June Swinburne

Genome Sequence, Comparative Analysis, and Population Genetics of the Domestic Horse

A Horse Is a Horse, of Course The history of horse domestication is closely tied to the history of the human society. Wade et al. (p. 865) report on the sequencing and provide a single nucleotide polymorphism map of the horse (Equus caballus) genome. Horses are a member of the order perissodactyla (odd-toed animals with hooves). The analysis reveals an evolutionarily new centromere on equine chromosome 11 that displays properties of an immature but fully functioning centromere and is devoid of centromeric satellite sequence. The findings clarify the nature of genetic diversity within and across horse breeds and suggest that the horse was domesticated from a relatively large number of females, but few males. The horse genome reveals an evolutionary new centromere and conserved chromosomal sequences relative to other mammals. We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

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.

Limited number of patrilines in horse domestication

Genetic studies using mitochondrial DNA (mtDNA) have identified extensive matrilinear diversity among domestic horses. Here, we show that this high degree of polymorphism is not matched by a corresponding patrilinear diversity of the male-specific Y chromosome. In fact, a screening for single-nucleotide polymorphisms (SNPs) in 14.3 kb of noncoding Y chromosome sequence among 52 male horses of 15 different breeds did not identify a single segregation site. These observations are consistent with a strong sex-bias in the domestication process, with few stallions contributing genetically to the domestic horse.

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.

International equine gene mapping workshop report: a comprehensive linkage map constructed with data from new markers and by merging four mapping resources

A comprehensive male linkage map was generated by adding 359 new, informative microsatellites to the International Equine Gene Map half-sibling reference families and by combining genotype data from three independent mapping resources: a full sibling family created at the Animal Health Trust in Newmarket, United Kingdom, eight half-sibling families from Sweden and two half-sibling families from the University of California, Davis. Because the combined data were derived primarily from half-sibling families, only autosomal markers were analyzed. The map was constructed from a total of 766 markers distributed on the 31 equine chromosomes. It has a higher marker density than that of previously reported maps, with 626 markers linearly ordered and 140 other markers assigned to a chromosomal region. Fifty-nine markers (7%) failed to meet the criteria for statistical evidence of linkage and remain unassigned. The map spans 3,740 cM with an average distance of 6.3 cM between markers. Fifty-five percent of the intervals are ≤5 cM and only 3% ≧20 cM. The present map demonstrates the cohesiveness of the different data sets and provides a single resource for genome scan analyses and integration with the radiation hybrid map.

Linkage disequilibrium and historical effective population size in the Thoroughbred horse

Many genomic methodologies rely on the presence and extent of linkage disequilibrium (LD) between markers and genetic variants underlying traits of interest, but the extent of LD in the horse has yet to be comprehensively characterized. In this study, we evaluate the extent and decay of LD in a sample of 817 Thoroughbreds. Horses were genotyped for over 50,000 single nucleotide polymorphism (SNP) markers across the genome, with 34,848 autosomal SNPs used in the final analysis. Linkage disequilibrium, as measured by the squared correlation coefficient (r(2)), was found to be relatively high between closely linked markers (>0.6 at 5 kb) and to extend over long distances, with average r(2) maintained above non-syntenic levels for single nucleotide polymorphisms (SNPs) up to 20 Mb apart. Using formulae which relate expected LD to effective population size (N(e)), and assuming a constant actual population size, N(e) was estimated to be 100 in our population. Values of historical N(e), calculated assuming linear population growth, suggested a decrease in N(e) since the distant past, reaching a minimum twenty generations ago, followed by a subsequent increase until the present time. The qualitative trends observed in N(e) can be rationalized by current knowledge of the history of the Thoroughbred breed, and inbreeding statistics obtained from published pedigree analyses are in agreement with observed values of N(e). Given the high LD observed and the small estimated N(e), genomic methodologies such as genomic selection could feasibly be applied to this population using the existing SNP marker set.

A region on equine chromosome 13 is linked to recurrent airway obstruction in horses

UNLABELLED REASONS FOR STUDY: Equine recurrent airway obstruction (RAO) is probably dependent on a complex interaction of genetic and environmental factors and shares many characteristic features with human asthma. Interleukin 4 receptor a chain (IL4RA) is a candidate gene because of its role in the development of human asthma, confirmation of this association is therefore required. METHODS The equine BAC clone containing the IL4RA gene was localised to ECA13q13 by the FISH method. Microsatellite markers in this region were investigated for possible association and linkage with RAO in 2 large Warmblood halfsib families. Based on a history of clinical signs (coughing, nasal discharge, abnormal breathing and poor performance), horses were classified in a horse owner assessed respiratory signs index (HOARSI 1-4: from healthy, mild, moderate to severe signs). Four microsatellite markers (AHT133, LEX041, VHL47, ASB037) were analysed in the offspring of Sire 1 (48 unaffected HOARSI 1 vs. 59 affected HOARSI 2-4) and Sire 2 (35 HOARSI 1 vs. 50 HOARSI 2-4), age 07 years. RESULTS For both sires haplotypes could be established in the order AHT133-LEXO47-VHL47-ASB37. The distances in this order were estimated to be 2.9, 0.9 and 2.3 centiMorgans, respectively. Haplotype association with mild to severe clinical signs of chronic lower airway disease (HOARSI 2-4) was significant in the offspring of Sire 1 (P = 0.026) but not significant for the offspring of Sire 2 (P = 0.32). Linkage analysis showed the ECA13q13 region containing IL4RA to be linked to equine chronic lower airway disease in one family (P<0.01), but not in the second family. CONCLUSIONS This supports a genetic background for equine RAO and indicates that IL4RA is a candidate gene with possible locus heterogeneity for this disease. POTENTIAL RELEVANCE Identification of major genes for RAO may provide a basis for breeding and individual prevention for this important disease.

Mixed inheritance of equine recurrent airway obstruction.

BACKGROUND Mode of inheritance of equine recurrent airway obstruction (RAO) is unknown. HYPOTHESIS Major genes are responsible for RAO. ANIMALS Direct offspring of 2 RAO-affected Warmblood stallions (n = 197; n = 163) and a representative sample of Swiss Warmbloods (n = 401). METHODS One environmental and 4 genetic models (general, mixed inheritance, major gene, and polygene) were tested for Horse Owner Assessed Respiratory Signs Index (1-4, unaffected to severely affected) by segregation analyses of the 2 half-sib sire families, both combined and separately, using prevalences estimated in a representative sample. RESULTS In all data sets the mixed inheritance model was most likely to explain the pattern of inheritance. In all 3 datasets the mixed inheritance model did not differ significantly from the general model (P= .62, P= 1.00, and P= .27) but was always better than the major gene model (P < .01) and the polygene model (P < .01). The frequency of the deleterious allele differed considerably between the 2 sire families (P= .23 and P= .06). In both sire families the displacement was large (t= 17.52 and t= 12.24) and the heritability extremely large (h(2)= 1). CONCLUSIONS AND CLINICAL RELEVANCE Segregation analyses clearly reveal the presence of a major gene playing a role in RAO. In 1 family, the mode of inheritance was autosomal dominant, whereas in the other family it was autosomal recessive. Although the expression of RAO is influenced by exposure to hay, these findings suggest a strong, complex genetic background for RAO.

A horse whole-genome-radiation hybrid panel: chromosome 1 and 10 preliminary maps.

In recent years there has been increasing interest in mapping thehorse genome, particularly to identify disease and performance-enhancing genes. Although a number of horse mapping tools havebeen developed and have proved very useful (genetic maps, so-matic cell hybrid panels, and a BAC library), the benefits ofwhole-genome–radiation hybrid (WG-RH) mapping have not beenavailable. Its advantages over genetic linkage mapping are: (i) theresolving power is not lost in regions of the genome with a lowrecombination rate, because it does not rely on meiotic recombi-nation events; (ii) it is especially useful in animals such as thehorse with relatively long generation times and single births; and(iii) genetic and physical maps can be integrated, as both poly-morphic and non-polymorphic markers can be placed on the samemap. The WG-RH panel constructed in this study is the first suchpanel to be reported for the horse, and its preliminary character-ization demonstrates its usefulness for horse genome mapping.The panel was constructed by the fusion of horse embryonicendothelial primary lung cells (male) to the established hamsterfibroblast cell line A23 (Westerveld et al. 1971) by using themethod described in McCarthy et al. (1997). A series of fusions,involving irradiation (3000 rads) of donor cells prior to fusion withequal numbers of recipient cells, generated ∼160 hybrids in total.From these 160 hybrids, 94 were selected at random and screenedby using 20 widely distributed markers (representing 17 chromo-somes). Any hybrids that did not produce an amplification productwith these markers were screened by FISH to determine whetherthey contained horse DNA from other chromosomes or regions ofchromosomes (not represented by the 20 markers); hybrids foundto be negative by the FISH screen were replaced at random fromthe remaining unused hybrids, and the same PCR and FISH screen-ing procedure was repeated until 94 hybrids were assembled.These 94 hybrids were used for preliminary characterization as amapping panel. It is expected that the majority of the hybrids(>95%) characterized here will be represented in the TM99 panelof 94 hybrids that is being grown on a large scale by ResearchGenetics Inc. (Huntsville, Ala. 35801).The amount of horse DNA retained by the hybrids in the panelwas evaluated by examining the retention of the 20 widely dis-tributed horse markers. On average each marker was retained in27.8% of the hybrids (ranging from 10.6% to 71.3%; Table 1),implying that the panel as a whole retains the equivalent of ap-proximately 26 horse genomes. These retention frequencies com-pare well with those found for the human and mouse RH panels,which have been used successfully for creating whole-genomemaps (Gyapay et al. 1996; McCarthy et al. 1997).The mapping ability of the panel was assessed by producingRH maps for the two horse chromosomes with the most markersavailable, and comparing these with the latest genetic maps (Swin-burne et al. 2000a). In total, 39 markers on Chromosome (Chr) 1and 15 markers on Chr 10 were analyzed (Table 1). The averageretention of markers was 15.4% (ranging from 5.3% to 29.8%) onChr 1, and was higher, 25.4% (ranging from 16.0% to 44.7%) onChr 10. An increase in the retention frequency on smaller chro-mosomes was also noted in human and mouse RH panels (Gyapayet al. 1996; McCarthy et al. 1997). For each chromosome, linkagegroups with at least 4-LOD units support were identified; four suchgroups were found on Chr 1 and two were detected on Chr 10(Figs. 1 and 2). Within each of these linkage groups, frameworkmarkers were ordered with 3-LOD units support, and most of thenon-framework markers were ordered with 2-LOD units support.Some non-framework markers had lower statistical support fortheir order (shown in italics, Figs. 1 and 2). Similarly, the relativeorder of some linkage groups was suggested by linkage analysisbut had low statistical support [Chr 1 (groups B and C) and Chr 10(groups A and B)]. The relative order of the other RH linkagegroups was determined by comparison with the genetic map andFISH localizations.The genetic map can give a misleading impression of markerdensity because genetic distances may be small owing to regionshaving a low meiotic recombination rate. In such regions the mark-ers may actually be physically far apart and, therefore, at a lowerdensity than predicted by the genetic map. Since RH panels requirea high density of markers, this might account for the low statisticalsupport for the ordering of some markers and linkage groups. Alow density of available markers could also explain why two mark-ers (HLM5, ICA22) could not be placed on the RH map. HLM5 hasalready been shown to be 24 cM from its nearest neighboringmarker on the genetic map. Three other markers could not beplaced on the RH map (UM004, HTG12, and HMS15), but wereable to be placed on the genetic map in a region equivalent to RHlinkage group D; this discrepancy between the two maps should beresolved as both maps are characterized further.This study has demonstrated the ability of the horse WG-RHpanel to produce an accurate genome map as there is good agree-ment of the genetic and physical maps with the RH maps for Chrs1 and 10 (Figs. 1 and 2). Only a few differences between the mapswere observed, and experiences with human mapping suggest thatsuch differences are common and are resolved as more markers areincorporated into the maps (Walter et al. 1994). The WG-RH panelwas able to order some markers that co-segregate on the geneticlinkage map, i.e., Chr 1 markers LEX39 and ICA18, and ICA41 andICA32, and Chr 10 markers LEX8 and COR015, and NVHEQ18