF8). The aim of this study we tested 3 of these loci for their asso- work was to develop a fast test to genotype a ciation with the 3 ORF-variants. For 573 Ger- large number of samples comparing one classi- man breeding sheep from important breeds cal and two innovatives method for the identi- (Dorper, Gotland, Isle de France, German me- fication of SNPs. The first one was represented rino, East Frisien milk, German black headed by a simple PCR-based allele detection system mutton, Suffolk, Texel) these 3 new loci were with fluorescent genotyping technology on 377 genotyped in addition to the 3 ORF-positions. DNA Sequencer (ABI Prism-Applied Biosys- Only breeds of which at least 60 samples had tems), which required several manipulations been obtained were included in analysis of and was time consuming. The second one used variance. In German merino the association of a SNaPshot methodology by single nucleotide the new markers with risk levels deduced from primer extension executed in different steps. ORF genotypes was only weak (r2 = 7,6 %). In For the last one an allelic discrimination in contrast more than 75 % of the risk variance in PCR Real-Time was employed in only one East Frisien milk, Suffolk and Texel could be step with a MGB TaqMan, a specific short explained by the 3 new loci. Independent from probe to identify a single base mismatch. breed the ORF-genotype ARR/ARR, the only Piedmontese animals (324 semen or hair sam- genotype in the lowest risk group, was always ples) were analized using the three methods: (n = 76) associated with a specific threefold 315 homozigotes and 9 heterozigotes for the homozygeous combination of alleles at the mutation were found. The PCR Real-Time new sites. Thus the new variants might be used shown the following advantages: easy execu- for predicting scrapie susceptibility at least in tion, less time for manipulations and analysis, some breeds. The precision of risk assessment therefore represents the faster test to apply to a could be further increased by taking additional large number of samples. informative markers into account. A similar approach could be used in cattle, for which no D107 linkage of genetic markers inside or outside the Association between new variants in the ORF of the PrP gene with BSE incidence is ovine PrP gene and Scrapie susceptibility known so far. Ref.: Dawson, M., Hoinville, L.J., Hosie, B.D., SIEGFRIED PREUSS, ANDREAS W. KUSS, Hunter, N. (1998): Vet. Rec. 142: 623-625 HONGZHE HE, HEINZ BARTENSCHLA- GER, HERMANN GELDERMANN D108 University of Hohenheim, Institute of Animal Variants of CSN3 in Chinese yak (Bos Husbandry and Breeding, Department of Ani- grunniens) mal Breeding and Biotechnology, Stuttgart, Germany EVA-MARIA PRINZENBERG1, HAN JIAN- Scrapie is a fatal neurodegenerative disease of LIN2, GEORG ERHARDT1 sheep which belongs to the group of trans- 1Justus-Liebig-University of Giessen, Depart- missible spongiform encephalopathies (TSE). ment of Animal Breeding and Genetics, Gies- The host encoded prion protein (PrP) plays a sen, Germany; 2Gansu Agricultural University, central role in the disease process. Genetic International Yak Information Centre, Lanzhou susceptibility to scrapie and pathogenesis are 730070, China associated with polymorphisms in three diffe- Variants of κ-casein (CSN3) have been exten- rent codons (136, 154 and 171) of the ovine sively studied in cattle and up to 9 alleles have PrP gene. Up to 15 different genotypes can be been simultaneously shown by SSCP analysis observed in several breeds. These genotypes in Bos taurus and Bos indicus breeds so far. have been grouped in 5 levels of scrapie risk Evolution of these alleles and a possible com- (Dawson et al. 1998). Efforts have been made mon ancestor still remain unclear in some to improve resistance against Scrapie by bree- cases. PCR-SSCP analysis of domesticated yak ding for resistant genotypes. However the de- CSN3 exon IV revealed a two allele polymor- termination of these ORF-variants is time con- phism showing intermediate migration patterns suming and expensive. In a separate investiga- compared to the cattle CSN3*A and B alleles. tion several polymorphic sites outside the PCR products of both yak CSN3 alleles were ovine PrP-ORF were identified which can be cloned and sequenced. All yak had nucleotide determined more easily and cost-saving. In this sequences corresponding to Thr in amino acid

118 Section D: Marker, Polymorphism and Biodiversity position 136 (identical to CSN3*A) and Ala in ies organic method for DNA extraction from position 148 (identical to CSN3*B). This is in biological stains (single hair) allows for non – accordance with a sequence reported from invasive analysis of anonymous genomes, Bison bonasus (CSN3*GBison) and may repre- where limited DNA is available. Purified PCR sent the common ancestor of CSN3*A and B products (313 length) were se- variants of cattle. Moreover, a 12 bp insertion quenced with ABI Prism Dye Primer Cycle resulting in a duplicated nucleotide and amino Sequencing Ready Reaction Kit (PE Biosys- acid motive was found in one yak allele com- tems) according to the user’s manual. The se- pared to the other. Position of the insertion quencing products were separated in a DNA could not be unequivocally assigned. The du- sequencer ABI PRISM 377 (PE Biosystems). plication is either corresponding to the codons The electrophoretic data were analysed by the for amino acids 147 to 150 or 148 to 151 Sequence Navigator v. 1.0.1 software (PE Bio- which are repeated identically. In 18 yak typed systems). Performed studies revealed that de- by SSCP analysis, the long variant was found rived sequences of cytb gene can be considered with frequencies about 70% and the short vari- as representative for investigated species and ant about 30%, implicating the longer variant can be used for diagnostic species identifica- being the predominant and probably the older tion from anonymous biological stain. allele in yak. The loss of the insertion may have led to the ancestral CSN3 allele from D110 which all today known variants in Bos indicus Fine mapping of the intestinal receptor and Bos taurus evolved. locus for enterotoxigenic Escherichia coli F4ab and F4ac on chromosome 13 in pigs D109 Sequence analysis of cytb gene in some spe- PASCAL PYTHON1, HANNES JÖRG1, STE- cies conserved in Poland (Bison Bonasus, FAN NEUENSCHWANDER1, CHRISTIAN Lynx Lynx, Canis Lupus) HAGGER1, CHRISTIAN STRICKER2, ESTER BÜRGI3, HANS ULRICH BERT- BEATA PRUSAK1, J. REKLEWSKI2, A. SCHINGER1, GERALD STRANZINGER1, KRZYWIŃSKI3 PETER VÖGELI1 1Institute of Genetics and Animal Breeding, 1Swiss Federal Institute of Technology, Insti- Jastrzebiec, Poland; 2Kampinos National tute of Animal Sciences, Zurich, Switzerland; Park, Poland; 3Wild Animals Park, 2Applied Genetics Network, Altendorf, Swit- Kadzidłowo, Poland zerland; 3University of Zurich, Department of The preservation of genetic diversity both Internal Veterinary Medicine, Switzerland within and among natural populations is a fun- Enterotoxigenic E. coli (ETEC) with fimbriae damental goal of conservation biology. Vari- of the F4 (K88) family are frequently associ- ous molecular markers and PCR techniques ated with diarrhoea in neonatal and weaned have been used in a wide variety of studies in pigs. A linkage analysis was performed on a an effort to attain this diversity. Cytochrome b pedigree of 200 Swiss Large White pigs to gene is encoded by mtDNA and contains spe- refine the localization of receptor loci F4ab cies – specific information. Because of no re- and F4ac. Small intestinal enterocyte prepara- combination between different mtDNAs and tions from 171 eight-weeks-old pigs were phe- maternal inheritance, mtDNA can shed light on notyped by an in vitro adhesion test using two the evolutionary history of species. Analysis of strains of E. coli representing the variants F4ab cytb gene sequence can be used to determine and F4ac. The serum transferrin (TF) gene and taxonomic identity, phylogenetic relationships 10 microsatellites on chromosome 13 were and genetic distance between species under linked with F4ac receptor locus (F4acR) (re- investigations as well as to monitor number combination rates (θ) between 0.00 and 0.11 and migrations of threatened species. The aim and lod score values (Z) between 11.4 and of this study was to analyse the cytb gene se- 40.4). The multipoint analysis revealed S0222- quence in some wild species (Bison bonasus, TF-Sw2459-S0068-F4acR-S0075-Sw1030- Lynx lynx, Canis lupus) being under conserva- Sw520-Sw398 as the most likely gene order. tion in Poland. The next step was to compare The F4acR locus was located between S0068 derived sequences with adequate sequences and Sw1030, with a recombination rate (θ) of existing in GenBank. Applied in present stud- 0.05 between S0068 and F4acR, and 0.03 with

119 Section D: Marker, Polymorphism and Biodiversity

Sw1030. A weak and a strong adhesion recep- monomorphic and 269 polymorphic bands tor were observed for F4ab. The weak adhe- were present for the 18 primers. Heterozygos- sion receptor for F4ab was detectable only in ity of polymorphic bands varied from 0.01 to pigs lacking F4acR and the strong adhesion 0.50. receptor for F4ab coincided with the presence of F4acR. No pigs were found expressing only D112 F4acR and lacking F4abR. Therefore, we con- Genetic diversity in clude that the receptor for F4ac binds F4ab breed bacteria as well, controlled by one gene local- ized between S0068 and Sw1030. However, R. RASERO, P. SACCHI, S. MAIONE, S. due to the limited number of informative ani- SARTORE, E. CAUVIN mals, the inheritance of the weak adhesion University of Torino, Department of Animal receptor F4ab could not be shown. Production, Grugliasco, Italy The Piedmontese cattle, originally a dual pur- D111 pose breed, became the main Italian beef cattle Estimation of genetic variability in owing to the presence of muscular hypertro- Mazandaran native fowls using RAPD phy. In spite of this in the last years its number markers decreased. Furthermore, the adoption of AI is quite widespread and a reduced number of GHODRAT RAHIMI1, ALIREZA KHA- BLUP selected bulls is commonly used. This NAHMADI1, ARDESHIR NEJATI- study is concerned with the analysis of genetic JAVAREMI2, SAEID ESMAEILKHANIAN2 variation to conserve future selection options. 1Univ. Mazandaran, Dept. Anim. Sci., Sari, Thirty-two microsatellites mapping on 21 dif- Iran; 2Anim. Sci. Res. Inst., Karaj, Iran ferent chromosomes were chosen according to Polymorphism at RAPD marker loci is char- their technical properties and potential for de- acterized by the presence (+) or absence (-) of tecting polymorphism. A preliminary investi- specific DNA sequences across the genome. If gation of 29 unrelated animals allowed to find the individual carries the sequence on at least 22 markers (15 in 2 multiplex PCR) having one of its homolog chromosomes it will show heterozygosity >0.6 by which 233 more indi- a band. Therefore, RAPD markers follow a viduals were typed. The HW proportions were complete dominant mode of inheritance and tested using an exact test, overall heterozygos- only two phenotypes are visible for each ity excess or deficiency was estimated using marker locus. Separation of the dominant phe- an asymptotic test. Five loci showed disagree- notypes into the two constituting genotypes is ment with HW proportions but only one of only possible by further analysis of the pedi- them (TGLA53) showed significant deficiency gree. In this study blood samples were col- of heterozygosity. The observed and effective lected from 10 males and 90 females of average numbers of alleles/locus were 9.5±3.5 Mazandaran Native Fowl Breeding Station and and 4.7±1.6 respectively, the average expected were treated with EDTA. Twenty random heterozygosity was 0.765±0.069. The prob- decamer primers were used in RAPD-PCR ability of identity between randomly chosen analysis. Any inept DNA pattern generated due pairs was 2.4E-11 and 1.8E-7 for the 2 multi- to unsatisfactory amplification was excluded plex PCR respectively. These informations will from the analysis and only reproducible bands be used to study the genetic evolution of the in multiple runs were taken into consideration breed under the selection pressure. and scored as present or absent. Eighteen primers yielded satisfactory amplification D113 products. Banding data was analyzed by the The allele ea - a rare mutation in the MC1R RAPD-MGA computer program developed by gene in horse (EQUUS CABALLUS) one of the authors. The program assumed HWE to estimate allelic frequencies. The MONIKA REIßMANN1, HEINZ-JÜRGEN number of bands displayed for each primer WAGNER2, LÁSZLÓ GULYÁZ3, SOPHIE ranged from 6 to 29. Only those bands absent SCHUSTER1 in at least one individual were considered as 1Humboldt-University, Institute of Animal Sci- polymorphic and those present for all birds ence, Berlin, Germany; 2Free University, In- were taken as monomorphic. In total 12 stitute of Veterinary Biochemistry, Berlin,

120 Section D: Marker, Polymorphism and Biodiversity

Germany; 3University of West-Hungary, Insti- of the Spanish Fighting Bull (FB) founding tute of Animal Breeding and Husbandry, Mo- castes. Isolated from the remaining castes sonmagyaróvár, Hungary during centuries, its ethnic characteristics are The coat colour of horses is considerably de- different from the others. On the other hand, termined by the MC1R gene. A single point the Betizu (BET) is considered as one of the mutation in this gene leads to the chestnut al- most endangered cattle breed in Spain and it is lele e. We found another point mutation in the one of the few feral breeds in Europe. Its origin MC1R gene that is spread in the Black Forest is uncertain, one hypothesis places the BET /1/. This mutation leads to the allele ea and it breed as the precursor of CN but it seems more has no association to the melanin synthesis but realistic to consider the BET as the low devel- it falsifies the differentiation between E and e oped and distant relative of Pyrinean (PYR) using the widespread Taq I - RFLP test. breed. Fifty animals from CN and BET were Here we report the detection of the allele ea in selected for population characterisation using another horse breed. The tests were carried out 30 microsatellites, showing both breeds a het- in 457 individual horses belonging to 18 erozygosity deficiency. The genetic relation- breeds, amongst them Ardennais, , ships between these breeds and PYR and FB Black Forest, Hungarian Coldblood, as well as populations were also analysed. The genotype in 18 Przewalski individuals. Confirming our data from 21 microsatellites analysed in these former result the allele ea was found in the four populations was used as a baseline for Black Forest and furthermore only in the Hun- identifying the breed of 300 animals classified garian Coldblood. Obviously within the spe- as CN and BET. CN cattle belonged to six cies horse the allele ea exists exceptionally different farms, as FB is traditionally bred on rare, whereas within the both breeds it is farms which impose reproductive isolation, we spread frequently: have analysed the genetic relationships be- Allelic frequency in the Black Forest (n=75): E tween the different CN subpopulations and the = 0.01; e = 0.80; ea = 0.19 and BET, PYR and FB populations. Phylogenetic allelic frequency in the Hungarian Coldblood trees constructed on the basis of different ge- (n=73): E = 0.07; e = 0.80; ea = 0.13. netic distance measures (Da, Gst, Dc) cluster Because there is no evidence of a direct rela- together the different CN farms and BF. Fi- tion between these two breeds it is remarkably nally, the genetic relationships between 339 that the ea allele has not been found in Arden- CN and 44 FB individual cattle were also nais and Noriker – two breeds which took part studied showing genetic differences between in the development of the named breeds. From subpopulations. an evolutionary point of view we suggest that the single base mutation ea in the MC1R gene D115 occurred in a very late time. Its origin remains Genetic mapping porcine EST sequences to be elucidated. using length polymorphisms /1/ Wagner, H.-J.; Reissmann, M. (2000), Anim. Genet. 31, 289-290 GARY A. ROHRER, BRAD A. FREKING, DAN J. NONNEMAN D114 USDA, ARS, U.S. Meat Animal Research Cen- Genetic characterisation of two Northern ter, Clay Center, NE, USA Spanish endangered cattle breeds (Betizu The current priority of the MARC swine ge- and Casta Navarra) using microsatellites: nome group is to identify and map SNPs (sin- Population assignment of individuals and gle nucleotide polymorphisms) associated with subpopulation relationships EST sequences to develop the comparative map and provide a large number of validated INMACULADA MARTÍN-BURRIEL, SNP markers for the porcine genome. Our CLEMEN RODELLAR, EMILIO GARCÍA- approach is based on sequencing amplicons MURO, ISAÍAS ZARAGOZA, PILAR from animal genomic DNA spanning introns ZARAGOZA and evaluate the sequence files for polymor- University of Zaragoza, Biochemical Genetics phisms. While collecting this sequence data, it and Blood Groups Laboratory, Faculty of was noted that approximately 20% of the am- Veterinary, Zaragoza, Spain plicons possess polymorphic insertion/deletion The Casta Navarra (CN) is considered as one events (Fahrenkrug, et al., Anim. Genet.,

121 Section D: Marker, Polymorphism and Biodiversity

2002). Some of these insertion/deletion events king the defensin-gene region have been sub- were actually caused by the presence of a cloned. In order to establish a contig, the CA/GT dinucleotide repeat. A cost effective inserts of the BAC-clones have been analysed method for genotyping these polymorphisms is using BAC-end sequencing and oligonucleoti- to design primers flanking the polymorphism, des have been designed from each end of amplify genomic DNA incorporating radioac- insert. Further, we designed primers based on tive nucleotides, separate the fragments with published bovine EST-sequences, which are polyacrylamide gel electrophoresis and expo- homologous to hBD-3. All BACs have been sing the gels to film overnight. With this me- characterized by PCR to identify overlapping thodology, seven informative microsatellite regions. A preliminary contig, based on 9 BAC markers and 36 informative insertion/deletion clones, has been established. markers were developed for 42 genes (one intron contained informative microsatellite and D117 insertion/deletion markers). Forty of these Isolation and mapping of the Mitochondrial genes were placed on the porcine genetic map Glycerol-3-Phosphate Acyltransferase as two markers did not have enough informati- (GPAM) gene in Cattle ve meioses to detect significant linkage. These genes were distributed across 14 of the 19 R. ROY1, M. GAUTIER2, H. HAYES2, P. chromosomes of the porcine genome and de- LAURENT2, P. ZARAGOZA1, A. EGGEN2, velop additional ties between the human and C. RODELLAR1 porcine genomes. 1Laboratorio de Genética Bioquímica y Grupos Sanguíneos, Facultad de Veterinaria, D116 Universidad de Zaragoza, Zaragoza, Spain; Genomic characterization of bovine beta- 2Laboratoire de Génétique biochimique et de defensin genes Cytogénétique, INRA-CRJ, Jouy-en-Josas, France SUSANNE ROOSEN, ERNST KALM, Glycerol-3-phosphate acyltransferase catalizes CHRISTIAN LOOFT the first step of glycerolipid biosynthesis. It Institute of Animal Breeding and Husbandry, plays a key role in the regulation of cellular Christian Albrechts University, Olshausenstra- triacylglycerol and phospholipid levels.There sse 40, D-24118 Kiel, Germany are two isoforms of Glycerol-3- phosphate Defensins are cationic peptides of 38-42 amino acyltransferase in mammals, a mitochondrial acids and are part of the innate, unspecific host and a cytosolic form. The mitochondrial form defense mechanisms of mammals. Within the (GPAM) prefers saturated fatty acyl-CoA as a scope of the research project ″Inquiries of the substrate, whereas the cytosolic enzyme uses importance of antimicrobial peptides in the both saturated and unsatured fatty acyl-Co. We bovine udder″ the relevance of bovine beta- used the mouse GPAM gene sequence to defensin genes concerning udder health is to be design specific primers in order to partially analysed. First own studies provided evidence amplify the bovine gene. We assigned GPAM of gene expression of several defensins in epit- to BTA26 using the INRA hamster-bovine helial tissue of bovine mammary glands. In somatic cell hybrid panel and confirmed this order to characterize the genomic organization assignment by analysis of a 3000rad radiation of bovine beta-defensin genes, we designed panel. The primers were used to screen a primers based on published beta-defensin con- bovine BAC library and two BAC clones were sensus sequences and screened the primary identified (0522B11 and 0784H09). Both were pools of two bov. BAC libraries. 18 defensin- mapped to BTA 26q22 by fluorescence in situ positive BACs have been isolated by PCR hybridization. This is in agreement with the analysis and characterized by digestion with corresponding human and mouse localizations restriction endonuclease and pulsed-field gel on HSA 10q24-q26 and MMU 19 respectively. electrophoresis. A NotI digestion revealed an Two polymorphic microsatellites were isolated average insert size of 83 kb. The digested from each BAC clone after subcloning and BACs were adapter-ligated and sequenced hybridation with a poly (AC) probe. within the defensin-gene area. Up to now, 11 of 18 BACs have been analysed this way. PCR-products established with primers flan-

122 Section D: Marker, Polymorphism and Biodiversity

D118 amplified a specific PCR product in Alpine Assessment of genetic diversity in Mazurian Ibex. Seven loci were polymorphic in two red deer populations by DNA fingerprinting populations (A and B), six in the third. The analysis average number of alleles per locus was 2.42, 2.42 and 2.14 for populations A, B and C re- J. RUTKOWSKI1, M. SACHARCZUK2, K. spectively (ranging from 1 to 3 allele per lo- JASZCZAK2, M. ŻURKOWSKI1 cus). The average heterozigosity was 0.41, 1Research Station for Ecological, Agriculture 0.40 and 0.39 respectively. In populations A and Preserve Animal Breeding; 2Institute of and B all loci were in Hardy - Weinberg equi- Genetics and Animal Breeding of Polish librium. In C population one locus showed Academy of Sciences significant deviation from Hardy - Weinberg Multilocus DNA fingerprinting technique proportions towards an excess of heterozigotes. based on screening of many polymorphic loci Calculated Fst values revealed a low degree of in the genome is effective tool for determina- differentiation in allelic frequencies between tion of genetic heterogeneity and differentia- populations. The reduced genetic variation tion of population. A study involving the use observed in the three populations confirms the of DNA fingerprinting was conducted to asses historic data of a severe population bottlenek. the genetic variation and diversity in Mazurian red deer population. D120 High molecular weight DNA was prepared PDME in Brown Swiss cattle: an improved from blood samples by proteinase K digestion molecular test for Italian livestock and phenol/chloroform extraction. The restric- tion endonuclease Hinf I was used to digest STEFANO SANGALLI1, SUE DENISE2, individual and pooled DNA samples. Hybridi- EMILY OBERG2, MARIO BERTOLETTI1, sation was performed with the 33.6 probe. PAOLO AJMONE-MARSAN3 The individual and representative DNA finger- 1Laboratorio Gruppi Sanguigni, Cremona, printing profiles were analysed with a compu- Italy; 2Department of Animal Sciences, Uni- ter program and band sharing (BS) were mea- versity of Arizona, Tucson, USA; 3Universita’ sured within four regions and between them. Cattolica del Sacro Cuore, Piacenza, Italy Higher values of BS between individuals within regions than between regions were ob- Weaver syndrome or PDME (Progressive De- served. generative Myeloencephalopathy) is an auto- somal recessive disease causing severe neuro- D119 logical defects in Brown Swiss cattle. In Italy, Microsatellite analysis of genetic diversity in Brown Swiss individuals belonging to carrier Alpine ibex (CAPRA IBEX) families have been tested routinely since 1996, using TGLA 116 and MAF 50 microsatellites P. SACCHI1, R. RASERO1, E. CAUVIN1, S. that permitted to reach a diagnosis with a like- SARTORE1, S. MAIONE1, B. BASSANO2, G. lihood of at least 90% in approximately 50% of MENEGUZ1, M. BLASI3 the cases investigated. To improve the preci- 1University of Torino, Dipartimento di Pro- sion of the diagnosis, we tested the informa- duzioni Animali, Grugliasco, Italy; 2Parco tiveness of 9 additional markers flanking the Nazionale Gran Paradiso, Italy; 3Laboratorio PDME gene on chromosome 4. Six of these Gruppi Sanguigni, Potenza, Italy belong to the official set used for diagnostic Genetic diversity at microsatellite loci was purposes in the United States, while three have examined in three populations of Alpine Ibex never been tested before. We set up two PCR (Capra ibex) from different geographic loca- multiplexes: a) MAF 50, TGLA 116, BMS tions in Italian Alpes. Sixty individuals were 2172, BMS 885, DIK 008, INRA 072, BM captured in Gran Paradiso area, namely 46 in 6458 and b) BM 1224, BM 6437, RM 232, Val Savarance (A) and 14 in Valle Orco (B); BMS 779. We genotyped the most important other 21 individuals were captured in Parco carrier bulls used in Italy. All carriers tested so Naturale delle Alpi Marittime (C). Thirty mi- far share the same haplotype linked to the dis- crosatellite primer pairs designed in domestic ease gene at 8 microsatellite loci that span a cattle, goat and sheep were chosen. From the region of 11.9 cM. This result suggests a recent set of selected markers twenty-two (73,33 %) origin of the mutation and the inheritance of

123 Section D: Marker, Polymorphism and Biodiversity the recessive PDME gene from a common D122 ancestor, as observed in other bovine genetic Analysis of genetic structure of Japanese Black defects (CVM, BLAD, SMA). All affected cattle of Hyogo using microsatellite makers animals tested so far are homozygous for the same haplotype. Recombinant animals allowed SHINJI SASAZAKI1, TAKESHI HONDA1, to locate the PDME gene between BM 1224 MORIYUKI FUKUSHIMA2, KENJI OYAMA3, and INRA 072. HIDEYUKI MANNEN4, FUMIO MUKAI4, SOICHI TSUJI4 D121 1Kobe University, Graduate school of Science and Analysis of genetic variation in Agerolese Technology, Kobe, Japan; 2Northern Hyogo cattle breed Prefecture Institute of Agriculture, Wadayama, Japan; 3Kobe University, Experimental Farm, S. SARTORE1, P. SACCHI1, L. DI STASIO2, Kasai, Japan; 4Kobe University, Faculty of R. RASERO1, V. PERETTI3, F. CIOTOLA3, Agriculture, Kobe, Japan V. BARBIERI3, G. SARTORE1 Japanese Black cattle of Hyogo prefecture (Tajima 1University of Torino, Dipartimento di Pro- strain) is famous as high quality meat producer and duzioni Animali, Grugliasco, Italy; 2University has been maintained as a closed system more than of Torino, Dipartimento di Scienze Zootec- 80 years with a few sires. The average inbreeding niche, Grugliasco, Italy; 3University of Napoli, coefficient of the strain reaches over 0.2 and is still Dipartimento Scienze Zootecniche, Napoli, increasing year by year. Serious inbreeding Italy depressions will be expected. To avoid inbreeding The Agerolese cattle is an endangered breed, depression, we investigated genetic structure of the with less than 100 animals, reared in Naples strain using microsatellite markers. Here we province. It derives from crosses between Po- analyzed representative 252 cows which are kept at dolian, Brown Swiss, Italian Friesian and Jer- Northern Hyogo Prefecture Institute of Agriculture sey during the XX century. It is a dual purpose with 21 out-group animals which have lower kinship breed, well adapted to a mountainous country with Tajima strain. Relationship coefficients were and fed with products of pruning and under- calculated from their pedigree information as each growth. This study is concerned with the char- Japanese Black cow or bull has complete pedigree acterization of local genetic resources. Sixty information. The genetic distances between animals were genotyped for sixteen microsat- individuals were calculated using the polymorphic ellites (chosen according to their possibility of information of microsatellite as well as pedigree multiplexing and potential for detecting poly- records. Two dendrograms were drawn, one from morphism) and 2 coding genes, POU1F1 and pedigree record and another from polymorphic GH1, analysed by PCR-RFLP and AS-PCR information of DNA markers, using UPGMA respectively. Deviations from the HW frequen- method. The dendrogram drawn by using DNA cies were evaluated with an exact test and markers is consistent well with that from relations- overall heterozygosity excess or deficiency coefficients. In both dendrograms, all 21 was estimated using an asymptotic test. All the individuals of outgroup constructed one group. selected loci showed polymorphism. The ob- Within Tajima strain several sub-groups were served and effective average numbers of al- clearly detected, so that using this information we leles/microsatellite locus were 8.2 and 4.0 re- can make plan to maintain the strain. This result spectively. The POU1F1 and GH1 loci were showed that DNA polymorphic information using biallelic. The average expected heterozygosity microsatellite markers well reflects the pedigree was 0.686. Although statistically significant record even in an unusually high inbreeding herd of deviations from HW equilibrium were ob- cattle, from which it is suggested that analysis using served, they occurred at 3/18 loci and only 2 of microsatellite markers is useful tool to reveal the them showed significant lack of heterozygotes. genetic structure without pedigree information. The results show that, in spite of its very re- duced size, the Agerolese maintains a rela- D123 tively high variability, likely depending on its Paternity test development in dromedary composite origin. The genotypic information racing camels with DNA microsatellites will be used to plan individual mating systems aimed to the maximal maintenance of the ex- JÜRGEN SASSE1, REKHA SHAH1, SAN- isting genetic variability. JEEV PULLENAYEGUM2

124 Section D: Marker, Polymorphism and Biodiversity

1Central Veterinary Research Laboratory, PO evolutionary change in function, the cycle was Box 597, Dubai, UAE; 2Present address: Win- thought not to be expressed in reptiles and terbourne Drive, Oakville, Ontario, Canada birds. In a previous study, we reported that Dromedary camel racing used to be a traditio- OTC was expressed in negligible quantities in nal pastime of the Bedouin. Today it has de- chicken kidney and cDNA sequence suggests veloped into a highly organised sport compa- that it was highly conserved compared to rable to . Large sums are paid for mammals. In this study, we provide evidence exceptional animals. The high monetary value that functional OTC is expressed in reptiles of the camels and the increased use of artificial and birds. All uricotelic animal OTC genes insemination and embryo transfer has necessi- appear to have the necessary substrate-binding tated a paternity and individual certification sites and mitochondrial targeted signal sequen- test. This poster details the compilation of ces. The phylogenetic tree constructed from microsatellite markers from two previous pu- mature OTC coding sequence was largely con- blications into one parentage test. Six micro- sistent with vertebrate evolution. However, it satellite markers from New World camelids was found that the leader peptide regions for and four developed in dromedaries were poo- OTCs are different in length and show little led into two multiplex reactions of five similarity to one another. Therefore, we inve- markers each (LCA33, YWLL44, CVRL04, stigated subcellular localization of OTC using LCA18, CVRL07 / VOLP03, YWLL08, immunofluorescence analysis and a conversion CVRL02, CVRL05, YWLL38). The PCR pro- from precursor form to mature form using ducts were combined into one fragment analy- mitochondrial import assay in uricotelic ani- sis on an ABI PRISM™310 Genetic Analyser. mals. These results showed that precursor The system was evaluated in 10 Local, 9 OTC was transferred to mitochondria and was crossbreed and 10 Sudani camels. These ca- processed to mature form in uricotelic animals. mels are raced or used for breeding in the These results suggest that OTC gene products UAE. The combined exclusion probability for have been expressed in uricotelic animals and all animals was 0.997, while 0.996, 0.986 and function as a urea cycle enzyme. 0.987 for the Local, crossbreed and Sudani, respectively. These markers may also be useful D125 for identifying the breeds of individual animals Molecular analysis of relatedness among in order to be entered into the correct race ca- five turkey strains tegories. Currently, the test is being evaluated in known dromedary families and larger ED SMITH, ELIZABETH LONG, DEDRA sample sizes of the respective breeds are under WRIGHT, ISRA ELRAYAH, WILLIAM study. PIERSON, PHILIP SPONENBERG Virginia Tech, Blacksburg, VA 24061, USA D124 Over the last five years, we have led efforts to Molecular evolution of ornithine transcar- develop resources essential for increasing our bamylase gene in uricotelic animals understanding of the turkey genome. These resources have been used to establish genetic TAKESHI SHIMOGIRI1, KATSUYOSHI differences between wild turkeys of different KOYANAGI2, KOTARO KAWABE1, HI- origins and backgrounds and between com- DEYUKI MANNEN2, SHIN OKAMOTO1, mercial turkeys. However, there has been very YOSHIZANE MAEDA1, YASUO KITAGA- little analysis of non-commercial turkey varie- WA3, SOICHI TSUJI2 ties. Additionally, the relatedness of different 1Kagoshima University, Faculty of Agriculture, non-commercial turkey strains remains pri- Kagoshima, Japan; 2Kobe University, Faculty marily based on phenotypic information and of Agriculture, Kobe, Japan; 3Nagoya Univer- speculation. Here, we describe results of mo- sity, BioScience Center, Nagoya, Japan lecular analysis of relatedness among five tur- Ornithine transcarbamylase (OTC) is one of key varieties including Blue slate, Spanish ornithine-urea cycle enzymes. During verte- black, Bourbon red, Royal palm, and Narra- brate evolution, the cycle enzymes have consi- gansett. The molecular resources used in the derably altered expression patterns and func- analyses included microsatellites, sequence tion physiologically in ammonia detoxification tagged sites, and RAPD primers. Evidence and osmotic regulation. From a viewpoint of from these analyses suggests that the royal

125 Section D: Marker, Polymorphism and Biodiversity palm and narragansett are more closely related D127 but more distant from the other three varieties. Genetic differentiation of the three hare When combined with phenotypic information, species from the Iberian Peninsula using this data may finally help us understand the microsatellite DNA value of these varieties to the on going efforts in turkey genome analysis and mapping as well AINHOA SOLIS1, MIKEL IRIONDO1, as to resource population development for FERNANDO PALACIOS2, ANDONE QTL mapping. ESTONBA1 1University of the Basque Country, Dpt. of D126 Animal Biology and Genetics, Bilbao, Spain; Large scale linkage mapping of bovine ESTs 2Ministery of Science and Technology, CSIC, using SNPs Department of biodiversity and evolutive bio- logy, Madrid, Spain TIMOTHY P. L. SMITH, ROGER T. STONE, We studied genetic variation in 52 individuals GARY L. BENNETT, EDUARDO CASAS, of three hare species of the Iberian Peninsula: WARREN M. SNELLING, JOHN W. KEELE Lepus europaeus (brown hare), Lepus grana- USDA, ARS, U.S. Meat Animal Research Cen- tensis (Iberian hare) and Lepus castroviejoi ter, Clay Center, NE, USA (broom hare). Although L. europaeus inhabit Considerable variation in production traits in all Europe, in the Peninsula appears only in exists within commercial populations of cattle, the north. L. castroviejoi and L. granatensis are suggesting the presence of allelic variation in endemic to the Iberian Peninsula and while L. important genes that could be exploited using castroviejoi is present only in the northwest, L. genomics. However, this possibility requires granatensis can be found in the whole Penin- the development of appropriate research tools. sula. Seven microsatellite DNA markers In the absence of an effort to completely se- (SOL8, SOL30, SAT2, SAT8, SOL33, SAT12 quence the genome, a realistic approach to and SAT5) were studied automatically by an genome science in mammalian livestock spe- ABI 310 Genetic Analyzer and their fragment cies is construction of dense comparative maps size by Genescan software. A tree diagram with human and mouse. To construct such showing the genetic relationships among the maps, we have undertaken a program of EST sampled hare species was built up using sequencing using pooled-tissue, normalized UPGMA method based on the Nei genetic libraries. The EST sequences are aligned with distance. The robustness of the tree was eva- the human genome via BLASTN analysis to luated by carrying out 1000 bootstrap iterations identify probable introns and human map posi- with the PHYLIP statistical package. The tion. This data is used to design primers that dendrogram shows that L. europaeus is the amplify intronic sequence within genes corre- most differentiated species. Simple allele sha- sponding to the ESTs. Currently 2342 primer ring statistics were applied to investigate the pairs have been tested on bovine genomic genetic structure of the species. The neighbor- DNA, of which 1619 (69%) produced ampli- joining phylogenetic tree shows clear differen- cons appearing to be of sufficient quality for tiation among the three hare species. The ge- sequencing. Successful sequences were de- netic diversity is considerably higher in L. rived from at least one end of 1340 amplicons europaeus than in L. granatensis and L. (83%), and revealed heterozygous positions castroviejoi. The observed low level of genetic within the four sires of the mapping population variability in L. castroviejoi can be explained for 839 amplicons (63% of successful sequen- by its limited geographical distribution, while ces). Primer-extension assays for over 500 of the high diversity of L. europaeus could be these SNPs have been designed, and genotype attributed to the fact of being the original spe- data for 465 has been collected by MALDI- cies. L. europaeus is the oldest species of the TOF mass spec genotyping. Presently, linkage three, as pointed out by Pérez-Suárez (1994) mapping has been performed for 399 bovine and our results suggest that L. granatensis and ESTs, making a substantial contribution to the L. castroviejoi are closed species, descendent bovine linkage map and comparative maps from a single common ancestor. with other species.

126 Section D: Marker, Polymorphism and Biodiversity

D128 M. ZAJAC1,2, A. CHMURZYNSKA1, M. Characterization of polymorphism and SWITONSKI1 mapping of the porcine SKI gene 1Department of Genetics and Animal Breeding, August Cieszkowski Agricultural University of ANTONIN STRATIL1, GERALD REINER2, Poznan, Poland; 2Institue of Human Genetics, LUC J. PEELMAN3, ROBERTA DAVOLI4, Polish Academy of Sciences, Poznan, Poland MARIO VAN POUCKE3, PAOLO ZAMBO- Two exons (2 and 3) of the leptin gene of four NELLI4, HERMANN GELDERMANN2 species belonging to the family Canidae: the 1Institute of Animal Physiology and Genetics, dog (Canis familiaris), the Chinese raccoon Academy of Sciences of the Czech Republic, dog (Nyctereutes procyonides procyonides), Libechov, Czech Republic; 2Institute of Animal the red fox (Vulpes vulpes) and the arctic fox Husbandry and Breeding, University of Ho- (Alopex lagopus) were screened for intra- and henheim, Stuttgart, Germany; 3Ghent Univer- interspecies variants with the use of the SSCP sity, Department of Animal Nutrition, Genetics, and DNA sequencing approaches. Neither in Breeding and Ethology, Merelbeke, Belgium; the exon 2 nor in the exon 3 any polymorphic 4Sezione di Allevamenti Zootecnici, University SSCP patterns among 16 canine breeds, in- of Bologna, Reggio Emilia, Italy cluded in this study, were found. The patterns The SKI oncoprotein (avian sarcoma viral of the exon 2 were identical in all studied spe- oncogene homolog) is involved in neural tube cies, but were distinctly different in case of the development and muscle differentiation. SKI exon 3. Thus, DNA sequencing of the exon 3 affects muscle mass, apparently via activation was performed and several single nucleotide of MRF genes. In man the SKI gene was first substitutions were identified. Comparison of localized to HSA1q22-q24 and is recently the DNA sequences revealed existence of the reassigned to HSA1p36.33. To study the following number of the subsitutions, distin- porcine SKI gene we designed PCR primers guishing the studied species: one between the using the human sequence (EMBL X15218). A dog and the raccoon dog, four between the dog single fragment was amplified. The fragments and the arctic fox, three between the raccoon from the Pietrain and Meishan pigs were dog and the arctic fox. Data for the red fox are cloned and sequenced. The sequence (364 bp) yet incomplete. The obtained results suggest was 97 % identical to the human sequence. that exon 2 of the leptin gene is highly conser- The two pig sequences differed in two bases vative in the family Canidae, but some muta- (positions 325-326: Pietrain, TC; Meishan, tions in the exon 3 were established during the CG). The Meishan fragment was cut by re- evolution of this family. Moreover, our studies striction enzyme Alw26I (BsmAI) (allele B), support previous conclusions concerning spe- but not the Pietrain one (allele A). The base cies divergence in the family Canidae. substitutions result in amino acid replacement (This study was supported by the Foundation (serine – arginine). Codominant inheritance of for Polish Science, contract 13/2000) this polymorphism was confirmed in the Ho- henheim Meishan x Pietrain pedigree. SKI was D132 polymorphic in Pietrain, Black Pied Prestice, Mitochondrial differentiation in Northern Large White, Landrace, Czech Meat Pig, European sheep Hampshire and Duroc breeds, while Meishan was monomorphic for allele B. By linkage MIIKA TAPIO1, ILMA GRIGALIUNAITE2, analysis in the Hohenheim pedigree SKI was LARS-ERIK HOLM3, SVEN JEPPSSON4, assigned to chromosome 6 and the gene order JUHA KANTANEN1, ILONA MICEIKIENE2, was: S0087-RYR-LIPE-EAH-A1BG-SKI- INGRID OLSAKER5, HALDJA VI- FABP3-S0146. In radiation hybrid mapping INALASS6, EMMA EYTHORSDOTTIR7 (using IMpRH panel) no marker gave a LOD 1MTT Agrifood Research Finland, Jokioinen, score >4.8 (this value is the treshold level for Finland; 2Lithuanian Veterinary Academy, significant assignment). (Supported by Grant Kaunas, Lithuania; 3Danish Institute of no. 523/00/0669) Agricultural Science, Tjele, Denmark; 4Swedish Board of Agriculture, Jönköping, D129 Sweden; 5The Norwegian School of Veterinary Evolution of the leptin gene in the family Science, Oslo, Norway; 6Institute of Animal Canidae Science of Estonian Agricultural University,

127 Section D: Marker, Polymorphism and Biodiversity

Tartu, Estonia; 7Agricultural Research ence of ecological stress-factor. The heterozy- Institute, Reykjavik, Iceland gosity observed in this study with ISSR- mark- Differentiation in 32 short- and long-tailed ers was comparable with those revealed using sheep breeds in Northern Europe was studied biochemical markers in earlier studies. It is by sequencing of mitochondrial control region. probably that under of low irradiation dose Breeds were grouped into four categories: At- influence conditions the heterozygous animals lantic short-tailed breeds, Atlantic long-tailed have advantages for reproduction. breeds, Baltic Sea region short-tailed breeds and Baltic Sea region long-tailed breeds. D136 Analysis of molecular variation showed sig- Development of single nucleotide poly- nificant differentiation between the four groups morphism (SNP) markers adjacent to

(ΦCT=0.036) and between populations within existing microsatellites to increase the groups (ΦSC=0.289). Pairwise comparisons marker informativeness for detailed showed that the Baltic long-tailed breed group QTL analysis was not differentiated from the Baltic short- 1 1 tailed group or the Atlantic long-tailed group. YOKO UCHIDA , NAOE KANAYA , 2 This agrees with prior knowledge about ances- ATSUSHI HORIUCHI , HIROHIDE UEN- 3 3 try of breeds belonging to Baltic long-tailed ISHI , TAKESHI HAYASHI , TAKASHI group. The results encourage further dissection AWATA3 1 of mtDNA variation on Northern European STAFF Institute, 446-1 Ippaizuka, Kami- 2 sheep. yokoba, Tsukuba, Ibaraki, Japan; Shizuoka Prefectural Swine & Poultry Experiment D135 Station, 2780 Nishikata, Kikugawa, Shizu- Cattle polymorphism revealed using ISSR- oka 439-0037, Japan; 3National Institute of PCR at animals from 10-km alienation zone Agrobiological Sciences, 2-1-2 Kannondai, of Chernobyl NPS Tsukuba, Ibaraki 305-8602, Japan QTL analyses have been performed in various N. V. TRYAPITCINA, V. I. GLAZKO animal populations to reveal genetic regions Institute of Agriecology and Biotechnology of responsible for quantitative economic traits. UAAS,Kiev,Ukraine However, there are some regions where poly- Family analysis of heredity of amplicon (ISSR- morphic microsatellite (MS) markers are diffi- PCR markers) in 4 generations of Holstein cult to be located, particularly in resource fa- cattle reproduced in alienation zone of Cher- milies derived from the cross between closely nobyl's accident (200ci/km2) was carried out. related breeds, and it may be an obstacle to the The analysis covered 40 animals from 8 fami- precise location of the QTL. Polymorphic lies. Among them 3 families are derived from markers adjacent to non-polymorphic MS 3 cows survived accident in 1986 in this local- markers may be useful instead of the MS ity and the rest - from cows supplied in al- markers in such cases. We report a method for ienation zone after accident in1992-94. All development of single nucleotide polymor- animals of F1,F2,F3 generations were born phism (SNP) markers in a particular genomic from one bull also survived accident in the region using BAC library. As an example, we same place. It was revealed and exposed to tried to detect SNPs in pig chromosome 16 family analysis 124 loci of anonymous DNA (SSC16), where QTL (palmitic acid content) sequences obtained using three dinucleotide has been detected in Jinhua x Large White and seven trinucleotide microsatellite repeat resource family. BAC clones containing three motifs as primers. 57 loci among them were MS markers around the QTL, SWR2480, polymorphous. More polymorphic spectra SW1897 and S0061, which were non- were obtained in 3 families originated from polymorphic in the family, were obtained from cows survived accident in this locality The a swine BAC library. Sequences adjacent to family analysis was not revealed any muta- the MS markers and both ends of the BAC tional event at animals investigated. It was inserts were determined, so that PCR primers found that for 7 out of 10 primers studied the for detection of SNPs were designed inside the quantity of loci in F2 and F3 generation were sequences. SNPs were detected by comparison increased in comparison with F0 generation, of sequences of the PCR products derived from that may be considered as response to influ- the parents of the family using these primers,

128 Section D: Marker, Polymorphism and Biodiversity and analyses on the family were performed School of Veterinary Science, Oslo, Norway; with SNaPshot (Applied Biosystems). Utili- 4Yakutian State Agricultural Academy, Ya- zing these SNPs as markers, a sufficient num- kutsk, Russia; 5Lithuanian Veterinary Aca- ber of informative markers could be provided demy, Kaunas, Lithuania; 6Latvia University of on SSC16 and the region of the QTL could be Agriculture, Jelgava, Latvia; 7Nordic Gene- more precisely restricted. bank for Farm Animals, Ås, Norway; 8Roslin Institute (Edinburgh), Roslin, UK; 9Agrifood D137 Research Finland, Jokioinen, Finland Mapping of 10 genes to porcine chromo- Genetic variation of 24 erythrocyte antigens, some 13 and refining the regions of con- each treated as an independent diallelic locus, served synteny with human chromosome 3 was studied to determine the genetic differ- ences between 71 cattle populations. The data MARIO VAN POUCKE1, MARTINE from Nordic, Baltic and Polish cattle breeds YERLE2, FRANÇOIS PIUMI3, KATHLEEN (31) were pooled with European (37) and Ya- JACOBS1, CARINE GENÊT2, MARC MAT- kutian (3) populations. An iterative procedure THEEUWS1, ALEX VAN ZEVEREN1, LUC was carried out to estimate the allele frequen- J. PEELMAN1 cies. Genetic distances were calculated and a 1Department of Animal Nutrition, Genetics, neighbor-joining tree was constructed from the Breeding and Ethology, Ghent University, distance matrix. The robustness of the tree 9820 Merelbeke, Belgium; 2Laboratoire de topology was evaluated by bootstrapping Génétique Cellulaire, INRA, 31326 Castanet- (1000 replicates over loci). The known histori- Tolosan, France; 3Laboratoire de Radiobiolo- cal relationships between breed groups were gie et d' étude du génome, INRA CEA, 78352 confirmed. Correspondence analysis was used Jouy-en-Josas, France to reveal the major patterns of genetic variation We report here the localization of BAIAP1, among the breeds, based on the allele fre- HTR1F, PTPRG and UBE1C by FISH, quency data at the antigen markers. A two- B4GALT4, BAIAP1, GATA2, IL5RA, LMCD1, dimensional scaling plot was used to illustrate MME and RYK by RH mapping and B4GALT4 the breed relationships. The plot area of the by linkage mapping to Sscr13. The mapping of European breeds did not overlap with the area these 10 different genes (all mapped to Hsap3) of Baltic-Nordic breeds. not only confirms the extended conservation of synteny between Hsap3 and Sscr13, but also D139 defines more precisely the regions with con- Genetic diversity at microsatellite level of served linkage. The syntenic region of the the Brazilian Pantaneiro Horse centromeric part of Sscr13 was determined by isolating BAC clones using primers amplifying FABIANA T. SERENO1, JOSÉ R. SERENO2, porcine microsatellite markers S0219 and PEDRO P. RODRÍGUEZ-GALLARDO3, JO- S0076 (mapped to this region). Sequence com- SÉ L. VEGA-PLA3, JUAN V. DELGADO1 parison of the BAC end STS sequences with 1Departamento de Genética, Universidad de the human working draft sequence showed that Córdoba, España; 2EMBRAPA Pantanal, the centromeric part of Sscr13 is syntenic with Corumbá, Brasi; 3Laboratorio de Grupos San- Hsap3p24. guíneos, Servicio de Cría Caballar, Córdoba, España D138 An analysis of 13 microsatellite loci in 101 Patterns of blood group antigen diversity in animals has been used to define the genetic European and Yakutian cattle populations structure of the Pantaneiro Horse from Brasil. This breed was originated form horses intro- SIRJE VÄRV1, HALDJA VIINALASS1, SA- duced by Spanish and Portuguese colons about RAH BLOTT2, INGRID OLSAKER3, ZOYA three centuries ago. Population was selected to IVANOVA4, ILONA MICEIKIENE5, ZIEDO- resist the adverse conditions of the enviroment NIS GRISLIS6, ERLING FIMLAND7, JOHN L. and now is submitted to a conservation pro- WILLIAMS8, JUHA KANTANEN9 gram where DNA typing will be an important 1Estonian Agricultural University, Tartu, Esto- tool to help to identity and paternity control. nia; 2University of Cambridge and Sygen In- The genetic variation was estimated by allele ternational, Cambridge, UK; 3Norwegian frequencies and average breed heterocygosity.

129 Section D: Marker, Polymorphism and Biodiversity

Nei´s DA distances from Thoroughbred, Ara- ted across all loci was 5.4 in the Gannan bian, Spanish Pure Breed (Andalusian) and sample and 5.5 in the Datong sample. Uruguay Creole horses were calculated show- Heterozygosity estimates for the two yak ing a minimum distance with Spanish Pure populations were comparable to those Breed (0.228) and similar distance from Thor- calculated for the European cattle breeds. oughbred and Arabian (0.355 and 0.332). Dis- Estimated Genetic distance (DA) between the tances were used to construct an UPGMA den- two yak populations was less than the distance drogram. Also an individual tree was obtained between the most closely related of 15 from distances based in shared alleles algo- European cattle breeds suggesting that the two rithm. Results indicate a great diversity level, Chinese yak populations are very closely rela- clear distancing respect to the other breeds and ted. genetic uniformity inside the Pantaneiro Horse. D141 D140 Standardised presentation of SNP- Using bovine microsatellite primers for as- genotypes for paternity testing, individual sessment of genetic diversity in two Chinese identification and genetic distance analysis yak (Bos grunniens) populations FABIAN A. O. WERNER1, GREGOR DUR- WANG MINQIANG1, STEFFEN STEWITZ1,2, GEORG THALLER1, JÖRN WEIGEND2, ASILI BARRE-DIRIE2, JOSEPH MOSNER3, RUEDI FRIES1 W. CARNWATH2, LOU ZHONGLIN3, 1Technische Universität München, Lehrstuhl HEINER NIEMANN2 für Tierzucht, Freising-Weihenstephan, Ger- 1College of Chemical Engineering and Tech- many; 2Agrobiogen GmbH, Hilgertshausen, nology, Biologic Science, Biological Engi- Germany; 3G.A.G BioScience GmbH, Bremen, neering, Applied Chemistry, Yantai University, Germany P.R. China; 2Institute for Animal Science and We propose a standardised set of single Animal Behaviour (FAL); 3Lanzhou Institute of nucleotide polymorphisms (SNPs) as an alter- Animal Science and Pharmaceutic, Chinese native to microsatellites for verification of Academy of Agricultural Sciences, Lanzhou, identity, paternity testing and the analysis of P.R.China genetic distances in animals. Advantages of DNA samples of 48 Chinese domestic yaks SNPs over microsatellites are lower mutation were obtained from two separate herds. 20 rates, no need for a specific typing platform, animals came from a population, classified as suitability for standardisation, straightforward “Plateau type”, maintained at the Gannan data management and availability of high Liqiaru Stock Breeding Farm in Gansu throughput genotyping systems. Standardisati- Province, while 28 animals were sampled from on is achieved by selecting appropriate SNP a herd of “Huanhu type” yaks maintained at loci for a species and defining an order in the Datong Yak Farm in Qinghai Province. A which the genotypes are represented. Each subset of 13 bovine microsatellite markers was SNP position is queried for the presence or selected from the cattle diversity database, absence of a specific base. The three possible CaDBase genotypes are coded in a digital form (homo- (www.ri.bbsrc.ac.uk/cdiv_www/homepage.ht zygous allele 1: ‘10’, heterozygous: ‘11’, ho- m), and the corresponding bovine PCR primers mozygous allele 2: ‘01’). The resulting string were applied to the yak samples. The results of digits is termed digital DNA signature. The indicated that priming sites in the flanking first position is assigned to a sex-specific SNP regions and the microsatellite repeat sequences for gender testing. So far we have developed a themselves are identical in both species. The set of 60 SNP loci in cattle with allele frequen- yak data were compared with data available in cies of at least 0.2 for the less frequent allele the CaDBase for 15 European cattle breeds. within the breeds Holstein Friesian, Brown All loci were polymorphic in yaks and frag- Swiss and Simmental. These markers allow ment sizes overlapped those reported for exclusion powers exceeding 99.99% for pa- European cattle. The allele size distribution in rentage testing and probabilities of identity yaks fit a stepwise mutation pattern. The total lower than 10–11 for individual identification. number of yak alleles ranged from 4 to 8 per We will extend the digital DNA signature to 96 locus; and the mean number of alleles calcula- positions for the bovine species. This signature

130 Section D: Marker, Polymorphism and Biodiversity will also be useful for the assessment of breed 4Department of Veterinary Microbiology and specificity and genetic distance analysis. Parasitology, Sokoine University of Agri- culture, P. 0. Box 3019, Morogoro, Tanzania D142 The diversity of Tanzania's livestock populati- Genome-wide scan of Anal Atresia in pig on provides important and valuable resources (Sus Scofa) for local farmers and food security. However, information on small ruminants'indigenous SABINE WIEDEMANN, RUEDI FRIES, genetic resources remains largely incomplete. GEORG THALLER Two genotyping techniques were used to stu- TU München - Weihenstephan, Chair of Ani- dy the genetic diversity and differentiation of mal Breeding, Germany five Tanzanian local sheep populations from Anal Atresia is a rare disorder with an inci- geographically separated regions (Arusha, dence of 0.1-1.0% in swine. The mode of inhe- Mwanza, Mtwara, Dodoma and Coast). Four ritance is not known, but previous studies indi- primers were used for RAPD amplification cate that the genetic model involves a single and six microsatellite loci were studied in 94 locus in addition to polygenic effects. To iden- unrelated Tanzanian sheep. Two reference tify susceptibility loci for Anal Atresia we breeds from West Africa (West African Dwarf have performed a genome scan using an af- sheep) and England (North Ronaldsay sheep) fected half-sib design. After editing, a total of were also included in the microsatellite study. 27 paternal families consisting of 72 affected A total of 30 RAPD bands and 198 microsa- piglets were included in the analysis. The ge- tellite alleles were detected in the Tanzanian nome scan was carried out with 130 fluo- populations. RAPD average heterozygosity rescent labelled microsatellite markers with an values ranged from 0.137 (Dodoma) to 0.203 average spacing of 20-25 cM over the porcine (Arusha) whereas the microsatellite expected genome. The PCR amplification products were heterozygosity values (H0) ranged from 0.702 run on ABI 377 instruments and genotypes (Mtwara) to 0.763 (Dodoma). The smaller analyzed by GeneScan 3.1 and Genotyper 2.5 microsatellite genetic distance (Ds), observed software. Two-point and multi-point nonpara- within the Tanzania sheep, was between the metric linkage analysis of the pedigrees were Mwanza and the Arusha population (0.019); performed using the computer package Allegro whereas the largest one was between the 1.0. Three chromosomal regions with a nomi- Mtwara and the Mwanza population (0.221). nal significance level of p = 0.05 showed ma- The highest RAPD band-sharing value was ximum NPL scores of 2.57 (p = 0.006), 1.94 (p obtained between the Mwanza and Arusha = 0.028) and 1.90 (p = 0.031), respectively. population (0.552) and the lowest one between The transmission disequilibrium test (TDT) the Mtwara and the Mwanza population was applied to markers in these regions. Signi- (0.302). Overall, genetic relationships among ficant χ2 -test statistics with p-values of 7 x 10-7 the Tanzanian sheep population reflects their and 3 x 10-3 were found for two markers, geographical locations. which proved to be significant after accounting for multiple testing. D144 Characterisation of new variants and geno- D143 typing of the Caprine Kappa Casein gene Assessment of genetic relationship of Tanzanian sheep ecotypes using RAPD MOHAMED H. YAHYAOUI1, and microsatellite DNA markers ANTONELLA ANGIOLILLO2, FABIO PILLA2, ARMAND SANCHEZ1, JOSEP M. JOHN STEPHEN1, CLEMENS B. A. FOLCH1 WOLLNY2, RAMNI JAMNADASS3, OLI- 1Departament de Ciència Animal i dels Ali- VIER HANOTTE3, PAUL S. GWAKISA4 ments, Facultat de Veterinària, Universitat 1Mogabiri Farm Extension Centre, P. 0. Box Autònoma de Barcelona, 08193 Bellaterra, 134, Tarime, Tanzania; 2Institute of Animal Spain; 2Department S.A.V.A., Università del Breeding and Genetics, Georg-August- Molise, Campobasso, Italy University, 37075 Göttingen, Germany; The genetic polymorphism of goat caseins is of 3International Livestock Research Institute, interest due to its relationship with composi- P. 0. Box 30709, Nairobi, Kenya; tion and technological characteristics of milk.

131 Section D: Marker, Polymorphism and Biodiversity

Kappa casein (KCN) is the protein that deter- mapping analysis showed conserved synteny mines the size and specific function of milk between this region of chromosome 2q and the micelles, and its cleavage by chymosin is re- segment of human chromosome 8q, genes sponsible for milk coagulation. We have pre- located on human chromosome 8q are possible viously characterised three variants (A, B, and candidates for this disease. However, there are C) of the KCN in Spanish and French goat few available chicken DNA markers to con- breeds by screening the major part of the cod- struct the high density map for this resion. In ing region in exon 4. Two other variants have this study, we attempted to map more functio- been recently detected in German and Italian nal genes, which located on human chromo- breeds. The full coding region of the KCN some 8q, to chicken chromosomes. Segments gene (exons 3 and 4) has been analysed for of chicken orthologues of human selected ge- polymorphism by the sequencing method. No nes were amplified from parental DNA of the additional mutations were found, with the ex- KU resource family, and the parental alleles ception of a single nucleotide substitution in were sequenced. Sequence polymorphism was exon 3, with no amino acid change. However, identified between resource family parental the analysis of the association between the DNAs. The polymorphism was genotyped the different mutations resulted in two new haplo- backcross panel by PCR RFLP to place genes types, designed F and G. A protocol for rapid on the chicken linkage map. Thirteen genes and simultaneously genotyping of all KCN were mapped to chicken chromosome 2q, indi- variants using the primer extension method cating the high level of conserved synteny was described. A total of 176 animals among between human chromosome 8q21-24 and six European breeds were genotyped. The chicken chromosome 2q. The AM locus was novel variants are detected only in Teramane mapped between DECR1 (6.9cM, 8q21) and and Murciano-Granadina breeds. Alleles A and SDC2 (11.5cM, 8q22). These results suggested B are the most frequent variants in the majority that more genes located on human chromoso- of breeds with a prevalence of the B variant, me 8q21-22 should to be mapped to chicken except for the Canaria breed where allele A is chromosomes in order to identify the causative more frequent. The C variant is present espe- gene of chicken muscular dystrophy. cially in Saanen breed. All other alleles are found at low frequencies and are specific for D146 some breeds. Polymorphism of 12 microsatellites in the Silesian and Thoroughbred horses raised in D145 Poland Functional gene mapping of chicken chro- mosome 2q to identify the causative gene of TOMASZ ZĄBEK, MARIAN DUNIEC, chicken muscular dystrophy EWA SŁOTA National Institute of Animal Production, De- KANAKO YOSHIZAWA1, EUN JUN LEE1, partment of Immuno- and Cytogenetic, Poland KYOKO INABA1, TAKEKI KIKUCHI2, The polymorphism of 12 microsatellites was MAKOTO MIZUTANI3, HIDEYUKI MAN- investigated in the Polish population of 106 NEN4, SOICHI TSUJI4 Silesian and 114 Thoroughbred horses. The 1Kobe University, Graduate School of Science Silesian breed derives from the heavy German and Technology, Kobe, Japan; 2Department of draft horses with the main influence of the Animal Models for Human Disease, National Oldenburg stallions. The Thoroughbred horses Institute of Neuroscience, Tokyo, Japan; had a great influence on the Silesian breed as 3Nippon Institute of Biological Science, Ko- the result of broad crossing the Silesian dams buchizawa, Japan; 4Kobe University, Faculty with the Thoroughbred stallions during the 60s of Agriculture, Kobe, Japan and 70s in the past age. Muscular dystrophy of chicken has been stu- In the study of microsatellite polymorphism died since 1950’s, but the causative gene is not the automated DNA sizing was applied. PCR yet known. Recent our studies revealed that products of 12 microsatellite markers (VHL20, genetic locus for chicken muscular dystrophy HTG4, HTG6, HTG7, HTG10, AHT4, AHT5, of abnormal muscle (AM) was mapped to HMS2, HMS3, HMS6, HMS7, ASB2) were chromosome 2q using the Kobe University amplified with the fluorescently labelled pri- (KU) resource family. Because comparative mers and then analyzed on the automated DNA

132 Section D: Marker, Polymorphism and Biodiversity sequencer (ABI 377). 94 alleles were identified taurus, Bison bonasus and Bison bison re- in the Silesian breed and 73 in the Tho- spectively. Among 33 biochemical systems roughbred horses. The frequencies of most investigated 15 polymorphic ones were re- variants in both breeds were similar, despite vealed. The quantity of polymorphic loci for B. the ones specific for the Silesian breed, which taurus, B.bonasus and B.bison were 9, 6 and occurred very rarely. The mean heterozygosity 6 respectively. Ceruloplasmin, amylase-I and values were close in both breeds (0.740 for the peptidase B were polymorphous in all species Silesian horse and 0.680 for the Thoroughbred) tested. The genetic distances calculated on suggesting the near level of genetic variation base of different RAPD-PCR, ISSR-PCR of studied populations. Calculated standard markers were higher than ones calculated on Nei's genetic distance (0.28) indicate the small the base of biochemical markers. The evalua- degree of differences in the genetic structure of tion of interspecies genetic relationships in both horse breeds, which remains in agreement subfamily Bovinae was essentially depended with historical and studbook data. The com- from the peculiarities of separate molecular bined exclusion probability (PE>0.999) in both genetic marker (locus), included in analysis, in breeds confirms the usefulness of this set of comparison with type of marker (protein markers in parentage verification. polymorphism, variability of DNA repeat dis- tribution). D147 Survey of prion gene polymorphisms influ- D150 encing scrapie resistance in Hungarian Recommendation for allele calling for high flocks throughput genotyping of horses

ATTILA ZSOLNAI, ISTVAN ANTON, SEAN CORLEY1, IAN FINDLEY2, IVAN LASZLO FESUS BIROS1 Research Institute for Animal Breeding and 1The University of Queensland, Australian Nutrition, Herceghalom, Hungary Equine Genetics Research Centre, Brisbane, Our aim is to survey prion gene polymor- Australia; 2The University of Queensland, phisms in different flocks and to identify ani- Australian Genome Research Facility, mals having natural resistance against scrapie. Brisbane, Australia This task requires analysing the known genetic DNA-based genetic profiling using microsa- variances. As for the analysis we would like to tellite markers has become the internationally present a faster and more robust method than accepted method for individual identification PCR-RFLP. Moreover we hope, breeders will and parentage verification both in humans and use our data and initial results in selection. many other wild and domesticated animals including horses. For horse typing, we have D149 adopted laboratory protocols and two panels of The study of genetic differentiation of some microsatellite markers developed by VGL, representatives of subfamily Bovinae using University of Davis, California. Over a 7 different types of markers month period, we have tested the accuracy and reliability of these markers by comparing allele V. I. GLAZKO calling in both control DNA samples and Institute of Agriecology and Biotechnology of 10,000 test samples on the 3700 DNA Analy- UAAS, Kiev, Ukraine ser (Applied Biosystems). We also compared The comparative analysis of genetic differen- the accuracy of allele calling in 400 tested tiation between three species of subfamily samples on three different DNA analysers Bovinae - Bos taurus, Bison bonasus, Bison (3700 DNA Analyser, 377 DNA Analyser bison with the use of different types of mo- (Applied Biosystems) and MegaBACE (Amer- lecular-genetic markers - biochemical (35 loci) sham Biosciences). Data have been statistically and DNA markers (RAPD-PCR, ISSR-PCR) analysed using standard methods (arithmetical was carried out. The part of polymorphic loci average, standard deviation and student t-test). revealed on the base of biochemical markers Our data showed that : in investigated species were 28,5; 17,3 and - variation in allele calling was < 0.1 sD in 17,1 and level of heterozygosity in average per control DNA samples over 7 months locus were 0,131; 0,065; and 0,071 for Bos

133 Section D: Marker, Polymorphism and Biodiversity

- variation in allele calling was < 0.1 sD for Maremmano horse, Thoroughbred and Sarci- all alleles in each test marker dano horse) was analysed. Five unrelated - there is a statistically significant difference horses were chosen for each breed. The mito- (P<0.001, up to 1.5 bp) between empirical chondrial DNA from peripheral blood was and theoretical allele calling between extracted by standard methods. Two primers in smaller and larger alleles the conserved region flanking d-loop were - that both capillary systems (3700 and chosen to specifically amplify the polymorphic MegaBACE) have noticeable allele cal- region using the kit ReadyMixTM (SIGMA). lingvariation compared to the slab gel- Direct sequence of amplicons, carried out by based ABI 377 Big Dye Terminators chemicals (Applied Bio- Our results demonstrate the importance of systems) and automatically analysed by ABI defining the range of sizes of individual alleles PRISM 377 equipped with GeneScan® and across different DNA analyzers particularly for Navigator® softwares (Applied Biosystems), parentage verification and sharing results be- allowed to obtain a consensus sequence of tween 397-bp between sites 15382 and 15778 (Gen- Bank X79547). The obtained sequences were D151 aligned and compared with a reference se- Sequence variation in the mitochondrial quence (GenBank X79547) by BLAST and dloop region of Dahomey and other cattle DNASIS programs. A phylogenetic tree was breeds constructed using d-loop sequence differences using DNAMLK software of PHYLIP pack- SIBYLLE HAHN, INGER VOELKEL, INA age. Only for Giara horses we could find a PFEIFFER & BERTRAM BRENIG high degree of similarity, which can be as- University of Goettingen, Institute of Veteri- cribed to one maternal line. On the contrary, nary Medicine, Goettingen, Germany horse, and The dloop region belongs to the most evolving showed at least two different patterns which segment in the mitochondrial genome. Its se- can be ascribed to two main maternal lines. quence provides a sensitive assay of residual The remaining breeds showed a wider diversity genetic variation in cattle breeds. 400 bp from suggesting the presence of many maternal this region were sequenced and analysed for 10 lines. Dahomeys. These sequences were compared with published sequences from other cattle D153 breeds (Bos indicus and Bos taurus). We Identification, characterisation and linkage found only a small intra-specific genetic mapping of SNPs in porcine skeletal muscle variation in Dahomeys. However, variation genes was present and the haplotypes of the Da- homeys correspond more to Bos indicus than ROBERTA DAVOLI, LUCA FONTANESI, to Bos taurus. In the examined region we were SILVIA BRAGLIA, MAURIZIO CORSARO, able to detect four diagnostic nucleotide posi- BARBARA LAMA, MASSIMO CAG- tions. Two of this nucleotide positions might NAZZO, VINCENZO RUSSO suggest a convergent evolution of bos indicus DIPROVAL – Sezione di Allevamenti Zootec- and Dahomey. nici, Faculty of Agriculture, University of Bo- logna, Reggio Emilia, Italy D152 Porcine skeletal muscle genes can be consid- Study of mitochondrial d-loop DNA se- ered candidates for meat quality and produc- quence variation in some Italian horses tion traits as meat derives mainly from skeletal muscle tissues. With the aim to identify these MARIA C. COZZI, MARIA G. STRIL- genes, we isolated about 1000 ESTs from an LACCI, MARIA LONGERI, MARTA ZA- adult porcine skeletal muscle cDNA library. NOTTI Then, to develop DNA markers that could be Istituto di Zootecnica, Faculty of Veterinary useful in association studies with meat quality Medicine, Milan, Italy and production traits, we started a sistematic The genetic variability of mitochondrial d-loop approach to identify single nucleotide poly- DNA sequence in seven horse breeds (Giara morphisms (SNPs) in these ESTs. PCR prim- horse, Haflinger, Italian trotter, Lipizzan horse, ers were designed to amplify fragments of 34

134 Section D: Marker, Polymorphism and Biodiversity genes expressed in this tissue. To search for venteen loci are combined and amplified in a polymorphisms, we carried out PCR-SSCP single PCR reaction, dramatically improving analyses on genomic DNA pooled from 20 the power of statistical tests for pedigree ana- pigs and on individual DNA. Moreover se- lysis while reducing the time and labor requi- quencing was performed to confirm the pres- red to perform such tests. ence of DNA mutations. On the whole we analyzed ~6100 bp and identified SSCPs in 13 D155 genes. So far, sequencing was performed for Critical parameters for the optimization of 12 genes (ATP1A2, CA3, CTSL, DECR1, short tandem repeat multiplex polymerase HUMMLC2B, MYH4, Myopalladin, PSMA4, chain reactions SLN, TNNT3, TTN, and ZASP) and we identi- fied 15 SNPs, 1 insertion/deletion and 2 micro- ANTHONY FISHBACK, MATTHEW satellites. Allele frequencies at these loci were SHAUNESSY, TYLER ARCHBOLD, WAY- studied in six different pig breeds (Large NE MURRAY White, Landrace, Duroc, Belgian Landrace, Maxxam Analytics, Genetic Identification Piétrain and Hampshire). Moreover, linkage Division, Guelph, Canada mapping was obtained for TNNT3 (Sscr 2), Short Tandem Repeat (STR) loci have become HUMMLC2B (Sscr 3), ATP1A2 and DECR1 the genetic marker of choice for parentage (Sscr 4), PSMA4 (Sscr 7), CRYAB and SLN determination and population differentiation. (Sscr 9) and MYH4 (Sscr 12). Several STR loci are required for such studies in order to obtain an appropriate amount of D154 genetic polymorphism. Fortunately, genotypic Development of a 17-plex Microsatellite data collection has become efficient via the co- PCR Kit for the Genotyping of Horses amplification of multiple loci (multiplexing) in a single Polymerase Chain Reaction (PCR) PERO DIMSOSKI using fluorescently labeled DNA primers. Applied Biosystems, Foster City, USA However, the development of a robust multi- Microsatellite-based molecular markers are plex system presents many challenges. Allele- used for the genotyping of horses, primarily for scoring difficulties can result due to the pro- pedigree verification. The only commercially duction of stutter bands during the amplificati- available kit, StockMarks for Horses (Applied on of dinucleotide repeat STR loci, and Biosystems, Foster City, USA), consists of 12 through variability in “plus A” modifications primer sets labeled with 3 different fluorescent (non-templated adenylation of the 3’ end of the dyes, which are amplified in two multiplex amplified sequence by Taq polymerase) of PCR reactions. The recent introduction of PCR products. Nucleotide substitutions in the medium- and high-throughput genotyping in- DNA sequence of a primer-binding site can  struments (ABI PRISM 3100 DNA Genetic also result in allele scoring difficulties. The  Analyzer and ABI PRISM 3700 DNA Genetic manual editing required to correct misidenti- Analyzer) required the development of a new fied alleles greatly inhibits the efficiency of equine genotyping kit that would match the automated genotyping systems. We will show increased performance of the instruments. that adjusting thermocycling parameters and This new equine kit contains five extra loci various concentrations of PCR reagents can (ASB17, LEX3, HMS1, CA425, and ASB23) eliminate the production of misidentified alle- in addition to the twelve original ISAG- les in PCR multiplex systems containing up to recommended loci (VHL20, HTG4, AHT4, 15 STR loci. HMS7, HTG6, HMS6, HTG7, HMS3, AHT5, ASB2, HTG10, and HMS2). This is accom- D156 plished through the use of a new set of five Radiation hybrid map of bovine genome fluorescent dyes developed by Applied Biosy- using microsatellites and AFLPs 1 1 stems (DS-31) with four of the dyes being used C. GORNI , R. NEGRINI , M. J. T. VAN 2 2 3 to label the forward amplification primers (6- EIJK , H. HEUVEN , A. VALENTINI , P. 1 4 FAMTM, VICTM, NEDTM, and PETTM) in each AJMONE-MARSAN , J. L. WILLIAMS 1 primer set. An in-lane size standard labeled Istituto di Zootecnica, Università Cattolica 2 with the fifth dye (LIZTM) provides accurate del S. Cuore, Piacenza, Italy; Keygene N. V., 3 size determination for genotyping. These se- Wageningen, The Netherlands; Dipartimento

135 Section D: Marker, Polymorphism and Biodiversity di Zootecnica, Università della Tuscia, sheep than other animals. A comparison of the Viterbo, Italy; 4Roslin Institute (Edinburgh), cDNA sequence among cattle revealed ten Roslin, United Kingdom single nucleotide polymorphisms (SNPs) in the Radiation hybrid (RH) cell panels are a very encoding region and one nucleotide deletion in efficient tool for ordering monomorphic mark- 3’-untranslated region. Among these SNPs, a ers therefore they are particularly useful for G to A substitution in the pro-fragment region mapping genes and ESTs and building com- and a change from G to C in the heavy-chain parative maps. A 3000 Rad bovine-hamster region resulted in a glycine to serine and a RH panel has been recently developed through glycine to alanine substitution, respectively. an EC sponsored collaboration. We have in- Nine different cattle breeds were screened for vestigated the use of AFLP technology to in- the presence of both polymorphisms using crease the number of markers available on the PCR-RFLP analysis. Three Japanese beef RH map as anchor points for the construction breeds (Japanese Black, Japanese Shorthorn of a physical map of BAC or YAC contigs. and Japanese Brown) and two zebu breeds The 94 cell lines of the RH panel were typed (Brahman and Santa Gertrudis) contained these with 37 AFLP EcoRI/TaqI primer pairs carry- polymorphisms, whereas four European breeds ing respectively 4 and 3 selective nucleotides (Holstein-Friesian, Jersey, Aberdeen-Angus at their 3' end. A total of 740 bovine specific and Hereford) did not. AFLP bands were clearly distinguishable from the hamster profiles. Their retention frequency D159 averaged 0,179 and ranged from 0,011 to A genetic map for Atlantic salmon (Salmo 0,775. AFLP markers were incorporated into salar). the existing 1200 microsatellite RH framework map using the program Carthagene. More than KATRINE HANES1, SIGBJ∅RN LUNNER1, 600 AFLPs were linked to the framework map JOHN TAGGART2, RICHARD POWELL3, at LOD 6 or more. Unlinked AFLP markers LARS-ERIK HOLM4, RENÉ GUYOMARD5, may represent i) bands heterozygous in the ROY DANZMANN6 & BJ∅RN H∅YHEIM1 donor bovine cell line and therefore only a 1Norwegian School of Veterinary Science, single allele is detected; ii) bands originating MGA-Genetics, Oslo, Norway, 2University of from repetitive DNA templates; iii) bands too Stirling, Institute of Aquaculture, Stirling, far away from nearby mapped markers to show Scotland, 3National University of Ireland, significant linkage in a high resolution map, Department of Microbiology, Galway, Ireland, which is unlikely, or iv) bands that are hard to 4Danish Institute of Agricultural Sciences, distinguish and hence with high typing errors. Department of Animal Breeding and Genetics, Tjele, Denmark, 5INRA, Laboratoire de géné- D158 tique des poissons, Jouy-en-Josas, France and The cDNA sequence and polymorphisms of 6University of Guelph, Department of Zoology, bovine procathepsin-D Guelph, Canada Building a genetic map in salmonids requires 1 MIKITO HIGUCHI , NORIKAZU special attention when compared to developing MIYASHITA2, YOSHITAKA NAGAMINE1, a map in mammals. The salmonid genome has 1 2 AKIRA WATANABE , TAKASHI AWATA undergone duplication in relatively recent 1 National Agricultural Research Center for evolutionary time and therefore shows residual Tohoku Region, Department of Animal Pro- tetraploidity. Although many genes have been duction and Grassland Farming, Morioka, extinguished after the duplication event the Japan; 2National Institute of Agrobiological fact that areas of the genome is duplicated in Sciences, Institute of Insect and Animal Scien- an almost identical manner makes the deve- ces, Tsukuba, Japan lopment of a genetic map challenging. Atlantic The cDNA sequence of the bovine cathepsin-D salmon also show a considerable difference precursor, procathepsin-D was determined by with respect to recombination frequencies bet- RT-PCR with primers designed according to ween the male and female map. There is little the human procathepsin-D cDNA sequence. or no recombination in the males while the Both the bovine procathepsin-D cDNA se- females seems to exhibit normal recombinati- quence and deduced protein sequence exhibi- on. This makes assigning a specific marker to a ted higher homology to their counterparts in linkage-group relatively easy using male data

136 Section D: Marker, Polymorphism and Biodiversity while the data from females must be used to not mapped microsatellites are being genoty- construct a linkage-map. A first generation ped. With the addition of these 1100 microsa- genetic map for Atlantic salmon was con- tellites alone, the USDA-MARC cattle linkage structed in the EU-project: Generation of hig- map will contain at least 2300 DNA markers hly informative DNA markers and genetic with 1.3 cM average intervals. marker maps of salmonid fishes (SALMAP). In this project a total of 447 microsatellite loci D161 was fully charcterised (363 from Atlantic sal- Development and typing of Single Nucleoti- mon, 57 from rainbow trout and 27 from de Polymorphism markers in a QTL region brown trout). 300 markers (299 DNA-markers for fatness traits on porcine chromosome 2 + sex) were genotyped in the reference fami- lies and subsequently mapped by linkage- BART JUNGERIUS1, ANNEMIEKE RAT- analysis. We have expanded this map by de- TINK1, BARBARA HARLIZIUS1,2, BER- veloping 520 new microsatellite markers and NARD VAN OOST3, MARINUS TE PAS4, to date we have added 150 of these to the map. MARTIEN GROENEN1 1Wageningen University, Animal breeding and D160 Genetics Group, Wageningen Institute of Ani- Mapping of over 1100 bovine polymorphic mal Sciences, Wageningen, The Netherlands; microsatellite markers to the USDA-MARC 2Present adress: Institute for Pig Genetics, cattle linkage map Beuningen, The Netherlands; 3Utrecht Univer- sity, Department of Clinical Sciences of Com- NAOYA IHARA1, AKIKO TAKASUGA1, panion Animals, Faculty of Veterinary Medici- KAZUNORI MIZOSHITA2, HARUKO TA- ne, The Netherlands; 4Institute for Animal Sci- KEDA1, MAYUMI SUGIMOTO3, GARY L. ence and Health (ID-Lelystad), The Nether- BENNETT4, KENT M. REED5, CRAIG W. lands BEATTIE6, YOSHIKAZU SUGIMOTO1 A maternally imprinted quantative trait locus 1Shirakawa Institute of Animal Genetics, Fu- (QTL) for backfat thickness (BFT) has been kushima, Japan; 2Cattle Breeding Develop- identified on the p-arm of porcine chromosome ment Institute Kagoshima, Kagoshima, Japan; 2 (SSC2p). To reduce the size of the interval of 3National Livestock Breeding Center, Fukus- the QTL and eventually be able to identify the hima, Japan; 4USDA-ARS, U.S. Meat Animal underlying genes responsible for the observed Research Center, Clay Center, NE, USA; effects additional DNA markers are needed. In 5University of Minnesota, Department of Vete- order to increase the marker density on SSC2 rinary PathoBiology, St. Paul, MN, USA; single nucleotide polymorphism (SNP) 6University of Nevada, Department of Animal markers were developed by sequencing PCR Biotechnology, Reno, NV, USA products amplified from genomic DNA of 8 Polymorphic microsatellite markers are parti- individuals from different breeds. Until now cularly important to construct a genetic linkage over 200 SNPs were identified in 36 sequence map. In cattle, a second-generation linkage tagged sites (STS) covering 80 cM of SSC2 map has been constructed with 1236 DNA including the entire P-arm. Currently these markers (Kappes et al., 1997). The linkage SNP markers are being used for detailed map has been useful for mapping loci of gene- haplotyping of animals from the experimental tic diseases as well as economically important QTL mapping cross. The SNPs are assayed in traits in cattle. Furthermore, an important as- multiplex single base extension reactions using pect of strategy for physical map construction, the ABI Prism SnapShot kit and analysed on such as an RH map, is the use of many, orde- an automated sequencer (ABI377). After gel red DNA markers on a linkage map to deter- analysis using Genescan 3.1.2 and creating a mine the relative orders of linkage groups. To table containing all signal peaks, the genotypes further facilitate the power of linkage map, we are assigned in a highly automated way in a have developed microsatellites and mapped MS Excel workbook designed for this purpose. them to the USDA-MARC cattle linkage map. The output from this workbook can be easily We have newly developed over 800 poly- formatted to match database import file or files morphic microsatellites and mapped approxi- for linkage analysis using CRIMAP. mately 400 microsatellites. The remaining 400 microsatellites and 300 already developed, but

137 Section D: Marker, Polymorphism and Biodiversity

D162 between HSA1p35-p36 and SSC6 by using Screening of Bos indicus and Bos taurus human STS markers directly for RH map- cattle breeds for DGAT1 polymorphism ping

B. KAUPE1, A. WINTER5, E. IBEAGHA1, C. SACHIKO KIUCHI1, YING ZHANG CHEN2, ÖZBEYAZ2, J. L. WILLIAMS3, P. AJMONE- HIROHIDE UENISHI1, TAKESHI HA- MARSAN4, R. FRIES5, G. ERHARDT1 YASHI1, EIICHI SOEDA2, HIROSHI YA- 1Justus-Liebig-University of Giessen, Depart- SUE1 ment of Animal Breeding and Genetics, Gies- 1National Institute of Agrobiological Sciences, sen, Germany; 2Department of Zootechnics, Genome Research Department, 2 Ikenodai, University of Ankara, Turkey; 3Roslin Institute Kukizaki, Inashiki, Ibaraki 305-0901, Japan; Edinburgh, Scotland, United Kongdom; 2Institute of Physical and Chemical Research 4Institute of Zootechnics, Catholic University (RIKEN), Tsukuba Institute, Gene Bank, 3-1-1 of S. Cuore Piacenza, Italy; 5Technische Uni- Koyadai, Tsukuba, Ibaraki 305-0074, Japan versität München, Lehrstuhl für Tierzucht, Since the human genome organization was Freising-Weihenstephan, Germany elucidated, much effort has been made to con- In cattle the diacylglycerol O-acyltransferase struct comparative map between swine and gene (DGAT1), which maps to the centromeric human. The determination of the synthenic end of BTA14, is suspected to be a positional relationship between swine and human chro- candidate gene with a major effect on milk fat mosomes is extremely important for the selec- content and other milk characteristics, as re- tion of candidate genes responsible for traits. ported recently for German Simmental and In the present study, we have attempted to use Dutch/ New Zealand Holstein Friesian popu- 910 STSs in HSA1p35-p36 (35Mb) for con- lations. We genotyped 821 DNA samples from struction of a denser comparative map between 17 cattle breeds including Bos taurus and Bos human and swine chromosomes. Thirteen pri- indicus from 5 different countries on 3 conti- mer pairs of 910 STSs, which are evenly loca- nents (Europe, Africa, Asia), by applying a ted in the HSA1p35-p36 spanning 35Mb, PCR-RFLP test based on the K232A could be used for assignment of the correspon- (AAG→GCG) substitution. The lowest fre- ding STSs to the IMpRH. Eleven STSs were quency of the AAG allele was found in Bos assigned to the framework markers of the taurus type cattle e.g. Hereford (0.00), while IMpRH framework map in SSC6 with lod highest AAG allele frequencies were harbored scores greater than 5 by two-point analysis. by Bos indicus type cattle, like Banyo Gudali WI-20819 was revealed to link to SW1044 (0.86) and White Fulani (0.92). Beef breeds localized in SSCX with a lod score of 7.40; had lower AAG allele frequencies, ranging this result was consistent with that obtained by from Hereford (0.00) through Piemontese analysis using somatic cell hybrid panel. The (0.03) to Chianina (0.38) cattle, compared to remaining one, R91D18R, was suggested not dairy breeds with range from Ayrshire (0.02), to link to the framework markers of SSC6 but British Friesian (0.03) through Holstein Frie- to link to MX1 localized in SSC13 with a lod sian (0.56) to Jersey (0.69). Non-selected score of 2.37. In order to elucidate most likely breeds like South Anatolian Red (0.20), East position of the 11 STSs in the framework map Anatolian Red (0.25), Turkish Grey Steppe of SSC6, RH mapping score data of the 11 (0.36) and Anatolian Black (0.51) had interme- STSs together with those of SSC6 framework diate allele frequencies. It remains to be seen if markers were subjected to the calculation using the frequency of haplotypes carrying the Lysi- Carthagene software. This revealed that the ne residue increased in recent years due to alignment of the 11 STSs with a direction from changes in selection criteria. It is conceivable SSC6cen to q-arm telomere was conserved in that the AAG allele could have been intro- HSA1 with a direction from pter to centrome- gressed from Bos indicus cattle into Bos taurus re. cattle after domestication and more recently through introduction of zebu cattle into D164 southern Europe. Integrated physical and linkage mapping of bovine chromosome 24 D163 Construction a dense comparative map ERCAN KURAR1,2, JAMES E. WOMACK3,

138 Section D: Marker, Polymorphism and Biodiversity

BRIAN W. KIRKPATRICK1 genotypes in order to estimate genetic vari- 1University of Wisconsin-Madison, Department ability within and between 8 cattle breeds from of Animal Sciences, Madison, WI, USA; Italy (Chianina and Grigio-Alpina), France 2Faculty of Veterinary Medicine, University of (Limousine -Italian strain- and Normande), Selcuk, Konya, Turkey; 3Department of Veteri- Spain (Betizu and Menorquina), Norway nary Pathobiology, Texas A&M University, (Telemark) and Denmark (Jutland). Expected College Station, TX, USA heterozygosities were relatively high in Italian We present herein a bovine chromosome 24 Limousine, Chianina and Grigio-Alpina (BTA24) radiation hybrid (RH) map using 40 (Het=0.24, 0.23 and 0.23, respectively), aver- markers scored on a panel of 90 radiation hy- age in Normande and Betizu (0.21), and rela- brids. Of these markers, 29 loci were ordered tively low in Jutland, Menorquina and with odds of at least 1000:1 in a framework Telemark (0.19, 0.19 and 0.17, respectively). map. An average retention frequency of 17.4% Gst value indicates that 77 % of the AFLP was observed, with relatively higher frequen- variation is maintained within breeds. Da dis- cies near the centromere. The length of the tances between breeds range from 0.04 comprehensive map was 640 cR5000 with an (Telemark-Jutland) to 0.10 Chianina-Betizu average marker interval of approximately 17.3 and in general reflect geographic distances cR5000. The observed locus order is generally between areas of origin. PCOOA based on consistent with currently published bovine Jaccard distance between individuals groups linkage and physical maps. Nineteen markers animals according to their breed of origin. were either Type I loci or closely associated However, it also indicates the genetic original- with expressed sequences and thus could be ity of the Chianina, which is probably the most used to compare the BTA24 RH map with ancient Italian autochtonous cattle breed, and human mapping information. All genes located of the Spanish Betizu, which is autochtonous on BTA24 were located on human chromoso- in the Basque and Navarra regions. me 18 and supported the previously reported regions of conserved synteny. The comparative D166 data revealed the presence of at least six con- Analysis of polymorphism of two horse ge- served regions between these chromosomes. nes potentially important in genetic resi- stance to intracellular bacteria D165 Genetic diversity of Italian, French, Spanish JÁN MATIAŠOVIC, LEONA KŘENKOVÁ, and Nordic cattle breeds as assessed by PETR HOŘÍN AFLP markers University of Veterinary and Pharmaceutical Sciences, Institute of Animal Breeding and R. NEGRINI1, E. MILANESI1, M. ZA- Genetics, Brno, Czech Republic NOTTI2, K. MOAZAMI-GOUDARZI3, C. For further association and expression analysis, RODELLAR4, I. OLSAKER5, L.-E. HOLM6, two genes involved in resistance to intracellu- I. J. NIJMAN7, J. A. LENSTRA7, P. lar bacteria were screened for polymorphic AJMONE-MARSAN1 markers. The 5' UTR, 3' UTR and a part of the 1Institute of Zootechnics, Catholic University CDS region were screened within the CD14 of S. Cuore, Piacenza, Italy; 2Institute of gene, encoding the LPS receptor. No polymor- Zootechnics, University of Milano, Italy; phism was found using PCR-SSCP and/or 3INRA, Jouy-en-Josas, France; 4Facultad Vet- sequencing. The second gene, iNOS, was te- erinaria, Zaragoza, Spain; 5Division of Ge- sted using PCR-SSCP, PCR-RFLP and se- netics, The Norwegian School of Veterinary quence analysis. A part of this gene comprising Science, Oslo, Norway; 6Danish Veterinary intron 8 down to exon 10 was analyzed. A Laboratory, Copenhagen, Denmark; 7Faculty PCR-RFLP polymorphism for MspI due to a of Veterinary Medicine, Utrecht Univerity, The C/T substitution was found in intron 9. Homo- Netherlands zygotes for both alleles as well as heterozygo- The EU-sponsored Resgen project (CT98-118) tes were identified. Inter-breed differences is investigating genetic diversity in European between Thoroughbred and Old Kladruber in cattle by assessing molecular markers and allelic frequencies were observed. variation in selected genes. A total of 98 AFLP markers have been assayed on 160 cattle

139 Section D: Marker, Polymorphism and Biodiversity

D167 D168 Integration of Genetic and Radiation Hy- Evaluation of paternity by microsatellite brid maps of the pig: The second generation analysis over two breeding seasons in IMpRH maps American bison bulls (Bison Bison) held under semi-natural conditions DENIS MILAN1A, THOMAS SCHIEX1B, MARTINE YERLE1A, ANETTE RINK2, CATHERINE RODEN1,2, GUY MOMMENS3, CRAIG BEATTIE2, LEE ALEXANDER3, HILDE VERVAECKE1,2, LINDA VAN EL- RACHEL HAWKEN3, LARRY SCHOOK3, SACKER1,2 HIROSHI YASUE4, DANIEL POMP5, ME- 1Centre for Research and Conservation, Royal RETE FREDHOLM6, GARY ROHRER7 Zoological Society of Antwerp, Antwerp, Bel- 1Cellular Genetics Lab & Biometry and Artifi- gium; 2University of Antwerp, Department of cial Intelligence Lab., INRA, Castanet- Biology, Wilrijk, Belgium; 3PolyGenLab, Tolosan, France; 2University of Reno, USA; Malle, Belgium 3University of Minnesota, USA; 4NIAS & Extreme sexual dimorphism -shown by bison- STAFF, Tsukuba, Ibaraki, Japan; 5University has been theoretically related to a high vari- of Nebraska-Lincoln, USA; 6Royal Veterinary ance in male reproductive success. The as- and Agricultural University, Frederiksberg, sumption that only some males breed a very Denmark; 7USDA-MARC, Clay Center, Ne- large part of the female herd has been raised by braska, USA studies in the wild, but has never been sup- More than 4500 markers, ESTs and genes have ported by genetic data. The American bison been mapped on IMpRH radiation hybrid panel (Bison bison) became a near-extinction species and submitted to IMpRH Server before 30 at the beginning of the 20th century and knew a March 2002, whereas 757 markers only were revival mostly through bison farming. Al- mapped on the first generation map (Hawken though the species has become economically et al, 1999). To take advantage of the different important, there is still a great lack of informa- resolutions observed on the genetic and the RH tion on their reproductive behaviour and ca- maps, maps were constructed with Carthagene pacity. During the breeding season, bison software using SIMULTANEOUSLY genetic herds, consisting of females and calves, are (MARC USDA families) and RH (3800 joined by several adult males in search of markers mapped on IMpRH panel) data. For mating partners. This natural pattern is mim- each chromosome, a framework map was pro- icked under semi-natural conditions. The aim duced using a step-wise locus adding strategy of this study is to assess the assumption of (SWLA). The resulting map was tested by extreme male reproductive variance for bison various procedures (flips, simulated annealing bulls on a bison farm. We present paternity ...) to try to find alternate better maps and to data of 4 groups (yr 1: n=19, 31, 38 and 52; yr check if the difference of LOD with the second 2: n=27, 36, 41 and 43) of American bison most likely map is at least of 3. Frequently, over two years held under semi-natural condi- using checking procedures, we were able to tions in Belgium, Europe. By analysing blood, identify maps demonstrating that the frame- hair and tissue samples using eight to ten work map proposed by the SWLA strategy was polymorphic microsatellites, the annual repro- not a framework map. Framework maps were ductive ‘success’ of the bulls is successfully also manually improved to ensure the best determined. We compare the annual success possible coverage of each chromosome. Addi- between years and individuals. The use of mi- tional markers were mapped relatively to the crosatellites for paternity assignment has framework maps, and comprehensive maps proven to be very useful in bison in spite of were established. their relative low genetic variance due to their The aim is to propose soon reliable second historic genetic bottleneck. generation maps to assign and map additional markers and genes. Data and maps will be D169 available on IMpRH Server Development of microsatellite multiplexes (http://imprh.toulouse.inra.fr). Groups which for use in a Swiss alpine chamois (Rupicapra contributes with at least 100 markers share r. rupicapra) population study using primers authorship of this poster. A total of 19 groups designed from domestic Bovidae submitted data on IMpRH Server.

140 Section D: Marker, Polymorphism and Biodiversity

JEANNETTE MUNTWYLER, GABRIELA Justus Liebig Universität, Giessen, Germany; OBEXER-RUFF, CLAUDE GAILLARD, 10Institute of Zootechnics, Catholic University of MARIE-LOUISE GLOWATZKI-MULLIS S. Cuore, Piacenza, Italy; 11Istituto di Zootecnica, University of Berne, Institute of Animal Gene- Universitá della Tuscia, Viterbo, Italy; 12Instituto tics, Nutrition and Housing, Berne, Switzer- di Zootecnica, University of Milano, Italy; land 13Facoltà di Agragria, Campobasso, Italy; In order to enable a rapid and low-cost method 14Division of Genetics, The Norwegian School of to conduct population genetic studies in Swiss Veterinary Science, Oslo, Norway; 15Gyvunu alpine chamois, we developed multiplex-PCR Genetikos Laboratorija, Kaunas, Lithuania reactions. The molecular tool of choice for the We present a preliminary analysis of the diver- genetic analysis in this study are microsatellite sity of European cattle based on Amplified DNA markers designed from domestic Bovi- Fragment Length Polymorphisms (AFLP). As dae that amplified successfully in a cross- part of an EU project (Resgen CT 98-118), species approach. We have evaluated various animals (n=20) of 30 cattle breeds covering microsatellite primers that demonstrate poly- Europe were sampled with West African morphism in biodiversity and parentage studies N'Dama and Indian zebu (Bos indicus) as out- in small ruminants (M.-L.G., personal commu- groups. This yielded two datasets: (1) 239 nications; Saitbekova et al., 1999 & 2001). In AFLP fragments, of which 165 were polymor- addition, we are taking part in the 2001/2002 phic in 20 animals per breed and (2) 329 frag- ISAG comparison test of sheep and goats. ments, of which 309 were polymorphic in a From all markers tested, we have selected 30 subset of 5 animals per breed. Genetic dis- loci rendering sufficient polymorphism in a tances expressed as (1- Jaccard index or band sample of 30 Swiss alpine chamois which can sharing) and PCO plots clearly showed a sepa- be amplified in multiplex reactions. Genoty- ration between the zebu and cattle breeds. Dis- ping analysis was undertaken using an ABI tances between zebu and taurine breeds were 3100 genetic analyser. The multiplexes de- about twice the distances between taurine scribed may also be of interest for other gene- breeds, but from the taurine breeds the West tic epidemiologic studies focused on alpine African N’Dama as well as the Hungarian Grey chamois (Rupicapra r. rupicapra) species. are closer to the zebus. Distances between breeds from the same country were often rela- D171 tively low, suggesting an effect of the geo- Genetic diversity of European cattle breeds graphical distances. Intrabreed distance values inferred from AFLP fingerprinting of taurine breeds are comparable, indicating a similar genetic variability within each breed. THE EUROPEAN GENETIC CATTLE DI- However, in some breeds we found a high VERSITY CONSORTIUM (I. J. NIJMAN1, J. spread of the individual distances, which was A. LENSTRA1, G. MOMMENS2, G. DOLF3, also apparent from PCO plots and may be an P. WIENER4, D. BURTON4, J. L. WIL- indication of stratification. Intrabreed values LIAMS4, K. MOAZAMI-GOUDARZI5, D. are about 80 % of the interbreed values. This LALOË5, S. DUNNER6, J. CANON6, P. ZA- suggests that the heavy selection on production RAGOZA7, C. RODELLAR7, A. SANCHEZ8, traits or other phenotypes in some of the breeds G. ERHARDT9, O. JANN9, C. WEIMANN9, may have homogenized only a small part of the P. AJMONE-MARSAN10, R. NEGRINI10, A. genome, while much of the total genetic variety VALENTINI11, M. ZANOTTI12, F. PILLA13, still has been retained. A. BRUZZONE13, D. IAMARTINO13, I. OL- SAKER14, I. MICEIKIENE15) D172 1Faculty of Veterinary Medicine, Utrecht Unive- Linkage mapping of 4 calpain genes to chik- rity, The Netherlands; 2PolyGenLab, Malle, ken chromosomes Belgium; 3Institute of Animal Breeding, Nutrition and Housing, University of Berne, Switzerland; FUMIHIKO OKUMURA1, YOKO SHINBO1, 4Roslin Institute (Edinburgh), Roslin, UK; 5INRA, KANAKO YOSHIZAWA2, KOTARO KA- Jouy-en-Josas, France; 6Facultad Veterinaria, WABE1, TAKESHI SHIMOGIRI1, HIDEYU- Madrid, Spain; 7Facultad Veterinaria, Zaragoza, KI MANNEN2, SHIN OKAMOTO1, HANS H. Spain; 8Facultad Veterinaria, Barcelona, Spain; CHENG3, YOSHIZANE MAEDA1 9Department of Animal Breeding and Genetics, 1Kagoshima University, Faculty of Agriculture,

141 Section D: Marker, Polymorphism and Biodiversity

Kagoshima, Japan; 2Kobe University, Faculty 3.8% of SCID carriers (9/238). Homozygous of Agriculture, Kobe, Japan; 3USDA/ARS, recessive animals were not found because all Avian Disease and Oncology Laboratory, East horses analyzed were older than four month, Lansing, USA and at this age the affected animals were al- Calpain is a Ca2+-requiring cysteine protease. ready dead. Checking the carriers' pedigrees it In animals, calpain forms a large gene family was possible to confirm the participation of comprising more than 15 members. In recent one stallion identified as the possible disease studies, it is found that mu-calpain linked to disseminator. Considering the economic losses postmortem tenderization of muscle in bovine. that could come from the unknowing use of Our studies show mu/m-calpain activity diffe- carrier stallions, the results show the impor- rentiates between quail lines selected by body tance of the adoption of a control program by weight. As mentioned above, calpains are va- the Brazilian Arabian Horse Registry (AB- riable enzymes in livestock industry. Many CCA). calpain genes have already mapped to mam- malian chromosomes, while only p94 gene D174 mapped to chicken chromosome 5 (GGA5). Efficacy and reliability of nine DNA micro- Here, we developed PCR-RFLP of 4 calpain satellite analysis in Brazilian Zebuin Cattle genes expressed in chicken muscle (mu- calpain, mu/m-calpain, m-calpain and p94), KÁTIA T. SOUZA, CRISTIANE L. OLIVEI- and located their markers on 2 backcross fami- RA, VALÉRIA P. M. CALIJORNE, MÁRCIA lies (East Lansing reference population and C. B. N. CAMPOS1, CARLA L. P. MENE- Kobe University resource family). In human, 3 ZES1,2 calpain genes (mu-calpain, m-calpain and p94) 1Biocod Biotecnologia Ltda.; 2FAPEMIG mapped to HSA1, HSA11, and HSA15, re- It is important for bovine DNA testing labora- spectively. mu/m-calpain gene is not identified tories to provide the cattle industry with accu- in human. As a result, we could develop 3 rate estimates of the efficacy and reliability of calpain makers and obtain their segregation DNA tests offered. To address these issues for data from 2 families. These results showed that genealogy e registry service of the Cattle m-calpain and mu/m-calpain genes mapped to Breeders, discriminate power were obtained GGA3, and that p94 gene mapped to GGA5 as from breed panel data. previously described. Map distance from m- We have tested multiplex combinations over calpain gene to mu/m-calpain gene was about our Zebuin breed panels to provide an indica- 30cM. It was found that m-calpain gene was tion of the efficacies of selected markers for located on the syntenic region of HSA1 pre- parentage and paternity testing. The Zebuin viously reported. We found that p94 gene si- Breed panels consist of 80 samples from unre- gnificantly linked to RYR3 (ryanodine recep- lated animals of Brahman, Nelore and Guzera tor 3) located on HSA15. Now, m-calpain gene breeds. is mapping to chicken chromosome. The nine loci are amplified in two PCR reac- tions: a multiplex with 05 microsatellites D173 (TGLA 227, TGLA 126, TGLA 122, M 1824 Prevalence of the Severe Combined Immu- and ETH 10) and a multiplex with 04 (TGLA nodeficiency Disease (SCID) in Arabian 57, ETH 225, TGLA 53, BM 1818). horses raised in Brazil The PCR reactions were performed in 10 µl containing: 1 µl of STR 10x buffer, 0,5U of Taq DENISE A. A. OLIVEIRA, CLÁUDIA S. polymerase (Promega Corp.),working solutions TEIXEIRA, MARCELO Y. KUABARA of primers in variable concentrations (1 a 2,5 Escola de Veterinária da UFMG, µM/sample) and 50 ng of DNA [email protected], Belo Horizonte-MG, PCR cycles were 95ºC 1 min, 55ºC 1 min, 72ºC 2 Brazil min at 35 times. Gel electrophoresis and genotype In the present study, 238 Arabian horses raised determination were performed on the ALF in Brazil, were genotyped using the PCR tech- express sequencer (Amersham Science Corp.). nique to detect the presence of the mutant gene We find out that Power of Discrimination of two responsible for the SCID (Severe Combined combined multiplex is 0,99999999995 in Brasil- Immunodeficiency Disease). The results ian Zebuin Cattle. showed 96.2% of normal horses (229/238) and

142 Section D: Marker, Polymorphism and Biodiversity

D175 D176 The polymorphism in the 5’-flanking region Contribution to the rabbit R-banded karyo- of chicken prolactin gene type nomenclature by FISH localization of 23 chromosome specific genes on both G- YONG LIANG, GUANFU YANG, XIQUAN and R-banded chromosomes ZHANG College of Animal Science, South China H. HAYES1, C. ROGEL-GAILLARD2, C. Agricultural University, Guangzhou, 510642, ZIJLSTRA3, N. A. DE HAAN3, C. URIEN2, China N. BOURGEAUX2, M. BERTAUD1, A. Prolactin is a peptide hormone secreted by BOSMA3 pituitary glands and plays important roles in 1Laboratoire de Génétique biochimique et reproductive and growing processes of the Cytogénétique; 2Laboratoire de Radiobiologie individual. The cleavage site of et Etude du Génome, INRA, Jouy-en-Josas, deduced amino acids from preprolactin cDNA France; 3Department of Cell Biology and Hi- of chicken prolactin (cPRL) changed from stology, Faculty of Veterinary Medicine, Leu-Pro-Ile-Cys in quality-type Yuehuang and Utrecht University, Utrecht, The Netherlands Taihe Silkies to Pro-Pro-Ile-Cys in layer-type Banding patterns of rabbit chromosomes have Isa Brown (Zou et al., 2001). The mutation of been described in detail using different G-, Q- the site made signal peptidase to have no effect and R-banding techniques, however, both in- on the preprolactin, which could cause Isa ternational nomenclatures (1976 & 1981) are Brown to have no broodiness. Other nucleotide based on G-banded chromosomes. The INRA changes between the chicken cDNAs were laboratory has launched a project on the rabbit often present in the 5’- flanking region, signal genome map involving large-scale localization peptide, and 3’-flanking region. The polymor- of type I and II markers by FISH on R-banded phism in 5’-flanking regions of cPRL was chromosomes. Therefore, an R-banded rabbit studied in quality-type Yuehuang chickens, karyotype nomenclature in perfect agreement Taihe Silkies and layer-type Leghorn chickens with the 1981 G-banded nomenclature is a in the present study. Three pairs of primers prerequisite. In order to establish unambiguous were designed based on the sequence in 5’- correlations between G and R-banded rabbit flanking region of broiler prolactin gene (Oh- chromosomes we have defined 23 marker kuo et al., 2000). The sequences of these pri- genes, one per chromosome, and localized mers were primer pair A them precisely on G and R-banded chromo- 5’TTACCTCCTGGCCTTTGTG3’ and somes. The choice of these genes was based on 5’GTTCTGGGCCTCTCACTT3’, primer pair reciprocal chromosome painting data between B 5’TCTCCCACTAGACTCTTTC3’ and man and rabbit (Korstanje et al 1999). A 3- 5’CTGTGTGTTTGTCTCCAT3’, and primer genome equivalent rabbit BAC library con- pair C 5’CTGTCCCTGTTTCTCAAC3’ and structed by Rogel-Gaillard et al (2001) was 5’GATGACTTGCTCTACCAG3’. The poly- screened by PCR with specific primers to merase chain reactions (PCR) were conducted identify and isolate clones for each gene. The in Mastercycler (Enppendorf Co. Ltd.) accor- BAC clones were then labelled with biotin, ding to the following procedures: 94ºC 7 min, hybridised to rabbit chromosomes treated ei- 30 cycles of 94ºC 50s, 59.5ºC (54ºC for primer ther for G or R bands and hybridisation sites pair B and 62ºC for primer pair C) 50s and were revealed by immunofluorescence detec- 72ºC 2.5 min. PCR products were directly used tion. Here, we present the 23 chromosome for sequencing. The homology of the sequen- specific genes, their localization by FISH and ces from three types of chickens was analyzed updated schematic representations of R-banded with DNASIS (version 3.0). The analysis of rabbit chromosomes. homology showed that there were great diffe- rences between various types of chickens. A D177 24bp insert in 5’-flanking region of prolactin Effect of POU1F1 polymorphism on meat gene was found in layer-type Leghorn but not quality traits in Piemontese cattle found in quality-type Yuehuang chickens or Taihe Silkies. The above result was confirmed LILIANA DI STASIO1, STEFANO SARTO- with SSCP method. RE2, GIANLUIGI DESTEFANIS1, ALBERTO BRUGIAPAGLIA1

143 Section D: Marker, Polymorphism and Biodiversity

1University of Torino, Department of Animal breeds. To determine phylogenetic relationship Sciences, Torino, Italy; 2University of Torino, between the different mutation, we used an Department of Animal Production, Torino, SNP based approach, identifying SNPs from Italy BAC clones (RPCI81 library) associated with The POU1F1 gene encodes a transcription markers in a 30 cM interval, which includes factor regulating the expression of growth the 500 kb XLPRA zero recombination region. hormone and therefore it has been suggested as 10 identified SNPs were screened in 86 dogs a putative candidate gene for genetic variation from breeds identified with XLPRA, and about of meat production traits. A preliminary inves- 50 DANN samples collected from other Cana- tigation in Piemontese cattle suggested that diae species to serve as outgroups, discrimi- POU1F1 polymorphism could affect the vari- nating different alleles by either restriction ability of some qualitative characteristics of the digestion or using the SnaPshot ™ Multiplex meat. To verify the results on a larger sample, Kit. Resulting haplotypes were analyzed to we examined 105 subjects of both sexes, infer history of the observed RPGR ORF 15 slaughtered in two slaughterhouses. For each mutations. Here we present data showing that subject the POU1F1 genotype was analysed by each of the observed deletions in ORF 15 is PCR-RFLP with HinfI. In addition, sarcomer linked to a distinct haplotype background in length, drip losses, cooking losses and tender- the canine XLPRA interval concluding that the ness (as Warner-Bratzler shear) at 1, 3, 7 and three different types of mutations identified in 11 d post mortem were determined on LTL canine RPGR ORF 15 arose independently in muscle. The data were analysed by GLM pro- husky and Samoyed dogs originated in a com- cedure, with POU1F1 genotype, sex and mon ancestor. Furthermore, SNP analysis of slaughterhouse as fixed effects. Two alleles the RP3 region in several Canadiae species were found and the frequencies of the geno- demonstrates phylogenetic evolution of this types were 3.8% for AA, 52.4% for AB and chromosomal segment. 43.8% for BB. Significant (P<0.05) differences among genotypes were observed for WBs at D180 day 3 (6.6, 9.1, 10.8 kg for AA, AB and BB Hungarian Gray Cattle Blood Group and respectively), day 7 (5.8, 7.7, 8.8 kg) and day DANN Allele Frequency in Hungary 11 (4.5, 6.7 and 7.6 kg). These results confirm the effect of POU1F1 locus on meat tender- ALICE GYURMAN; BEÁTA BÁN ness, with the AA genotype associated to more National Institute for Agricluture Quality favourable values. Also the sarcomer length Control was affected by POU1F1 genotype (1.63, 1.75 A Hungarian gray cattle is a native breed in and 1.84 µm for AA, AB and BB; P<0.05). On Hungary. The number of adult animals is less the contrary, the relationships with cooking than 5000. In the last 40 years we have been losses were not confirmed. The effect of the examining the parentage of each offspring slaughterhouse was always highly significant. yearly on the basis of the blood group determi- nation to avoid the inbreeding as much as pos- D179 sible. On the basis of B blood system this Independent origin of three microdeletions breed shows big similarity, we have found in RPGR exon ORF15 of canids altogether 16 different B alleles. The degree of homozigosity was 16% calculated on the basis BARBARA ZANGERL, QI ZHANG, JENNI- of this system ( it is much higher than in other FER JOHNSON, GREGORY M. ACLAND, breeds in Hungary). GUSTAVO D. AGUIRRE We started to examine the DANN microsatel- Cornell University, James A. Baker Institute lites last year, using a StockMarks Paternity for Animal Health, Ithaca NY, USA Typing System bovine II. version 2, and we We investigated the possible ancestral origin of calculated the gene frequencies of the different mutations previously identified in the RPGR microsatellite loci (N=57). The most polymor- exon ORF15, two of which, a five and two phic locus was TGLA53 (we found 11 alleles) base pair deletion , were causally associated most homogenous one was the BM 1824 (only with the retinal degenerations XLPRA1 and four alleles). XLPRA2, respectively. The XLPRa1 deletion We summarize the gene frequencies of trans- is shared between al least two different dog ferrin, haemoglobin, 8 blood group and 11

144 Section D: Marker, Polymorphism and Biodiversity microsatellite system in a table. aimed to determine the genetic relationship of Loxia curvirostra rubrifasciata to other Loxia D182 species. Ornithologists disagree whether Loxia Characterization of two SNPs (single c. rubrifasciata is a subspecies of Loxia, a hy- nucleotide polymorphisms) in the porcine brid of the common crossbill with the two- INSL3 gene and their exclusion as a com- barred crossbill or just an aberration of the mon genetic basis of hernia inguina- common crossbill. There are morphological lis/scrotalis in pigs indications for all three assumptions. We tested sample material of five Loxia species for alle- CHRISTOPH KNORR, ULRIKE PETERS, lic variations in highly polymorphic microsat- BERTRAM BRENIG ellite loci and for mitochondrial DNA se- Georg-August-University of Goettingen, quence variations Institute of Veterinary Medicine, Goettingen, Germany D184 The INSL3 gene encoding Leydig cell insulin Species determination using mtDNA se- like hormone is an important candidate gene quencing and PCR-RFLP of DNA from for congenital disorders of the reproductive forensic material tract in pigs. Comparative sequencing using phenotypically hernia inguinalis/scrotalis af- INA PFEIFFER(1), JOACHIM BURGER(2) & fected and unaffected animals showed that the BERTRAM BRENIG(1) porcine gene is remarkably conserved. No (1) University of Goettingen, Institute of Veteri- polymorphisms were found in the two exons or nary Medicine, Goettingen, Germany(2) Johan- in the intron. Two SNPs were detected in the nes Gutenberg University, Institute of Anthro- promoter region (G-224A and A-164C) and pology, Mainz, Germany fast screening methods were developed for Determination of the species origin of un- large scale studies. The A allele at position - known material, e.g. dry blood drop on a leaf, 224 is newly described and represents a rare is sometimes a task in forensic science. Usu- allele with a frequency of 5% (n=375). The C ally, short fragments of conserved regions of allele at position -164 shows an equal distribu- mitochondrial DNA are amplified using PCR tion to the previously known A allele at that and then sequenced. In our case a detailed position. Screening of the two SNPs in a phylogenetic analysis of a blood drop was population of hernia inguinalis/scrotalis af- necessary. The required asservates normally fected pigs (n=223) revealed that the SNPs can contain particulary degraded DNA. Therefore, be excluded as a common genetic basis for this we have developed a PCR with mtDNA for congenital disorder. sequencing and a less expensive PCR-RFLP method. For the interspecific sequence poly- D183 morphism we chose a part of the D-loop region Molecular Variance In Continental Cross- for sequencing and a 200 bp fragment of the bill Species (Loxia) mt-cytochrome b gene. Both PCR methods allowed a reliable species identification from ANNETTE MUELLER(1), INA PFEIFFER(1) forensic material, e.g. a blood drop on a leaf, & BERTRAM BRENIG(1) even those containing degraded DNA. (1)University of Goettingen, Institute of Veteri- nary Medicine, Goettingen, Germany D185 Crossbills are birds, whose habitat ranges from Genetic differentiation of several dog breeds the forest belt of north-east Europe and north- by analysis of mitochondrial DNA central Asia to North America and the Western Palaearctic. Their diet consists mainly of coni- INGER VOELKEL , INA PFEIFFER & BER- fer seeds and species are defined by their bill TRAM BRENIG depth and colouring of feathers. The aim of University of Goettingen, Institute of Veteri- this study was to determine the genetic diver- nary Medicine, Goettingen, Germany gence of continental crossbill species (Loxia) Because of the dog`s increasing importance as and their phylogenetic relationship. Tested “man´s best friend”, there is growing interest material included 46 blood or organ samples of in assigning blood, hair or saliva samples to five Loxia species. Our special interest was the particular dog breed. This could not only

145 Section D: Marker, Polymorphism and Biodiversity be a significant progress for forensic science but would also facilitate the differentiation between purebreds and crossbreds. We investi- gated the mtDNA of several wolves and 10 different dog breeds. For our study we chose wolf-like breeds as well as breeds with antipo- dal phenotypes, e.g. dachshund or boxer, in- cluding at least 30 non-related individuals. The sequence analysed was part of the mitochon- drial D-loop region, with a length of about 250 bp. So far, we have sequenced mtDNA of 137 dogs belonging to different breeds. In the ex- amined region we were able to identify at least 4 diagnostic nucleotide positions. Future stud- ies will include the analysis of the complete D- loop region for each dog to enable a breed- screening.

D186 The Polymorphism of β-Lactoglobulin Gene in Several Sheep Breeds by PCR-RFLP

NASSIRY M.R.1* , MARZANOV N.S. 2 Animal science Dept., College of Agric., Fer- dowsi Univ., P.O.Box: 91775-1163, Mashad, Iran, All-Union Research Institue of Animal Husbandary RF-142012 P.O. Dubrovitsy Po- dolsk District, Russia In the last years there has been a considerable interest in milk protein gene polymorphisms because of their potential use as genetic markers to improve the efficiency of selection for quantitative traits. The aim of this work was to analyze the genotype distribution of β- Lactoglobulin in sheep. A polymerase chain reaction assay has been used for genotyping β- Lactoglobulin A and B variants in Oparinsky, Sovite merino and Karakol sheep. DNA was extracted from blood of 58 animals. For PCR of genomic DNA , primers, wich amplified a 452 bp region of the ovine β-Lactoglobulin gene from intron II were chosen. The genetic variants A and B differ in an amino acid at position 38 (Tyr to Hys) and this base substi- tution gives rise to a RsaI polymorphism. The genotypes distribution in Oparinsky. Sovite merino and Karakol sheep were 20%, 30%, and 44% for AA, 40%, 45% and 56% for AB and 40%, 25% and 00% for BB respectively. The populations were in Hardy-Weinberg equilibrium. Key Words. β-Lactoglobulin, Russian sheep, PCR-RFLP.

146 Section E: Associations between Markers and Traits

Section E: simultaneously found in both lines, a feature that may indicate that they are the result of Associations between markers linkage disequilibrium. No association was and traits found between the identified polymorphisms and plasma IGF-I concentration.

E001 E002 Identification of three single nucleotide po- Mapping susceptibility to anal atresia in lymorphisms in the chicken IGF1 and IGF2 the pig genes and their association with growth traits PAMELA CASSINI1, PAUL BOETTCHER2, 3 3 1 1 TETSUO HORI , HARUO OHKAWA ; MARCEL AMILLS , NEUS JIMÉNEZ , DA- 2 2 2 BIANCA CASTIGLIONI , LEIF ANDERS- NIEL VILLALBA , MARC TOR , ESTER 4 1 2 2 SON , ELISABETTA GIUFFRA MOLINA , DOLORS CUBILÓ , CARINA 1 1 3 FPTP-CERSA, Centro Studi Ricerche Agro- MARCOS , AMADEU FRANCESCH , AR- alimentari, Segrate, Italy; 2CNR-IBBA, Insti- MAND SÁNCHEZ1, JOAN ESTANY2 1 tuto de Biologia e Biotecnologia Agraria, Departament de Ciència Animal i dels Ali- Segrate, Italy; 3University of Tsukuba, Dept. of ments, Facultat de Veterinària, Universitat Pediatric Surgery, Tsukuba, Japan; 4Swedish Autònoma de Barcelona, Bellaterra 08193, 2 University of Agricultural Sciences, Dept. of Spain; Departament de Producció Animal, Animal Breeding and Genetics, Uppsala, Universitat de Lleida, Lleida 25008, Spain; 3 Sweden Unitat de Genètica Avícola, Centre Mas Bové, Anal atresia is a multi-factorial congenital IRTA, Reus 43280, Spain disease, affecting approximately 1 in 5000 live The polymorphism of the chicken IGF1 and births in humans. Am similar malformation IGF2 genes has been analysed in two geneti- naturally occurs in pigs, which can be consid- cally diverse chicken lines (PN and MN) of the ered a suitable model of investigation. A pedi- Penedesenca breed. The PN and MN lines gree with an increased of the disease has pre- reach a live weight of 2.5 and 1.3 kg at 11 viously been develped by selective breeding weeks. Live weight, average daily gain and (Hori et al. 2001, J. Pediatr. Surg. 36:1370-4). feed efficiency were recorded at 44, 73 and Simulation was used to empirically estimate 107 days. Plasmatic IGF1 concentrations were the likelihoods of different genetic models determined at 73 days by using an enzyme- underlying the abnormality. The primary moti- linked immunoassay. We sequenced the 5’end vation of trying to establish the most likely of the IGF1 gene and exon 2, intron 2 and genetic model was to aid the statistical analysis exon 3 of the IGF2 gene in three individuals of the genome scan experiment. Each model from each line. Moreover, we amplified the was used in an attempt to reconstruct all of the chicken IGF1 and IGF2 cDNAs from reverse phenotype observed in the pedigree. Ten mil- transcribed total RNA and sequenced them lion replicates were generated for each model forward and reverse. We identified one re- and the number of correct reconstructions of → placement A C at the 5’end (SNP1) of the the phenotypes was taken as the measure of the IGF1 gene. In addition, two silent mutations relative likelihood of the respective model. were found at the IGF2 gene. The first one was Genetic models differed in terms of numbers a substitution C → T at exon 3 (SNP2), of loci, distributions of allelic effects, levels of whereas the second one was a G → A re- dominance and penetrance, and importance of placement at intron 2 (SNP3). The IGF1 and non-genetic effects. Preliminary results con- IGF2 polymorphisms were typed in both lines firm indication of recessive allelic effects and (n = 60, in each line) by using the Hinf I suggest the presence of multiple loci, with (SNP1) and Hsp92 II (SNP2) restriction en- incomplete penetrance. The existing microsat- zymes, respectively. A primer-extension based ellite dataset was implemented with new mark- protocol was employed to detect SNP3. Sig- ers to fully cover the genome and analysed nificant associations (p < 0.05) were found under the new assumptions. Preliminary results between SNP1 and average daily gain at 107 from physical mapping of candidate genes days and feed efficiency at 44 days, 73 days belonging to the Sonic Hedgehog transduction and 107 days. Any of these associations were system are reported.

147 Section E: Associations between Markers and Traits

E003 CURTIS P. TASSELL1, HARRIS A. LEWIN2 A proposed model for the interaction of the 1USDA-ARS, Gene Evaluation and Mapping scurred and polled/horned loci Laboratory, Beltsville, Maryland, USA; 2Department of Animal Sciences, University of MIKA ASAI-COAKWELL1, SHEILA M. Illinois at Urbana-Champaign, Urbana, Illi- SCHMUTZ2 nois, USA 1Swiss Federal Institute of Technology, Insti- Originally two research groups conducted in- tute of Animal Sciences, Zurich, Switzerland; dependent genome scans in Dairy Bull DNA 2University of Saskatchewan, Department of Repository grandsire families to identify quan- Animal and Poultry Science, Saskatoon, titative trait loci (QTL) affecting economically Canada important traits. Each group selected eight Scurs are bony growths found in the area of families for study, six that were common horn development in Bos taurus breeds. They across both studies. We report putative QTL are undesirable to the cow-calf and feedlot affecting milk production traits using the mer- industries, which are moving towards a polled ged data from the two groups. The six common population due to economic losses caused by families were genotyped at 367 microsatellite horns. However, scurs have been difficult to markers. Genome coverage was estimated to eradicate because of their complex inheritance. be 2713.5cM (90%), with an average spacing Scurs are sex-influenced and masked by the of 7.4cM. QTL Express software presence of horns. Homozygosity of the polled (http://qtl.cap.ed.ac.uk) was used for regression allele has also been suggested to mask scurs in interval mapping within each family. Phenoty- the heterozygous condition. We were able to pic traits included daughter deviations for verify that scurs are masked using two polled milk, protein and fat yields, protein and fat male calves that were offspring of a scurred percentages, somatic cell score and productive dam, an obligate homozygote for scurs. Our life, weighted by their respective reliabilities. data suggest that homozygosity of the polled Permutation was used to calculate chromoso- allele masks scurs in the homozygous conditi- me-wide P < 0.05 and P < 0.01 significance on as well. Eight scurred females in our study thresholds. One hundred seven putative marker could be shown to carry the horned allele effects were identified at P < 0.05, 36 of these through a horned parent or horned offspring, at P < 0.01. Highly significant effects (P << and no scurred animals were shown to be ho- 0.01, F-statistic > 15) were found on chromo- mozygous polled. Following a genome scan some (BTA) 3 affecting fat percentage and using 17 embryo transfer families, the scur protein yield, BTA6 affecting protein percen- locus was mapped to bovine chromosome 19, tage, and BTA14 affecting fat percentage and whereas the polled/horned locus was mapped yield. Interval analysis of the merged dataset to the centromeric region of BTA1. The locus identified putative QTL not detected in the was further fine mapped to a location between separate studies. QTL identified in this study microsatellite markers BMS2142 (LOD =4.46) may be useful for marker-assisted selection to and IDGVA46 (LOD =2.56) using 27 offspring improve milk production and manipulate milk in 5 families. We therefore postulate a recep- protein and fat components. tor-ligand model to explain the interaction between the polled and scurred loci, which also E005 accounts for the sex-influenced nature of the Construction of a physical map along the scurred phenotype. porcine chromosome 7 q . Detection of QTL affecting milk production in 6 Dairy Bull DNA Repository grandsire fami- ANGELA BARBOSA1, CHRISTINE RE- lies NARD1, CELINE URIEN1, NOËLLE BOUR- GEAUX1, JEAN CLAUDE SAVE1, CARINE E004 GENÊT2, DENIS MILAN2, FRANÇOIS PI- Detection of QTL affecting milk production UMI1, CLAIRE ROGEL-GAILLARD1, PAT- in 6 Diary Bull DANN Repository grandsire RICK CHARDON1 families 1Laboratoire de Radiobiologie et d'Etude du Génome INRA CEA Jouy en Josas, France and MELISSA S. ASHWELL1, DAVID W. HEY- 2Laboratoire de Génétique Cellulaire, INRA, EN2, YANG DA2, TAD S. SONSTEGARD1, Castanet Tolosan, France

148 Section E: Associations between Markers and Traits

Several QTLs in the pig have been recently known effects of the MHS-genotype on pro- fixed between SLA and the SO102 markers on duction traits, we investigated the influence of the long arm of SSC7. In order to find new the FUT1 variation on these traits. We geno- markers and precise the QTL locations, a typed 813 DL (German Landrace), 576 PI physical map of the chromosomal segment was (Piétrain) and 68 DE (German Large White) extended from the SLA class II region toward from 332 boars. The genotype effect on pro- the telomere. PCR screening of a BAC library duction traits were estimated with PROC GLM with primer pairs designed from human genes of the SAS software package using two differ- known to map to HSA6 and with chromosome ent regression models. For the stress resistant walking primers, lead to a sorting of 111 BACs DL and DE we applied a model including ef- distributed in 6 contigs. Several primers used fects of test year, test month, sire, test station for BAC screening were tested on the ImpRH and the FUT1-genotype. When analysing the panel to ensure the relative location of the PI animals, the effect of MHS-genotype were clones. The closest contig from SLA encom- added to the model. No significant effects were passed 50 BACs representing about 1.3 MB. It found for the growth trait ADG (average daily harbors 10 loci, including the TAPASIN locus gain), the carcass composition traits SF (sidefat and the SW1856 and SW2019 markers. thickness), FA (fat area) and CL (carcass Whereas in humans the DNA segment between length) and for the meat quality trait TAPASINE and HLA DPA is 150 kb long, the PH24MLD (pH24 musculus longissimus dorsi). orthologous porcine DNA segment appears to However, the PH1MLD (pH1 musculus longis- be larger than 1 MB. One of the synthenic simus dorsi) showed a significant FUT1 effect breakpoints has been assigned close to (p=0,026) for the PI breed. As expected, we SW2019. A second 1.3 MB long contig made found high significant effects of the RYR re- of 22 clones contains ZNF76 plus 8 additional ceptor (p<0,001) on the meat quality traits, FA loci. Alignment of BAC-end sequences on the and CL, whereas there was no effect on SF and human draft sequence revealed a straight con- ADG. servation of the distance between genes for this region. Further along the chromosome, we E007 mapped successively 4 contigs of 16, 13, 4 and Genes controlling appetite show interde- 7 BACs characterized by the NDR, PIM, pendencies in allele frequency in beef cattle NFYA and GLO loci, respectively. New mark- ers have been characterized for all contigs. FIONA C. BUCHANAN, TRACEY D.THUE, Thus with BACs covering the SLA region we SHEILA M. SCHMUTZ have now assigned about 230 BACs in the Department of Animal and Poultry Science, peri-centromeric region of SSC7. University of Saskatchewan, Saskatoon, Can- ada E006 Previously we have identified SNPs in two Effects of variation in the FUT1-Gene on genes, leptin (LEP) and corticotrophin- various Traits in Swine releasing hormone (CRH). These genes are associated with the control of appetite. They STEFAN BINDER1, KAY-UWE GÖTZ2, comprise only a small part of elaborate path- GEORG THALLER1, RUEDI FRIES1 ways. For example, CRH indirectly releases 1Lehrstuhl für Tierzucht, Technische Univer- glucocorticoids which stimulates the release of sität München, Freising-Weihenstephan, Ger- leptin that in turn decreases appetite. CRH has many been shown to stimulate expression of alpha 2Bayerische Landesanstalt für Tierzucht, Grub, melanocyte stimulating hormone ( MSH) the Germany agonist for melanocortin-4 receptor (MC4R) Oedema disease is caused by the adherence of which reduces appetite. We genotyped 116 E. coli strains to ECF18R receptors in the por- steers with DNA tests for LEP (BTA4), CRH cine intestinal tract. The FUT1-gene was iden- (BTA14) and MC4R (BTA24). We propose tified as a candidate for the expression of these that genotypes at unlinked genes from path- receptors. Resistant and susceptible animals ways controlling body weight may well be can be distinguished by a variation in this coadaptive. Their allele frequencies are gene. Because of strong linkage disequilibrium not independent of each other which is between the FUT1- and MHS-locus and the referred to as interchromosomal coadaptation.

149 Section E: Associations between Markers and Traits

Genotypic frequencies not in Hardy-Weinberg regions involved in FP behaviour and cortico- Equilibrium (HWE) would support this. sterone response to manual restraint. Genotype combinations were analysed by chi- square to determine whether or not the ob- E010 served were significantly different from ex- Quantitative trait loci for birth weight, lon- pected HWE ratios. Genotype frequencies gissimus muscle area, and marbling on bo- were not in HWE between CRH and LEP (P = vine chromosome 5. 0.0001) or LEP and MC4R (P = 0.0001). This suggests that selection for carcass composition EDUARDO CASAS, JOHN W. KEELE, and/or growth has altered gene frequencies at STEVEN D. SHACKELFORD, ROGER T. several related genes in current North Ameri- STONE, MOHAMMAD KOOHMARAIE can beef cattle. USDA-ARS, U. S. Meat Animal Research Center, Clay Center, Nebraska, U.S.A. E008 A study to detect quantitative trait loci (QTL) Feather pecking behaviour and stress re- on bovine chromosome 5 (BTA5) affecting sponse in Laying hens: a QTL-analysis growth, carcass composition and meat quality traits was pursued. Thirteen microsatellite A.J. BUITENHUIS1, T.B. RODENBURG2, markers were genotyped on 547 progeny from Y.M. VAN HIERDEN4, B. ASK1, M. SI- a Brahman X Hereford sire mated to mostly WEK1, S.J.B. CORNELISSEN1, M.G.B. composite (MARC III) dams. Traits analyzed NIEUWLAND3, H. VOS1, P. DE GROOT1, were birth weight (kg), marbling, and longis- S.M. KORTE4, P. KOENE2, H. BOVEN- simus muscle area (cm2). Significant QTL HUIS1, J.J. VAN DER POEL1 were detected when the expected number of 1Animal Breeding and Genetics Group; false positives (ENFP) was less than .05 (F- 2Ethology Group; 3Adaptation Physiology statistic greater than 16.6), and suggestive Group, Wageningen Institute of Animal Scien- when the ENFP was less than 1 (F-statistic ces, Wageningen University, Wageningen, The between 10.0 and 16.59). The effect of the Netherlands; 4Department of Behaviour, QTL on the traits was measured in standard Stress Physiology and Management, Institute deviation units (SD). Significant QTL were for Animal Science and Health (ID-Lelystad detected for birth weight (ENFP= .0007) at 57 BV), Lelystad, The Netherlands cM from the beginning of the linkage map, and The concern for animal welfare in the West for longissimus muscle area (ENFP= .049), at European countries results in a change in hou- cM 53. Suggestive evidence for the presence sing systems for laying hens from battery ca- of a QTL for marbling (ENFP= .2) was detec- ges to free-range systems. Feather pecking ted at cM 75. The effect of the QTL were 0.5 (FP) behaviour is a major problem in free ran- SD, 0.4 SD, and 0.36 SD, for birth weight, ge housing systems. In order to look for genes longissimus muscle area, and marbling, re- involved in FP behaviour a QTL experiment spectively. Before implementing marker- was designed. The F2 population originates assisted selection programs, the effect of the from a cross between randomly chosen birds of QTL on BTA5 needs to be estimated in addi- a high FP line (HFP) and a low FP line (LFP). tional target populations. Reciprocal crosses were made between the HFP and the LFP line to generate the F1 ani- E011 mals. From the F1 animals 7 males were se- Association of genetic markers in candidate lected and mated to 28 females to produce 650 genes with growth and carcass traits in Ko- F2 hens. The birds have been tested for FP rean cattle (Hanwoo) behaviour in a social FP test at 6 weeks and 30 weeks of age, respectively. The corticosterone E.R. CHUNG1, Y.S. KIM1, W.T. KIM1, response to manual restraint was measured at S.K. HAN2 32 weeks of age as a measure for coping stra- 1Laboratory of Animal Molecular Genetics, tegy. To date we have finished the genotyping Division of Applied Animal Science, College of of 180 microsatellite markers and phenotyping Life Science and Natural Resources, Sangji of the F2 population. By combining the geno- University, Seoul, Korea; 2Laboratory of Mo- typic and phenotypic data from this experiment lecular Genetics, Department of Dairy Sci- we should be able to identify the chromosomal ence, College of Animal and Life Science,

150 Section E: Associations between Markers and Traits

Konkuk University, Seoul, Korea QTL was localized to a confidence interval of Candidate genes are targets for genetic marker 4 cM near the center of bovine chromosome 6 studies because of their biological significance by interval mapping. A total of 6,524 ESTs on the quantitative traits of interest. The possi- expressed preferentially in the mammary gland ble association between growth and carcass of mice were bioinformatically selected from traits differences and genotype variations in 52,000 clones of two mammary gland cDNA growth hormone(GH), growth hormone re- libraries. Of these ESTs, only kiaa0914 was ceptor(GHR), growth hormone releasing hor- included in the genes identified from the hu- mone(GHRH), leptin(Lep), myogenic factor man physical map of the region syntenic to the 5(Myf5), heart-fatty acid binding protein(H- QTL confidence interval. A total of 388 Is- FABP) and calpain(Cal) genes was examined raeli-Holstein bulls were genotyped for a SNP in 430 Korean cattle from National Livestock located in intron 9 of this gene, in which Research Institute, R.D.A. Performance traits Adenosine was replaced by Guanine. The analyzed were growth traits(birth weight, 6, 12 frequency of the G allele was 44%. The effect and 18 month weights and average daily gain) of this polymorphism on the sire genetic and carcass traits(carcass weight, carcass per- evaluations was analyzed by a model that also centage, backfat thickness, eye muscle area, included the effect of sire birth date to account marbling score and grade of meat quality). for genetic trend. The effect allele substitution Genetic markers of candidate genes were de- was significant (p<0.01) for fat concentration, termined by PCR-RFLP or SSCP techniques. and highly significant (p<0.0001) for protein Preliminary data analysis was performed using concentration, but did not significantly affect the GLM procedure of SAS computer program milk, fat, or protein yield. The substitution with a linear model. Allele frequencies were effects were 0.045% fat, and 0.035% protein. compared between high and low grade of meat The A allele was associated in increased fat quality using contingency to the chi-square and protein concentration. The profile of ef- tests. There were no significant associations fects observed was similar to the effects found between genotype frequencies of candidate previously by the daughter-design analysis, but genes and any of the above traits. However, accounts for only half of the observed QTL significant differences(p<0.05) in allele fre- effect. Thus this mutation is closely linked to quencies for the GH, Lep and Myf5 genes the QTL polymorphism. were observed between the two groups se- lected for high and low grade of meat quality. E013 Differences in gene frequencies of A and B Mapping quantitative trait loci for twinning alleles between high and low groups were 0.88 in Holstein dairy cattle and 0.12 and 0.76 and 0.24 for GH, 0.51 and 0.49 and 0.64 and 0.36 for Lep and 0.42 and JENIFER CRUICKSHANK1, MARGARET 0.58 and 0.56 and 0.44 for Myf5, respectively. DENTINE1, P. JEFFREY BERGER3, BRAIN Further studies are need to confirm the signifi- W. KIRKPATRICK2 cant associations seen in this study. 1Department of Dairy Science, University of Wisconsin-Madison, Madison, Wisconsin, E012 USA; 2Department of Animal Sciences, Uni- Population-wide linkage disequlibrium be- versity of Wisconsin-Madison, Madison, tween a SNP and a OTL affecting milk pro- Wisconsin, USA; 3Department of Animal Sci- tein production one bovine chromosome 6 in ence, Iowa State University, Ames, Iowa, USA Israeli-Holsteins Twinning in dairy cattle has been associated with many negative health and reproductive MIRI COHEN, EYAL SERROUSSI, MICHA events that cause economic loss to the produ- RON, MISHA REICHENSTEIN, ALLA cer. Reports have suggested that twinning PLIS-FINAROV, MOSHE SHANI, JOEL I. rates are increasing and that there may be a WELLER positive relationship between milk production Institute of Animal Sciences, ARO, The Volcani and twinning frequency. Quantitative trait loci Center, Bet Daga, Israel (QTL) for twinning rate on bovine chromoso- A QTL affecting protein content and percent- mes 5, 7, 19, and 23 have been previously age was detected in the Israeli Holstein popu- identified. The objectives of this study were to lation by a daughter design analysis. This detect and confirm the existence and effects of

151 Section E: Associations between Markers and Traits these QTL in dairy cattle. This project utilized Total body fat percentage was estimated in the sire predicted transmitting ability (PTA) values IH x IL F2 (using chemical extraction) and in for twinning rate estimated from North Ameri- the AIL at F11 (using dual-energy X-ray ab- can Holstein calving data. Half-sib families of sorptiometry). Microsatellite markers have 25 sires with high twinning rate PTA compri- been genotyped in the MH x ML (n=149) and sed the population under investigation. DNA IH x IL (n=92) F2 populations. Summaries of extracted from semen samples was analyzed phenotypic evaluation and initial QTL map- using 45 microsatellite markers on four chro- ping results in the F2 intercrosses will be pre- mosomes. Marker heterozygosity of the patri- sented. This study will facilitate enhanced archs averaged 56%. Some of these families understanding of the polygenic genetic archi- are related and will be combined into larger, tecture of energy balance and body composi- multi-generation families for additional analy- tion. sis. For twinning QTL identified in Holsteins, chromosomal positions will be more narrowly E016 defined. Frequencies of haplotypes associated Population-wide analysis of a QTL affecting with twinning will be estimated in elite Hol- milk-fat production in the Israeli Holstein stein cow populations. The effect of these population QTL on milk, fat, and protein production, pro- ductive life, and somatic cell score will also be MARINA GOLIK1, EYAL SERROUSSI1, estimated. Suggestive evidence of twinning JOEL I. WELLER1, EPHRAIM EZRA2, QTL was found on chromosomes 5 and 23 in MICHA RON1 families analyzed to date. 1Institute of Animal Sciences, ARO, The Volca- ni Center, Bet Daga, Israel; 2Israel Cattle E015 Breeders Association, Caesaria Industrial Fine mapping of genes regulating heat loss Park, Israel in mice Several studies have shown that a missense mutation in the bovine DGAT1 gene located at KARI ELO1, MERLYN NIELSEN1, DALE the centromeric end of bovine chromosome 14 VAN VLECK2, DANIEL POMP1 has a major effect on milk yield and composi- 1University of Nebraska-Lincoln, Department tion. A total of 374 Israeli-Holstein sires were of Animal Science, Lincoln, Nebraska USA; genotyped for this polymorphism by Fluo- 2Roman L. Hruska U.S. Meat Animal Research rescent Allele-specific PCR. Of these 10 were Center, ARS, USDA, Lincoln, Nebraska USA homozygous for the mutation and 80 were Three mapping populations have been pro- heterozygous. The frequency of the mutant duced from lines of mice that have undergone allele was 13.4%. The effect of this mutation 16 generations of divergent selection for high on the sire genetic evaluations was analyzed by and low heat loss using direct calorimetry. a model that also included the effect of sire Two populations consisted of F2 intercrosses birth date to account for genetic trend. The originating from either outbred (MH x ML, effect of DGAT1 allele substitution was highly n=560) or inbred (IH x IL, n=640) high and significant (p<0.0001) for milk and fat yield, low selection lines. The IH x IL F2 animals and fat and protein concentration. For all five were further intermated to produce an ad- traits, the effects associated with cows homo- vanced intercross line (AIL) that has been phe- zygous for the mutant allele was approximately notyped at F11 (n=2,080). The main aims of twice the effects associated with cows hetero- this study are to identify QTL underlying en- zygous for the mutant allele, relative to cows ergy balance and body composition and, using without the mutant allele. Thus the effect of fine-mapping results from the AIL in conjunc- this QTL is approximately codominant. The tion with the human and mouse whole-genome allele substitution effects for the mutant allele sequences, identify candidate polygenes af- were -308 kg milk, 7.8 kg fat, -2.9 kg protein, fecting these traits. Maintenance heat loss 0.17% fat, and 0.06% protein. A total of 1747 (kcal/kg0.75/day) and feed intake (g/kg0.75/day), cows and 407 bulls were also genotyped for body weights (3 and 6 wks and at tissue har- microsatellite ILSTS039, which is tightly lin- vest) and tissue weights (brown adipose, sub- ked to DGAT1, and strong population-wide cutaneous and gonadal fat depots, liver and linkage disequilibrium was observed. Allele heart) were measured in all three populations. 225, which was the shortest of eight alleles

152 Section E: Associations between Markers and Traits observed, was closely associated with the E018 mutant DGAT1 allele. Frequency of this allele Associations of beta-lactoglobulin alleles was 12% in cows and 16.8% in bulls. with milk productions traits in Churra sheep E017 Positional candidate cloning of a QTL in BEATRIZ GUTIÉRREZ, JUAN J. ARRANZ, dairy cattle: Identification of a missense MOHAMMED EL ZAREI, YOLANDA mutation in the bovine DGAT1 gene with BAYÓN, FERNANDO DE LA FUENTE, major effect on milk yield and composition FERMIN SAN PRIMITIVO Various associations between milk protein BERNARD GRISART1, WOUTER COPPIE- polymorphisms and milk production trains TERS1, FRÉDÉRIC FARNIR1, LATIFA KA- have been described in different ruminant spe- RIM1, CHRISTINE FORD2, PAULETTE cies. In sheep, the effect of the three alleles at BERZI1, NADINE CAMBISANO1, MYRIAM the beta-lactoglobulin locus (LGB) on milk MNI 1, SUZANNE REID2, PATRICIA SI- traits has revealed differences among dairy MON1, RICHARD SPELMAN3, MICHEL breeds. The objective of this study was to es- GEORGES1, RUSSELL SNELL2 timate the effects of the varians at the LGB 1Department of Genetics, Faculty of Veterinary locus on milk traits in Churra sheep, an indige- Medicine, University of Liège (B43), 4000- nous Spanish breed. Beta-lactoglobulin alleles Liège, Belgium;2ViaLactia Biosciences (NZ) were determined using PCR-RFLP on DANN Ltd., University of Auckland Medical School, isolated from frozen blood. Samples were col- Auckland, New Zealand; 3Livestock Improve- lected from 750 ewes belonging to the selec- ment Corp.Hamilton, New Zealand tion nucleus of Churra sheep, corresponding to We recently mapped a QTL with major effect 14 flocks connected via artificial insemination. on milk yield and composition to the centro- Available milk traits were milk protein and fat meric end of bovine chromosome BTA14 yield as well as protein and fat percentage. within a 5 cM interval. A strong candidate Date were analysed using a mixed linear gene called DGAT1 (Diacyl Glycerol Acyl model, considering as random effect the factor Transferase 1), was identified in the BAC “individual lactation” which includes the ge- contig covering this interval. Maximum likeli- netic and permanent environ mental effect hood LD analysis suggested the existence of associated with each animal. Allele frequencies two “young” QTL alleles (Q1 and Q2, associ- obtained were LGBA = 0.31 and LGBB = 0.69. ated with an increased milk fat content) em- Regarding milk production effects, AA homo- bedded in distinct haplotypes and several q zygous animals showed higher concentrations alleles. DGAT1 was completely sequenced for of milk solids (mainly fat content), indicating individuals with known QTL phenotype. Four that milk from these ewes could be more valu- polymorphisms were observed : two in introns, able for cheese processing. one in 3’UTR and one in exon 8. For the lat- ter, the Q1 and Q2 alleles were shown to be E019 identical, and they differed from all q alleles Genetic predisposition of Thoroughbred by the substitution of a highly conserved racehorses to exercise-induced pulmonary amino acid (K232A). Surprisingly, the amino haemorrhage in South Africa acid of the Q1 and Q2 alleles was shown to correspond to the ancestral state (phylogenetic CINDY HARPER1, ALAN GUTHRIE1, consideration). Allele specific RT-PCR ex- DUNCAN McDONALD2, KOOS VAN DEN periments allowed us to demonstrate that the K BERG3, ENETTE VAN DYK3, PAUL MOR- allele is characterized by an increased propor- LEY4, KENNETH HINCHCLIFF5 tion of alternatively spliced transcripts. Inves- 1Equine Research Centre, Faculty of Veterina- tigations of the activity of the corresponding K, ry Science, University of Pretoria, Onderte- A and alternatively spliced allozymes are un- poort, South Africa; 2Jockey Club of Southern derway. Around 2,000 progeny-tested indi- Africa, Turffontein, South Africa; 3Department viduals were genotyped for DGAT1 polymor- of Companion Animal Studies, Faculty of Vete- phisms. An association study allowed us to rinary Science, University of Pretoria, Onder- evaluate that K232A accounts for 50% of the tepoort, South Africa; 4Department of Envi- variation in breeding value for fat content. ronmental Health, College of Veterinary Me-

153 Section E: Associations between Markers and Traits dicine and Biomedical Sciences, Colorado sity of Jerusalem, 91904 Jerusalem, Israel; State University, Fort Collins, CO, USA; 2Dept. of Animal Science, Faculty of Agricultu- 5Department of Veterinary Clinical Sciences, re, Rehovoth, Israel College of Veterinary Medicine, The Ohio A full-sib intercross line (FSIL) is a mapping State University, Columbus, OH, USA population constructed to provide the QTL Exercise-induced pulmonary haemorrhage mapping advantages of an F2 population for an (EIPH) is a problem that occurs worldwide in outcrossing species. In the FSIL design, two horses that participate in strenuous exercise. parent individuals, from the same or different EIPH does not appear to affect all horses to a populations, are mated to produce a large full- similar extent, with the degree of haemorrhage sib family. The full-sibs are intercrossed at varying from barely noticeable to severe. It has random to produce the first FSIL1 generation; been speculated that EIPH may have a genetic the FSIL1 progeny are randomly intercrossed basis but the occurrence of this condition is in turn, to produce the FSIL2 generation, etc. influenced by environmental factors. In this The high degree of linkage disequilibrium study EIPH was defined as epistaxis or blood introduced by the limitation to two founder at one or both nostrils following racing. South individuals gives the FSIL its statistical power. African racing authorities do not allow the use The Jerusalem Resource Population, now at of furosemide to treat or prevent the condition, FSIL15, was constructed by crossing a single therefore, the data collected from Jockey Club White Rock heavy breed male with five semi- records in this country is a realistic reflection inbred Leghorn layer females. The population of a proposed predisposition in the South Afri- was phenotyped in each generation for growth can racing Thoroughbred population for the rate, anatomical and egg production traits, and period examined. Individual horses that in some generations for immune response. showed epistaxis as a result of EIPH while Biometrical analysis of the first nine genera- racing between 1990 and 2001 were examined. tions showed that inbreeding accumulated at a The incidence of haemorrhage was directly rate of 0.012 per generation, following the first related to the following factors, age, sex, full-sib generation. The population did not weight carried, racing district, distance run, show any obvious effects of inbreeding. Time environmental temperature and humidity, track trends in trait value were not found, and phe- surface and track condition and the signifi- notypic variation, heritabilities and genetic cance of each factor established. The sire, dam correlations were similar to those found for and sire of the dam of each case were deter- normal populations. Thus, the JRP appears to mined and each sire and dam sire were exam- represent a genetically normal chicken popula- ined for the number of offspring that devel- tion, within which QTL affecting a variety of oped EIPH within the period under investiga- traits of economic importance are segregating. tion. These data were corrected for the total number of offspring produced by each stallion E021 during this period. Sires and dam sires with Fine-mapping of a QTL affecting egg white more than 50 offspring were compared and it thinning in chicken was found that certain of these sires produced significantly more offspring with EIPH. Blood MERVI HONKATUKIA1, MARIA TUISKU- samples from these animals were collected and LA-HAAVISTO1, JOHANNA VILKKI1, allele frequencies of a number of microsatellite NINA SCHULMAN1, DIRK-JAN DE markers were determined. KONING3, ANNELI VIRTA2, ASKO MÄKI- TANILA1 E020 1Animal Production Research, MTT Agrifood The Jerusalem Resource Population: A Research Finland, 31600 Jokioinen, Finland, multi-generation quasi-Full-sib Intercross 2Food Research, MTT Agrifood Research Population for High Power and High Reso- Finland, 31600 Jokioinen, Finland, 3Roslin lution QTL Mapping in Poultry. Biometri- Institute,Roslin, EH25 9PS, U.K cal characteristics Our previous genome scan of chicken revealed three putative areas affecting egg white thin- 1 1 2 E. HEIFETZ , H. KHATIB , D. HELLER , Y. ning. The F2 population was derived from a EITAN1, M. SOLLER1 cross between two genetically and phenotypi- 1Department of Genetics, The Hebrew Univer- cally extreme egg layer lines. Egg white qual-

154 Section E: Associations between Markers and Traits ity was measured as albumen height in Haugh from 0 to 10 weeks, abdominal fat, and plasma units at ages of 40 and 60 weeks, HU40 and concentrations of insulin and glucagon. A ge- HU60, respectively. Because the confidence nome scan using 100 markers on 25 linkage intervals for the detected QTL were over 50 groups in the chicken genome has been com- cM wide, a method to improve the QTL loca- pleted. The scan covers 85% of the chicken tions after initial detection was needed. We genome and an additional 50 markers are being chose the most significant QTL on chromo- typed to reach a coverage of 90%. Results of some 2 for further analyses. For fine-mapping QTL analyses will be reported. a backcross design with multiple marker re- gression was used together with denser micro- E023 satellite marker intervals. Both poor and supe- Chicken fatness QTL mapping using an rior F2 hens in egg white quality were selected Advanced Intercross Line for the female parents of backcross generation. Adding three microsatellite markers on the DANYEL G.J. JENNEN1, ADDIE L.J. QTL area narrowed its position from 55 to 31 VEREIJKEN2, RICHARD P.M.A. CROOI- cM. Moreover, a grid search fitting two QTL JMANS 1, TINEKE VEENENDAAL1, HENK was performed on the chromosome to test BOVENHUIS1, JAN J. VAN DER POEL1, whether there could be more than one QTL on GERARD A.A. ALBERS2, MARTIEN A.M. the chromosome affecting egg white thinning. GROENEN1 It was tested with a standard F-test whether the 1Animal Breeding and Genetics, Wageningen best two QTL model explained significantly Institute of Animal Sciences, Wageningen Uni- more variance than the best single QTL. The versity, Wageningen, The Netherlands; result suggested that instead of one QTL there 2Nutreco, Boxmeer, The Netherlands are two distinct QTL areas affecting egg white We have performed a total genome scan in chik- thinning. In addition, the results from the ken resulting in the localisation of QTLs for backcross generation indicated that one of the several economically important traits including QTL areas predominantly affects HU40 and fat deposition. Briefly, 480 F2 animals were the other HU60. individually genotyped for 284 microsatellite markers, and 3 different batches of 2000 F3 E022 animals each were phenotyped for fatness traits. Mapping of Quantitative Trait Loci for Deposited abdominal fat was measured in 2000 growth and fatness in chickens F3 animals at 7, 9 and 10 weeks of age. QTL analysis, using a regression interval mapping LINA U. JACOBSSON1, HEE-BOK PARK1, approach, were performed for total fat and fat PAUL B. SIEGEL2 , LEIF ANDERSSON1 percentage for the three groups of animals. Six 1Department of Animal Breeding and Genetics, different regions, located on six different chro- Swedish University of Agricultural Sciences mosomes, were identified that showed highly Uppsala, Sweden; 2Virginia Polytechnic Insti- significant F-statistics for several of the fatness tute and State University, Department of Ani- traits, indicating the presence of (a) gene(s) at mal and Poultry Sciences, Blacksburg, USA these locations that control the amount of fat Two chicken lines divergently selected for deposition in chicken. For the fine mapping of growth have been used to create an F2 inter- the regions containing the fatness QTLs an cross mapping population. The parental lines Advanced Intercross Line (AIL) has been pro- show a 9-fold difference in body weight at 8 duced. Over 3000 F9 animals were produced weeks of age and differ markedly in traits re- and measured for the fatness traits and typed lated to appetite and deposition of fat. These with markers from the six identified QTL lo- two extreme chicken lines have been esta- cations. blished by selecting for high and low body weight at 8 weeks of age for over 40 genera- E024 tions by P. B. Siegel. The mapping population Frequency of ER and RYR1 alleles and their was generated by crossing 30 individuals from association with reproductive performance each parental line reciprocally to generate 8 F1 in primiparous sows of synthetic line 990 males and 76 F1 females. A total of 974 F2 individuals were phenotyped for growth and MARIAN KAMYCZEK1, MARIAN RÓŻY- body composition traits including body weight CKI2, ANDRZEJ JANIK2, ANNA

155 Section E: Associations between Markers and Traits

KWACZYŃSKA1, MONIKA SIKORA1, AN- Dummerstorf, Germany NA RADKO2, TOMASZ ZABEK2 In an effort to improve the mapping resolution 1National Research Institute of Animal Pro- of a quantitative trait loci (QTL) controlling duction,Experimental Station,Pawłowice, Po- resistance to trypanosomosis on Bta7, a pre- land; 2National Research Institute of Animal viously unknown region of homology between Production, Kraków/Balice, Poland Bta7 and the murine Tir1 trypanotolerance Estrogene receptor (ER) and ryanodine recep- QTL region on Mmu17 was identified using a tor (RYR1) genes were evaluated for their as- combination of comparative and radiation hy- sociations with litter size in primiparous sows brid (RH) mapping. We report here the buil- line 990. Total of 548 sows were genotyped ding up of a bovine BAC contig spanning this for both loci. Relationship between ER, RYR1 region. Four BAC clones from the RPCI 42 genotypes and litter size were evaluated using bovine library were identified with two BAC analysis of variance. The polymorphism of the clones covering the entire homologous seg- ER gene was determined by the PCR/RFLP ment between the two QTL. The size of the method. The PCR product was digested by the homologous region is estimated to be around restriction endonuclease AvaI. It was found 300kb. One of the BAC clones includes a that observed allele frequency was 0,924 for chromosomal breakpoint of conserved synteny allel ERA and 0,076 for allel ERB. Low fre- between Bta7 and mouse Mmu17 and Mmu8. quency of ERB allele causes that in presented All BAC clones were physically located to the material only 7 homozygous ERB/B sows was Bta7 QTL region by fluorescent in situ hybri- found. In primiparous sows average number disation. The region of homology contains of pigs total born (8,80 and 9,20) and average eight genes, which were identified using com- number of pigs born alive ( 8,60 and 8,80 ) parative mapping, and ordered in cattle using a were respectively for ERA/A and ERA/B geno- 12,000rad RH panel (Texas A&M). PCR am- type. The polymorphism of the RYR1 gene plification using the four BAC clones as tem- was also determined by using PCR/RFLP plates confirms this gene repertoire and order. method. It was found that observed allele fre- Sequencing of the BAC clones may identify quency was 0,620 for allel RYR1C and 0,380 new genes and provide information on the for allel RYR1T. Average number of total comparative genome organisation of the region born pigs ( 9,81, 9,87 and 9,95 ) and average of homology. number of pigs born alive (8,71, 8,77 and 8,62) were respectively for RYR1C/C, RYR1C/T and E026 RYR1T/T genotype. Positive effect of ERB Identification of QTL affecting production allele on litter size in primiparous sows line traits in a cross between Red Jungle fowl 990 was small. It was found that genotype in and White Leghorn chicken RYR1 locus give no influence on litter size at first litter. Research is still continued and more SUSANNE KERJE1, ÖRJAN CARLBORG1, data for next litters will be collected soon. KARIN SCHÜTZ2, PER JENSEN2, LEIF ANDERSSON1 E025 1Swedish University of Agricultural Sciences, Construction of a BAC contig at a cattle Department of Animal Breeding and Geneics, QTL region controlling resistance to try- Uppsala, Sweden; 2Swedish University of Ag- panosomosis on BTA7 that is homologous to ricultural Sciences, Department of Animal the murine TIR1 region on MMU17 Environment and Health, Section of Ethology, Skara, Sweden SIMON KANG‘A1,2, PHILOMEEN NILS- SON1, TOM GOLDAMMER3, JOEL MWA- Red Jungle fowl chicken are considered to be KAYA1, ELIUD N.M. NJAGI2, MANFRED the wild ancestor to our modern chicken. We SCHWERIN3 , OLIVER HANOTTE1 have crossed one Red Jungle fowl male with 1The International Livestock Research Institute four White Leghorn females to generate a three (ILRI), Genetics & Genomics, Nairobi Kenya; generation mapping population. 41 F1 off- 2Kenyatta University, Biochemistry Depart- spring were intercrossed and 853 F2 individu- ment, Nairobi, Kenya; 3Forschungsinstitut für als were hatched. A genome scan including die Biologie landwirtschaftlicher Nutztiere 105 genetic markers distributed over 25 chro- (FBN), Forschungsbereich Molekularbiologie, mosomes/linkage groups was carried out and

156 Section E: Associations between Markers and Traits the resulting linkage map were used for QTL ses of these SNPs. Phenotypic associations of analysis. Data were collected for weight at 1, these polymorphisms were also found to be 8, 46, 112 and 200 days and growth were cal- present in several commercial populations. culated between the different recordings. Total These combined results suggest that these ge- and average egg weight were also recorded. A nes are good candidates for the growth and total of 9 traits were used for the QTL analysis fatness QTLs reported on pig chromosomes and resulted in 15 chromosomal regions sig- 1, 7, and 13. The results obtained to date nificant at the 5% genome wide level. When suggest that polymorphisms identified in searching for suggestive QTLs two more re- these genes will be useful for marker- gions were discovered. Six of the detected assisted selection for growth and fatness QTL had an effect on several traits. For body traits in the pig. weight at 200 days we found six significant QTL and all of them showed an additive in- E028 heritance with no significant dominance ef- Detection of QTL for birth weight in Cha- fects. The observed QTL explains a large por- rolais within the SEGFAM resource popula- tion of the two-fold difference in growth be- tion tween the two parental lines. CHRISTA KÜHN1, FRANK BECKER2, E027 HANS-JÜRGEN PAPSTEIN3, JOCHEN Investigation of candidate genes for the WEGNER3, OLAF BELLMANN3, JÜRGEN growth and fatness QTL on pig chromoso- VOIGT4, VOLKER GUIARD5, MANFRED mes 1, 7, and 13 in a Berkshire x Yorkshire SCHWERIN1 family and commercial populations 1Res. Unit Molecular Biology; 2Res. Unit Re- 3 1 1 productive Biology; Res. Unit Muscle Biology KWAN-SUK KIM , HAUKE THOMSEN , 4 2 and Growth; Res. Unit Nutritional Physiology JOHN BASTIAANSEN , YUANDAN 5 1 1 “Oskar Kellner”, Res. Unit Genetics and ZHANG , JACK C.M. DEKKERS , GRA- 2 Biometry, Research Institute for the Biology of HAM S. PLASTOW , MAX F. 1 Farm Animals, Dummerstorf, Germany ROTHSCHILD 1 To investigate the genetic and physiological Department of Animal Science, Iowa State 2 background of divergent nutrient transforma- University, Ames, IA, USA; Sygen Internatio- tion for growth and lactation in dairy and meat nal, Berkeley, CA, USA type cattle a resource population (SEGFAM) is Quantitative trait loci (QTL) analyses using established in the Research Institute for the molecular markers have detected several im- Biology of Farm Animals. In SEGFAM the portant genomic regions for growth and fatness Charolais and the German Holstein breed serve traits in pigs. The improved comparative map as representatives of the accretion and secre- between human and pig chromosomes and tion metabolic type, respectively. Substantial knowledge of the biological mechanisms of differences between those breeds exist for a these traits suggest several candidate genes in variety of physiological traits. However, there the identified QTL regions. To further investi- was indication, that unlike the situation in in- gate the identified growth and backfat QTL, bred lines of model organisms QTL with effect we have studied three positional and biological on these traits still segregate within selected candidate genes, MC4R, HMGA1 and GHRL, cattle breeds. This hypothesis of segregating which mapped to pig chromosomes 1, 7 and 13 QTL within a phenotypically extreme breed respectively. We have identified single was tested for the trait birth weight by a whole nucleotide polymorphisms (SNPs) within these genome scan in a half sib design originating genes and used them for QTL and association from the Charolais founder sires of the SEG- analyses. All three genes were mapped to po- FAM population. Five families each consisting sitions within the backfat and growth QTL of a Charolais sire, which was mated to Cha- regions that were identified in a Berkshire x rolais and German Holstein cows, as well as Yorkshire family. The identified SNPs were dams and offspring were genotyped with 198 significantly associated with observed variati- microsatellite markers distributed across all on in F2 animals for backfat and growth traits chromosomes. Variance analysis indicated using single marker analyses. Some interacti- putative QTL for birth weight on chromosomes ons were also detected from combined analy-

157 Section E: Associations between Markers and Traits

4, 5, 6, 14, 15, 16, 19, und 23. For the QTL References; Ehrmann,S., H.Bartenschlager positions on BTA4, 5, 6, 19, and 23 corre- and H.Geldermann: J.Arnim.Breed.Genet. sponding results for birth weight or related 144, 49-53 1997a. and 114 121-132, 1997b. traits have been found in other populations. Our results indicate, that in spite of extreme E030 phenotype segregating QTL for the respective QTL population to investigate the genetics traits can be detected within high selection of the pale, soft and exudative (PSE) meat in cattle breeds. chickens

E029 MÔNICA C. LEDUR1, JORGE A.F. LARA2, Associations between polymorphisms in KERLI NINOV1, CRISTINA A. BONASSI1, 5’ flanking regions of milk protein genes MASSAMI SHIMOKOMAKI2, ERALDO L. ZANELLA1, GIOVANNI R. BERTANI1, and traits of individual milk proteins in 3 cattle ALEXANDRE L. NEPOMUCENA 1Embrapa Swine and Poultry, Concórdia, Bra- 2 ANDREAS W. KUSS, JOCHEN GOGOL, zil; Londrina State University, Department of Food and Drugs Technology, Londrina, HEINZ BARTENSCHLAGER, HERMANN 3 GELDERMANN Brazil; Embrapa Soybean, Londrina, Brazil University of Hohenheim, Institute of Animal An F3 resource population was developed to Husbandry and Breeding, Department of Ani- identify quantitative trait loci (QTL) affecting mal Breeding and Biotechnology, Stuttgart, the functional properties of chicken meat, in- Germany cluding PSE. This population was originated Bovine milk protein yield as well as the rela- by crossing two divergent lines, a broiler (TT) tive composition of protein fractions were and a layer (CC) line, developed at Embrapa found to be associated with variants of the milk Swine and Poultry Research Center. 1497 F3 protein encoding genes (Ehrmann et al. 1994a animals were raised as broilers up to 42 days, & b). The aim of the study presented here was when were slaughtered and the following phe- to quantify milk proteins coded by specific loci notypic traits were measured: body weight, and investigate their individual association abdominal fat, breast fillet, parts and carcass with polymorphic gene variants. The study was weight, and pH 15 min after slaughter. The based on material from the two breeds German color (L* value), water-holding capacity, and Holstein Friesian and Simmental. A number of pH were measured 24 hours postmortem in milk samples per cow and lactation was ana- triplicate breast fillet samples. Blood samples lysed. Isoelectric focussing was used for allelic from parental, F1, F2 and F3 populations were identification of polymorphic milk proteins collected for DNA analyses. After the slaugh- and alkaline Urea-PAGE in combination with ter, thigh muscle samples were removed and densitometry was applied for quantification of stored in liquid nitrogen for RNA analyses. milk protein fractions (α-s1-Casein, β-Casein, Genome scan and candidate gene analyses will κ-Casein, α- Lactalbumin, β-Lactoglobulin). be performed to identify QTL regions related DNA variants were analysed in the 5’-flanking to these meat quality traits. We evaluated the regions of the β-Lactoglobulin and κ-Casein incidence of pale, soft and exudative meat in genes by PCR-RFLPs. Experimental data, our population. PSE meat results in low yield together with milk performance and pedigree of industrial products and unacceptable ap- data were submitted to analysis of variance. pearance by consumers. PSE meat was consid- The results reveal associations between spe- ered when the pH 24 h postmortem was below cific DNA variants and yield as well as the 5.8 and the L* value (color measurement) was relative portion of the individual milk proteins above 52.0. The incidence of PSE was encoded by the respective loci. These findings 532/1497 animals (35,5%). We are now inves- are interpreted as distingt influence of the vari- tigating two candidate genes for PSE, the RYR1 and RYR3, both related with mechanisms able gene positions on the investigated milk- 2+ parameters and are of relevance for further regulating myoplasmatic Ca concentration genetic analysis of milk protein yield and and excitation-contraction coupling. composition.

158 Section E: Associations between Markers and Traits

E031 FRIEDMANN, MOSHE SOLLER Associations of bovine leptin polymor- Department of Genetics, Hebrew University of phisms with circulating leptin concentra- Jerusalem, Jerusalem 91904, Israel tions The daughter design for mapping QTL uses only the information that is provided by the SILVIA C. LIEFERS1,2, MARINUS F.W. TE alleles of the sire of the family. However, a PAS1, ROEL F. VEERKAMP1, CAROLE relatively small number of maternal grandsires DELAVAUD3, YVES CHILLIARD3, TETTE (MGS) or great grandsires (MGGS) are active VAN DER LENDE2 in any given years, and these provide the bulk 1ID-Lelystad, Division of Animal Sciences, of the dams; in addition, the dams are chosen Lelystad, The Netherlands; 2Wageningen Uni- so that they are not in close relationship with versity, Animal Breeding and Genetics Group, the sire, limiting the selection of MGS. Thus, Wageningen, The Netherlands; 3INRA-Theix, there is much opportunity for linkage disequi- Herbivores Research Group, St-Genes- librium among the dams of the daughters. In- Champanelle, France cluding the dam alleles in QTL mapping will: Leptin is a protein involved in the regulation of (i) increase statistical significant of the results, feed intake, fertility and immune functions. To (ii) expand the genetic map, due to additional investigate the association of leptin polymor- generations of recombination between marker phisms with circulating leptin levels, 323 Hol- allele and QTL among the dam chromosomes, stein heifers were typed for three polymor- allowing more accurate determination of QTL phisms at the bovine leptin gene locus. The location, and, (iii) provide information in regi- Sau3AI RFLP is located in the intron, the ons where the sires are not informative, due to HphI RFLP at the beginning of the second homozygosity of the sire at the QTL or at the exon and the BM1500 microsatellite is located markers. To test this possibility, densitometric 3,6 kb downstream of the leptin gene. The data on dam allele frequency in pooled milk HphI polymorphism causes an Ala to Val sub- samples were obtained during a daughter de- stitution in the coding region of the gene. From sign analysis of milk production traits by se- late pregnancy until 80 days after calving lective DNA pooling. Within each sire-marker- blood samples were taken every two weeks. trait combination, alleles originating from the Leptin concentrations were determined using a dam only (e.g., all except the sire alleles), were RIA. During pregnancy the HphI-BB genotype tested for significance of the allele frequency showed higher (P<0.05) leptin levels compared difference between high and low pools. There to the AA and AB genotypes, and the BM1500 was a good agreement with the results obtained A-allele showed lower (P<0.05) leptin levels by analysis of sire alleles. In addition, as ex- compared to the presence of the BM1500 B- or pected if the genetic map has expanded, C-allele. During pregnancy and lactation the markers showing statistical significance and presence of the BM1500 B-allele caused peaks of significance for three different milk higher (P<0.05) leptin levels than the presence production traits (protein percent, protein yield of the other two alleles. The two exons in- and milk yield) were more tightly clustered cluding the exon/intron boundaries of the than found for the sire marker alleles. leptin gene of 90 animals were sequenced to investigate linkage of the polymorphisms to E033 mutations at the coding region. Several muta- QTL analysis for milk production traits tions were found and it seems that the BM1500 and SCS on chromosome 23 in the Spanish A-allele is linked to an Arg to Cys mutation in Holstein-Friesian population exon 1. Furthermore it seems that the recombi- nation frequency is high for this region. SAGRARIO MARCOS1, DELFINO HERNÁNDEZ2, MALENA SERRANO1, E032 MARÍA J. CARABAÑO1 Using information on segration of dam 1INIA, Departamento de Mejora Genética marker alleles within a daughter design; for Animal, Madrid, España; 2CONAFE, Ma- mapping QTL affecting milk production drid,España traits in Israel Holstein dairy cattle As a way to improve the validity of previously detected QTL's on BTA 23, we present an EHUD LIPKIN, GILI GRUZMAN, ADAM analysis of that chromosome for milk produc-

159 Section E: Associations between Markers and Traits tion traits and somatic cell score (SCS) in the age map of the whole genome by using the Spanish Holstein-Friesian cattle to compare CRI-MAP version 2.4 software. The QTL results between studies from different popula- analysis was performed by a linear regression tions. This study is being performed in a grand method using either the carcass weight (model daughter desing (GDD) comprising 742 sons 1) or the backfat thickness (model 2) as a co- distributed over twenty six paternal half sib variate. Model 1 showed significant QTL in families.The number of sons per sire ranges chromosome 4 (LIN, DBI and PI) 6 (DBI and from 10 to 59 and is on average 28,5.The phe- UI), 8 (PA, PAL and ACL), 10 (MYR) and 12 notypic units of measurement for statistical (LINL and GAD). Model 2 showed significant analisys are daughter yield deviation (DYD) effects on chromosomes 8 (PA, PAL and based on the national database and provided by ACL), 10 (MYR) and 12 (LINL, GAD, ACL, the National Association of Spanish Holstein- PI). QTL on chromosomes 8 (PA, PAL and Friesian (CONAFE) for milk yield (Kg), fat ACL), 10 (MYR) and 12 (LINL and GAD) yield (Kg), protein yield (Kg), fat percentage, showed significant F-value in both models at protein percentage and SCS. Eleven microsa- genomewide level of significance. The results tellites markers covering 61 cM of BTA 23 of this study suggest that some QTL have ef- are being genotyped. Multiple-marker interval fect for both fat deposition and fatty acid com- mapping with both regression and maximum- position, and other QTL only affect fatty acid likelihood methods will be applied. composition, independently of the fat deposi- tion traits. An adequate choice of the covariate E035 used in the model of analysis plays a crucial QTL for fatty acid composition in pigs using role. either the backfat thickness or the carcass weight as a covariate in a regession method E036 Segregation of resistance to nematode infec- ALEX CLOP1, ANNA MERCADE1, CRIS- tion in F2 lambs of Suffolk x Gulf Coast TINA ÓVILO2, MIGUEL PEREZ-ENCISO3, Native sheep and associated QTL ALBERT CERCOS1, ANNA TOMAS1, ANA FERNANDEZ2, AGUSTINA COLL1, JOSEP JAMES E. MILLER1, NOELLE E. COCKET2, M. FOLCH1, CARMEN BARRAGAN2, GRANT A. WALLING3, TRACY L. SHAY2, MARIA A. OLIVER4, ISABEL DIAZ4, LUIS ROYAL A. McGRAW4, STEVEN C. BIS- VARONA5, LUIS SILIO2, JOSE R. HOP3, CHRIS A. HALEY3 NOGUERA5, ARMAND SANCHEZ1 1Louisiana State University, Department of 1Department de Ciènca Animal i dels Aliments, Pathobiological Sciences, School of Veterinary Facultat de Veterinàra, Universitat Autònoma Medicine, Baton Rouge, Louisiana USA, 2Utah de Barcelona, Bellaterra 08193, Spain; State University, Biotechnology Center, Logan 2Departamento de Mejora Genética y Biotec- Utah USA, 3Roslin Institute, Edinburgh, Scot- nologia, INIA, Madrid 28040, Spain; 3SAGA- land, 4University of Georgia, Department of INRA, Castanet-Tolosan 31326, France; 4 Physiology and Pharmacology, College of IRTA-CTC, Monells 17121, Spain; 5Area de Veterinary Medicine, Athens, Georgia USA Producció Animal, Centre UdL-IRTA, Lleida Sixty-two, 86 and 84 F2 lambs from Suffolk x 25198, Spain Gulf Cost Native (Native) F1 ewes were born We have analized a F2 pedigree obtained by in 1998, 1999 and 2000, respectively. Lambs mating 3 Iberian boars to 31 Landrace sows in grazed until weaned, at which time they were order to detect OTL for fatty acid composition dewormed and maintained on concrete for 6 in backfat. We recorded the percentages of the weeks. Subsequently, they grazed for six most relevant fatty acids by gas chromatogra- weeks and nematode infection level was eva- phy. These fatty acids were Myristic (MYR), luated by fecal egg count (FEC) and blood Palmitic (PA), Palmitoleic (PAL), Stearic packed cell volume (PCV). For 1998 lambs, (STE), Vaccenic (VAC), Oleic (OLE), Lino- mean FEC and PCV ranged from 933-19,433 leic (LIN), Linolenic (LINL), Gadoleic (GAD) eggs per gram (EPG) and 14.7-30.7%, respec- and Eicosadienoic (EIC). We also calculated tively. For 1999 lambs, mean FEC and PCV their average chain length (ACL), double bond ranged from 633-31,267 EPG and 7.3-27.0%, index (DBI), unsaturated index (UI) and per- respectively. For 2000 lambs, mean FEC and oxidability index (PI). We constructed a link- PCV ranged from 50-21,650 EPG and

160 Section E: Associations between Markers and Traits

15.0-33.0%, respectively. Typically, under and epistatic effects and four QTLs with epi- similar challenge, the mean FEC of the parent static effects only, were significantly identified Suffolk and Native breeds is in the 7-15,000 among all analyzed traits. The QTL on Chr. 7 EPG and 500-3,000 EPG range, respectively. (Nidd1/of ) had a main effect (p < 0.05) on six Similarly, mean PCV is in the 15-19% and 24- traits and an epistatic effect (p < 0.10) on one 28% range, respectively. The range of FEC trait. For the plasma glucose level at 0 and 120 and PCV indicated that resistance to infection min, adjusted for body weight, the optimal segregated. A selective genotyping strategy models were able to account for a total of 75% was used in which 50 microsatellite markers and 59% of the phenotypic variance, respecti- were screened across 40% of the F2 lambs vely. Because both multiple QTLs and epista- (20% highest FEC and 20% lowest FEC) and ses are included in the model simultaneously, their parents. In total, 80 lambs (including 26 MCIM allows not only for the accurate identi- lambs of Sire 1, 25 lambs of Sire 2 and 29 fication of QTLs but also the correct partition lambs of Sire 3), 3 sires and 56 dams were of the phenotypic variance into the various genotyped. Within-family linkage analysis for genetic components. marker effects suggest QTL for FEC on chro- mosomes 1, 3 and 19. Associations with these E038 markers will be further explored using interval Distribution of Haplotypes in BTA 6, 14, 19, mapping. and 21 Within One Commercial Line of Bos taurus and Their Associations With Growth E037 Traits Multiple QTL mapping with epistatic inter- actions for non-insulin-dependent diabetes JENIFER L. KNEELAND1, CHANGXI LI1, mellitus in rat BRENDA MURDOCH1 , JOHN BASARAB2, WARREN SNELLING3, BERNHARD BEN- TAKESHI MIYAKE1,2, AKIRA NARITA2, KEL4, STEPHEN S. MOORE1 TAKAHISA YAMADA2, GAUDENZ DOLF 1Department of Agricultural, Food and Nutri- 1, CLAUDE GAILLARD1, KOZO MATSU- tional Science, University of Alberta, Edmon- MOTO3, YOSHOYUKI SASAKI2 ton, Alberta, Canada; 2Alberta Agriculture, 1Institute of Animal Genetics, Nutrition and Food and Rural Development, Lacombe Rese- Housing, University of Berne, 3012 Berne, arch Center, Lacombe, Alberta, Canada; Switzerland; 3USDA, ARS, US Meat Animal Research Cen- 2Graduate School of Agriculture, Kyoto Uni- ter, Clay Center, Nebraska, USA; 4Agriculture versity, Kyoto 606-8502, Japan; 3University of and Agri-Food Canada, Lethbridge Research Tokushima School of Medicine, Tokushima Center, Lethbridge, Alberta, Canada 770, Japan The objective of this study was to verify and The Otsuka Long-Evans Tokushima Fatty rat fine map quantitative trait loci (QTL), for is an animal model for obese-type non-insulin- growth in industry beef cattle herds, that have dependent diabetes mellitus (NIDDM) in hu- been previously identified in experimental mans. The newly developed multiple QTL reference herds. By verifying and fine mapping mapping method, Monte Carlo Interaction QTLs that affect growth one can identify posi- Mapping (MCIM), was applied to map QTLs tional candidate genes and gain a greater un- and epistatic interactions between two QTLs derstanding of their biology and function. Co- for NIDDM in 160 F2 crossbred rats with 213 segregation between a particular genetic mar- informative markers. As indicators of NIDDM ker and a QTL in a population is critical for susceptibility, the plasma glucose levels at 0, successfully mapping QTLs. Common haplo- 30, 60, 90 and 120 min (mg/dl x min) were types are expected to carry on and segregate determined by using the oral glucose tolerance among individuals of commercial breeding test. For each trait, the optimal model was de- lines. Genes underlying QTL of interest may termined by forward model-selection procedu- be present in these common haplotypes, thus re. Threshold values for significance tests were making it possible to map them to a particular determined by permutation tests, and they were chromosomal location. We report here the severer than those obtained by Chi-square va- identification and mapping of QTLs for birth lues with Bonferroni correction. Seven QTLs weight, pre-weaning average daily gain, and with main effects only, four QTLs with main average daily gain on feed in a commercial line

161 Section E: Associations between Markers and Traits of Bos taurus using the identical-by-descent Results of QTL analysis showed that chromo- haplotype sharing method. One hundred and some 1 comprise of three significant loci, seventy-six calves of twelve bulls were typed which 95% confidence interval (CI) between using forty-nine microsatellite markers chosen 3-6cM, while chromosome 5 has 2 loci with a from BTA6, 14, 19 & 21 (8 – 18 markers from 95% CI of 1cM and 2cM. The confidence ran- each chromosome). For each calf the alleles ge obtained can now facilitate positional inherited from each parent were identified. cloning of genes underlying the QTL. Haplotypes of each calf can then be identified along the length of each chromosome. Statisti- E041 cal analysis using the General Linear Model Association of GH and IGF-1 polymor- (GLM) procedure in SAS (version 8) identified phisms to growth traits in a synthetic beef twenty-one chromosomal regions that have cattle breed association with growth traits at a significance threshold of p<0.05. ANDRÉA P. PEREIRA1, MAURÍCIO M. ALENCAR2, HENRIQUE N. OLIVEIRA3, E039 LUCIANA C.A. REGITANO2 High-resolution mapping of trypanotoler- 1Federal University of São Carlos, Genetics ance QTL Tir2 and 3 using F12 advanced and Evolution Department, São Carlos, Brazil; intercross lines 2Embrapa Southeast Cattle Research Center, São Carlos, Brazi;, 3São Paulo State Univer- JOSEPH K. NGANGA1,2, JOHN P. GIBSON1, sity, Animal Breeding and Nutrition Depart- STEPHEN J. KEMP3, FUAD A. IRAQI1 ment, Botucatu, Brazil. This work was sup- 1International Livestock Research Institute, P. ported by FAPESP and CNPq O. Box 30709, Nairobi, Kenya; 2Jomo Ke- The Canchim beef cattle (5/8 Charolais + 3/8 nyatta University of Agriculture and Techno- Zebu), has been selected for meat production logy P. O. Box 62000, Nairobi, Kenya; 3School in Brazil since 1953.In the present work the of Biological Sciences, Donnan Laborato- effect of candidate genes polymorphisms was ries,University of Liverpool UK investigated in 688 animals born between 1998 Different mouse strains show variation in re- and 2000. From these 307 belonged to the sponse to trypanosoma congolense infection original Canchim population (GG1) that was (Trypanosomosis). In previous studies, we formed in 1953 and the remaining belong to a have mapped quantitative trait loci (QTL) for Canchim population formed by recent crosses trypanosomosis resistance (trypanotolerance) (GG2). DNA extracted from blood samples in two F2 populations to chromosomes 17, 5 using a salting out procedure was amplified for and 1 and designated as Tir1, Tir2 and Tir3 a 223 bp fragment, spanning intron IV and respectively. Using advanced intercross lines exon V of the growth hormone (GH) gene, and (AIL) approach, only Tir1 has been fine map- for type 1 insuline like growth factor (IGF1) ped to a confidence interval less than 1cM. In microsatellite in 25 µl reactions. GH products order to fine map Tir2 and 3, F12 C57BL/6J x were digested with AluI and fragments were A/J AIL fixed for the susceptible and resistan- resolved in 3% agarosis electrophoresis. IGF1 ce alleles at Tir1 locus were generated. The products were separated by electrophoresis in 8 two AIL populations and the parental controls % non denaturing polyacrylamide gels and were then challenged with T. congolense at 12 silver stained to identify the alleles. Genotype weeks of age and followed for survival times effects on breeding values for birth weight over 180 days. Mice from the two survival (BW), weaning weight (WW) and yearling extremes of the population fixed for the weight (YW) were investigated by the SAS susceptible alleles were genotyped with a panel GLM procedure. The statistical model included of microsatellite markers across the previously the effects of genetic group and GH and IGF- mapped regions. The data was analyzed with 1 genotypes. Significant effects were found for maximum likelihood and least square interval- GH genotype on YW (P ≤ 0.05), with positive mapping methods. The mean survival time of effects associated to the V allele, and for IGF1 AIL fixed for the susceptible and resistant on BW (P ≤ 0.01) and YW (P ≤ 0.01). QTL on chromosome 17 was 95 and 125 days respectively whereas that of AJ and C57BL parental lines was 60 and 80 days respectively.

162 Section E: Associations between Markers and Traits

E042 E043 Combined approaches at INRA to fine map QTL research on duration of tonic immo- QTL influencing growth, fatness and car- bility in quail cass composition traits in pig. ODILE ROUSSOT1, KATIA FÈVE1, FLO- JULIETTE RIQUET1, MARIE-PIERRE RENCE PLISSON-PETIT1, FRÉDÉRIQUE SANCHEZ2, OLIVIER DEMEURE1, NA- PITEL1, ALAIN VIGNAL1, JEAN-MICHEL THALIE IANNUCCELLI1, KATIA FEVE1, FAURE2, ANDREW D. MILLS2, DANIEL HÉLÈNE GILBERT2, CARINE GENÊT1, GUÉMENÉ2, CHRISTINE LETERRIER2, MICHÈLE BONNET1, CHRISTOPHE PE- SANDRINE MIGNON-GRASTEAU2, RY2, YVON BILLON2, JEAN GOGUÉ2, PASCALE LE ROY3, MIGUEL PEREZ- JEAN-CLAUDE CARITEZ2, PASCALE LE ENCISO4, CATHERINE BEAUMONT2 ROY2, CHRISTINE RENARD3, JEAN- 1Laboratoire de Génétique Cellulaire, INRA, PIERRE BIDANEL2, DENIS MILAN1 Castanet-Tolosan, France; 2Station de Recher- INRA, France: 1Laboratoire de Génétique ches Avicoles, INRA, Nouzilly, France; Cellulaire, Castanet-Tolosan; 2Station de 3Station de Génétique Quantitative et Appli- Génétique Quantitative et Appliquée, Jouy-en- quée, INRA, Jouy-en-Josas, France; 4Station Josas; 3Laboratoire de Radiobiologie et d’Amélioration Génétique des Animaux, INRA, d’Etude du Génome, Jouy-en-Josas Castanet-Tolosan, France A whole genome QTL analysis of growth, The study of behavioural traits is essential to fatness and carcass composition data from a F2 the assessment of animals stress and welfare. A experimental cross between 6 Meishan (MS) QTL detection study was initiated to identify sows and 6 Large White (LW) males has been the genome regions involved in the control of performed at INRA. Highly significant QTL fearfulness, an important component of ani- effects were detected in several chromosomal mals capacity of adaptation. An F2 cross was regions: the telomeric regions of SSC 1q carried out between two quail lines that had (growth, fatness, lean cuts weights) and SSC been divergently selected for more than 25 2p (lean and fat cuts weights), SSC 4 (growth, generations for or against duration of tonic fatness) and SSC 7 (growth, fatness, lean and immobility (DTI), a catatonic-like state of fat cuts weights) were selected for further reduced responsiveness to external stressful analyses. Unlike other studies, our analysis did stimulation. A total of 1048 animals were ob- not reveal any imprinting effect for the SSC2 tained and measured for several traits including QTL. Further investigations are underway to DTI and NI, the number of inductions neces- dissect chromosomal regions containing QTLs: sary to induce immobility reaction. The differ- (1) backcross animals produced from F1 origi- ence (DIFF) between DTI and NI (both centred nal animals are being studied; (2) synthetic and reduced) was analyzed. Segregation analy- commercial lines related to the INRA Meishan sis led to expect the existence of a gene ex- population are being analysed to identify re- plaining more than one standard deviation of combinant chromosomes interesting for fine DIFF. As no genetic map of quail was avail- mapping of the QTL by progeny testing, and to able yet, a set of 432 AFLP™ markers was study the effects of selection on haplotype developed. Using variance analysis, significant frequencies in QTL regions; (3) the effects of effects were observed for alleles of several QTL regions are also being investigated in markers belonging to one linkage group. A other experimental populations (pure Large QTL analysis tends to confirm these results so White, LW x Piétrain). In parallel we are de- that the existence of a QTL within this group is veloping in QTL regions (1) a physical map highly suspected. based on radiation hybrid maps and BAC con- tigs, and its comparison with human genome E044 map; (2) a denser genetic map with new micro- Association of bovine butyrophilin encoding satellites and SNP. A study of the transcrip- gene polymorphism with milk fat percent- tome of animals with various QTL genotypes age in Polish Black-and-White and German is also planned to better define QTL effects. Holstein-Friesian cattle

CHANDRA SHEKHAR PAREEK1, RAVI SHEKHAR PAREEK1, KRZYSZTOF

163 Section E: Associations between Markers and Traits

WALAWSKI1, HANS-MARTIN SEYFERT2 on. The syndrome is inherited as an autosomal 1Department of Animal Genetics, University of recessive disease exclusively expressed in Warmia and Mazury, Olsztyn, Poland; male individuals as shorter sperm tail length 2Division of Molecular Biology, Research In- and immotile spermatozoa. Currently 61 boars stitute for the Biology of Farm Animals, Dum- are known to have the syndrome. Based on the merstrof, Germany assumption of a recent common origin of the Butyrophilin is a type I membrane protein of disease-causing mutation, a genome-wide se- immunoglobulin super-family that is secreted arch was performed with 228 evenly spaced in association with the milk fat globule mem- microsatellites by homozygosity mapping of brane from mammary epithelial cells. A pro- affected and unaffected DNA pools. One locus, tein sequence polymorphism concerning aa SW2411 on Chr 16, demonstrated a signifi- position 35 (Pro vs Glu) was identified and cantly skewed allele distribution between the verified in the active Polish Black-and-White two pools. Linkage analysis of five markers in and German HF breeding population. The ob- this region mapped the disease-causing gene served allele A encodes for aa Pro at position within a 6-cM confidence interval region with 35 in contrast to Glu encoded by allele B. The a highest LOD score of 7.7 at marker SW419. association of these allelic variants with milk Based on haplotype data, the mutation lies production traits among Polish Black-and- between markers SW2411 and SW419, and White and German HF population was investi- therefore the confidence interval determined gated. A total of 88 and 298 breeding bulls with linkage analysis can be reduced to a 3 cM from Polish Black-and-White and German HF region proximal to SW419. It appears that was genotyped for A and B alleles by PCR- three-marker haplotypes can be used for mar- RFLP method. In addition, a half-sib pedigree ker-assisted selection within analyzed pe- from Polish-Black-and-White bull was geno- digrees, but pedigree data is still needed for typed for A and B alleles, since animals re- MAS. Furthermore, current fine mapping may vealed significant differences in the parame- reveal a more precise population-wide asso- ters, viz., milk, fat, protein yields and their ciated haplotype and facilitate identification of percentages. The frequencies of alleles A and a new gene affecting sperm tail development. B were observed as 25.3%, 74.7% in 83 half- sib progenies from Polish-Black-and White E046 cattle and 57.9%, 42.1% in German HF bulls, QTL mapping experiment in F2 cross of respectively. A significant association was chickens divergently selected for antibody found between this protein sequence polymor- response to SRBC phism and milk fat percentage. Allele B corre- lates with a significantly increased milk fat M. SIWEK1, S.J.B. CORNELISSEN1, M.G.B. percentage without affecting the milk yield. NIEUWLAND2, A.J. BUITENHUIS1, H. BO- VENHUIS1, J.J. VAN DER POEL1; H.K. E045 PARMENTIER2 Mapping of an immotile short tail sperm 1Animal Breeding and Genetics Group, defect in Finnish Yorkshire on porcine 2Adaptation Physiology Group, Wageningen chromosome 16 Institute of Animal Sciences, Wageningen Uni- versity, P.O. Box 338, NL – 6700 AH Wa- ANU I. SIRONEN1, MAGNUS ANDERS- geningen, The Netherlands SON2, PEKKA UIMARI3, JOHANNA VILK- The aim of this paper is to present a first data KI1 of experiment of mapping QTL associated with 1MTT Agrifood Research Finland, Animal immune response. The experimental F2 po- Production Research, Animal Breeding, FIN- pulation originates from a cross (ISA Warren) 31600 Jokioinen, Finland; 2University of Hel- between two divergently selected lines for sinki, Department of Clinical Veterinary Sci- either high (H line) or low (L line) primary ences, Helsinki, Finland; 3University of Hel- antibody response to sheep red blood cells sinki, Department of Biosciences, Helsinki, (SRBC) at 5 days after primary intramuscular Finland immunisation with SRBC at 37 days of age. An immotile short tail sperm defect (ISTS) has For the QTL experiment reciprocal crosses recently been identified as a hereditary disor- have been made to produce 1240 F2 animals. der within the Finnish Yorkshire pig populati- The phenotypic data for the immunological

164 Section E: Associations between Markers and Traits responses have been determined. Blood sire. The number of progeny with both marker samples were collected from birds in the day 0 data and phenotypes ranged from 2 to 14 in and the test day for relevant antigens i.e.: pri- each of the 43 sire families. A total of 175 of mary antibody response against SRBC at 35 37,539 tested effects were significant within days, E.coli at 55 days, KLH-DNP (keyhole family (P<0.001), and 17 marker by trait com- limpet haemocyanin – dinitrophenyl) at 82 binations on seven chromosomes out of 905 days, Mycobacterium butyricum at 103 days, tested were significant (P<0.01) in across- secondary antibody response against SRBC at family F-tests. In both analyses, adjacent 127 days and cellular response against ConA markers were found to be significant. A per- (ConcanavalinA) at 132 days of age. For the mutation test is under development to account genotypic analysis the Whole Genome Scan for non-normality of the EPG data and mul- has been performed, 174 microsatellite tiple testing. markers were chosen, distributed over the chicken genome, approximately 20 centiMor- E048 gan (cM) apart, to type in total 722 animals. In A deletion of HSPA1B causes hereditary QTL analysis the half sib model will be used myopathy of diaphragmatic muscles in Hol- for each of 24 half sib families (12 per recipro- stein-Friesian cattle cal cross). MAYUMI SUGIMOTO1, HIDEFUMI FU- E047 RUOKA2 , YOSHIKAZU SUGIMOTO3 A genome scan for gastrointestinal nemato- 1National Livestock Breeding Center, Nishigo, de resistance in cattle Japan, 2Obihiro University of Agriculture and Veterinary Medicine, Department of Veterina- TAD S. SONSTEGARD1, LOUIS C. GAS- ry Pathology, Obihiro, Japan; 3Shirakawa BARRE1, CURTIS P. VAN TASSELL1, R. Institute of Animal Genetics, Nishigo, Japan MARK THALLMAN2, TEREZINHA PA- Hereditary myopathy in Holstein-Friesian DILHA1,3 cattle is an autosomal recessive disease cha- 1United States Dept. of Agriculture, Agricultu- racterized by late onset, ruminal tympany, and ral Research Service, Beltsville, MD, USA; central core-like structures in the diaphragma- 2United States Dept. of Agriculture, Agricultu- tic muscles. A whole-genome scan of 26 fa- ral Research Service, Clay Center, NE, USA; mily members, including 12 affected, showed 3EMBRAPA-ARS Labex Program, Beltsville, significant linkage of a hereditary myopathy MD, USA locus to a 0.6-cM interval in BTA23q21, equi- Breeding for host resistance offers an alternati- valent to HSA6p21.3. The maximum LOD ve method to control disease caused by ga- score, 7.67, was obtained for marker CYP21, strointestinal (GI) parasites. However, the ge- near the genes that encodes heat-shock 70-kd netic factors affecting host resistance and protein 1A (HSPA1A) and 1B (HSPA1B). transmission in cattle have yet to be identified. HSPA1A and 1B are duplicated heat-shock Using a population of linebred Angus cattle protein, 70-kd, (HSP70) loci within the bovine that was divergently selected for resistance to major histocompatibility complex on 23q21, 8 GI parasites, we performed a genome-wide kb apart from each other, known to be the ma- scan to identify QTL for traits related to infec- jor mammalian chaperones. Sequencing of tion. Genotypes for 196 microsatellite markers affected samples revealed a complete deletion were generated from ~300 progeny with phe- of HSPA1B. Molecular chaperones prevent notypic records for parasite challenge, parents, aggregation and refold unfolded proteins. Sin- and over 70 sires from the historic pedigree. ce central core-like structures in affected dia- Traits analyzed were mean, peak, and final phragmatic muscles shows strong immunore- numbers of nematode eggs/gram (EPG) of activity for actin and , impaired pro- feces and mean and final serum pepsinogen tein clearance by incomplete HSP70 may un- levels. Marker segregations were determined derlie the pathogenesis of myopathy. using Genoprob. The analysis model included sex of calf, age of dam at calving, age of calf at parasite challenge, calving season, sire, and within-sire regression on the probability of inheriting one of the two QTL alleles of the

165 Section E: Associations between Markers and Traits

E049 and its important role in prenatal development Genetic mapping of quantitative trait loci in mammals. This QTL is segregating in our for fatness in chickens Wild Boar/Large White intercross and pre- vious studies did not reveal any sequence dif- KEN TATSUDA, KUNINORI FUJINAKA, ference in the IGF2 coding sequence. Thus, we YUMIKO KURODA assume that the QTL may be due to one or Hyogo Prefectural Agricultural Institute, more regulatory mutations. We have now de- Kasai, Japan termined 32 kb contiguous genomic sequence To identify the quantitative trait loci (QTL) containing the Insulin-IGF2 region. This regi- affecting abdominal fat deposition and lipid on contains a very high GC content, many content in chickens, QTL analysis was carried CpG islands and very few interspersed repeats. out using a resource population of 222 F2 pro- A comparative sequence analysis with the ho- geny from a cross between a Satsumadori (a mologous region in humans and mouse revea- Japanese native breed showing little fat depo- led that all coding sequences are well conser- sition) male and a White Plymouth Rock (a ved between species but also that a large pro- broiler breed showing significant fat depositi- portion of the non-coding sequence in this on) female. We chose 80 microsatellite loci region is surprisingly well conserved between from 575 loci publicly available from 16 lin- species. The results will facilitate the further kage groups, based on their utility and locati- characterization of this important QTL in the on. One QTL affecting the ratio of abdominal pig and we are currently doing comparative fat weight to live body weight at 16 weeks of sequence analysis of chromosomes with age was mapped at 149 cM on chromosome 7, “known” QTL status in the search for causati- with a LOD score of 8.9, and accounted for ve mutation(s). 33.3% of the variance. The closest loci to the QTL were MCW0316 and ADL0169. Two E051 QTLs affecting the lipid content in thigh meat Mapping economic trait loci for exterior at 16 weeks of age were mapped at 111 cM on traits in a commercial pig population chromosome 3 with a LOD score of 6.0, ac- counting for 75.1% of the variance and at 194 XIAOLING GUO1, CHRISTIAN LOOFT1, cM on chromosome 5 with a LOD score of 5.7, ESTELA FABUEL PEÑALVER1, NORBERT accounting for 69.5% of the variance. The REINSCH1, HOLGER LOOFT2, ERNST closest loci to the QTLs were MCW0212 on KALM1 chromosome 3 and ADL0298 on chromosome 1Christian-Albrechts-University of Kiel, Insti- 5. The difference in these two traits in F2 was tute of Animal Breeding and Husbandry, Kiel, associated with the allele types of each marker, Germany; 2PIC Germany Ltd, Schleswig, which suggests the possibility of using marker- Germany assisted selection for fatness in chickens. The aim of our project was to map economic trait loci for porcine exterior traits. The design E050 of the project is based on a five line cross. 19 Comparative sequence analysis of the Insu- hybrid boars (Piétrain+ x (Hampshire x Pié- lin-IGF2 region harbouring a major QTL train)) were mated to 52 hybrid sows (Leicoma for muscle development in the pig x (Large White x Landrace). Their 333 proge- nies were fattened on our experimental farm ANNE-SOPHIE VAN LAERE, VALERIE and slaughtered at 115 kg live weight. For AMARGER, LEIF ANDERSSON these animals 23 exterior traits as for example Swedish University of Agricultural Sciences leg scores were scored by a trained person (SLU), Department of Animal Breeding and following a subjective linear scale. A genome Genetics, Uppsala, Sweden scan covering all chromosomes with 159 mi- We and others have identified a paternally crosatellite markers and 3 class-I-markers expressed Quantitative Trait Locus (QTL) (Ryr1, RN, Pit1) was performed. Marker link- primarily affecting muscle development at the age maps were computed with CriMap and distal tip of pig chromosome 2p. IGF2 (Insu- used for interval mapping using a least-squares lin-like Growth Factor 2) was identified as the regression model in parental half-sib families. major candidate gene due to its perfect coloca- Effects for five exterior traits, i.e. the fore legs lisation with the QTL, its paternal imprinting, angle, ear shape, the fore feet position, the leg

166 Section E: Associations between Markers and Traits status and the hind legs step, were detected on E053 chromosomes 4 (Pexp. < .01), 6 (Pexp. < .01), 6 Detection of QTL for resistance to gastro- (Pexp. < .10), 7 (Pexp. < .01) and 13 (Pexp. < .10), intestinal nematode infection in mice respectively. 1 1,2 FUAD A. IRAQI , DAVID M. MENGE , E052 JERZY M. BEHNKE3, ANN LOWE3, ALAN Gene mapping progress in cattle and upda- J. TEALE1,4, JOHN P. GIBSON1, LEY- 1 3 ted comparative map with man, mouse, rat DEN R. BAKER , DAREK WAKELIN and pig 1International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya; H. HAYES, C. ELDUQUE, M. GAUTIER, L. 2Jomo Kenyatta University of Agriculture and SCHIBLER, E. CRIBIU, A. EGGEN Technology P. O. Box 62000, Nairobi Kenya, Laboratoire de Génétique biochimique et 3School of Biological Sciences, University of Cytogénétique, INRA, Jouy-en-Josas, France Nottingham, NG7 2RD, U.K.; 4Present Our on-going goal is to improve and update the address, Institute of Aquaculture, University of comparative genome organization between Sterling, FK9 4LA, U.K. cattle and man but also among the most de- Gastro-intestinal (GI) nematodes are arguably tailed mammalian species genomes ie cattle, the most important disease in domestic rumi- mouse, rat and pig. In the past years, we have nants. Current control mainly relies on che- localized by FISH over 250 genes on the bo- motherapy, but resistance to all major anthel- vine chromosomes using bovine BAC and mintics is now widespread. There is a consid- YAC probes and caprine BAC probes. In this erable interest in understanding the naturally work, we have compiled all the genes mapped occurring genetic resistance to GI nematodes in cattle, goat, sheep and pig (linkage mapping, in sheep. This can be done more rapidly in somatic cell hybrid and radiation hybrid map- well-defined genetic mouse model. Different ping, FISH) and for which the human ortholog mouse strains show variation in response to GI is known and mapped (1646 genes). When nematode infection. Here we report results of a possible the corresponding data in mouse and genome-wide search for QTL in an F2 cross rat were included. For each human chromo- between the resistance, SWR and the suscepti- some the genes were ordered precisely from ble CBA parental mouse strains that was chal- telomere to telomere using the human genome lenged by trickle infection with Heligmoso- sequence data (UCSC GoldenPath). This com- moides polygyrus. Eight traits associated with parison of conserved syntenies among the hu- resistance to GI nematodes infection were man, bovine, mouse, rat and pig genomes pro- measured-i.e total worm count, three time vides an overall and detailed picture of the points of faecal egg count post infection, im- organization of the bovine genome relatively to munoglobulin G1 and E titers, granuloma the human genome, confirms that the degree of score and mucosal mast cell protease. Interval synteny conservation is higher between man mapping analysis after selective genotyping and cattle or pig than between man, cattle or detected 30 significant QTL affecting at least pig and mouse or rat and delineates 99 con- one of the resistance traits. Twenty-five QTL served chromosomal segments between man were associated with more than one resistance and cattle with a size ranging from 1 to 120 trait suggesting either pleiotropy or tight link- Mb. It also shows that the limits of the con- age as major mechanism of the QTL for gas- served chromosomal segments between man tro-intestinal resistance. Candidate genes and cattle are mainly located in G bands or at within these QTL were identified. Some of the the G/R bands junctions and that most of the genes appear to have mechanisms controlling regions for which no comparative mapping the intestinal immune and inflammatory re- data exists is located in G bands known to be sponses known to protect against gut parasites. gene poor. This compilation contributes to the High resolution mapping of these QTL is on- construction of comparative maps focusing on going using the advanced intercross lines the order of genes within segments of con- (AIL) approach. served synteny and constitutes a powerful tool for positional candidate gene cloning ap- proaches.

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E054 1University of Berne, Institute of Animal Ge- Chromosome-wide scanning of SSC3 for netics, Nutrition and Housing, Berne, Switzer- QTL associated with prolificacy in a land; 2CIRAD-EMVT, Petit-Bourg, Guade- Meishan x Large White population using a loupe, FWI, & Montpellier, France; 3AFFSA- comparative mapping approach Niort Laboratoire de Recherche Caprines, Niort, France J.S. MELVILLE1, Z. JIANG1, A.M. VER- We have established a set of 30 microsatellites RINDER GIBBINS1, J.A.B. ROBINSON1, J.P. of Bovidae origin for use in a biodiversity stu- GIBSON2 dy in Swiss and Creole goats (Saitbekova et al. 1CGIL, Department of Animal and Poultry 1999, Glowatzki-Mullis, personal communica- Science, University of Guelph, Guelph, Cana- tions). Additiona microsatellites located next to da; 2International Livestock Research Institute, “candidate” genes of interest, such as cytokine Nairobi, Kenya genes (IL4, IL12, INFgamma) and MHC class Previous genomic scans have identified por- II genes (DRB, DYA) were tested in the capri- cine chromosome 3 as harboring a suggestive ne species in order to detect possible associa- QTL associated with ovulation rate. Based on tions with two infectious caprine diseases. the published linkage, cytogenetic and radia- Microsatellite analysis was undertaken using tion hybrid (RH) maps, we have identified 41 an ABI373. In the first study, a total of 82 un- genes and gene family clusters that have been related Creole goats, 37 resistant and 45 placed on SSC3. Based on the location of susceptible to Heartwater disease (Camus et orthologues of these genes on the human se- al., 1995) were analysed. In this study, the two quence map, we found that SSC3 is homolo- loci DRBP1 (MHCII) and BOBT24 (IL4) were gous to the regions 0 - 155 Mb on HSA2, 0 - positively associated with disease susceptibi- 12 Mb and 45 - 130 Mb on HSA7, and 0 - 50 lity, demonstrating a corrected p Value of Mb on HSA16. Eighty-seven orthologous 0.002 and 0.005, respectively. In a second genes were selected as landmarks at approxi- investigation, we tested 36 goats, experimen- mately 3 Mb intervals in order to cover these tally infected with the nematode parasite homologous human segments. All primers for Trichostrongylus colubriformis (Chartier & these orthologous genes were designed based Hoste, 1997). These animals were devided in a on porcine gene or EST sequences and their “low”- and “high”-excreting group on the basis orthologous status was confirmed by direct of two independently recorded fecal egg sequencing of PCR products amplified from counts. For this parasite resistance study, we Meishan and Large White genomic DNA detected a significant association of one of the pools. The sequences from the two pools were alleles of the bovine microsatellite locus also compared to reveal any polymorphisms in SPS113 with “low”-excretion (resistance). The these genes. All of these markers were typed MHC class II locus DYA (P19), was weakly on a pig/hamster RH panel in order to con- associated with susceptibility in both diseases struct a high-resolution comparative map be- (pc=0.05). In future experiments, we will ex- tween SSC3 and the human genome. A tend the sample size in order to verify the de- Meishan x Large White population is being scribed associations. used to scan SSC3 for QTLs associated with sow productivity traits using high and low E056 pools based on phenotypic performance. Positional Cloning of a Novel Gene, LIM- BIN, Responsible for a Bovine Chondro- E055 dysplastic Dwarfism Association studies using random and “can- didate” microsatellite loci in infectious goat HARUKO TAKEDA1, MARIKA TAKAMI2, diseases TOMOKO OGUNI3, TAKEHITO TSUIJ4, KAZUHIRO YONEDA2, YUJI MISHINA5, GABRIELA OBEXER-RUFF1, URSULA HIROAKI SATO3, NAOYA IHARA1, TO- SATTLER1, DOMINIQUE MARTINEZ2, MOHITO ITOH1, YASUO MORITOMO6, JEAN-CHARLES MAILLARD2, CHRISTO- YOSHIKAZU SUGIMOTO1, TETSUO KU- PHE CHARTIER3 , NASIKAT SAITBEKO- NIEDA2 VA1, MARIE-LOUISE GLOWATZKI1, 1Shirakawa Institute of Animal Genetics, Nis- CLAUDE GAILLARD1 hi-shirakawa, Fukushima 961-8061, Japan;

168 Section E: Associations between Markers and Traits

2Graduate school of Natural Science and major QTL (FAT1) that has large effects on Technology, Okayama University, Tsushima- fatness and growth was detected on pig chro- naka, Okayama 700-8530, Japan; 3Animal mosome 4q (SSC4q) using a Wild Boar inter- Husbandry Research Institute, Kumamoto cross. We are pursuing both genetic and com- Prefectural Agricultural Research Center, parative mapping approaches in order to nar- Kikuchi-gun, Kumamoto 861-1113, Japan; row the QTL interval. We have defined the 4Department of Oral Morphology, Okayama QTL more precisely by tracking the inheritan- University Graduate School of Medicine and ce of the Wild Boar QTL allele through seven Dentistry, Shikata-cho, Okayama 700-8525, generations of backcross pigs. Pig chromoso- Japan; 5National Institute of Environmental me 4 is homologous to parts of human chro- Health Science, Research Triangle Park, mosome 1 and 8 (HSA1 and 8). We now have North Carolina, USA; 6Faculty of Agriculture, evidence that the QTL is located in a region Kyusyu Tokai University, Aso, Kumamoto homologous to HSA1q. Just outside the QTL 869-1404, Japan boundary the breakpoint to HSA8 is defined by Chondrodysplastic dwarfism in Japanese the gene VATPase. The homologues of the two brown cattle breed is an autosomal recessive markers flanking the QTL interval are both disorder characterized by short limbs. We located in the interval HSA1q23-q24. New have previously mapped the locus responsible genetic markers for eight genes in that region for the disease on a distal end of bovine chro- have been developed and genotyped in a three mosome 6. Here we narrowed the critical regi- generation intercross between the European on for approximately 2 cM by linkage analy- Wild Boar and the domestic Large White com- sis, constructed a BAC and YAC contig co- prising 236 animals. Single Nucleotide Poly- vering this region and identified a novel gene, morphisms (SNPs) were developed by am- LIMBIN (LBN), which possessed disease spe- plifying gene sequences located in the QTL cific mutations in the affected calves. One interval, using homologous human and/or mutation was single nucleotide substitution mouse sequences for primer design and micro- resulting in a creation of a cryptic splicing satellites were isolated from BACs correspon- donor site and the other was one-base deletion ding to coding sequences within the same QTL resulting in a frameshift mutation. The LBN region. A pig gene map was constructed and gene consists of 22 exons with 3,627-base shows a high degree of conserved synteny with open reading frame encoding 1,209 amino the human sequence map of HSA1q. The result acids. Strong expression of Lbn gene was ob- supports the utilisation of the human genome served in limb buds of developing mouse em- sequence draft in our search for potential can- bryos and in proliferating chondrocytes of didate genes for the QTL. epiphyseal growth plate. These findings indi- cate that LBN is responsible for bovine chon- E058 drodysplastic dwarfism and plays a role in Mapping QTL affecting milk composition skeletal development. traits in dairy cattle using a complex pedi- gree E057 Comparative genome analysis between pig AMANDA J. CHAMBERLAIN1, HELEN C. chromosome 4 and human chromosome 1 McPARTLAN2, THAMY BALASINGHAM2, and 8 MICHAEL J. CARRIK2, PHILIP J. BOW- MAN2, NICHOLAS A. ROBINSON2, MI- FRIDA BERG1, ALAN ARCHIBALD2, SU- CHAEL E. GODDARD1,2 SAN ANDERSON2, LEIF ANDERSSON1, 1Institute of Land and Food Resources, Uni- MARIA MOLLER1 versity of Melbourne, Australia; 2Victorian 1Department of Animal Breeding and Genetics, Institute of Animal Science, Melbourne, Aus- Swedish University of Agricultural Science, tralia Uppsala, Sweden; 2Department of Genomics The families of most progeny tested males are and Bioinformatics, Roslin Institute, Scotland, too small for a grand-daughter design, but UK there are many relationships between families The published draft human genome sequence that could be used in mapping QTL. The aims facilitates comparative genome analyses of less of this experiment were 1) to detect QTL in a analysed genomes such as the pig. Previously a complex real-life pedigree, and 2) to estimate

169 Section E: Associations between Markers and Traits

QTL allele effects for MAS. Variance compo- CT information in analysis of body composi- nents estimates and QTL allele effects were tion for QTL detection without the need for calculated using restricted maximum likeli- slaughter of animals opens the possibility to hood, after Gibbs sampling was used to esti- screen for QTL in growth rate and tissue yield mate the probability that QTL alleles are iden- at different stages of the growth phase. The tical by descent. A QTL was detected on presence of QTL for body composition is the chromosome 14, accounting for approximately first stage in gene identification and paves the 47% of the genetic variance for fat% , and on way for marker assisted selection or introgres- chromosome 20, accounting for approximately sion to be utilised in sheep breeding programs 45% of the genetic variance for protein%. The to improve body composition. standard deviation of predicted QTL transmit- ting ability for chromosome 14 was 67L for E060 milk, 0.08% for fat% and 0.02% for protein% Progress in QTL mapping for growth and and 67L, 0.05% fat% and 0.03% protein% for fatness traits on porcine chr 7 the QTL on chromosome 20. The correlations between the effect of QTL alleles on milk, O. DEMEURE1, N. IANNUCCELLI1, G. LA- fat% and protein% were also calculated and VAL1, C. RENARD2, J. RIQUET1, C. were found to be not significantly different GENÊT1, C. GASNIER3, J.P. BIDANEL4 , D. from +1 or –1 which is consistent with the MILAN1 presence of only 2 alleles at each QTL. 1Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, France; 2Laboratoire de E059 Radiobiologie et d’Etude du Génome, INRA, Identification of quantitative trait loci Jouy-en-Josas, France ; 3Gene+, Erin, Fran- (QTL) in sheep for live body composition ce; 4Station de Génétique Quantitative et using x-ray computed tomography Appliquée, INRA, Jouy-en-Josas, France The porcine chromosome 7 contains QTLs COLIN R. CAVANAGH, MARILYN JONES, influencing growth, back fat thickness and GINA ATTARD, IMKE TAMMEN, HER- intramuscular fat content. To precise the QTLs MANN W. RAADSMA position, we followed various approaches. ReproGen, Centre for Advanced Technologies Among them we performed comparative map- in Reproduction and Genetic, The University ping analysis in the chromosomal region con- of Sydney, Camden NSW 2570, Australia taining QTLs, we also analysed additional A whole genome scan was implemented to animal populations. 23 genes selected in a detect quantitative trait loci (QTL) for body human region covering 40 Mb were mapped composition using an experimental back-cross on IMpRH panel. We observed a high conser- between Awassi and Merino sheep. Computer vation of gene order and distances between our Tomography (CT) was used to predict yield for framework map and the UCSC human genome body composition in 164 live wethers. The assembly, excepting for three genes, found 20 genome scan involved 117 informative micro- Mb upper than their human position. In paral- satellite markers covering 26 autosomes and lel, to precise effects of QTL, we studied a provided around 70% coverage of the genome. synthetic LW x MS population under selection. Preliminary results indicate the existence of Considering that if QTL have a significant thirty-one QTL identified (un-adjusted for effect on trait, the frequency of favourable body weight at the time of scanning) for car- alleles should increase in selected animals, we cass lean, carcass fat, internal fat, carcass studied evolution of allele frequency for mark- weight, and bone quantity (P<0.05). Of these, ers covering the genome on animals from F0 seven reached chromosome wide significance and F2 to F5 generations. Simulations have (P<0.01) for predicted carcass lean, and bone been done for each marker, to verify that evo- content. Adjustment for body weight as a co- lution of frequencies could not be explained by variate, resulted in a different subset of thirteen genetic drift only. On chr7, one Meishan hap- QTL of which four were in common with un- lotype was highly selected. Results also sug- adjusted yield traits described above and seven gest that all Meishan alleles do not have the reached chromosome wide significance same effect. Comparing these results with re- (P<0.01) for internal fat, carcass fat, carcass sults obtained on an other synthetic line, we lean and bone content. The procedure to use are trying to identify an IBD segment contain-

170 Section E: Associations between Markers and Traits ing QTL(s). These combined approaches MARIAN KAMYCZEK2, DANUTA CIEŚ- should allow us to better map genes involved, LAK1, MARIUSZ PIERZCHAŁA1, JO- and to identify markers of interest for Marker LANTA KURYŁ1 Assisted Selection in synthetic lines. 1Polish Academy of Sciences, Institute of Ge- netics and Animal Breeding, Jastrzębiec,05- E063 552 Wólka Kosowska,Poland; 2National Rese- Use of radiation hybrid mapping to locate arch Institute of Animal Production, Pig Hy- candidate genes for female reproductive bridization Centre, 64-122 Pawłowice,Poland traits on porcine chromosome 8 The following genes: FSHB, OPN, PRL and PRLR, LEP and LEPR, were investigated as a ANNEMARIE H. KING1, ZHIHUA H. candidate genes for reproduction traits. The JIANG2, GARY A. ROHRER3, JOHN P. material consisted of 519 sows from Polish GIBSON2, DAVE WADDINGTON1, ALAN Synthetic 990 line with differentiated level of L. ARCHIBALD1 reproduction indicators. Several performance 1Roslin Institute, Roslin, Midlothian, UK; traits were recorded: total number born 2University of Guelph, Guelph, Canada; (ILUR), number born alive (ILURZ) number 3USDA-MARC, Clay Center, Nebraska, USA piglets at age of 21 days (IL21), number Improvement of porcine reproductive perform- weaning piglets (ILODS), litter weight at age ance by traditional selective breeding programs of 21 days (WAG21), litter weight at weaning has been limited by the low heritabilities of (WAGODS). The relationship between each traits such as ovulation rate, prenatal survival gene genotypes and reproductive traits was and litter size. Therefore, the identification of evaluated according to the last squares method. candidate genes for use in marker-assisted In PRL locus no significant effect was ob- selection programmes would be particularly served in first parity for litter size but in later desirable. Quantitative trait loci (QTL) for parities the 2/3 animals produce more piglets prenatal survival, litter size, teat number and than 1/1, 3/3 and 1/2 sows. For traits ILUR, ovulation rate have been mapped to chromo- ILURZ, and IL21 this effects are significant at some 8 in earlier studies. The number of genes P<0.01. In locus PRLR, amplified according to mapped to pig chromosome 8 and thus (Vincent 1998), significant effect for number positional candidate genes is limited. We have born alive (ILURZ) of the BB genotype was developed a radiation hybrid map of pig chro- observed but only in first parity. In the current mosome 8, in order to determine whether spe- study the LEP and LEPR regions were ampli- cific genes map within the QTL. A skeleton fied according to Stratil et al 1997 (LEP) and RH map based on 44 markers (including 15 1998 (LEPR). In first parity they were no dif- genes) has been established using the Cam- ferences between genotypes for reproduction bridge-Roslin pig-hamster whole genome RH traits but in later parities the significant effect panel (http://www.ri.bbsrc.ac.uk/radhyb/) and was observed for the traits ILUR and ILURZ the Carthagene software (LEP) also WAG21 and WAGODS (LEPR). In (http://www.inra.fr/bia/T/CarthaGene/). The OPN locus, in later parities significant effect mapped genes include the pig homologues of was observed for the traits IL21, ILODS, and the Booroola (BMPRIB) and SPARC-like 1 WAG21, in all cases value of the traits was (SPARCL1) genes. The resulting gene map highest for homozygote AA. No significant confirms the earlier observations of extensive associations of the FSHB locus with litter size conservation of gene order between human and weight of the piglets were observed. chromosome 4 and pig chromosome 8, but with at least one major rearrangement. AHK is E065 supported by a BBSRC industrial CASE stu- Genetic correlations among libido, scrotal dentship with Sygen as the industrial sponsors. circumference and reproductive female traits in Nelore breed E064 The estimation of the candidate genes poly- C.R.QUIRINO1, J.A.G. BERGMANN2, M.Y. morphism effect on the reproductive traits KUABARA2 , C.G. FONSECA2, V.R.VALE in line 990 sows FILHO2, V.J. ANDRADE2, S.R. REIS2, R. MENDONÇA2 AGNIESZKA KORWIN-KOSSAKOWSKA1, 1State University of North Fluminense-RJ,

171 Section E: Associations between Markers and Traits

Brazil; 2Federal University of Minas Gerais Normal allele (D) and mutant allele (d) were State, Veterinary Faculty, Belo Horizonte-MG, diagnosed in 464 fattened offspring of three Brazil carrier sires (Sire 1-Sire 3: Dd), and 1,207 The objective of this study was to estimate reproduction cows of Sire 1. The fattened genetic correlation between libido and scrotal offspring were 230 normal (DD) and 234 car- circumference of yearling Nelore bulls and age rier (Dd), and the reproduction cows were 577 at first calving and days to first calving of their DD and 630 Dd. Sire, sex, farm and age (lin- half sib sisters. Days to first calving were cal- ear and quadratic) were considered as the culated as the number of days between the source of variance in order to evaluate factors beginning of the breeding season and the date affecting carcass traits. of the first calving. Least squares means and The significant association of the CL-16 standard errors were 2.4±.5 and 32.9±2.1 cm genotypes was not seen in the carcass traits for libido and scrotal circumference, respec- when the effects of environmental factors were tively. Least squares means and standard errors eliminated (p > 0.05). Moreover, a significant for age at first calving and days to first calving association of the genotypes was not seen with were, respectively, 37.8±3.9 months and the predicted breeding value for carcass traits 298.3±20.9 days. Genetic correlation between in reproduction cows of Sire 1. Furthermore, libido and age at first calving and days to first the carcass traits had no significant linkage calving were -.21±.32 and -.18±.11 and, - with the approximately 30 cM region including .66±.58 and -.42±.25 for sister and daughter, CL-16 locus in a half-sib family of Sire 1 respectively. Genetic correlation between comprising 136 offspring (p > 0.05). There- scrotal circumference and age at first calving fore, exclusion of the mutant allele d from and days to first calving were -.09 ± .30 and - Japanese Black cattle population would not .10±.23, respectively. Results suggested that deteriorate the carcass traits. selection for libido would be more effective than selection for scrotal circumference when E067 trying to improve female reproduction. The relationship between polymorphisms of the porcine myogenin, MYF3 and MYF5 E066 genes and microstructural characteristics of Association analysis between the deletion Longissimus muscle. mutant allele of Claudin-16 deficiency and carcass traits in Japanese Black cattle JOLANTA KURYL1, DANUTA KLO- SOWSKA2, DANUTA CIESLAK1, GABRIE- NAOHIKO KOBAYASHI1, TAKASHI HI- LA ELMINOWSKA-WENDA2, WOJCIECH RANO2, TAKAYUKI IBI3, TUYOSHI KAPELANSKI3, MARIUSZ PIERZCHALA1, OHTANI1, YOSHIYUKI SASAKI3, YOSHI- KONRAD WALASIK2 KAZU SUGIMOTO2 1Polish Academy of Sciences, Institute of Ge- 1Gifu Prefectural Livestock Research Institute netics and Animal Breeding, Jastrzebiec, Po- Hida Beef Cattle Department, Gifu, Ja- land; 2University of Technology and Agricul- pan;2Shirakawa Institute of Animal Genetics , ture, Department of Histology, Bydgoszcz, Fukushima, Japan; 3Graduate School of Agri- Poland; 3University of Technology and Agri- culture, Kyoto University, Kyoto, Japan culture, Department of Pig Breeding, By- We have reported that Claudin-16 (CL-16) dgoszcz, Poland deficiency, an autosomal recessive disease in The effect of polymorphisms in the MyoD Japanese Black cattle, is caused by a deletion family genes on microstructural characteristics mutation of 37-kb region including exon 1 to 4 of porcine Longissimus muscle was investi- of CL-16, resulting in lack of the CL-16 ex- gated in 115 crossbreds [Pietrain x (Large pression. The CL-16 DNA-based diagnostic White x Landrace)]. The porkers were slaugh- tests revealed that the mutant allele has widely tered at about 105 kg live body weight. Muscle been distributed among sires highly evaluated. samples taken from M. longissimus lumborum Whether the mutant allele is tightly linked to for histological examinations were cut in cry- economically important trait loci became an ostat and subjected to double reaction for ac- important issue. We thus analyzed the asso- tivity of NADH-TR oxidoreductase and myo- ciation of the mutant allele with carcass traits fibrillar ATPase to identify muscle fiber types available. (STO, FTO, FTG). Ten muscle bundless were

172 Section E: Associations between Markers and Traits randomly selected to evaluate the proportion of ferences between groups. Of critical impor- muscle fibers and a content of pathological tance is the accurate quantification of DNA. It fibers. The diameters of fibers were measured is proposed that quantitative competitive PCR using a Leica Q 500 MC image analysis sys- (QC-PCR) offers the best prospects in this tem. A polymorphism in the myogenin gene regard. By using a suitable correction proce- (MYOG) was identified at its 3’ end (Soumil- dure, we have also shown that accurate esti- lion et al., 1997) and that in the MYF3 gene in mates of allele frequencies can be obtained intron 1 (Knoll et al., 1997). Two polymor- from a pool. phisms in the MYF5 gene were identified with HinfI and DdeI according to te Pas et al. (1999) E071 and Stratil & Cepica (1999), respectively. The Major genes for high resistance to Haemon- diameter of FTO fibers and content of both chus contortus and Fasciola gigantica in STO and FTO fibers in the bundle were af- Indonesian Thin Tail (ITT) sheep fected with the MYF5/HinfI genotype. The content of giant fibers in the bundle was sig- H.W. RAADSMA1, E.T. MARGAWATI2, D. nificantly dependent on MYOG genotype PIEDRAFITA3, E. ESTUNINGSIH4, S. WID- (P<0.05) and MYF5/HinfI genotype (P<0.01). JAJANTI4, BERIAJAYA4, SUBANDRIYO5, Significantly (P<0.05) lowest content of P. THOMSON1, T.S. SPITHILL3 pathological fibers in the bundle was observed 1Reprogen, The University of Sydney, Camden, in Longissimus muscle of porkers of AA Australia; 2Research and Development Centre genotype at the MYF3/DdeI locus. We con- for Biotechnology-LIPI, Cibinong, Indonesia; cluded that the typed polymorphisms are 3Department of Biochemistry and Molecular probably linked to causal mutations influenc- Biology, The University of Monash, Australia; ing microstructural characteristics of porcine 4 Veterinary Research Institute Balitnak, Bogor Longissimus muscle. Indonesia and 5Central Research Institute for Animal Science, Bogor, Indonesia E068 We have undertaken a molecular characterisa- Selective DNA pooling as a rapid screen for tion of a putative major gene for resistance marker allele frequency differences between Fasciola gigantica and Haemonchus contortus two groups: An approach for improved sen- in Indonesian Thin Tail (ITT). Our gene map- sitivity ping approach uses 10 resource families (900 progeny), which are a backcross between resi- MAXY MARIASEGARAM1, NICHOLAS A. stant (ITT) and susceptible (Merino) genotypes ROBINSON2, MICHAEL E. GOODARD1,2 in a susceptible (Merino) background. All pro- 1Institute of Land and Food Resources, Uni- geny are being phenotyped for expression of versity of Melbourne, Australia; 2Victorian resistance by challenge with immature parasi- Institute of Animal Science, Melbourne, tes. Preliminary segregation analysis in 400 Australia progeny from 7 sires, confirmed the presence DNA pooling is a strategy where individual of a gene with large effect for resistance to DNA samples from each of two test groups are both F gigantica (FGR ) and H contortus pooled and amplified in a PCR reaction. This (HCR)in this population. A full genome scan enables rapid screening of a large number of using a panel of 85 highly polymorphic micro- markers for linkage with polygenic traits. The satellite markers provided support for 3 QTL technique is combined with selective geno- and a chromosomal location of putative major typing, whereby subjects will only be added to resistance genes FGR and HCR for a positional the pool if their scores for the trait under study candidate cloning approach. Preliminary evi- fall at the extremes of the phenotypic distribu- dence of the puative QTL for FGR and HCR tion. DNA from the “high” and “low” groups mapped to different chromosomes, suggesting respectively can then be pooled separately. We that resistance to these two parasitic diseases is have utilized such a method to identify genes not under control of the same gene. affecting milk yield and composition in Aus- tralian dairy cattle using a genome wide scan. This poster focuses on techniques to make selective DNA pooling reproducible and sen- sitive to small changes in allele frequency dif-

173 Section E: Associations between Markers and Traits

E072 cology, Athens, Georgia USA and (4) Roslin Fine mapping of quantitative trait loci Institute, Edinburgh, Scotland (QTL) affecting susceptibility to ascites Gastrointestinal parasites have a profound in chicken effect on sheep production. In this study, a genome-wide QTL scan was implemented to TARIK RABIE1, RICHARD CROOIJMANS1, identify chromosomal regions in the ovine ADDIE VEREIJKEN2, GERARD AL- genome that play a role in resistance to ga- BERS2,TINEKE VEENENDAAL1, ROSILDE strointestinal parasites. A sheep population DIJKHOF1, JAN VAN DER POEL1, HENK segregating for parasite burden (measured by BOVENHUIS1, MARTIEN GROENEN1 fecal egg count) was constructed at Louisiana 1Wageningen University, Animal Breeding and State University and included F2 offspring of Genetics Group,Wageningen Institute of Ani- F1 parents produced from Gulf Coast Native mal Sciences, Wageningen, The Netherlands; (resistant) and Suffolk (susceptible) crosses. A 2Nutreco, Boxmeer, The Netherlands selected group of these F2 lambs and their An experimental population was created for parents were genotyped for 50 microsatellite mapping of loci that are involved in the resis- markers. Preliminary QTLs for parasite resi- tance/susceptibility of broilers to develop pul- stance were identified on ovine chromosomes monary hypertension syndrome (PHS) also 1 and 3. Interval mapping was then applied known as ascites. A total genome scan with using all markers genotyped on each of these microsatellite markers was performed on the F1 chromosomes. The most significant results and F2 generations (10 full-sib families with a were detected on chromosome 1, with a putati- total of 476 individuals),whereas an F3 genera- ve QTL localized to the center of this chromo- tion (4202 animals) was phenotyped. A regres- some. sion analysis identified three genomewise sig- nificant QTL on chromosomes 2, 4 and 6 (ge- E074 nomewise p values of 0.01, 0.04 and 0.02 re- QTL for birth weight and daily gain at spectively) and suggestive QTL on chromo- weaning on chromosome 23 in German somes 1, 2, 3, 5, 8, 10, 13 and 28 (p values be- Angus cattle tween 0.11 and 0.77). To validate and to narrow down the QTL regions, the original cross was CHRISTINA WEIMANN, HORST BRANDT, used for breeding up to the F8 generation. Ap- MATTHIAS GAULY, GEORG ERHARDT proximately 4000 F8 animals were measured for Justus-Liebig-University of Giessen, Depart- the ascites related traits, and are currently being ment of Animal Breeding and Genetics, Gies- analysed with microsatellite and SNP markers sen, Germany on chromosomes 2, 4, 8, 10 and 28. Further- Five paternal half sib families of German more, extensive physical mapping (BAC contig Angus (n = 428) were typed (including dams) building) is used for the targeted development of with 9 microsatellites (INRA132, RM033, SNP markers and for the construction of detailed BM1815, BM1258, BOLA-DRB1, BM1818, gene maps for these QTL regions. BM1905, CSSM5, DYMS1) located on bovine chromosome 23. All dams were in the same E073 parities for both years. The family size varied Characterization of QTL associated with from 52 to 57 progenies for which individual resistance to nematode infection in sheep birth weight and daily gain at weaning were measured. Animals were weaned at an age of 9 TRACY L. SHAY(1), JAMES E. MILLER(2), to 10 months. The average birth weight was ± ROYAL A. McGRAW(3), GRANT A. WAL- 37.4 kg ( 5.5 kg) while average daily gain at LING(4), STEVEN C. BISHOP(4), CHRIS S. weaning was 954 g (± 117 g). The number of HALEY(4) & NOELLE E. COCKETT(1) alleles per marker ranged from 5 to 15 while (1)Utah State University, Department of Ani- heterozygosity ranged from 0.54 to 0.88. A sex mal, Dairy and Veterinary Sciences, Logan, averaged linkage map was constructed with the Utah USA, (2) Louisiana State University, De- BUILD option of the CRIMAP package (v. partment of Pathobiological Sciences, Baton 2.4) covering a distance of 88.0 cM. The Rouge, Louisiana USA, (3) University of Geor- marker distances varied from 5.1 to 18.0 cM. gia, Department of Physiology and Pharma- A QTL-analysis for birth weight and daily gain was done using the half sib analysis of the

174 Section E: Associations between Markers and Traits

QTL-express programme inbred line cross experiments C57Bl/6J x (http://qtl.cap.ed.ac.uk). Sex and year of birth NMRI/DKFZ and C57Bl/6J x Balb/cJ. 2014 F2 (1999 and 2000) were included as fixed effects animals could be used as base for the forma- in the model. Permutation test (1000 iterations) tion of performance groups after F1 intercross. was done to set chromosome wide significance This concerned the mice with extreme high (≥ thresholds. Within German Angus breed a 13 offspring) and extreme low litter size (≤ 5 QTL for birth weight could be detected at po- offspring) in both experiments. Altogether 112 sition 54 cM (p = 0.05). In addition a QTL for microsatellites were analysed with an average daily gain at weaning was detected at position distance of 29 cM. On base of estimations of 16 cM (p = 0.05) in one family. dominance degree the evaluation of relations between extent of heterozygosity of microsat- E075 ellites and litter size within the groups was A total genome scan for porcine hernia done by chi square test according to Pearson’s inguinalis and scrotalis table of two times two contingence. In the case of superdominance theory of heterosis asso- 1 1 K.BORNEMANN-KOLATZKI , C. KNORR , ciations to heterosis in litter size exist on 1 2 1 U. PETERS , B. HARLIZIUS , B. BRENIG chromosomes 2, 10, 11 ,17, 18 and 19. Domi- 1 University of Goettingen, Institute of Veteri- nance as reason of heterosis could be described nary Medicine, Goettingen, German; 2current on chromosomes 5, 6 and 13. These genome address: IPG Institute for Pig Genetics BV, regions are interesting for the development of Beuningen, Netherlands strategies in heterosis breeding. There are numerous references that genetic factors are involved in the development of E077 hernia inguinalis and scrotalis. Hernias result A significant QTL for the heel depth in from an abnormal wide inguinal canal, Holstein cattle whereby parts of the net and intestine descent into the inguinal canal (hernia inguinalis) or BERNHARD G BAUMGARTNER(1), into the scrotum (hernia scrotalis). About 3% BERND KRIEGESMANN(2), STEPHAN of all litters in pigs are affected. Neither in pigs JANSEN(2), HERMANN H. SWALVE(3), nor in man the mode of inheritance could be BERTRAM BRENIG(2) clarified so far. The goal of this project was the (1) University of Barcelona, Department of identification of genes, which increase the Biochemistry and Molecular Biology, Barce- susceptibility to develop hernia. For a genome lona, Spain, (2) University of Göttingen, De- scan, 71 full-sib pairs and their parents were partment of Veterinary Medicine, Göttingen, genotyped with 110 polymorphic markers. Germany, (3) Martin Luther University, Insti- Linkage analyses have been carried out with tute for Animal Breeding and Husbandry, the Genehunter package combining parametric Halle-Wittenberg, Germany and model free allele sharing methods. The Feet and leg problems are a major cause for an results showed that genetic heterogeneity is involuntary culling of cows in dairy herds. present, i.e. in different families different ge- Attempts to improve upon this situation com- nome regions with influence on susceptibility monly are limited to the use of selection deci- for the defect have been identified. sions based on scores for these traits within type score systems. Due to relatively low heri- E076 tabilities for feet and leg traits the chances for Superdominance and dominance as reasons a genetic improvement are very limited if con- of heterosis in litter size in mice. ventional breeding strategies are used. Marker assisted selection should offer greater benefits. C. BRUNSCH, I. STERNSTEIN Up to now, several QTLs have been identified Institute of Animal Sciences, Agricultural and with granddaughter-designs and whole genome Horticultural Faculty, Department of Breeding scans using microsatellites. However, none of Biology and Molecular Animal Breeding, these have been proven highly significant, nor Humboldt-University of Berlin, Germany. linked to important genes. The genetic reasons of heterosis in litter size In contrast, we chose a candidate gene ap- with regard to superdominance and dominance proach. We isolated and characterized the bo- effects were explained in two reciprocal mice vine bovine sulfate transporter Slc26a2gene

175 Section E: Associations between Markers and Traits and identified a polymorphism in the coding localisation of the Lacaune gene. Human genes region. Differences in the sulfate transport localised in this region and considered as pos- efficiencies of the 2 alleles were demonstrated sible positional and functional candidates will in cell culture. be tested. Leg scores were obtained from 3 independent AI studs in Germany and the polymorphism E079 was typed in 300 bulls. Using the PEST soft- Development of a resource population to ware contrasts between genotypes AA vs AB detect QTL affecting resistance to gastroin- and AA vs BB were estimated. Among other testinal parasites of cattle. findings, the B-allele appeared to be associated with higher heels in Holstein cattle and hard- LOUIS C. GASBARRE (1), TAD SON- ness criteria show favorable, although insig- STEGARD (2), CURTIS P. VAN TASSELL(2,3) nificant differences for the BB genotypes. & TEREZINHA PADILHA(4) Breeders desire higher heels and hence this (1) Immunology and Disease Resistance Labo- polymorphism may be very valuable to im- ratory, (2) Gene Evaluation and Mapping La- prove efficiency in breeding programs. boratory, (3) Animal Improvement Program Laboratory and (4) EMBRAPA-ARS LABEX E078 Program, Animal and Natural Resources In- Localisation and regional mapping of a ma- stitute, Beltsville Agricultural Research Cen- jor gene controlling ovulation rate in La- ter, Beltsville, USA caune sheep. Gastrointestinal nematodes severely reduce the efficiency of raising cattle on pasture. Genetic FRÉDÉRIC LECERF(1), LOYS BODIN(2), management of 15-25% of cattle can consider- JEAN-MICHEL ELSEN(2) & PHILIPPE ably reduce parasite transmission in cattle MULSANT(1). herds. A selection program was initiated using (1) INRA, Laboratoire de Génétique Cellulaire, parental stock from Wye Angus of the U of Castanet, France and (2) Station d'Amélioration MD. To date ≈350 progeny covering 4 gene- Génétique des Animaux, Castanet, France. rations have been phenotyped. Pedigree re- A new major gene increasing ovulation rate cords trace back to the founding animals of the (OR) has been evidenced in the Lacaune herd, and pedigree analysis reveals that >90% population. Using non-prolific dairy ewes as of the tested animals are paternally descended support, we generated half sib back-cross (BC) from a Wye bull born in 1944. DNA for gene- families issued of 7 F1 sons (heterozygous) of tic analysis has been acquired from all tested 3 sires. After excluding the Booroola and In- animals and from over 70 sires in the historic verdale regions, a full genome scan was per- pedigree. Phenotyping is accomplished by formed on these BC ewes. A first linkage of placing weaned calves on pastures infected the Lacaune prolificacy gene was first obtained with the 2 most common nematode parasites of with markers IDVGA46 and BM17132 on the US cattle. Calves are monitored weekly for 19 ovine chromosome 11 (OAR11). Ovine mark- physiologic measures. After at least 120 days ers from OAR11 were added to the map on test, calves are selected for replacement around the Lacaune locus and the closest breeders, re-challenge, or immediate kill. At flanking markers are CSAP and CSSM15 at kill an additional 23 immunologic or parasito- about 2 cM from the Lacaune locus. The lo- logic measures are recorded. Calves are as- calisation was confirmed by adding other ani- signed as: 1) Type I- always low parasite eggs mals (F1 and F1xBC). In order to define more per gram of feces (EPG) values, 2) Type II - precisely the human orthologous region and the increasing EPG values followed by a drop to existing breakpoints between HSA17 and levels of Type I calves, and 3) Type III - consi- BTA19 / OAR11, we have performed com- stent high EPG levels. Reinfected calves show parative mapping between these 3 species us- secondary EPG values consistent with those ing a bovine radiation hybrid panel. This per- observed during the primary exposure. The mitted us to define precisely the synteny be- selection has increased the percentage of Type tween these species. The orthologous regions I and Type III calves, and the range of EPD of bovine chromosome 19 and human chromo- values has been reduced to 0.5 of the mean some 17 are screened for additional polymor- EPG value. phic markers to improve the precision of the

176 Section E: Associations between Markers and Traits

E080 E081 Linkage and comparative mapping of the Age-dependent quantitative trait loci affec- gene responsible for susceptibility towards ting body weight in a cross between the se- E. coli F4ab/ac diarrhoea in pigs. lected mouse line NMR18 and DBA/2

CLAUS B. J∅RGENSEN(1), SUSANNA ERSIN KARATAYLI(1), ULLA RENNE(2), CIRERA(1), SUSAN ANDERSON(2), ALAN STEFANIE KARLE(1), OSWALD ROTT- ARCHIBALD(2), TERJE RAUDSEPP(3), MANN(3) & GUDRUN A. BROCKMANN(1) BHANU CHOWDHARY(1,3) LEIF ANDERS- (1)Research Institute for the Biology of Farm SON(4) & MERETE FREDHOLM(1) Animals, Department of Molecular Biology, (1) Royal Veterinary and Agricultutral Univer- Dummerstorf, Germany, (2)Research Institute sity, Department of Animal Science and Ani- for the Biology of Farm Animals, Department mal Health, Frederiksberg, Denmark, (2)Roslin of Genetics and Biometry, Dummerstorf, Ger- Institute, Department of Genomics and Bioin- many, (3)Technical University München- formatics, Midlothian, Scotland, United King- Weihenstephan, Institute of Animal Breeding, dom (3)Texas A&M University; Department of Freising, Germany Veterinary Anatomy and Public Health, Col- Quantitative trait loci (QTL) influencing body lege Station, Texas, USA (4) Swedish University weight at the age of 2, 3, 4, 5, and 6 weeks of Agricultural Sciences, Department of Ani- were mapped by linkage analysis in a F2- mal Breeding and Genetics, Uppsala, Sweden. population of a cross between the high growth In 1995, Edfors-Lilja and coworkers localised selected mouse line NMR18 and the control the locus for the E. coli K88ab (F4ab) and line DBA/2. In addition to body weight, QTLs K88ac (F4ac) intestinal receptor to pig chro- influencing abdominal fat weight and muscle mosome 13. Using the same family material weight were mapped at the age of 6 weeks. we have refined the map position to a region The two mouse lines descent from different between the microsatellites markers Sw207 genomic origin. They differ in body weight by and Sw225. Primers from these markers were 115% at 42 days. Genome-wide significant used to screen a pig BAC library and the posi- QTL effects were mapped for body weight in tive clones we used for FISH analysis. The at least one of the selected ages on chromo- results of the FISH analysis helped us to pro- somes 1, 2, 3, 4, 7, 9, and 14. A shift in the pose a new candidate gene region in the Sscr effects of QTLs was observed depending on 13q41-q44 interval. Shot-gun sequencing of the age of the animals. The QTLs affecting the BAC clones showed that the candidate body weight coincide with QTLs for abdomi- region contains an evolutionary break point nal fat weight on chromosomes 1, 2, 7, 14, and between human and pig. In order to further with QTLs for muscle weight on chromosomes characterise the rearrangements between 1, 7, 14. The effects found for all QTLs af- Sscr13 and Hsap3 detailed gene mapping of fecting body weight on different ages were Sscr13 was carried out. Consequently, a de- mostly additive, accounting together for 35-45 tailed comparative map between Sscr13 and % of the phenotypic variance of body weight Hsap3 was constructed and two candidate re- within the corresponding F2-population. The gions on human chromosome 3 were identified information on the age-dependent QTL effects for the gene responsible for susceptibility to- is useful for the elucidation of the underlying wards E. coli F4ab/ac diarrhoea in pigs. genes. Nomenclature: E. coli F4ab/ac diarrhoea in pigs, linkage mapping, comparative map- ping

177 Section F: Bioinformatics

Section F: proprietary sequence derived from whole- genome shotgun sequencing of livestock. Bioinformatics Contigs of assembled livestock genomic se- quences were blasted to the human draft ge- F002 nomic sequence and their locations, based on A Danish – Chinese collaborative project: human orientation, were stored in the compa- towards sequencing of the porcine genome rative database, thus providing a - bone for the livestock genomes. Gene indices THE DANISH PORCINE GENOME CON- were developed from public EST data from SORTIUM bovine, porcine and gallus species and consen- The Royal Veterinary and Agricultutral Uni- sus sequences of contig assemblies were stored versity, Department of Animal Science and in the database. The gene indices were linked Animal Health, Division of Animal Genetics, to the genomic sequences by blast homology. Frederiksberg, Denmark & Danish Institute of These gene indices were also compared to Agricultural Sciences, Department of Breeding NCBI Reference Sequence data and Genetics, Tjele, Denmark. (http://www.ncbi.nlm.nih.gov/LocusLink/ref An international consortium has been estab- seq. html) and RefSeq accession numbers lished between The Royal Veterinary and Ag- were stored as an attribute. The database ricultural University, The Danish Institute of contains public information from genetic lin- Agricultural Science and The National Com- kage maps and physical maps from radiation mittee of Pig Breeding, Health and Production, hybrid panel data and BAC sequencing. In on the Danish side, and Chinese Academy of addition, locations of quantitative trait loci, Science (CAS Human Genome Centre/Beijing identified by flanking sequences from the Genomics Institute) on the Chinese side. The scientific literature, have been uploaded into long-term goals of the consortium are to obtain the database. Visualization tools are being sequence information from the full comple- developed to allow multiple across-species and ment of pig genes and work towards deter- across-map comparisons. This unique resource mining the major part of the DNA sequence of will be used for comparative mapping of syn- the porcine genome. This work will greatly tenic regions among all species for discovering facilitate the elucidation of the function of economically important genes and understan- genes of phenotypic importance for traits that ding regulatory regions. are of interest to the pig industry. In the initial phase of the project we have contributed by F004 establishing 94 cDNA libraries representing SNP allele frequency estimation based on mRNA isolated from different tissues/different the analysis of sequencing traces developmental stages/different animals. From each of the libraries approximately 10.000 GREGOR DURSTEWITZ, ANDREAS WIN- ESTs will be generated by sequencing from the TER, RUEDI FRIES 5’ end. The status of the sequencing project Technische Universität München, Lehrstuhl für will be presented. Tierzucht, Freising-Weihenstephan, Germany The detection of single nucleotide variation is F003 most efficiently achieved by comparative fluo- Development of Human-Livestock Com- rescence-based sequencing of PCR-products. parative Database for Gene Discovery We evaluated the possibility of estimating allele frequencies during the process of SNP SUE DeNISE, DAVID ROSENFELD, KIM discovery through the analysis of sequencing MARSHALL, PHILIP CHEVALIER, TIM traces using the software suite phred, phrap, RULE, CRAIG KENT-BASHAM, DOBO- polyphred and the visualization tool consed for RAH SILER, LAURIE MOORE, KAREN semi-automated polymorphism detection. The BLOCK, BONNIE FERRIE, MICHELLE frequency estimates are based on the compari- HUTTON, MICHAEL SPENCER, MICHAEL son of normalized amplitude values of the two TORRES alternative bases resulting from pooled DNA MMI Genomics, Davis, California, USA with normalized amplitude values of the corre- A comparative human-livestock database has sponding bases resulting from DNA of homo- been developed from public information and zygous and heterozygous individuals. Ampli-

178 Section F: Bioinformatics tude values are extracted from the output gen- sequence data are integrated with genetic and erated by the base calling program phred. Best biological data. The MouseBLAST server normalization is achieved through optimised provides a sequence-level entry point into MGI binning of amplitude values. This is done by gene data. Cancer-related genetic and phenoty- varying the range of bases used for calculation pic data, including images for different strains of the average amplitude value for a given base of the , also are provided. as well as by varying the number of adjacent MGI collaboratively curates data with SWISS- bases excluded from average calculation. The PROT and NCBI/LocusLink and provides approach was automated using python scripts extensive links with these and other online and tested with several DNA pools with known resources such as GenBank, PubMed and frequencies. Predicted frequencies were usu- OMIM. MGI is supported by NIH grants ally within +/- 0.05 of the actual frequency. HG00330, HG02273, HD33745, CA89713 and For some polymorphisms it was necessary to DOE-FG02-ER62850. add DMSO to the PCR to ensure equal ampli- fication of both alleles. The described ap- F006 proach is particularly useful for simultaneous A bioinformatics pipeline and integrated SNP searching and preliminary allele fre- genomics database for beef cattle quency estimation in the context of association studies. C.A. GILL, S.R. XAVIER, C.A. ABBEY, D.L. ADELSON F005 Department of Animal Science, Texas A&M Mouse Genome Informatics University, College Station, TX, USA (http://www.informatics.jax.org): The A data management system has been develo- Comprehensive Integrated Online Mouse ped to maintain pre-existing genotypic and Information Resource phenotypic data, as well as newly generated DNA sequences. Data from the Texas A&M DAVID GARIPPA, JUDITH A. BLAKE, Angleton project, resulting from the generation CAROL J. BULT, JOEL E. RICHARDSON, of reciprocal backcross families between An- JIM A. KADIN, MARTIN RINGWALD, JA- gus and Brahman cattle, have been assembled NAN T. EPPIG AND THE MOUSE GENO- in a relational database. The database contains ME INFORMATICS GROUP entries for 712 individuals, 84 phenotypes and The Jackson Laboratory, Bar Harbor, ME genotypes for 406 markers. Web-based que- 04609 USA ries provide input for various statistical and The Mouse Genome Informatics (MGI) web mapping packages. We have also implemented site provides access to a comprehensive inte- a local BLAST and FASTA pipeline on a grated public information resource for mouse 48CPU SGI Origin 3800 supercomputer. A genetics, genomics and biology. The MGI collection of PERL scripts ports ABI traces database integrates information on over from a LINUX machine through the 30,000 mouse genes and experimental data Phred/Phrap suite of programs to assign quality from over 71,000 references. The database scores and remove any vector or E. coli se- provides extensive information on genes, in- quences. Cleaned sequences are sent to Repeat cluding nomenclature and function; genetic, Masker and then submitted to the supercom- physical, and comparative maps; allelic varia- puter for similarity searches against appro- tion; mouse mutant and strain phenotypes; and priate databases. Our continuing efforts to molecular segments and sequence clusters. positionally clone genes affecting carcass and Curated mouse orthologies with the human, rat growth traits have resulted in the generation of and 17 other mammalian species are available. several large sequence data sets (>5000 traces). The , Phenotype Classification Many of our sequences come from BACs and Terms and Mouse Anatomical Dictionary pro- similarity searches against the assembled hu- vide controlled vocabularies for annotation and man genome thereby provide comparative assistance in database searches. Many different positional information on these BACs. Parsed types of gene expression information are inte- BLAST output is then added to our relational grated into the database. Gene expression for database. Since we are constantly generating mouse development with over 23,000 referen- new sequence data and the public databases ces can be accessed. Emerging mouse genomic continue to expand, our BLAST results are

179 Section F: Bioinformatics refreshed on a monthly basis. among resources. Data changes daily, so the information system needs to frequently down- F007 load and integrate information from external Annotation and classification of porcine resources to remain current. The system needs ESTs to reconcile differences in data format. Multi- agent software systems have the ability to inte- NORIYUKI HAMASIMA1, KOHEI SU- grate information from many external resour- ZUKI1, HIDEAKI SUZUKI1, TAKASHI ces and reconcile differences in format. We AWATA2 are developing an autonomous multi-agent 1STAFF-Institute, Kamiyokoba, Tsukuba, Iba- system to download sequence, functional an- raki 305-0854, Japan; 2National Institute of notation, and mapping information from exter- Agrobiological Sciences, Kannondai, Tsukuba, nal sites and integrate it into comparative Ibaraki 305-8602, Japan maps. A data manager agent starts multiple In order to integrate porcine ESTs into a radia- pairs of data checker - downloader agents in tion hybrid map presendet at another poster response to messages from users or after session (Suzuki et al.), we have analyzed por- querying instructions from a database at initia- cine ESTs from cDNA libraries from the back lization. Each data checker agent detects fat tissue. On the other hand, as of the end of changes in a file date for an external resource. October 2001, 98,969 ESTs appeared in the When a change in date is detected, the data GenBank database. After removing porcine checker agent sends a message to its corre- mitochondral genome, repetitive sequences sponding data downloader agent to download and endogenous virus sequences with Repeat- the file. Once a download is complete, messa- Master program, we clustered these sequences ges are sent to agents controlling downstream with d2_cluster with stackPACK v 2.1 (Elec- processes indicating that new data is in the tric Genetics PTY Ltd.) and obtained 71,628 pipeline to be integrated into the information sequences. These sequences were annotated system. Downstream processes may include with BLAST search against non-redundant gunzip, formatdb, cross_match, blast and perl GenBank database. We have selected 4,380 data parsers. Agents can be distributed over sequences with the homology score value multiple platforms. >500. According to the human gene name, wie have localized these sequences to the human F009 High-Throughput Genomic Sequences data- A rapid screening method for SNP discov- base and classified them according to the Gene ery by MALDI-TOF mass spectrometry of Ontology Categories. RNase T1 digests

F008 STEFAN KREBS, IVICA MEĐUGORAC, Intelligent software agents for managing MARTIN FÖRSTER distributed genomics data University of Munich, Institute for Animal Breeding, Munich, Germany JOHN W. KEELE1, JIM E. WRAY1, MI- MALDI mass spectrometry is an established CHAEL N. HUHNS2, LARRY M. STE- platform for high-throughput genotyping of PHENS2, WARREN M: SNELLING1, GRE- single nucleotide polymorphisms (SNPs). For GORY P. HARHAY1, RANDAL R. BRAD- broad application of SNPs in animal genetics, LEY1 however, the number of SNPs is far from suf- 1U. S. Meat Animal Research Center, State ficient. We present a method for SNP discove- Spur 18D, Clay Center, Nebraska 68933, USA; ry that can use existing MALDI genotyping 2 University of South Carolina, Department of platforms and is automation-compatible. The Computer Science and Engineering, Swearin- method is based on in vitro RNA transcripts gen Engineering Center, Columbia, South from PCR products, that can be used to obtain Carolina 29208, USA highly informative sequence fingerprints by Maintaining a comparative genomics informa- digestion with the guanosine-specific ribo- tion system is challenging because data resour- nuclease T1. In these fingerprints a mutation ces are distributed, heterogeneous and dyna- can be detected as a mass shift or absence of a mic. Data originates from multiple resources wildtype peak or appearance of an additional distributed around the globe, and formats vary peak. Due to mass degeneracy of larger frag-

180 Section F: Bioinformatics ments and multiple presence of shorter frag- CluSTr is a database of clusters of SWISS- ments in a given sequence a certain fraction of PROT + TrEMBL proteins, based on pairwise possible mutations will remain undetected with sequence similarity. It is a useful resource for this method. Screening of both strands from whole genome analysis. one PCR product is possible by using both T3- and T7-tailed primers and the respective RNA F011 polymerases and markedly decreases the pro- SNP detection and characterisation from bability of missing a SNP. We have used a overlapping Bos taurus ESTs simulation of RNase digests from all possible mutations of a set of randomly generated se- JOHN C. McEWAN, ANNA SANTURE, quences that provides estimates for the general AMONIDA ZADISSA, MARK J. SCHREI- detection probability in dependence of PCR BER product length. A software package is provided AgResearch, Invermay Agricultural Centre, PB that helps to design PCR primers by plotting 50034 Mosgiel, New Zealand out regions with a high SNP discovery score Assembly of 367,811 public and private Bos and calculates expected mass fingerprints and taurus EST and mRNA sequences resulted in peaklists from the target sequence selected for 39,129 contiguous sequences (contigs) con- screening. The method was tested on known taining 2 or more ESTs and 62,232 singletons. polymorphisms and de novo sequenced geno- Putative SNPs were identified from the 18,508 mic BAC clones. contigs containing four or more ESTs. The following screening criteria were used: a minor F010 allele frequency greater than 15%, a minimum SWISS-PROT, TrEMBL, InterPro & of 2 sequences with the minor allele, no vari- CluSTr and animal genetics ants with more than 5% frequency in the sur- rounding 10 bases, and less than 1 SNP per MINNA H. LEHVASLAIHO; ROLF AP- 200 bp of contig length. A total of 4493 puta- WEILER tive SNPs were identified in 2262 contigs at a EMBL Outstation – European Bioinformatics frequency of one SNP per 728bp. The average Institute, Wellcome Trust Genome Campus, EST depth at the SNP site was 12.5 sequences. Hinxton, Cambridge, UK Using protein homology alignment, 1768 of SWISS-PROT Protein Knowledgebase is an these SNPs were identified as being in the annotated protein sequence database. It strives protein coding regions of 1158 contigs. These to provide entries with high-quality annotation, represented 1051 unique SwissProt/TrEMBL a high level of integration with other databases hits. Based on the derived protein coding and a minimal level of redundancy. SWISS- frame, 1127 of the putative SNPs were identi- PROT is accompanied by TrEMBL, a fied as coding for a change in amino acid. A computer-annotated protein sequence database number of criteria were used to assess the im- that can be considered as a preliminary section pact of the amino acid on the function of the of SWISS-PROT. TrEMBL contains the protein. SNPs were ranked using a weighted translations of all coding sequences present in index value, derived from amino acid similar- the EMBL Nucleotide Sequence Database, that ity and substitution scores. This method was have not yet been released as manually used as an initial automated screen to identify annotated, finished entry into SWISS-PROT. likely candidates for further study. This re- Together SWISS-PROT and TrEMBL provide source markedly increases the number of po- the user with a tool for various aspects of ani- tential type 1 markers available for Bos taurus mal genetics. The highest represented domestic and also identifies candidates that may have a animals in the databases are Gallus gallus functional impact on phenotypic traits. (1027 entries in SWISS-PROT/1776 entries in TrEMBL), Bos taurus (1332/1529), and Sus F012 scrofa (784/1479). InterPro is an integrated Analysis of genetic marker linkage without documentation resource for protein families, pedigree information domains and sites. It is a powerful diagnostic tool formed by a collaboration between ARDESHIR NEJATI-JAVAREMI1, GHO- SWISS-PROT + TrEMBL, , PRINTS, DRAT RAHIMI2 PROSITE, ProDom, SMART and TIGRFAMs. 1Anim.Sci.Res.Inst., Karaj, Iran; 2Univ. Ma-

181 Section F: Bioinformatics zandaran, Dept.Anim.Sci., Sari, Iran The SNP discovery pipeline is implemented on Increasing numbers of genetic markers are a Linux cluster (12 processors on 6 nodes) available in animals. Analysis of linkages be- which allows parallel processing of multiple tween marker loci requires that their distance sets of EST sequences. After sequencing on a be known. Estimating map distance between 96-well capillary instrument (MegaBace) marker loci is achieved by using genotypes of sequence traces are run through a pipeline of individuals at marker loci plus pedigree infor- programs that assign sequence quality values mation. Pedigree information is required to (Phred), mask contaminating vector sequence determine linkage phase and to infer any mei- (Crossmatch) and mask repeated elements otic recombination which takes place. Linkage (RepeatMasker). Subsequently, clustering is measure is then based on some functions of the performed on a DeCypher Supercomputer number of recombination. Although collecting based on the BlastN algorithm (Tera-BlastN) pedigree information in most animals is rela- and resulting lists of binned sequences are tively cheaper than obtaining marker geno- submitted to the contig assembly program types, there are many situations that the pedi- Phrap. Each contig is scanned for gree may not be known effectively. Popula- polymorphisms using PolyBayes. Resulting tions of fish reared in ponds or honeybees output files containing SNP information (e.g. which mate out of human control are just a few position, variation, template depth) are examples. However, in many of the species extracted into a database by in-house parsing where obtaining pedigree information is diffi- tools. Together with Blast information of the cult it is quite possible to keep separate lines contig consensi and/or individual sequences, and their crosses. When two marker loci are lists of candidate SNPs are generated that are linked it is possible to introduce gametic phase used to design assays for verification of disequilibrium (GPD) between them through polymorphisms and mendilian segregation in crossing two lines with differing allelic fre- appropriate family material. quencies at those loci. GPD across generations is a function of recombination rate between F014 loci and allelic frequency difference in two A bootstrap approach to reduce the bias of lines. In this study a simulation program has genetic distances estimated from population been developed to estimate recombination samples of different size rates by monitoring change in GPD across generations. The program does not require HENNER SIMIANER individual pedigree information and shows University of Göttingen, Institute of Animal performance of this simple methodology Breeding and Genetics, Göttingen, Germany across a range of population structures. Estimation of genetic distances between populations is based on estimated allele fre- F013 quencies derived from samples of the respec- Analysis pipeline for large-scale SNP decte- tive populations. It is generally recommended, tion in EST sequences that equal sample sizes are used in all popula- tions. If sample sizes are different, some FRANK PANITZ, LONE B. MADSEN, popular distance measures, like Nei’s (1972) HENRIK STENGAARD, CHRISTIAN standard genetic distance or Rogers’ (1972) BENDIXEN distance show a severe bias in form of an over- Danish Institute of Agricultural Sciences, Re- estimation of the distance of populations repre- search Centre Foulum, Dept. of Animal Bree- sented by small samples. This phenomenon is ding and Genetics, Tjele, Denmark demonstrated both with simulation results and As part of our participation in a number of with a real data set of 13 red cattle breeds, EST projects various tissue-specific cDNA where sample size was on average 64 animals libraries from different species were analyzed but one breed was only represented with 12 for polymorphic mutations that could modify animals. This bias can be reduced by estimat- gene function or gene expression. Focussing ing distances from bootstrap samples (Efron on identifying missense SNPs in coding and Tibshirani, 1993) of uniform size. Using regions of genes, we devised an integrated this approach in the red cattle data reduced the approach that detects candidate sites in tens of average distance of the breed with the smallest thousands of EST sequences in a few hours. sample to the other breeds by 54 per cent. The

182 Section F: Bioinformatics effectiveness of the suggested resampling ap- likely pedigree errors and problem markers proach is compared with other correction that needed additional analysis to generate methods suggested in the literature (Nei, correct marker data. Finally, inheritance prob- 1987). abilities calculated were used for a quantitative References: Efron, B., Tibshirani, R.J. 1993. trait locus analyses. An Introduction to the Bootstrap. Chapman and Hall, New York; Nei, M. 1972. American F016 Naturalist 106: 283 – 292; Nei, M. 1987. Mo- The Animal-Trust-Center approach in lecular Evolutionary Genetics. Columbia Uni- DNA-based traceability systems. versity Press, New York; Rogers, J. 1972. Measure of genetic similarities and genetic DAVID LÓPEZ HERRÁEZ1, HOLGER distances. Studies in Genetics VII, U. of Texas SCHÄFER1, EBERHARD MANZ2, VOLKER Pub WAGNER2, MICHAEL WINK1 1University of Heidelberg, Institut für Pharma- F015 zeutische Biologie; 2Generatio GmbH, Multilocus iterative allelic peeling of marker Heidelberg, Germany genotypes generated from a linebred Angus Traceability of animals and meat along the population divergently selected for resistan- entire production chain becomes a major appli- ce to gastrointestinal nematodes. cation of DNA-fingerprinting. ISAG provides support for laboratory setup by recommending CURTIS P. VAN TASSELL1, R. MARK markers and organising comparison tests. THALLMAN2, TAD S. SONSTEGARD1, Communicating identity data (DNA derived or LOUIS C. GASBARRE1, TEREZINHA, PA- other) across an open network requires tech- DILHA1,3 nology specified for digital identity serving. 1United States Dept. of Agriculture, Agricultu- For that purpose we introduce the Animal- ral Research Service, Beltsville, MD, USA; Trust-Center (ATC) as the core element of the 2United States Dept. of Agriculture, Agricultu- Animal-Trust-Infrastructure project, ATI* ral Research Service, Clay Center, NE, USA, (www.vernet-info.de). ATC employs techno- 3EMBRAPA-ARS Labex Program, Beltsville, logy known from digital signatures and gene- MD, USA rates true digital identities out of biological Gastrointestinal nematodes can dramatically identity data like DNA-fingerprints. This ap- affect efficiency of cattle raised on pasture, and proach guarantees uniqueness, integrity and therefore, a selection program was initiated to authenticity to every participant in an open investigate the role of host genetics on disease network communication. A prerequisite for and parasite transmission. The objective of this any comprehensive traceability and documen- study was to test the performance of GenoProb tation system. Furthermore, only parental ge- software for QTL analyses on this population. notypes need to be established since DNA- The population consists of over 300 progeny fingerprinting has been optimised for informa- representing four generations of divergent tive power in paternity exclusion. With labo- selection for resistance to parasite infection. ratory costs limited to reproductive animals the The parental animals were derived from a line- ATC might be useful in traceability systems bred Angus population descended from a sin- for pigs as well. Data on selected markers, gle bull born in 1944. This historic portion of laboratory standardization achievements and the pedigree spans another five generations. the ATC interface for data submission will be The complete pedigree included information presented. for 931 animals, and 383 of those had geno- *ATI is funded by the Bundesministerium für typic data recorded for 196 microsatellite Wirtschaft und Technologie [01MS102] Gene- markers. GenoProb provided a solution for this ratio GmbH and Institut für Pharmazeutische complex pedigree by implementing multilocus Biologie are members of the ATI consortium. iterative allelic peeling and was used to calcu- Further participants are: SECUDE GmbH, late genotype probabilities for each individual Darmstadt and Eurohandelsinstitut GmbH, represented in the pedigree. GenoProb uses an Köln. incomplete penetrance model for marker data and generates probabilities of scoring errors. These probabilities identified animals with

183 Index

AASHEIM, H.-CH. C005 BADER, A. C043 BINDER, S. E006 ABBEY, C.A. F006 BADIOLA, J. J. D085 BIROS, I. D150 ABRAMS, CH. C022 BAETTIE, C.W. A033 BISHOP, S.C. E036, E073 ACÍN, C. D085 BAKER, L.R. E053 BLAKE, J.A. F005 ACLAND, G. M. D179 BALASINGHAM, T. E058 BLANCO, I. C076 ADDIE, D.D. B005 BALDI, A. C048 BLASI, M. D007, D038, D119 ADELSON, D.L. D080, F006 BALLESTER, M. A002 BLOCK, K. F003 AERTS, J. A028 BÁN, B. D003, D180 BLOTT, S. D138 AGABA, M. C060 BAND, M.R. C002, C013, C014 BODIN, L. E078 AGRIMI, U. D035 BARALDI, F. A035 BOETTCHER, P. E002 AGUIRRE, G. D. D179 BARBIERI, V. D121 BOJCZUK, B. D057 AIDA, Y. B013 BARBOSA, A. E005 BOLLA, P. C007 AJMONE-MARSAN, P. D077, D120, BARDES, S. A027 BOLLONGINO, R. C073 D156, D162, D165, D171 BARDOLOI, T. D004 BONALDO, F. A036 AKYÜZ, B. D031 BARI, A. C013 BONASSI, CH.A. E030 AL-BAYATI, H. D067 BARNEWITZ, D. D061 BONDANELLI, P. D098 ALBERS, G. E023, E072 BARTHENSCHLAGER, H. C025 BONGIONI, G. D009, D106 ALDERBORN, A. D103 BARRAGAN, C. E035 BONNET, A. A036 ALENCAR, M.M. E041 BARRAO, R. C057 BONNET, M. E042 ALEX, M. B016 BARRE-DIRIE, A. D140 BØNSDORFF, T.B. C005 ALEXANDER, L. D167 BARTENSCHLAGER, H. D107, BORNEMANN-KOLATZKI, K. E075 ALLAIN, D. D016 E029 BORRMANN, L. A029 ALLEN, R. B006 BASARAB, J. E038 BOSDET, I. A012 ALT, K.W. C073 BASSANO, B. D119 BOSMA, A. D176 ALVAREZ, J. C066 BASTIAANSEN, J. E027 BOTTEMA, C. D. K. D080 ALVAREZ, M. C066 BAUMGARTNER, B.G. E077 BOURGEAUX, N. D016, D017, ALVES, E. D001 BAYÓN, Y. D100, E018 D042, D176, E005 ALZINGER, A. C063 BEATTIE, C.W. A020, D160, D167 BOVENHUIS, H. E008, E023, E046, AMANO, T. D071 BEAUMONT, C. E043 E072 AMARGER, V. E050 BEAVIS, W. C039 BOWMAN, PH.J. E058 AMILLS, M. C008, C038, C040, BECK, J. C072 BOZZINI, G. D007 D022, E001 BECKER, F. E028 BRADLEY, R.R. F008 ANDERSON, S. E057, E080 BECKER, K. A034, C047, C056 BRAGLIA, S. D153 ANDERSSON, L. A007, C006, C028, BEDOYA, G. D151 BRANDT, H. D090, E074 C053, D022, D103, E002, E022, E026, BEEVER, J.E. A014, A022 BRAUNSCHWEIG, M.H. C006 E050, E057, E080 BEHNKE, J.M. E053 BREM, G. A042 ANDERSSON, M. E045 BELL, T. K. D002, D046 BRENIG, B. A015, C029, C036, ANDO, A. B001 BELLMANN, O. E028 C042, C070, C071, C072, C074, C075, ANDRADE, V.J. E065 BENDIXEN, CH. A010, C024, D102, D066, D067, D151, D182, D183, ANDRÉ, C. A001, C031 F013 D184, D185, E075, E077 ANGEL, R. C001 BENDIXEN, E. C024 BRILES, W.E. B007 ANGIOLILLO, A. D144 BENÍTEZ, J. D097 BROCKMANN, G.A. E081 ANTON, I. D147 BENKEL, B. A012, E038 BROUWERS, B. A019 APARECIDA P. BRITO, M. A006 BENNE, F. A036 BROWN, J.J. B002 APWEILER, R. F010 BENNETT, G. L. D024, D126, D160 BRUGIAPAGLIA, A. D177 ARCHBOLD, T. D155 BENSIMON, A. A043 BRUNNER, R.M. C017 ARCHIBALD, A.L. C022, E057, BERG, F. E057 BRUNSCH, C. E076 E063, E080 BERGER, P.J. E013 BRUZZONE, A. D077, D096, D171 ARCIDIACONO, N. D044 BERGMANN, J.A.G. E065 BUCHANAN F.C., E007 ARNAL, N. A037 BERIAJAYA, E071 BUDELLI, E. C007 ARRANZ, J. J. D100, E018 BERNOCO, D. D006 BUITENHUIS, A.J. E008, E046 ARRUGA, M.V. C057 BERRYERE, T.G. C003, C034 BUITKAMP, J. A042, D010 ARTHUR, H. D002, D046 BERTANI, G.B. E030 BULL, L. C074 ASAI-COAKWELL, M. A011, E003 BERTAUD, M. D016, D176 BULLA, J. D056 ASHWELL, CH.A. C001 BERTOLETTI, M. D120 BULLERDIEK, J. A029, A034, A041, ASHWELL, M.S. D024, E004 BERTSCHINGER, H. U. D110 C047, C056 ASK, B. E008 BIDANEL, J.-P. E042, E060 BULT, C.J. F005 ATTARD, G. E059 BIDWELL, CH.A. A035, C004 BURGER, J. C073, D184 AWATA, T. B015, D136, D158 BILLAUT, A. A021 BÜRGI, E. D110 BACHMANN, S. A012 BILLON, Y. E042 BURT, D. D096, C022

199 Index

BURTON, D. D077, D171 CHO, C.Y. D021 DA MOTA, A.F. A006 BUYS, N. C067, D049 CHO, G.J. D019 DA, Y. E004 BYUN, H. D. D047 CHO, Y.-M. C045 DAMIAN, R. D074 CABAU, C. A036 CHOI, B.-W. C026 DAMIANI, G. C007 CABURET, S. A043 CHOLEWIŃSKI, G. D020 DANISH PORCINE GENOME CADIEU, E. A001 CHOMDEJ, S. C058 CONSORTIUM, F002 CAGNAZZO, M. C052, D153 CHOUDHURY, A. A003 DANZMANN, R. D159 CAIRNEY, M. A010 CHOWDHARY, B. E080 DAS, D. A003 CAIRNS, M. A017, D063 CHRASTINA, J. D056 DÁVALOS, G. C008 CALIJORNE, V. P. M. D174 CHUNG, E.R. D047, E011 DAVEY, G.C. A010 CALONGE, E. D011 CHUNG, H.Y. D021, D058 DAVIDSON, W.S. A025 CALVO, J.H. D012 CICOGNA, M. D089 DAVIS, E.E. C009 CAMBISANO, N. E017 CIESLAK, D. E067 DAVIS, M.E. D021, D052 CAMPOS, M.C.B.N. D174 CIEŚLAKD. E064 DAVOLI, R. C015, C052, D128, D153 CANALS, A. C076 CIOTOLA, F. D121 DE GROOT, P. E008 CAÑÓN, J. D011, D077, D099, D171 CIRERA, S. E080 DE HAAN, N. A. D176 CAO, H. C045 CIRIZA, J. A016 DE JONG, P.J. A012, A025 CAPLICE, N.C. A010 CLEGG, P.D. B002 DE KONING, D.-J. E021 CAPOTE, J. D022 CLEMENS, R. D090 DE KRUIF, A. C046 CAPPUCCIO, I. D013 CLOP, A. D022, E035 DE LA FUENTE, F. E018 CARITEZ, J.-C. E042 CNAANI, A. D023 DE ROCHAMBEAU, H. D016 CARLBORG, Ö. E026 COCKETT, N.E. A035, A043, C004, DE SILVA, U. D080 CARNWATH, J. W. D140 C009, E036, E073 DEBELJAK, M. C048 CAROLI, A. C007, D015 COHEN, M. E012 DEKA, K. D004 CARRIK, M.J. E058 COLL, A. E035 DEKKERS, J. C.M. E027 CARTER, S.D. B002, B005 COLLETTE, C. C067 DEKOJ, T.R. A012 CASAS, E. D126, E010 COLOMB, B. A021, D045 DELAVAUD, C. E031 CASAVANT, T. C039 COLOMBARI, P. D098 DELCROS, CH. A037 CASSINI, P. E002 COMINCINI, S. D035 DELGADO, J. V. D086, D139 CASTELLANOS, C. D034 CONNOR, E.E. A006, D024 DEMEURE, O. E042, E060 CASTIGLIONI, B. E002 CONTEL, E. P. B. D092 DeNISE, S. D120, F003 CAUVIN, E. D112, D119 COOK, R.W. C059 DENTINE, M. E013 CAVALCHINI, L.G. D105 COPPIETERS, W. D049, E017 DÉSERT, C. A036 CAVANAGH, C.R. E059 CORLEY, S. D150 DESTEFANIS, G. D177 CAVANAGH, J.A.L. C050 CORNELISSEN, S.J.B. A028, A030, DIEZ-TASCÓN, C. D080 CEPICA, S. D014 E008, E046 DI GUARDO, G. D035 CERCOS, A. E035 CORSARO, M. D153 DI SILVESTRO, F. A043 CERIOTTI, G. D015 COSTA, L.N. C015 DI STASIO, L. D121, D177 CEROLINI, S. B010 COTHRAN, E. G. D020, D025, D060 DI TOMMASO, A. D029 CERQUEIRA, F. A035 COUNTER, CH.M. A022 DIAZ, I. E035 CHAMBERLAIN, A.J. E058 COUTINHO, L.L. A004, A006 DÍAZ, S. B003 CHANG, K.-CH. C052 COWAN, C.M. C003 DICKSON, C.A. C060 CHANTRY-DARMON, C. D016 COZZI, M. C. D152 DIJKHOF, R. A031, E072 CHAPMAN, J. B006 CRAWFORD, A. M. D021 DIMITRIJEVIC, V. D059 CHARDON, P. B001, B009, D016, CREIGHTON, P. A005 DIMSOSKI, P. D154 D017, D042, E005 CREPALDI, P. D089 DISTL, O. C010, C012, C016, C027, CHARLIER, C. A035, A043, C004, CRIBIU, C. E052 C031, C036, C043 C009 CRIMELLA, C. D015 DIXON, L. C022 CHARON, K.M. D018 CRISÀ, A. D026, D083 DODD, J. N. D006 CHARTIER, CH. E055 CROOIJMANS, R. A028, A030, DODDS, K. D080 CHEN, Y. Z. D163 A031, A040, E023, E072 DOLF, G. A021, C011, D035, D040, CHENG, H.H. D172 CRUICKSHANK, J. E013 D045, D077, D171, E037 CHEONG, IL-CH. C026, C045, D058 CUBILÓ, D. E001 DONLON, J. A005 CHESSA, S. D015 CUBRIC-CURIK, V. D027, D033 DOPPLER, W. C048 CHEVALIER, PH. F003 CULMSEE, K. A041 DORNAN, S. C076 CHIARELLI, L. R. D035 CURIK, I. D027 DORROCH, U. C017 CHILLIARD, Y. E031 CZERNY; C.-P. B016 DOUAIRE, M. A036 CHIU, R. A012 DA, Yang E004 DOVC, P. C048 CHMURZYNSKA, A. D129 D’ANGELO, M. C057 DRÖGEMÜLLER, C. A015, C010, CHO, B.W. D019 DA COSTA, N. C052 C012, C027, C031, C036, C043

200 Index

DUNIEC, M. D146 FORD, S.P. A020 GODDARD, M.E. E058 DUNNER, S. D011, D077, D096, FÖRSTER, M. A008, D051, D074, GOGOL, J. E029 D099, D171 F009 GOGUÉ, J. E042 DURSTEWITZ, G. D141, F004 FOTI, M. G. D035 GOHMA, H. C044 EBATA, T. B014 FRAGHI, A. D078 GOLDAMMER, T. C014, C017, ECKERT, J. D030 FRANCESCH, A. E001 C041, E025 EDEAL, J.B. C039 FRANCESCHI, L. D089 GOLIK, M. E016 EDFORS-LILJA, I. C053 FRANKLIN, I. R. D080 GOMEZ-RAYA, L. D094 EGGEN, A. C005, D117, E052 FREDHOLM, M. E080, D167 GONGORA, J. D041 EGUCHI, T. B014, B015 FREKING, B. A. D115 GOODARD, M.E. E068 EITAN, Y. E020 FRIEDMANN, A. E032 GORBOVITSKAIA, M. D042 EL ZAREI, M. E018 FRIES, R. A009, C063, D141, D142, GORDON, L. A030 ELDUQUE, C. E052 D162, E006, . F004 GORDON, P. C019 ELLIS, N.A. C049 FU, A. C019 GORNI, C. D156 ELMINOWSKA-WENDA, G. E067 FUERBASS, R. C035 GOSWAMI, R.N. A003 ELO, K. E015 FUJIMOTO, J. B014 GOTO, R.M. B007 ELRAYAH, I. D125 FUJINAKA, K. E049 GÖTZ, K.-U. E006 ELSEN, J.-M. E078 FUKUI, E. D036 GRALAK, B. D020 ENNE, G. D027, D033 FUKUSHIMA, M. D122 GRAPHODATSKAYA, D.A. A011, EPING, H. C052 FURUOKA, H. E048 C018 ERHARDT, G. D077, D079, D096, GABLER, A. A008 GREEN, CH.A. A012, C014 D108, D162, D171, E074 GAGLIARDI, R. C057 GREEN, J.A. C039 ERTUĞRUL, O. D031, D072 GAILLARD, C. C011, D037, D040, GREPPI, G. F. D027, D033 ESMAEILKHANIAN, S. D111 D169, E037, E055 GRIGALIUNAITE, I. D043, D132 ESTANY, J. E001 GALIBERT, F. A001 GRISART, B. E017 ESTONBA, A. D091, D127 GALINA, L. C076 GRISLIS, Z. D081, D138 ESTRADA, L. D151 GALLI, A. D009, D106 GROBET, L. A019 ESTUNINGSIH, E. E071 GARCÍA-MURO, E. D114 GROENEN, M. A.M. A028, A030, EVANS, G. C076 GARIPPA, D. F005 A031, A040, D161, E023, E072 EVERTS, R.E. C002, C013, C014 GARNERY, L. D091 GRUZMAN, G. E032 EVERTS-VAN DEN WIND, A. A012, GARWOOD, K. C039 GUÉMENÉ, D. E043 C014 GASBARRE, L.C. E047, E079, F015 GUÉRIN, G. C016 EYER, K.E. A020 GASKELL, R.M. B005 GUIARD, V. E028 EYTHORSDOTTIR, E. D043, D132 GASNIER, C. E060 GUILLET, S. D044 EZRA, E. E016 GAULY, M. E074 GULYÁZ, L. D113 FABUEL PEÑALVER, E. E051 GAUSTAD, K.G. A018 GUSTAFSSON, V. C028 FARAUT, T. A027, D032 GAUTIER, M. C005, D117, E052 GUTHRIE, A. E019 FARNIR, F. E017, D049 GELDERMANN, H. D030, D107, GUTIÉRREZ, B. E018 FARSTAD, W. A018 D128, E029 GUTSCHER, M. A042 FAURE, J.-M. E043 GELFENBEYN, B. A001 GUYOMARD, R. D159 FEIL, R. A035 GENÊT, C. D137, E005, E042, E060 GUYON, R. (?) FELIGINI, M. D027, D033 GENZINI, E. D038 GUZIEWICZ, K. A021, D045 FELLAY, E. D037 GEORGES, M. A019, A035, A043, GWAKISA, P. S. D143 FENG, S. T. D058 C004, C006, C009, C067, D049, E017 GYURMAN, A. D003, D180 FERNÁNDEZ, A. D022, D034, E035 GIACCONE, P. C007 HABERMANN, F.A. A009, D067 FERRETTI, L. D035 GIBSON, J.P. E039, E053, E054, HAEGEMAN, A. D101 FERRIE, B. F003 E063 HAGEN-LARSEN, H. A010 FERRO, J. A. D092 GIESE, A. C015 HAGGER, C. D110 FESUS, L. D147 GILBERT, H. E042 HAHN,S. D151 FÈVE, K. A027, E042, E043 GILL, C.A. D080, F006 HAIDUCH, M. D010 FIMLAND, E. D081, D138 GILLES, M. C055, C061 HALA, K. B004 FINDLEY, I. D150 GIOVAMBATTISTA, G. B003 HALEY, CH.S. E036, E073 FINOCCHIARO, R. C007 GITSCHIER, J. C059 HALL, A. J. D046 FISHBACK, A. D155 GIUFFRA, E. E002 HAMASIMA, N. A023, F007 FITZSIMMONS, C. A007, C039 GLAZKO, V. I. D135, D149 HAN, S. K. D047, E011 FJALESTAD, K. T. D094 GLODEK, P. D090 HAN, Y. D030 FOLCH, J.M. A002, D144, E035 GLOUX, S. A001 HÅNES, K. D159 FONSECA, C.G. E065 GLOWATZKI-MULLIS, M.-L. D039, HANOTTE, O. C045, D048, D065, FONTANESI, L. C015, C052, D153 D169, E055 D143, E025 FORD, CH. E017 GMÜR, A. A021, D040 HANSEN, CH. C019

201 Index

HARBITZ, I. D035 IANNUCCELLI, N. E042, E060 KAJII, E. C068 HARHAY, G.P. F008 IANNUZZI, L. D066 KALM, E. D116, E051 HARKEN-JENSEN, CH. C009 IBÁÑEZ, E. A002 KALTWASSER, C. D090 HARLIZIUS, B. C052, D161, E075 IBEAGHA, E. D162 KAMPS, B. A040 HARMEGNIES, N. D049 IBI, T. E066 KAMYCZEK, M. D057, D076, E024, HARPER, C. E019 IHARA, N. A033, D160, E056, D145 E064 HARPER, G.S. C037 INOKO, H. B001 KANAYA, N. D136 HASEGAWA, T. A026, C020 INOUE-MURAYAMA, M. D053, KANG’A, S. E025 HASHIZUME, K. C044 D062 KANTANEN, J. D043, D081, D132, HASTINGS, N. C060 IRAQI, F.A. E039, E053 D138 HATEY, F. A036 IRIONDO, M. D091, D127 KAPELANSKI, W. E067 HAUKE, G. C027 IRPS, U. D010 KAPPES, S.M. A012 HAWKEN, R. D167 ISHIDA, M. D054 KARAKINE, E. D061 HAWTHORNE, W. B006 ITO, H. D053 KARATAYLI, E. E081 HAYASHI, T. D136, D163 ITO, S. D053, D062 KARLE, S. E081 HAYES, H. D016, D117, D176, E052 ITOH, T. A033, E056 KATA, S.R. C014, C017 HE, H. D107 ITOH, Y. D093 KATMANN, I. A021 HEDEGAARD, J. C024 IVANOVA, Z. D138 KAUPE, B. D162 HEIFETZ, E. E020 IWAŃCZYK, E. D020 KAWABE, K. D124, D172 HELLER, D. E020 IWASAKI, T. D053 KAYANG, B. B. D062 HEMMATIAN, K. A028 JACOBS, K. C025, D137 KEATING, A. F. D063 HENNE, H. C052 JACOBSSON, L.U. E022 KEELE, J.W. A012, D024, D126, HENNER, J. D050 JAGGY, A. C011 E010, F008 HÉRAULT, F. A036 JAKAB, F. D056 KEMP, S.J. E039 HERMANOWICZ-ŚWIERCZEK, A. JAKABOVÁ, D. D056 KENNEDY, L.J. B005 D018 JAMNADASS, R. D143 KENT-BASHAM, C. F003 HERNÁNDEZ, D. E033 JANAN T. EPPIG , J.T. F005 KERJE, S. E026 HERTNER, U. D051 JANETT, F. A011 KERSTEN, P. D095 HEUVEN, H. D0156 JANIK, A. D057, E024 KETCHUM, M. C066 HEYEN, D.W. E004 JANKE, A. C065 KHANAHMADI, A. D111 HIENDLEDER, S. C065 JANN, O. D077, D171 KHATIB, H. E020 HIGUCHI, M. D158 JANSEN, S. C074, E077 KHRABROVA, L.A. D064 HILLS, D. D035 JASZCZAK, K. D118 KIERSTEIN, G. D065, D066 HINCHCLIFF, K. E019 JENNEN, D. A040, E023 KIERSTEIN, S. D067 HINES, H. C. D021, D052 JENNIFER K. LOWE A001 KIKUCHI, T. D145 HIRANO, T. E066 JENSEN, P. E026 KIM, C.D. D021 HIRBO, J. D065 JEPPSSON, S. D043, D132 KIM, C.I. D058 HIROTA, K.-I. A026 JIANG, M. A013 KIM, I.C. D058 HITTE, CH. A001 JIANG, S. A013 KIM, K.-S. E027 HJÄLM, G. A007 JIANG, Z.H. E054, E063 KIM, L. A001 HOELZLE, K. B016 JIANLIN, H. D048, D108 KIM, N.-S. C026 HOLM, L.-E. A010, D043, D132, JIGUET-JIGLAIRE, C. A027 KIM, S.J. D019 D159, D165 JIMÉNEZ, N. E001 KIM, T.-H. C026 HONDA, T. D122 JIN, H. J. D021, D058 KIM, W.T. E011 HONKATUKIA, M. E021 JOERG, H.W. A 011, C018 KIM, Y.S. E011 HOPWOOD, P.A. C022 JOHANSSON, A. C053 KING, A.H. E063 HORI, T. E002 JOHNSON, J. D179 KIRKPATRICK, B.W. E013, D164 HOŘÍN, P. C023, D166 JONES, M. E059 KITAGAWA, H. D053 HORIUCHI, A. D136 JONES, S. A012 KITAGAWA, Y. D124 HORN, P. C024 JÖRG, H. W. D037, D110 KITZIS, A. D029 HOSHI, T. D062 JORGE, E. A004 KIUCHI, S. D163 HOYHEIM, B. A010, A025, D094, J∅RGENSEN, C.B. E080 KLEIM, A. C009 D159 JOVANOVIC, S. J. D059 KLOSOWSKA, D. D068, E067 HUANG, J. A013 JÓZSA, C. D003 KNEELAND, J.L. E038 HUANG, L.S: C076 JUDE, R. C016 KNOLL, A. D069 HUDECOVÁ, L. D056 JUNGERIUS, B. D161 KNORR, C. C072, C075, D182, E075 HUHNS, M.N. F008 JURADOL, J. J. D012 KOBAYASHI, N. E066 HULATA, G. D023 JURAS, R. D060 KOENE, P. E008 HUTTON, M. F003 KADIN, J.A. F005 KOGANEZAWA, M. D036 IAMARTINO, D. D038, D077, D171 KAISER, P. B004 KOHLMANN, K. D095

202 Index

KOLLERS, S. D067 LEEB, T. A015, C010, C012, C016, MARCOS, S. E033 KOMATSU, M. C037 C027, C031, C036, C043 MARELLI, S. D105 KOMMISRUD, E. A018 LEHVASLAIHO, M.H. F010 MARGAWATI, E.T. E071 KONIECZNY, M. C066 LEKHONG, S. A020 MARIANI, P. B010 KONO, M. D071 LENSTRA, J.A. C069, D077, D096, MARIASEGARAM, M. E068 KOOHMARAIE, M. E010 D165, D171 MARILLI, M. D089 KOOP, B. A025 LEONARDUZZI, R. D078 MARINEZ, M.L. A006 KOPAR, A. D031, D072 LEROUX, S. D032 MARKLUND, S. C028 KOPECNY, M. B004 LETERRIER, CH. E043 MARRA, M. A012 KORCZAK, C062 LEWIN, H.A. A012, C002, C013, MARRON, B.M. A014 KORTE, S.M. E008 C014, E004 MARSHALL, K. F003 KORWIN-KOSSAKOWSKA, A. LI, CH. C019, E038 MARTA OLIVERA, M. D151 E064 LI, N. D042 MARTIN, S.A. A010 KOYANAGI, K. D124 LI, Z. A013 MARTÍN-BURRIEL I. A016, D077, KOZLÍK, P. D056 LI, Z.D. D058 D085, D114 KRÁLÍC, P. C023 LIAN, Z. D042 MARTÍNEZ, A. M. D086 KRATZSCH, A. C062 LIANG, Y. D175 MARTINEZ, D. E055 KREBS, S. A008, D074, F009 LIEFERS, S.C. E031 MARTINS-WEß, F. A015 KREMPLER, A. C071 LILLEHOJ, H.S. C001 MARZANOV, N.S. D186 KŘENKOVÁ, L. D166 LIM, Y. J. D019 MASHIMA, S. A026 KRIEGESMANN, B. E077 LINARES, A. R. D151 MASOPUST, M. D014 KRÜGER, K. D073 LINGAAS, F. C005 MATHEWSON, C. A012 KRUTMUANG, P. C054 LIPKIN, E. E032 MATIAŠOVIC, J. C023, D166 KRZYWIŃSKI, A. D109 LIU, CH. A013 MATSUMOTO, K. E037 KUABARA, M.Y. D173, E065 LIU, L. C013, C014 MATSUO, K. D062 KÜHN, C.H. C041, D010, E028 LIU, Z. D017 MATSUTA, M. C020 KUIPER, H. C016, C027, C031, C036, LIU, Z.L. C002, C013 MATSUURA, N. D053 C043 LOKESZOVÁ, L. C023 MATTHEEUWS, M. C025, D137 KULISIEWICZ, J. D018 LONG, E. D125 MATTHEWS, J.B. B002 KUMAR, Ch.G. C013 LONGERI, M. D078, D105, D152 MAU, C. D050 KUNIEDA T, E056 LOOFT, C.H. D116, E051 McDONALD, D. E019 KUPPINGER, O. D074 LOOFT, H. E051 McEWAN, J.C. F011 KURAR, E. D164 LÓPEZ HERRÁEZ, D. F016 McGRAW, R. E036, E073 KURODA, Y. E049 LORENTZEN, T. A001 McKAY, S. C014, D088 KUROSAWA, M. A026 LOWDEN, S. C022 McPARTLAN, H.C. E058 KURYŁ, J. E064, E067 LOWE, A. E053 MEĐUGORAC, I. D051, F009 KUSS, A.W. D107, E029 LUDEWIG, P. D090 MEGGIOLARO, D. D089 KUTZER, T. C070 LÜHKEN, G. D079 MELÉNDEZ, B. D097 KUWANO, A. C020 LUKAC-HAVRANEK, J. D027 MELLENCAMP, M. C076 KWACZYŃSKA, A. D057, D076, LUKSCH, U. A042 MELVILLE, J.S. E054 E024 LUNDEBERG, J. A007 MENDONÇA, R. E065 LADEVEZE, V D029 LUNDÉN, A. C028 MENEGUZ, G. D119 LAHBIB-MANSAIS, Y. D032 LUNNER, S. D159 MENEZES, C. L. P. D174 LALOË, D. D077, D171 MAAK, S. C035 MENG, Y. C019 LAMA, B. C052, D153 MACHADO, M.A. A006 MENGE, D.M. E053 LAMETCH, R. C024 MADDOX, J. F. D080 MERCADE, A. E035 LANZA, A. D007, D038 MADSEN, L. B. D102, F013 MERKS, J.W.M. C052 LARA, J. A.F. E030 MAEDA, Y. D124, D172 MEYER, B. A034, C047, C056 LARKIN, D.M. A012 MAGNETTI, P. D105 MEYER, J.-N. D090 LATIFA KARIM, L. E017 MAILLARD, J.-CH. E055 MIANA-MENA, J. A016 LAURENT, P. D117 MAIONE, S. D112, D119 MICEIKIENE, I. D043, D077, D081, LAUVERGNE, J.-J. D037 MÄKI-TANILA, A. E021 D132, D138, D171 LAVAL, G. E060 MALCHENKO, S. C039 MIDDLETON, R. D046 LAW, A.S. B008 MALEVICIUTE, J. D081 MIGNON-GRASTEAU, S. E043 LE MEUTH-METZINGER, V. A036 MAMO, S.G. C055 MIGUEL, I. D091 LE ROY, P. E042, E043 MANNEN, H. C037, D054, D071, MIKAWA, A. A023 LECERF, F. E078 D082, D122, D124, D145, D172 MILAN, D. A020, A037, D017, D167, LEDUR, M. E030 MANZ, E. F016 E005, E042, E060 LEE, E. J. D145 MARCHITELLI, C. D026, D083 MILANESI, E. D165 LEE, J.-H. B006, C026 MARCOS, C. E001 MILLER, J.E. E036, E073

203 Index

MILLER, M.M. B007 NARITA, A. E037 PALACIOS, F. D127 MILLS, A.D. E043 NASSIRY, M.R. D186 PANITZ, F. A010, D102, F013 MILTIADOU, D.K. B008 NATARAJAN, SH. C014 PAPSTEIN, H.-J. E028 MINEZAWA, M. D062 NATTRASS, G. D080 PAPWORTH, R. D024 MÍNGUEZ, Y. D011 NEGRINI, R. D077, D156, D165, PARATI, K. D009 MINQIANG, W. D140 D171 PARISET, L. D096, D098 MIRETTI, M. M. D092 NEJATI-JAVAREMI, A. D111, F012 PARK, H.-B. E022 MISHINA, Y. E056 NEPOMUCENA, A.L. E030 PARK, J. C. D058 MITSUHASHI, M. A023 NETTLON, D. C032 PARKER, H. A001 MITSUO MORITA, M. B012 NEUENSCHWANDER, S. C029, PARMA, P. D027, D033 MIYAKE, T. C011, E037 C062, C068, D110 PARMENTIER, H.K. E046 MIYASHITA, N. D0158 NEZAMZADEH, R. C071 PARRA, D. D099 MIZOSHITA, K. D160 NEZER, C. C067 PATEL, T. B006 MIZUNO, S. D093 NGANGA, J.K. E039 PATRICIA SIMON, P. E017 MIZUTANI, M. D062, D082, D145 NGUYEN, M. C006, C067 PATRÍCIO, M. A004 MNI, M. D049, E017 NICHOLAS, F.W. C049, C050, C059 PAULETTE BERZI, P. E017 MOAZAMI-GOUDARZI, K. D077, NIELSEN, M. E015 PEDRETTI, K. C039 D096, D165, D171 NIEMANN, H. D140 PEDROSA, S. D100 MOEN, T. D094 NIEUWLAND, M.G.B. E008, E046 PEELMAN, L.J. C025, C046, D101, MOLINA, E. E001 NIJMAN, I. J. D077, D096, D165, D128, D137 MOLIST, J. A002 D171 PEINADO, B. D086 MOLLER, M. E057 NILSSON, PH. E025 PELED, J.U. C002; C013 MOMMENS, G. D077, D096, D168, NIMZYK, R. A029 PENA R.N. A002, C030 D171 NINOV, K. E030 PERAL-GARCÍA, P. B003 MOMPART, F. D032 NJAGI, E. N.M. E025 PEREIRA JR., H.A. D092 MONTEAGUDO, L.V. C057 NOGUERA, J.L. C008, E035 PEREIRA, A.P. E041 MONTEIRO-VITORELLO, C.B. NOLTE, I. A029, A034, A041, C047, PERETTI, V. D121 A004 C056 PEREZ-ENCISO, M. E035, E043 MOORE, L. F003 NOMURA, K. D071 PERY, CH. E042 MOORE, S. A012, C019, E038 NONNEMAN, D. J. D115 PETERS, U. D182, E075 MORAN, CH. B006, C026, D041, NOONE, C. A017 PETERSEN, A. H. D102 D046 NOWAK, Z. D018 PETTERSSON, M. D103 MORISSON, M. A027, A030, A031 NOZAWA, K. D054 PFEIFFER, I. C070, D151, D183, MORITA, M. D053 O’CONNELL, PH. B006 D184, D185 MORITOMOY. E056 OBERG, E. D120 PFITZINGER, H. D044 MORLEY, P. E019 OBEXER-RUFF, G. D169, E055 PIASECKA, A. A021 MOROZUMI, T. A023 OGUNI, T. E056 PIEDRAFITA, D. E071 MORRIS, B. G. D006 OHKAWA, H. E002 PIELBERG, G. C053, D103 MOSNER, J. D010, D141 OHTANI, T. E066 PIEŃKOWSKA, A. D104 MOTAVALIAN, M. D007 OKA, A. C037 PIERSON, W. D125 MOUSE GENOME INFORMATICS OKAMOTO, S. D124, D172 PIERZCHAŁA, M. E064, E067 GROUP, F005 OKINE, E. C019 PILLA, F. D038, D077, D078, D096, MUELLER, A. D183 OKUMURA, F. D172 D144, D171 MUKAI, F. D122 OLIVEIRA, C.L. D174 PINEROLI, B. A021 MULSANT, P. E078 OLIVEIRA, D.A.A. D173 PINTON, PH. A037 MUNCK, H. D094 OLIVEIRA, H.N. E041 PIROTTIN, D. A019 MUÑOZ, M.J. A016 OLIVER, M.A. E035 PITEL, F. E043 MUNTWYLER, J. D039, D169 OLIVER, R.A. B008, B011 PIUMI, F. C016, C025, D137, E005 MURAKAEVA, A. D095 OLLIER, W.E.R. B002, B005 PLASTOW, G.S. E027 MURANI, E. C052 OLSAKER, I. A009, A018, C005, PLIS-FINAROV, A. E012 MURATA, K. D082 D043, D077, D081, D132, D138, PLISSON-PETIT, F. E043 MURAYAMA, Y. D053 D165, D171 POLI, M. A. D092 MURDOCH, B. E038 OMI, T. C029, C068 POLLI, M. D105 MURPHY, E.F. A020 OSSA, J. D151 POMP, D. C039, D167, E015 MURRAY, W. D155 OSTA, R. A016 PONCELET, D. A019 MURUA ESCOBAR, H. A029, A034, OSTRANDER, E.A. A001 PONOMAREV, I. D061 A041, C047, C056 ÓVILO, C. D001, D034, D097, E035 PONSUKSILI, S. C052, C054, C055, MWAKAYA, J. E025 OYAMA, K. D122 C058, C061 NAGAMINE, Y. D158 ÖZBEYAZ, C. D162 PONZ, R. C057 NAGATA, S.-I. A026 PADILHA, T. E047, E079, F015 PORTOLANO, B. C007

204 Index

POSTIGLIONI, A. C057 RODENBURG, T.B. E008 SAVE, J.-C. D017, E005 POTO, A. D086 RODRÍGUEZ, B. A035 SAVIC, M. D059 POTTER, J. C003 RODRÍGUEZ, C. D001, D034 SAVOLAINEN, P. A007 POWELL, R. A010, D159 RODRÍGUEZ-GALLARDO, P. P. SCHÄFER, H. F016 POZA, J. A016 D139 SCHAMBONY, A. C016 POZZI, A. D009, D106 ROELOFS, B.M. A020 SCHEIN, J. A012 PRATHER, R.S. C039 ROGATCHEVA, M.B. A022 SCHELLANDER, K. C052, C054, PREUSS, S. D030, D107 ROGEL-GAILLARD, C. B001, D016, C055, C058, C061 PRIEM, J.M. C052 D017, D042, D176, E005 SCHELLING, C. A021, D045, D104 PRINZENBERG, E.-M. D108 ROHRER, G.A. C025, D014, D115, SCHIBLER, L. E052 PRUSAK, B. D109 D167, E063 SCHIEX, T. D167 PULLENAYEGUM, S. D123 RON, M. D023, E012, E016 SCHLÄPFER, J. A021, D035 PUTNOVÁ, L. D069 RØNNINGEN, K. C005 SCHMUTZ, SH.M. C003, C034, PYTHON, P. D110 ROOS, C. D029 E003, E007 QUIGNON, P. A001, C031 ROOSEN, S. D116 SCHNEIDER, M. P. D066 QUIRINO, C.R. E065 ROSATI, A. D007, D038 SCHOOK, L.B. A022, D167 RAADSMA, H.W. C049, C050, C059, ROSENFELD, D. F003 SCHREIBER, M.J. F011 E059, E071 ROSS, P. D063 SCHULMAN, N. E021 RABIE, T. A030, E072 ROTHSCHILD, M.F. C039, E027 SCHULZ-SCHÄFFER, W. C042 RADFORD, A.D. B005 ROTTMANN, O. E081 SCHUSTER, S. D113 RADKO, A. D057, E024 ROTUNDO, S. B010 SCHÜTZ, E. C042, C072 RAHIMI, G. D111, F012 ROUSSOT, O. E043 SCHÜTZ, K. E026 RAK, S.G., C031 ROY, R. D117 SCHWEITZER, P. A012 RAMON, C. D022 ROYO, L. A019 SCHWERIN, M. C017, C035, C041, RASERO, R. D078, D112, D119, RÓŻYCKI, M. D057, D076, E024 E025, E028 D121 RULE, T. F003 SCOTTI, E. C015 RATTINK, A. D161 RUND, L.A. A022 SEGERS, K. A035, C004 RAUDSEPP, T. E080 RUSSELL, G.C. B008, B011 SELTZER, P. C076 REBEIZ, M. A012, C014 RUSSO, V. C015, D153 SEMMER, J. A042, D010 REECY, J. C032 RUTKOWSKI, J. D118 SENSEN, CH.W. C019 REED, K. M. D160 RYVAR, R. B005 SERENO, F.T. D139 REGE, J. E. O. D048, D065 SACCHI, P. D112, D119, D121 SERENO, J.R. D139 REGITANO, L. C.A. E041 SACHARCZUK, M. D118 SEROUSSI, E. D023 REICHENSTEIN, M. E012 SAÏDI-MEHTAR, N. D012 SERRANO, M. E033 REICHERT, P. A009 SAITBEKOVA, N. E055 SERRANOL, M. D012 REID, S. E017 SAKOYAMA, R. C033 SERROUSSI, E. E012, E016 REINER, G. D128 SALTINI, G. B010 SEYFERT, H.-M. E044 REINSCH, N. E051 SAN PRIMITIVO, F. D100, E018 SHA, J. A013 REIS, S.R. E065 SÁNCHEZ, A. A002, C008, C038, SHACKELFORD, S.D. E010 REISSMANN, M. D113 C040, D022, D077, D144, D171, SHAH, R. D123 REKLEWSKI, J. D109 E001, E035 SHANI, M. E012 RENARD, CH. B009, E005, E042, SANCHEZ, M.-P. E042 SHARKEY, K.J. A020 E060 SANGALLI, S. D120 SHAUNESSY, M. D155 RENIER, C. A001 SANTALÓ, J. A002 SHAY, T.L. A035, C004, C009, E036, RENNE, U. E081 SANTSCHI, E.M. A020 E073 RICHARDSON , J.E. F005 SANTURE, A. F011 SHEKHAR PAREEK, CH. E044 RICHTER, A. A034, C047, C056 SARAC, M. D059 SHEKHAR PAREEK, R. E044 RICKLI, O. A021 SARGENT, C. C076 SHIGENARI, A. B001 RIEDER, S. J. D037, D050, D073 SARGENT, J.R. C060 SHIINA, T. B001 RINGWALD, M. F005 SARTORE, G. D121 SHIMAKURA, Y. C037 RINK, A. A020, D167 SARTORE, S. D112, D119, D121, SHIMOGIRI, T. D124, D172 RIQUET, J. E042, E060 D177 SHIMOKOMAKI, M. E030 ROBERTS, CH.A. C039 SASAKI, Y. C033, C044, E037, E066 SHINBO, Y. D172 ROBIE, A. A037 SASAZAKI, S. D122 SIEGEL, P.B. E022 ROBINSON NOLAN, E. C011 SASSE, J. D123 SIEME, H. C016 ROBINSON, J.A.B. E054 SATO, F. C020 SIERRA, I. C057 ROBINSON, N.A. E058, E068 SATO, H. E056 SIKORA, M. D057, E024 RODELLAR, C. A016, D077, D085, SATTLER, U. E055 SILER, D. F003 D096, D114, D117, D165, D171 SAVARESE, M. C. D077, D083, SILIÓ, L. D001, D034, E035 RODEN, C. D168 D096, D098 SILVA, A. D066

205 Index

SILVA, C.S. A004 TAKAMI, M. E056 VALENTINI, G. D035 SIMIANER, H. D090, F014 TAKASHI AWATA, T. F007 VALLINOTO, M. D066 SIMON, P. D049 TAKASUGA, A. D160 VAN DEN BERG, K. E019 SIMOND, D. B006 TAKEDA, H. D160, E056 VAN DER LENDE, T. E031 SIRONEN, A.I. E045 TAKESHIMA, SH.-N. B012, B013 VAN DER POEL, J. A028, A030, SIRONI, L. B010 TALBOT, R. C022 A031, A040, E008, E023, E046, E072 SIWEK, M. E008, E046 TAMMEN , I. . C049, C050, C059, VAN DYK, E. E019 SŁOTA, E. D146 E059 VAN EIJK, M.J.T. D156 SMIT, M.A. A035 TANIGUCHI, M. C037 VAN ELSACKER, L. D168 SMITH, D. B006 TANIGUCHI, Y. C033, C044 VAN HIERDEN, Y.M. E008 SMITH, E. D125 TAPIO, M. D043, D132 VAN LAERE, A.-S. E050 SMITH, J.A. B008, B011 TASSELL, C.P. E004 VAN OOST, B. D161 SMITH, T. A017, D063 TATSUDA, K. E049 VAN POUCKE, M. C025, D128, SMITH, T.P.L. D126 TE PAS, M. C052, D161, E031 D137 SNELL, R. E017 TEALE, A.J. C060, E053 VAN SOOM, A. C046 SNELLING, W.M. D126, E038, F008 TEISNER, B. C009 VAN TASSELL, C.P. A006, E004, SNOWDER, G.D. A035 TEIXEIRA, C. S. D173 E047, E079, F015 SOARES, M.B. A036, C039 TEJEDOR, M.T. C057 VAN VLECK, D. E015 SOEDA, E. D163 TESFAYE, D. C061 VAN ZEVEREN, A. C025, C046, SOLER, C. D029 THALLER, G. D141, D142, E006 D101, D137 SOLIS, A. D127 THALLMAN, R.M. E047, F015 VARLOTTA, C. D007 SOLLER, J.T. A041 THOMSEN, H. E027 VARONA, L. C040, E035 SOLLER, M. E020, E032 THOMSON, P. E071 VÄRV, S. D081, D138 SON, D.S. D058 THOMSON, W. B002, B005 VASICEK, D. B004 SONSTEGARD, T.S. A006, D024, THORSEN, J. A025 VASICKOVA, K. B004 E004, E047, E079, F015 THUE, T.D. E007 VEENENDAAL, T. A031, E023, SOUZA, K. T. D174 TIRELLA, G. D015 E072 SPEHR, V. C076 TOCHER, D.R. C060 VEERKAMP, R.F. E031 SPELMAN, R. E017 TOMÁS, A. C038, E035 VEGA-PLA, J. L. D086, D139 SPENCER, M. F003 TONOSAKI, K. D053 VERDINELLI, D. D035 ŠPILAR, S. C029, C068 TÖPFER-PETERSEN, E. C016 VERDOGLIA, L. D089 SPITHILL, T.S. E071 TOR, M. E001 VEREIJKEN, A. L.J. E023, E072 SPONENBERG, P. D125 TORO, M. A. D034 VERKAAR, E.L.C. C069 SPÖTTER, A. C036 TORRES, M. F003 VERRINDER GIBBINS, A.M. E054 SRINIVAS, K. C055 TOSSER-KLOPP, G. A036 VERVAECKE, H. D168 STEIGER, D. A011 TOZAKI, T. A026 VIDAL, O. C040 STENGAARD, H. D102, F013 TRAILOVIC, R. D059 VIGNAL, A. A027, A030, A031, STEPHEN, J. D143 TRANDŽÍK, J. D056 E043 STEPHENS, L.M. F008 TRANULIS, M. D035 VIGNAUX, F. A001 STERNSTEIN, I. E076 TRYAPITCINA, N. V. D135 VIINALASS, H. D043, D081, D132, STONE, R.T. D126, E010, TSUIJ, T. E056 D138 STRANZINGER, G. A011, C018, TSUJI, S. C037, D054, D071, D082, VILKKI, J. E021, E045 C029, C062, C068, D050, D073, D122, D124,. D145 VILLALBA, D. E001 D104, D110 TUGGLE, CH.K. C039 VILLEGER, S. A036 STRATIL, A. D128, C025 TUISKULA-HAAVISTO, M. E021 VIRTA, A. E021 STRICKER, C. D110 UCHIDA, Y. D136 VOELKEL, I. D151, D185 STRILLACCI, M.G. D078, D152 UEDA, Y. D053 VÖGELI, P. C062, C068, D110 STUBBS, L. A030 UENISHI, H. B015, D136, D163 VOIGT, J. E028 SUBANDRIYO, E071 UIMARI, P. E045 VONNAHME, K.A. A020 SUGIMOTO, M. D160, E048 ULLRICH, J. C076 VOS, H. E008 SUGIMOTO, Y. A033, D160, E048, URIEN, C. D016, D176, E005 VOß-NEMITZ, R. A015 E056, E066 v. CAPUCO, A. A006 WADDINGTON, D. E063 SUZUKI, H. A023, F007 v. RIPOLI, M. B003 WAFULA, J. D065 SUZUKI, K. F007 VAARA, I. C053 WAGNER, H.-J. D113 SWALVE, H.H. C042, E077 VACCARI, G. D035 WAGNER, V. F016 SWITONSKI, M. D068, D129 VAIMAN, D. D012, D016, D080 WAKELIN, D. E053 SZYDŁOWSKI, M. D068 VAIMAN, M. B009 WALASIK, K. E067 TAGGART, J. A010, D159 VALE FILHO, V.R. E065 WALAWSKI, K. E044 TAKAGAKI, Y. B014, B015 VALENTINI, A. D013, D026, D077, WALLING, G.A. E036, E073 TAKAHASHI, H. D062 D083, D096, D098, D156, D171 WALTERS, S. B006

206 Index

WANABE, H. C037 YUZBASIYAN-GURKAN, V. C011 WANG, N. A013 ZĄBEK, T. D057, D146, E024 WATANABE, A. D158 ZADISSA, A. F011 WATANABE, T. A033 ZAITCEV, A. M. D064 WEBB, G. D080 ZAJAC, M D129 WEGNER, J. E028 ZAMBONELLI, P. D128 WEIGEND, S. D140 ZANELLA, E.L. E030 WEIKARD, R. C041 ZANGERL, B. D179 WEIMANN, CH. D077, D096, D171, ZANOTTI, M. D077, D078, D096, E074 D105, D152, D165, D171 WELLER, J.I. E012, E016 ZARAGOZA, I. D114 WEMHEUER, W. C042 ZARAGOZA, P. A016, D077, D085, WERNER, F. A. O. D141 D114, D117, D171 WHITELAW, B. C030 ZAWADA, M. D104 WIDJAJANTI, S. E071 ZETOCHOVÁ, E. D056 WIEDEMANN, S. D142 ZHANG, F. C022, C039 WIENER, P. D077, D096, D171 ZHANG, Q. D179 WILKE, I. D061 ZHANG, X. D175 WILLIAMS, J.L. B008, B011, C027, ZHANG, Y. E027 D024, D035, D077, D096, D138, ZHAO, Q. D052 D156, D162, D171 ZHONGLIN, L. D140 WILLOUGHBY, K. B005 ZIJLSTRA, C. D176 WIMMERS, K. C052, C054, C055, ZSOLNAI, A. D147 C058, C061 ŻURKOWSKI, M. D118 WINDSOR, P.A. C050 WINK, M. F016 WINKLER, S. A041 WINTER, A. C063, D162, F004 WITTEMEIER, M. C042 WITTWER, CH. A021 WÖHLKE, A. C043 WOLF, C065 WOLLNY, C.B.A. D143 WOMACK, J.E. A012, C014, C017 WOMACK, J.E. D088, D164 WOODS, R. C039 WÖRNER, R. C052 WRAY, J.E. F008 WRIGHT, D. D125 WYE, N. A012 XAVIER, S.R. F006 XIAOLING GUO, X. E051 XUEBIN, Q. D048 YAHYAOUI, M. H. D144 YAMADA, T. C033, C044, E037 YAMAMOTO, R. B015 YANG, G. D175 YASUE, H. B001, B014, B015, C044, D163, D167 YEO, J. S. D071 YEON, S. H. D021 YERLE, M. A020, A027, A037, C025, D017, D032, D137, D167 YOKO AIDA, Y. B012 YONEDA, K. E056 YOO, C. H. D058 YOON, D.-Y. C045 YOSHIZAWA, K. D145, D172 YOSHIZAWA, M. D036 YUAN, Y.Q. C046

207 Index

28th ISAG Conference 2002

Lecture Halls in the Blue Tower

208