E. Bailey

A high density SNP array for the domestic horse and extant Perissodactyla: Utility for association mapping, genetic diversity, and phylogeny studies

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs within breeds ranged from 43,287 to 52,085. Genome-wide linkage disequilibrium (LD) in most breeds declined rapidly over the first 50–100 kb and reached background levels within 1–2 Mb. The extent of LD and the level of inbreeding were highest in the Thoroughbred and lowest in the Mongolian and Quarter Horse. Multidimensional scaling (MDS) analyses demonstrated the tight grouping of individuals within most breeds, close proximity of related breeds, and less tight grouping in admixed breeds. The close relationship between the Przewalskis Horse and the domestic horse was demonstrated by pair-wise genetic distance and MDS. Genotyping of other Perissodactyla (zebras, asses, tapirs, and rhinoceros) was variably successful, with call rates and the number of polymorphic loci varying across taxa. Parsimony analysis placed the modern horse as sister taxa to Equus przewalski. The utility of the SNP array in genome-wide association was confirmed by mapping the known recessive chestnut coat color locus (MC1R) and defining a conserved haplotype of ∼750 kb across all breeds. These results demonstrate the high quality of this SNP genotyping resource, its usefulness in diverse genome analyses of the horse, and potential use in related species.

Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

Genetic Diversity in the Modern Horse Illustrated from Genome-Wide SNP Data

Horses were domesticated from the Eurasian steppes 5,000–6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. FST calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection.

Mapping of 31 horse genes in BACs by FISH

An INRA equine genomic BAC library was screened by PCR using 24 primer sets developed for the 30UTR of EST clones from a 60-day horse embryo cDNA library [Brandon R., personal communication], 6 CATS primer sets [1] and 1 UM-STS primer set [2]. Clone identity was con¢rmed by cycle sequencing on an Applied Biosystems Prism Genetic Analyzer. Sequences were compared by BLAST searches to sequences in GenBank. Metaphase chromosome preparation and FISH were performed as previously described [3]. Information on the clones including INRA clone ID number, gene symbol, gene name, horse chromosome map position, clone GenBank accession number and the human gene homolog chromosome map position identi¢ed in the OMIM or NCBI databases is presented in Table 1. The locations of the thirty-one horse genes shown in Table 1 are consistent with human^horse homologies as predicted by ZOO^FISH and synteny mapping studies [4,5]. The position of TCRG on ECA4p de¢nes a new homology with HSA7p. The information in this report increases the resolution of the human^horse comparative gene map.

Horse alpha-1-antitrypsin, beta-lactoglobulins 1 and 2, and transferrin map to positions 24q15-q16, 28q18-qter, 28q18-qter and 16q23, respectively.

An equine genomic library in Lambda FIX II (Stratagene) was screened with DNA and PCR probes for alpha-1anti-trypsin (AAT), beta-lactoglobulins 1 and 2 (BLG1 and BLG2), and transferrin (TF) using random-primed P-labeled DNA, and clones isolated. AAT is a member of the protease inhibitor superfamily and a highly polymorphic glycoprotein. In the proposed classi®cation system for the equine AATs, the lambda clone used in ̄uorescence in situ hybridisation (FISH) experiments is Spi 2 [1]. BLG1 and BLG2 are polymorphic milk proteins. BLG clone identities were con®rmed by comparison with cDNA sequences. TF is a glycoprotein that carries iron to the cells of the body. The identity of the TF clone was con®rmed by direct sequencing of upstream sequence and exons 13, 15 and 16 and comparison with published cDNA horse sequence [2]. Metaphase chromosome preparation and FISH were done as previously described [3], using the horse clones for AAT, BLG1, BLG2, TF. Details of the probes and the results of the FISH mapping are listed in Table 1. Map positions are consistent with the Zoo-FISH map of Raudsepp et al. [4]. Placement of the equine Spi2 (AAT) con®rms the report of Goddard et al. [5].

Horse v-fes feline sarcoma viral oncogene homologue; pyruvate kinase, muscle type 2; plasminogen; beta spectrin, non-erythrocytic 1; thymidylate synthetase; and microsatellite LEX078 map to 1q14-q15, 1q21, 31q12-q14, 15q22, 8q12-q14, and 14q27, respectively.

An equine genomic BAC library was screened by PCR using universal sequence-tag site primers [1] speci¢c for the genes v-fes feline sarcoma viral oncogene homologue (FES); pyruvate kinase, muscle type 2 (PKM2); plasminogen (PLG); spectrin, beta, non-erythrocytic 1 (SPTBN1); thymidylate synthetase (TYMS); and primers for the horse microsatellite LEX078 [2]. FES may be involved in the initiation of cancer following chromosome translocation. PKM2, also known as ATP:pyruvate phosphotransferase, is a glycolytic enzyme involved in the formation of pyruvate and ATP. PLG is a zymogen in circulating blood from which plasmin is formed. TYMS catalyses the transfer of methyl groups during DNA synthesis. LEX078 is a polymorphic horse microsatellite that did not demonstrate linkage in the reference families of the Equine Gene Mapping Workshop [3]. Clone identity was con¢rmed by cycle sequencing (ABI/P-E Applied Biosystems) and sequences compared by BLAST searches or to published horse sequences. Metaphase chromosome preparation and FISH were done as previously described [4] using the horse clones for FES, PKM2, PLG, SPTBN1, TYMS and LEX078. The results of FISH mapping are listed in Table 1. Map positions for FES, PKM2, SPTBN1 and TYMS are consistent with the Zoo-FISH map [5]. Zoo-FISH with human painting probes did not demonstrate homology to horse chromosome 31; however, the map position for PLG agrees with its placement by synteny mapping [6].

Multiple alleles of ACAN associated with chondrodysplastic dwarfism in Miniature horses

Chondrodysplastic dwarfism in Miniature horses appeared to be a recessive genetic trait based on the occurrence of affected offspring by normal parents. Dwarf phenotypes vary and range from abnormal abortuses to viable offspring with evidence of skeletal dysplasia. A genome-wide association study implicated a region of ECA1 with dwarfism in Miniature horses. Aggrecan (ACAN) was a candidate gene in that region, and exons were sequenced to compare DNA sequences for dwarf and non-dwarf horses. Sequencing led to the discovery of variants in exons 2, 6, 7 and 15 associated with dwarfism. The four variants are identified with reference to Ecab 3.0 (GCF_002863925.1) as g.95291270del (rs1095048841), g.95284530C>T (ERP107353), g.95282140C>G (rs1095048823) and g.95257480_95257500del (rs1095048839) and designated here as D1, D2, D3* and D4 respectively. A previous study at another laboratory reported dwarfism associated with homozygosity for D3*. Homozygotes for those variants and compound heterozygotes for any combination of those variants always expressed a dwarfism phenotype. However, eight additional horses with dwarfism were found, seven of which were heterozygotes for D2, D3* or D4, suggesting the existence of additional ACAN alleles causing dwarfism. Among Miniature horses, the combined frequency of D1, D2, D3* and D4 was 0.163, suggesting a carrier rate of 26.2% for alleles causing chondrodysplastic dwarfism.