Physical and Comparative Mapping of Candidate Genes on the Horse Genome
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Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover ___________________________________________________________________ Physical and comparative mapping of candidate genes on the horse genome INAUGURAL-DISSERTATION zur Erlangung des Grades einer DOKTORIN DER VETERINÄRMEDIZIN (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover vorgelegt von Dorothee Stübs geb. Müller aus Heidelberg Hannover 2006 Scientific supervisor: Univ.-Prof. Dr. Dr. habil. O. Distl Examiner: Univ.-Prof. Dr. Dr. habil. O. Distl Co-Examiner: Prof. Dr. Dr. habil. H. Niemann Oral examination: 23.11.2006 This work was supported by grants from the German Research Council, DFG, Bonn, Germany (DI 333/12-1). To my beloved husband and our son Parts of this work have been accepted for publication or have been published in the following journals: 1. Animal Genetics 2. Cytogenetic and Genome Research 3. Archives for Animal Breeding Table of contents Chapter 1 Introduction 13 Chapter 2 Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits 17 Abstract 19 Zusammenfassung 20 Introduction 20 Organization of the horse genome 21 Cytogenetic map 22 Synteny map/ Radiation hybrid map 22 Comparative map 23 Linkage map 25 Chromosomal abnormalities 26 Coat colours 26 Equine genetic diseases and the development of gene tests 31 Monogenic equine diseases 31 Genetically complex equine diseases 33 Conclusions and perspectives 36 References 37 Chapter 3 Assignment of the COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping 53 Source/description 55 Primer sequences 56 Chromosomal location 56 Radiation hybrid mapping/ PCR conditions 57 Comment 57 Acknowledgements 57 References 57 Figure 58 Chapter 4 Physical mapping of the PTHR1 gene to equine chromosome 16q21.2 59 Source/description 61 Primer sequences 62 Chromosomal location 62 Radiation hybrid mapping/ PCR conditions 63 Comment 63 Acknowledgements 64 References 64 Figure 65 Chapter 5 Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 And SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping 67 Source/description 69 BAC library screening/ sequence analysis 71 Chromosomal location 72 Radiation hybrid mapping 72 Comment 73 Acknowledgements 73 References 73 Figures 74 Table 1 77 Table 2 80 Chapter 6 Physical mapping of the ATP2A2 gene to equine chromosome 8p14->p12 by FISH and confirmation by linkage and RH mapping 83 Rationale and significance 85 Materials and methods 85 BAC isolation and characterization of the equine ATP2A2 clone 85 PCR conditions and microsatellite analysis 87 Chromosome preparation 87 Fluorescence in situ hybridization 87 Radiation hybrid (RH) mapping 88 Linkage mapping 89 Results and discussion 89 Polymorphism 89 Chromosomal location 89 Acknowledgements 90 References 90 Figure 91 Table 92 Chapter 7 Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH mapping 93 Source/description 95 Primer sequences 96 Chromosome location 96 Radiation hybrid mapping 97 Comment 97 Acknowledgements 98 References 98 Figure 98 Chapter 8 Assignment of the TYK2 gene to equine chromosome 7q12-q13 by FISH and confirmation by RH mapping 99 Background 101 Procedures 101 Results 103 Acknowledgements 103 References 104 Figure 104 Chapter 9 Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping 105 Background 107 Procedures 109 Results 110 Acknowledgements 112 References 112 Figures 115 Tables 117 Chapter 10 General discussion 119 Chapter 11 Summary 127 Chapter 12 Zusammenfassung 131 Appendix 139 List of publications 149 Acknowledgements 152 List of abbreviations A adenine BAC bacterial artificial colony BLAST basic local alignment search tool BLASTN basic local alignment search tool nucleotide bp base pairs C cytosine cDNA complementary desoxyribonucleic acid cen centromer CHORI Children`s Hospital Oakland Research Institute cM centiMorgan cR centiRay DAPI 4`,6`-diaminidino-2-phenylindole DFG Deutsche Forschungsgemeinschaft (German Research Council) DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTPs deoxy nucleoside 5`triphosphates (N is A, C, G or T) ECA Equus caballus autosome EDM multiple epiphyseal dysplasia EMBL European Molecular Biology Laboratory F forward FISH fluorescence in situ hybridisation G guanine GTG giemsa trypsin giemsa HSA Homo sapiens autosome IMAGE integrated molecular analysis of genomes and their expression INRA Institut National de la Recherche Agronomique ISCNH international system for chromosome nomenclature of the domestic horse kb kilobase LOD logarithm of the odds Mb megabase MED multiple epiphyseal dysplasia mRNA messenger ribonucleic acid MS microsatellite NaH2PO4 Natriumdihydrogenphosphat Na2HPO4 Dinatriumhydrogenphosphat NCBI National Center for Biotechnology Information no. number NPL nonparametric linkage OC osteochondrosis OCD osteochondrosis dissecans OMIM online mendelian inheritance in man PCR polymerase chain reaction POS position QTL quantitative trait locus R reverse RET retention frequency RH radiation hybrid RZPD Resource Center/Primary Database, Berlin SSC sus scrofa chromosome STS sequence-tagged site T thymine TAMU Texas A & M University TBE tris-borate-ethylenediamine tetraacetic acid TE tris-ethylenediamine tetraacetic acid Ta annealing temperature UV ultraviolet X-Gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside Chapter 1 Introduction Introduction Introduction The horse gene map was - compared with other farm animals - still in its infancy at the beginning of this work, but it has quickly developed during the last few years. Today, genome investigation in horses is aimed at the decoding of the whole horse genome, as it has taken place for humans and several farm animals. Since the horse has not only been used as a helpful worker during the last centuries, but has developed into a dependable partner in equitation today, it is not surprising that the bone and cartilage metabolism and affected diseases like osteochondrosis and navicular disease play an interesting role in horse breeding in the meantime. Osteochondrosis is a developmental orthopaedic disorder of growing animals. The disease is accompanied by abnormal cartilage development in the joints of young growing horses causing subchondral fractures, cysts, wear lines, chondromalacia or cartilage flaps. It can also be manifested in the form of joint mice or free joint bodies (chips) and is known as osteochondrosis dissecans (OCD). A number of articulations can be affected, like the fetlock, hock, carpal, stifle, elbow, hip or vertebral joints. The accumulation of the disease in several horse breeds and families (prevalence in Warmblood and trotter horses between 10 and 79.5%) provides an indication of involved hereditary factors, but responsible genes still have not been found until today. Moreover, nutritional and endocrinological factors, skeletal growth rate and biomechanical trauma seem to play an important role in Osteochondrosis; so the disease appears to be multifactorial. To contribute new results to the comparative human-equine gene map, altogether 17 candidate genes have been chosen. The objective of the present study is the physical mapping of these candidate genes with the previously unknown localization on the horse genome for the first time. Ten genes have been picked due to their possible influence on osteochondrosis, five genes from different solute carrier families, one gene as a candidate gene for pastern dermatitis. This work describes the cytogenetical mapping of the candidate genes by FISH and the confirmation of the localizations by radiation hybrid mapping and offers new findings, which are partially surprising, in the end. - 15 - Introduction Overview of chapter contents The presented work consists of eleven chapters of which chapters 3-7 represent already published articles. Chapter 2 reviews the development and the actual situation of equine gene mapping including physical gene maps, comparative gene maps, RH-maps, linkage maps and describing the inheritance of coat colours and several hereditary diseases in horses. Chapters 3-8 describe the physical mapping of 16 genes on the horse genome for the first time. In Chapter 8 and 9 the mapped genes that have not yet been published and the mapping results are concluded and discussed. Chapter 10 provides conclusions recapitulating chapters 3 to 9 and a general discussion. In Chapter 11 the whole work is summarized concisely, while Chapter 12 is a detailed German summary. All tables and figures belonging to the different chapters can be found at the end of this work in the Appendix. - 16 - Chapter 2 Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits D. Stübs and O. Distl Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Germany Accepted for publication In press: Archives for Animal Breeding Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits Abstract Since the beginning of investigation in the horse genome in the early nineties, there has been a great progress, especially during the last five years. At the beginning the exploration of monogenic hereditary diseases was one of the main aims, and the causal