A New Gene Mapping Resource: Interspecies Hybrids Between Pere David’S Deer (Ehphurms Davidianus) and Red Deer (Cewus Ektphus)

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A New Gene Mapping Resource: Interspecies Hybrids Between Pere David’s Deer (Ehphurms davidianus) and Red Deer (Cewus ektphus)

M. L. Tate,* H. C. Mathias,” P. F. Fennessy, K. G. Dodds, J. M. Penty* and D. F. Hill* *AgResearch Molecular Biology Unit, Department of Biochemistry, University of Otago New Zealand and AgResearch, Invermay Apicultural Centre, Mosgiel, New Zealand Manuscript received May 2, 1994 Accepted for publication November 28, 1994

ABSTRACT Three male F1 hybrids between Pere David’s deer and red deer were mated to red deer to produce 143 backcross calves. The pedigrees a are rare exampleof a fertile hybrid between evolutionarily divergent species. We examined the use of these families for genetic mapping of evolutionarily conserved (Type I) loci by testing for genetic linkage between five speciesspecific protein variants 12and conserved DNA probes. Two probes were homologous, and the remainder syntenic, to the protein coding loci in cattle or humans. Using six restriction enzymes, eachDNA probe detected one or more restriction fragments specific to Pere David’s deer. Linkage analyses among the species-specific variants placed the loci into four linkage groups within which linkage between adjacent loci and gene order was supported by a LOD > 3. The linkage groups were( HPX, HBB) -FSHBACPZ, LDHA-W5-IGFZ, BMP3 ( CC, ALB) - ( KIT, PDGFRA) and LDLI”C3-FGFl. Southern and protein analysisof LDHA and ALB provided identical segregation data. These linkage groups were consistent with the cattle gene map and provide new information for comparing the gene maps of ruminants, humans and mice. The deer hybrids are an important new resource that can contribute to the comparative analysisof the mammalian genome.

N the 1970s, cytological analyses demonstrated that genes responsible for variation in phenotypic traits. How- I karyotypic banding patterns could be used to align ever, with the exception of the mouse map, veryfew chromosomes and chromosome segments between gen- of the markers on linkage maps are suitable for cross era, families and, in some cases, orders of mammals referencing and alignment of the various mammalian ( SUMNER1990). These data have been extended using genome maps. Typically linkage maps are composed of gene maps that compare the chromosomal location of highly polymorphic microsatellites(WEBER and MAY homologous loci among species (O’BRIENet al. 1985, 1989), which are usually anonymous and, with a few 1988) and such comparisons are most advanced in the notable exceptions (STALLINGet al. 1991 ) , not widely extensively mapped genomes ofmice and humans conserved among mammals. O’BRIENet al. ( 1993) has ( O’BRIEN1991; NADEAUet al. 1993). There is consider- formalized this distinction of marker types, defining the able interest in refining and extending the comparison evolutionarily conserved markers as “Type I” markers of gene maps to a wider variety of speciesin an attempt and the highly polymorphic but anonymous markers as to unify the genetic analysis of mammals and to provide “Type 11” markers. insights into mammalian genome organization and eve Only the mouse has a detailed linkage map of Type I lution (EDWARDS1991; FARRand GOODFELLOW 1992; markers and the key to the production of this map has O’BRIENet al. 1993). been the use of interspecies hybrid backcross mapping Genetic linkage mapping is currently the most produc- pedigrees ( COPELANDandJENKlNS 1991 ) . The principle tive technique in the refinement of gene maps of a wide example is an interspecies hybrid between Mus spetus variety of mammals, such as humans (WEISSENBACHet and M. musculus (the laboratory strain C57BL/ 6J) . al. 1992) , mice ( DIETRICHet al. 1992; COPELANDet al. These and othermouse interspecies hybrids continue to 1993) , rats ( SERIKAWAet al. 1992) , cattle ( BARENDSEet be the method of choice for mapping expressed genes aZ. 1994; BISHOP et al. 1994), sheep ( CRAWFORDet al. and refining the genetic map of the mouse ( COPELAND et al. 1993). The important feature of these interspecies 1994) and pigs ( ELLIGRENet al. 1994). Linkage maps provide the ability to rapidly determine the location and hybrids is that the genetic divergence between the species is such that species specific variants can be rapidly order of polymorphic genes and the potential to locate identified with virtuallyany DNA geneprobe. Each marker with species specific variants is heterozygous in Corresponding author: Mike Tate, AgResearch Molecular Biology Unit, Biochemistry Department, University of Otago, P.O. Box 56, the F1 hybrid and thus fully informative in backcross Dunedin, New Zealand. E-mail: [email protected] panels.

Genetics 139 1383-1391 (March, 1995) 1384 M. L. Tate et al.

Conceptually,this approach could beused to effi- gating in the backcross herd ( TATEet al. 1992; EMERSON ciently generate linkage maps of TypeI loci from a wide and TATE1993). We have used the term “variant” for variety of mammalian genera. Parallels havebeen drawn the fixed differences observed between the species and between the mouse hybrids and crosses created for ge- reserved the word “polymorphism” for variation within netic mapping in domestic animals suchas the Bos taurus a species. The five protein variants were assumed to re- X B. indicus hybrid (ROBERTS1990) and crosses among flect variation in the coding gene loci, namely, C3 (com- divergent strains of domestic pig Sus scrofa (ARCHBALD plement component 3), GC ( vitamin D bindingprotein) , ALB et al. 1991) . Although these crossesintroduce some addi- (albumin), LDHA (lactate dehydrogenase A) and HPX tional polymorphism into mapping panels, linkage maps (hemopexin). Loci were chosen for RFLV investigations of Type I markers comparable with those produced with on the basis of homologyor conserved synteny with these M. spetus hybrids (eg: BUCHBERGet al. 1989) have not protein loci in the human and cattle gene maps (Table been forthcoming suggesting these large mammal hy- 1 ) . Three of the proteins, (HPX, LDHA and C3) are brids do not share the wide divergence of the mouse close toproposed evolutionary break points between hu- hybrids. Breeding of wider hybrids for genetic linkage man and cattle chromosomes (O’BRIENet al. 1993) so mapping has been attempted in sheep (sheep X goat we selected gene probes flanking these breakpoints (Ta- hybrids; HILL andBROAD 1991) and cats (Asian leopard ble l ) . X domestic cat; LYONSand O’BRIEN1994) butfew backcross animals havebeen produced. In marsupials, a sub MATERIALSAND METHODS specificbackcross between tammar wallabiesis being evaluated for linkage mapping ( MCKENZEEet al. 1993). Pedigrees and sampling: Three F1 stags, produced from The interspecies hybrid we have developed is between artificial insemination of red deer hinds with Pere David’s the P6re David’s deer or Milu, Elaphurus davidianus, and deer semen (ASHER et al. 1988; FENNESSY et al. 1991), were mated to red deer from 1989 to 1991 on three New Zealand red deer, Cmus elaphus. The deer species show high deer farms. During this period, the threestags naturally mated level of genetic divergence, similar to that between M. with 91, 21 and 32 red deer, respectively, and semen from spetus and M. musculus ( BONHOMMEet al. 1984). Esti- the lattertwo stags was used to artificially inseminate 31 and 83 mates of the genetic distance (NEI 1972) between the red deer,respectively, using methods described by FENNESSY et deer species are 0.35 from 22 protein loci (EMERSON al. ( 1991 ) . All animals were maintained outdoors onpasture. Blood samples (50- 100 ml ) were taken from all the available and TATE1993) and 0.46 from 45 protein loci (TATEet parents and progeny of these matings. The parents of the al. 1992) . Red deer are the common large deer of Eu- sires had either died soon after mating or were not available rope and closely related subspecies and species are natu- for sampling. However, reference samples were available from rally distributed throughout Europe, Asia and North five P&reDavid’s deer from the grandsires’ herd. America (WHITEHEAD1972). In contrast, P6re David’s Protein variation: Five proteins known to distinguish red deer andP6re David’s deer were scored inthe pedigrees using deer, originally from China, are one of the world’s rarest previously described methods ( TATEet al. 1992; EMERSONand deer species (JONES et al. 1983) . The two species have TATE 1993). Briefly, LDHA was examined by native starch very large differences in their appearance and biology, gel electrophoresis of red blood cell lysates followed by histo- the most notable being the antler, foot and tail morphol- chemical staining. GC, C3 and HPX were analyzed by native ogy ( WEMMER1983), seasonality ( LOUDONet al. 1989), polyacrylamide gel electrophoresis of plasma followed by west- ern analysis using antibodies specific to the human form of disease resistance ( ORRand MACKINTOSH 1988) and be- these proteins (Dako, Carpenteria, CA). ALBwas analyzed havior (ALTMANN and SCHEEL1980). However, both by isoelectric focusing at pH 3- 10 with 6 M urea followed by species havethe same number of chromosomes and very general protein staining. similar karyotypes( WANC1988) andfertile hybrids have Selection of DNA probes: Large numbers of cDNA probes from a variety of mammalian sources had been previously occasionally been produced ( BEDFORD1951 ) . Recently, screened for hybridization with red deer DNA on “zooblots,” in New Zealand, Phe David’s deer males that were in which included EcoR1-restricted DNA from human, sheep, excess to the breeding requirements of the Pbe David goat, cow, reddeer, pig and possum (Trichurus vulpecula; population have been successfully crossed to red deer J. M. PENTY and H.C. MATHIAS, unpublished data). From females using artificialinsemination ( ASHER et al. 1988) . this information, we selected 12 mammalian probes which In this study,we report on the fertility of the F1 hybrid showed good homology with red deer DNA (Table 1) . The probes were selected from analysis of published comparative males and the production of large backcross families. gene maps of human, mouse, and cattle (see Introduction). The aim of this study was to evaluate these pedigrees They were not preselected on the basis ofDNA polymor- as a resource for linkage mapping Type I markers and phisms. comparing the chromosomal arrangement of lociin Restriction fragment length variation: DNA was extracted deer with other species. The experimental approach was using the salt method, blotting and hybridization protocols described by MONTGOMERYand SISE (1990) with the excep- to use restriction fragment length variants ( RFLVs) de- tion that probes were labeled using a “megaprime” random tected by conserved coding sequences to build linkage primelabeling system (Amersham, UK) . Probes were groups around five protein variants known to be segre- screened for variation on filters, which contained DNA from Deer Interspecies Hybrids 1385

TABLE 1 Loci examined in the deer interspecies pedigrees; comparative map locations, methods, restriction enzymes and DNA probes used

Chromosomal Deer Clone used in RFLV Location species Location Locus specific Name Genbank Name specific Locus name Symbol Humanb Mouse' Cattled variants" (species) account Reference

Bone morphogenetic Bh4P3 4pl4q21 5.50 RFLVHindIII, BMP3-315 (human) M22491 WOZNEYet al. protein 3 MspI, POUII (1988) Albumin ALB" 4qllq13 5.46 Protein and SSAl (ovine) X17055 BROWNet al. RFLV (1989) HindIII, MspI Vitamin D binding GC 4q12q13 5 SYn Protein protein variation only Hardy-Zuckerman 4 rn 4pllq22 5.37 RFLV M, hckit-171 (human) M16592 YARDENet al. feline sarcoma POUII (1987) viraloncogene Insulin-like growth ZGF2" llp15.5 7.74 RFLV PmII B5 (bovine) X53553 BROWNet al. factor I1 ( 1990) Hemoglobin, beta HBB" llp15.5 7.49 RFLV MspI, G4EC3HA3 (caprine) TOWNESet al. TaqI ( 1984) Hemopexin HPX 11p15.515.4 - Protein variation only Lactate LDHA" llp15.1-pl4 7.23 Protein andLDHl2 (bovine) D90143 ISHICURO dehydrogenase A RFLV. MspI, et al. (1990) hII, Tap1 Follicle stimulating BHB" llp13 2.42 RFLV HindIII, bovFSH3l (bovine) M14853 MAURER and hormone, beta BglII, MspI, BECK (1986) polypeptide Tap1 Acid phosphatase 2, ACP2" llpll 2 RFLV Hindm CT29-8 (human) X53061 POHLMANN lysosomal et al. (1988) CD5 antigen cD5" llq13 19.08 RFLV BgZII, BCD5 (bovine) X12548 RJ et al. (1990) T@ Lowdensity LDLR 19~13.3 9.04 RFLV HindIII, LDLR-1 (bovine) M11341 HOBBSel al. lipoprotein hII (1985) receptor Complement c3 19~13.3~13.2 17.28 Protein component 3 variation only Fibroblast growth FGFl 5q31.3q33.2 18.18 RFLV BglII, JC3-5 (human) M13361' JAW et al. factor 1 (acidic) WI, TaqI (1986) Comparative loci from the list compiled by O'BRIENet al. (1993). * Data from the Human Genome Database (GDB) , March 1994. 'Data from HILLYARDet al. (1993) and NADEAUet al. (1993). Data from WOMACKet al. (1993) and MEZZELANIet al. (1994), bracketed assignments were made by interpolation from the comparative maps presented by O'BRIEN et al. (1993). 'Details of screening procedure are given in the text; the enzyme(s) in bold type were used to genotype family samples. rThe sequence is from independently isolated clone of the same gene. two or three Pere David's deer, an F, sire, and three red deer and analysed usingthe computer package MAPMAKER version each cut with six restriction endonucleases (BgZII, HindIII, 3.0 (LANDER et uZ. 1987) with data entered in the backcross MspI, PstI, AruII, TaqI). RFLVs were then examined on family format. Twepoint linkage analysis was used to identify linkage filters that contained DNA from all progeny, sires and dams between loci, and thus construct linkage groups (LOD > 3). digested with one of the six endonucleases identified from The relative likelihoods of all possible orders of loci in each the screening filters. linkage group were then compared anda maximum likelihood Linkage auaiysis All experimentalresults were indepen- map for the most likely gene order was calculated using the dently scoredby two people and any equivocal results excluded MAP function of MAPMAKER. The error detection optionwas from the analyses. Where a polymorphism was identified in red used initially with the probability for error set at 1% for each deer, alleles were named alphabetically inorder of decreasing locus. Potentiallyincorrect genotypes were checkedand in two fragment length whereas the Pcre David allele was designated cases a mistaken scoring was corrected. The analysis was then ' P'irrespective of the size. The full genotypicdata was used to repeated without using error detection. check pedigree information. For linkage analysis, genotypic RESULTS data in the backcross were simplified to a single score indicat- ing the presence (in heterozygous form) or absence of the Pedigrees: The three F, P2re David X red deer hy- Pere David allele. Data from the three sires were combined brids stags produced 58, 34, 51 living progeny respec- 1386 M. L. Tate et al. tively in the three years of matings, 43 of these were TABLE 2 from artificial inseminations and the remainder from Linkage groups (LOD > 3) identified by two-point analysis natural mating. In total, 123 of these backcross animals were available for sampling. The dams of 79 of these No. recombinants/no. calves were sampled, while the dams of the remaining Loci informative meosis. rf LOD 44 calves wereeither not individually identified in pedi- Linkage Group 1 gree records or could not be located for sampling. No HPY-HBB 0/30 0.00 9.03 twin calveswere recorded but nineof the reddeer dams HPX"-FSHB 3/31 0.10 5.05 had hybrid calves in two different years. HBB-FSHB 8/122 0.07 23.9 Molecularmarkers: Eachof the DNA probes HBB-ACP2 35/121 0.29 4.82 screened for fragment lengthvariation identified differ- FSHB-ACP2 28/122 0.22 8.72 Linkage Group 2 ences between P6re David's deer and red deer with at LDW-LDHA 0/31 0.00 9.33 least one of the six restriction enzymes used (Table 1) . LDHA-cD5 24/122 0.20 10.5 For all but the ACP2 (lysosomal acid phosphatase) and cD5-IGFZ 19/122 0.16 13.8 ZGF2 (insulin-like growth factor ZI) probes, two or more Linkage Group 3 restriction enzymes identified an RFLV. For each probe ALB"-ALB o/ 122 0.00 36.7 the restriction enzyme producing theclearest RFLV dif- ALEGC 0/123 0.00 37.0 ference between P&e David's deer and red deer with ALB-BMP3 13/122 0.11 18.8 ALB-KIT 6/123 0.05 26.6 the lowest number of additional bands was chosen for ALB-PDWRA 6/123 0.05 26.6 further study. The restriction enzyme (s) used are indi- GC-BMP3 13/122 0.11 18.8 cated in boldface type in Table 1. GGKIT 6/123 0.05 26.6 When family samples were analyzedthe validity of the GC-PDGFRA 6/ 123 0.05 26.6 species differences identified in screening was strongly BMP3-KIT 19/122 0.16 13.8 supported for all protein and DNA markers. The red BMT3-PDWRA 19/122 0.16 13.8 deer dams were monomorphic for 10 of the markers KIT-PDGFRA 0/123 0.00 37.0 Linkage Group 4 but showed polymorphism in the remaining seven loci, C3"-LDLR 7/121 0.06 24.8 namely the protein loci ALB, C3 and GC and the RFLVs C3"-FGFl 25/121 0.21 9.65 detected with FGFl, FSHB, LDHA and LDB. In each LDLK-FWl 31/121 0.26 6.52 case, the polymorphism within red deer was clearly dis- Loci genotyped using protein variation. tinct from the Pbe David variant. No genetic variation was detected in the P2re David's deer. For all markers, the F1 sires showed a heterozygous type with one allele four groups of linked loci (Table 2), namely, ACP2, being the unique Pkre David allele and the other, an FSHB, HBB and HPX; W5, IGF2 and LDHA; ALB, allele found in red deer. In each case, the backcross BM3, GC, KIT, PDGFRA; and C3,FGFl and LDLR. calves wereeither heterozygous for the P6re Davidallele Among loci from different linkage groups the highest or had only the red deer alleles. The frequency of het- LOD score was 1.1 giving little evidence of any associa- erozygotes in the calves for the PPre David allele did tion between the groups. In two cases, ALB and LDHA, not differ significantly from the expected proportion the same locus was genotyped using both protein varia- of 0.5 for any marker. The protein variants in GC, C3, tion and RFLVs. In these comparisons, no recombina- ALB and HPX wereinherited codominantly in the back- tion was detected between theprotein variants and cross. LDHA had a band ofactivity present inPPre RFLVs ( Table 2 ) providing confirmation of the identity David's deer that was not present in red deer. Hence of the locus and the segregation pattern in the back- whether an individual was homozygous or heterozygous cross pedigrees. For subsequent analyses, data from the for the LDHA' allele could only be distinguished on RFLVs were used as these had no missing data. the intensity of staining. Comparison of the likelihood of all possible orders Linkage analysis: Linkage was examined by testing of loci within each linkage group gave strong support for cosegregation between species-specific markers. forthe following orders: (HPX,HBB)-FSHBACP2, The HPX and LDH protein variants, which required LDHA-CD5-ZGF2, BMP3 ( GC, ALB)- ( KIT, PDGFRA) fresh blood samples, were genotyped in the 31 progeny and LDLR-C3-FGFl. Markers withinbrackets showed no of only one sire. The remainder of the loci were tested recombinants and therefore could not be ordered.The in all 123 available progeny and unequivocal genotypic next most likely gene orders had relative LOD scores data was obtained for either 123 or, in eight loci, 122 of -3.8, -8.0, -4.9 and -3.6 for groups 1, 2, 3 and 4, progeny. Table 2 shows the significantly linked pairs of respectively. The maximum likelihood map produced loci from pair-wise tests of all combinations of the 17 for each of these gene orders isshown in Figure 1. markers tested. The two point linkage data identified Typically each backcross individual was informative for Deer Interspecies Hybrids 1387

HBB H PX FSHB ACP FSHB HPX I I 1 -77.9 ’ 6.5cM I 23.8cM

LDHA CD5 IGFZI FIGURE1 .-Graphical representation 1 I I -86.7 of the maximumlikelihood map for 21.2cM 16.4cM eachlinkage group derivedfrom the MAPMAKER analysis. The map shows the chromosomal order and distance ALB KIT between loci in Kosambi centi-Morgans BMP3PDGFRA GC (cM). The absolute likelihood of the I I I map is given on the right. 1 I -65.5 1 O.OcM I 4.9cM

LDLR c3 FGFA 1 -75.5 5.8cM 21.8cM I 5.8cM all markers so the location of recombination events in each individual could be placed. Figure 2 summarizes all the genotypic data for each linkage group giving the number of animals with no recombination across a linkage group ( or ) or with a recombination event n=41 45 3 5 18 9 in a particular marker interval in a linkage group (a transition from to 0).This presentation of the data confirms the stated gene order, which was the only order in which no double recombination events occurred.

DISCUSSION n=40 39 11 13 8 11 The value of interspecies hybrid backcrosses for linkage mapping has been convincingly demonstrated in the mouse, a “map rich” species where interspecies backcrosses playan ongoingrole in the construction of a genetic map ( COPELANDet aZ. 1993). Similar genetic mapping resources are currently lacking in other ma- malian orders and, compared with the mouse, there are few alternative resources for mapping and ordering n= 55 48 6 7 5 1 “comparative” or Type I markers. We have identified a wide interspecies hybrid in deer, a ruminant species with no previous genetic mapping information,and successfully used it to build linkage groups and orderevolu- tionarily conserved loci. We evaluate the deer interspecies hybrids as a gene mapping resource and discuss n= 52 37 5 2 10 14 their use in a comparative approach to gene mapping FIGURE2.-Summary of the results of the linkage analysis and genetic analysis of traits in deer and other mam- in the Pere David’s deer X red deer backcross families. Each mals. column represents the chromosome inherited from the F1 Genetic divergence and fertiliq The key feature of Pere David X red deer sire in the backcross progeny. The interspecies hybrid pedigrees is that they contain useful order of markers along the chromosome derived from our genetic variation in coding regions that are typically linkage analysis is shown on the left. The presence of Pere David alleles (M) and red deer alleles (0)are indicated. The conserved within a species. The expectation, from pro- number of each type of chromosome observed inthe linkage tein data, of a wide molecular genetic divergence be- analysis is listed below. Sevenchromosomes with missing data tween Pere David’s deer and red deer( TATE et d.1992 ) points are excluded. 1388 M. L. Tate et al. was confirmed by the RFLV data. P6re David’s deer and all available parents (E = 70) and assumed that the red deer showed fixed allelic differences with each of parents not available for typing showed the absolute the 12 coding sequence gene probes chosen for study. species-specificity of alleles found in the genotyped ani- The loci were not preselected on the basis of polymor- mals. This assumption was essential to determine both phism and so we expect that virtually any singlecopy the phase of linkage and the segregation of sire alleles probe that hybridizes to deer DNA will identify a differ- in some progeny and introduces aslight possibility, first, ence between Pere David’s deer and red deer.Zooblot that the P (Pere David) allele in one FI hybrid was data including >200 cDNA probes (J. M. PENTY and actually inherited from its red deer dam and, second, H. C. MATHIAS, unpublished data) show a very large that a backcross calf whose mother was not available number of useful probes are available for deer map- for genotyping inherited a Pallele from its dam and a ping. In these data, 80% of ovine and bovine cDNA red deer allele from its Pere David sire. Clearly this is probes hybridized strongly to deer DNA at moderate very unlikely as 70 red deer and five P6re David’s deer stringency, whereas under similar conditions 60% of showedfixed differences. We calculated that, even porcine and human probes hybridized. The recent his- given an unlikely distribution of gene frequency, the tory of P6re David’sdeer may contribute to the number probability of the first possibility was <1 in 3000 per of fixed allelic differences between the species. Pere locus and the second possibility <1 in 150 per locus, David’s deer have undergone repeated founder effects per calf. Low frequency errors, such as these, would be and a bottleneck of small population size possibly last- likely to be detected by analysis of double recombinants ing >lo00 yr (JONES et al. 1983). and other procedures used to identify genotyping er- A wide evolutionary genetic divergence between two rors in the data set (LINCOLN andLANDER 1992) but species and the ability to produce fertile hybrids are no anomalous results were identified in these analysis. usually mutually exclusive. In M. spetus hybrids, which In future pedigrees, we suggest it will not be necessary show a similar level of genetic divergence to the deer to routinely genotype the red deer dam of each back- hybrids ( BONHOMMEet al. 1984) , only females, the ho- cross but it would be preferable to have the dams avail- mogametic sex, are fertile ( COPELANDet al. 1993). In able for genotyping if an unlikely distribution of geno- contrast, both male and female FI hybrids are fertile types is detected. in the Pere David’s deer X red deer (FENNESSY and Interspecies hybrids can show unusual patterns of MACKINTOSH 1992) . Female deer, typically have only inheritance thatmay affect linkage analysis. Segregation one offspring per year, so the fertility of male hybrids distortion has occasionally been reported in mouse in- is essential to the rapid production of large numbers terspecies hybrids ( SIRACUSAet al. 1989) and recombi- of backcross hybrids for linkage analysis. We found no nation suppression, caused by small chromosomal in- evidence of infertility in the three F1 males used in this versions that have accumulated between diverged study as their calving results were within the normal species, is also a concern (HAMMERet al. 1989) . The range expectedfor red deermated under similar condi- linkage analysisin deerdemonstrated stable inheri- tions. We are currently producing a further 200 fully tance and segregation of markers in the hybrid pedi- recorded backcross animals and, in addition, hybrids grees, at least for the chromosome segments covered are being produced on commercial deer farms because by the present markers. Also, recombination rates ap- they offer the potential of introducing desirable P6re peared to be compatible with map information from David’s deer traits into New Zealand’s farmed deer other species.Loci showing no recombination have herd, which includes >1,000,000 red deer and wapiti been mapped very close to each other in other species. ( C. elaphus ssp.). We know of no other large mammal For example, KIT and PDGFRA show no recombination where such large numbers of a wide hybrid have been in interspecies hybrid mouse panels ( KOSAKand STE- produced. PHENSON 1992 ) and ALB and GC are very closely linked Genetic segregationand linkage analysis: In the pres- in a wide variety of mammals ( O’BRIEN1993) . We ent deer backcross, only male meioses are examined so could not find any comparative linkage data for HPX segregation among Xchromosome markers cannot be and HBB but these loci do map to the same chromo- tested. For linkage analysis of autosomal markers with some band in humans (Table 1 ). species-specific alleles, we see no important differences Comparative gene maps: The basic tenet of compar- between the half-sib deer and the full-sib mouse inter- ative gene mapping is that the high degree of similarity species backcrosses. One practical aspect of using half- between the gene maps of mammals can be used to sib families is that there are a relatively large number unify many aspects of mammalian genetics. This has of parental animals. DODDS et al. (1993) have shown beendemonstrated repeatedly in comparisons of that in manyinstances little or no extra linkage informa- mouse and human gene maps and in the use of mouse tion is gained from genotyping the dams of large pater- models for human disease (HILL andVAN HEININGIN nal half-sib families. In the present analysis, we typed 1992) . The use of comparative maps within a mamma- Deer Interspecies Hybrids Interspecies Deer 1389 lian order such as the ruminants (towhich cattle, sheep shape) and physiological traits (eg. gestation length, and deer belong) islikely to be even more robust. seasonality and disease resistance). Hybrids show mea- Strong similarities between the genemaps of cattle and surable variation in some of these traits ( FENNESSYand deer are expected considering the close evolutionary MACKINTOSH 1992). The same features that make the relationships of the families Cervidae and Bovidae and pedigrees useful for linkage mapping, also make them the similarities in the G banding patternof P2re David’s advantageous for the genetic analysis of these complex deer and cattle chromosomes (BUCKLANDand EVANS traits (LANDERand BOTSTEIN 1989) .

1978). Our data are a good illustration of the use of We thank PETERBOWMAR and WHITLEY OTWAYand JOHN PATENE the cattle map to construct linkage groups in deer. The for providing their calving records and for assistance with sampling loci examined fell into linkage groups which are identi- the deer. We also thank SUEGALLOWAY and other staff in the AgRe- cal to their syntenic grouping in cattle (Table 1) . The search Molecular Biology Unit for advice and training in laboratory five loci not mapped in cattle (BW3, C3, CD5, HPX techniques. DNA clones were received American TypeCulture Collec- tion (CT29-8 and hckit-171), J. Abraham (JG3-5), D. BOWEN-POPE and PDGFRA) were placed into a group predictedfrom (17B), W. BROWN(SSA1, B5), H. HOBBS(LDLR-I), J. LINCREL human-bovine comparative mapping data (Table 1) . (G4EC3HA3), L. LISHER(BMP3-315), R. MAURER (bovFSH31), N. The deer linkage data, therefore, provide support for ISHIGURO(LDH12) , Q. YU (BCD5). This work was supported by a the gene assignments in cattle. With the exception of grant from the New Zealand Foundation for Research Science and FSHB and HBB, the loci examined in this study are Technology to AgResearch. either not assigned in cattle or assigned by only one LITERATURECITED method in a single study ( WOMACKet al. 1993) . Further- more even in this limited data set the new gene order ALTMANN,V. D., and H. SCHEEL,1980 Geburt, beginn des sozialver- haltens und erstes lernen beim Milu, Elaphurus davidianus. Milu information from thedeer indicates additional re- 5 146-156. arrangement within a syntenic group that is conserved ARCHBALD,A. L., S. COUPERWITEand C. S. HALEY,1991 Genetic between human and ruminantsbecause in the HSAllp mapping in meishan/large white pig families. Genet. Res. 58: 75. loci IGF2 and LDHA are separated by CD5 whichis ASHER,G. W.,J. L. ADAM, W. OTWAY, BOWMAR,P. G. VAN REENEN et al., from HSAllq (Figure 1) . 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