An endothelial regulatory module links blood pressure regulation with elite athletic performance.

The control of transcription is crucial for homeostasis in mammals. A previous selective sweep analysis of horse racing performance revealed a 19.6 kb candidate regulatory region 50 kb downstream of the Endothelin3 (EDN3) gene. Here, the region was narrowed to a 5.5 kb span of 14 SNVs, with elite and sub-elite haplotypes analyzed for association to racing performance, blood pressure and plasma levels of EDN3 in Coldblooded trotters and Standardbreds. Comparative analysis of human HiCap data identified the span as an enhancer cluster active in endothelial cells, interacting with genes relevant to blood pressure regulation. Coldblooded trotters with the sub-elite haplotype had significantly higher blood pressure compared to horses with the elite performing haplotype during exercise. Alleles within the elite haplotype were part of the standing variation in pre-domestication horses, and have risen in frequency during the era of breed development and selection. These results advance our understanding of the molecular genetics of athletic performance and vascular traits in both horses and humans.

Genetic diversity and signatures of selection in four indigenous horse breeds of Iran.

Indigenous Iranian horse breeds were evolutionarily affected by natural and artificial selection in distinct phylogeographic clades, which shaped their genomes in several unique ways. The aims of this study were to evaluate the genetic diversity and genomewide selection signatures in four indigenous Iranian horse breeds. We evaluated 169 horses from Caspian (n = 21), Turkmen (n = 29), Kurdish (n = 67), and Persian Arabian (n = 52) populations, using genomewide genotyping data. The contemporary effective population sizes were 59, 98, 102, and 113 for Turkmen, Caspian, Persian Arabian, and Kurdish breeds, respectively. By analysis of the population genetic structure, we classified the north breeds (Caspian and Turkmen) and west/southwest breeds (Persian Arabian and Kurdish) into two phylogeographic clades reflecting their geographic origin. Using the de-correlated composite of multiple selection signal statistics based on pairwise comparisons, we detected a different number of significant SNPs under putative selection from 13 to 28 for the six pairwise comparisons (FDR < 0.05). The identified SNPs under putative selection coincided with genes previously associated with known QTLs for morphological, adaptation, and fitness traits. Our results showed HMGA2 and LLPH as strong candidate genes for height variation between Caspian horses with a small size and the other studied breeds with a medium size. Using the results of studies on human height retrieved from the GWAS catalog, we suggested 38 new putative candidate genes under selection. These results provide a genomewide map of selection signatures in the studied breeds, which represent valuable information for formulating genetic conservation and improved breeding strategies for the breeds.

The genetics of gaits in Icelandic horses goes beyond DMRT3, with RELN and STAU2 identified as two new candidate genes.

BACKGROUND: In domesticated animals, many important traits are complex and regulated by a large number of genes, genetic interactions, and environmental influences. The ability of Icelandic horses to perform the gait 'pace' is largely influenced by a single mutation in the DMRT3 gene, but genetic modifiers likely exist. The aim of this study was to identify novel genetic factors that influence pacing ability and quality of the gait through a genome-wide association study (GWAS) and correlate new findings to previously identified quantitative trait loci (QTL) and mutations. RESULTS: Three hundred and seventy-two Icelandic horses were genotyped with the 670 K+ Axiom Equine Genotyping Array, of which 362 had gait scores from breeding field tests. A GWAS revealed several SNPs on Equus caballus chromosomes (ECA) 4, 9, and 20 that were associated (p < 1.0 × 10-5) with the breeding field test score for pace. The two novel QTL on ECA4 and 9 were located within the RELN and STAU2 genes, respectively, which have previously been associated with locomotor behavior in mice. Haplotypes were identified and the most frequent one for each of these two QTL had a large favorable effect on pace score. The second most frequent haplotype for the RELN gene was positively correlated with scores for tölt, trot, gallop, and canter. Similarly, the second most frequent haplotype for the STAU2 gene had favorable effects on scores for trot and gallop. Different genotype ratios of the haplotypes in the RELN and STAU2 genes were also observed in groups of horses with different levels of pacing ability. Furthermore, interactions (p < 0.05) were detected for the QTL in the RELN and STAU2 genes with the DMRT3 gene. The novel QTL on ECA4, 9, and 20, along with the effects of the DMRT3 variant, were estimated to account jointly for 27.4% of the phenotypic variance of the gait pace. CONCLUSIONS: Our findings provide valuable information about the genetic architecture of pace beyond the contribution of the DMRT3 gene and indicate genetic interactions that contribute to the complexity of this trait. Further investigation is needed to fully understand the underlying genetic factors and interactions.

The Enrichment of Specific Hair Follicle-Associated Cell Populations in Plucked Hairs Offers an Opportunity to Study Gene Expression Underlying Hair Traits.

Gene expression differences can assist in characterizing important underlying genetic mechanisms between different phenotypic traits. However, when population-dense tissues are studied, the signals from scarce populations are diluted. Therefore, appropriately choosing a sample collection method that enriches a particular type of effector cells might yield more specific results. To address this issue, we performed a polyA-selected RNA-seq experiment of domestic horse (Equus ferus caballus) plucked-hair samples and skin biopsies. Then, we layered the horse gene abundance results against cell type-specific marker genes generated from a scRNA-seq supported with spatial mapping of laboratory mouse (Mus musculus) skin to identify the captured populations. The hair-plucking and skin-biopsy sample-collection methods yielded comparable quality and quantity of RNA-seq results. Keratin-related genes, such as KRT84 and KRT75, were among the genes that showed higher abundance in plucked hairs, while genes involved in cellular processes and enzymatic activities, such as MGST1, had higher abundance in skin biopsies. We found an enrichment of hair-follicle keratinocytes in plucked hairs, but detected an enrichment of other populations, including epidermis keratinocytes, in skin biopsies. In mammalian models, biopsies are often the method of choice for a plethora of gene expression studies and to our knowledge, this is a novel study that compares the cell-type enrichment between the non-invasive hair-plucking and the invasive skin-biopsy sample-collection methods. Here, we show that the non-invasive and ethically uncontroversial plucked-hair method is recommended depending on the research question. In conclusion, our study will allow downstream -omics approaches to better understand integumentary conditions in both health and disease in horses as well as other mammals.

A QTL for conformation of back and croup influences lateral gait quality in Icelandic horses.

BACKGROUND: The back plays a vital role in horse locomotion, where the spine functions as a spring during the stride cycle. A complex interaction between the spine and the muscles of the back contribute to locomotion soundness, gait ability, and performance of riding and racehorses. Conformation is commonly used to select horses for breeding and performance in multiple horse breeds, where the back and croup conformation plays a significant role. The conformation of back and croup plays an important role on riding ability in Icelandic horses. However, the genes behind this trait are still unknown. Therefore, the aim of this study was to identify genomic regions associated with conformation of back and croup in Icelandic horses and to investigate their effects on riding ability. One hundred seventy-seven assessed Icelandic horses were included in the study. A genome-wide association analysis was performed using the 670 K+ Axiom Equine Genotyping Array, and the effects of different haplotypes in the top associated region were estimated for riding ability and additional conformation traits assessed during breeding field tests. RESULTS: A suggestive quantitative trait loci (QTL) for the score of back and croup was detected on Equus caballus (ECA) 22 (p-value = 2.67 × 10- 7). Haplotype analysis revealed two opposite haplotypes, which resulted in higher and lower scores of the back and croup, respectively (p-value < 0.001). Horses with the favorable haplotype were more inclined to have a well-balanced backline with an uphill conformation and had, on average, higher scores for the lateral gaits tölt (p-value = 0.02) and pace (p-value = 0.004). This genomic region harbors three genes: C20orf85, ANKRD60 and LOC100056167. ANKRD60 is associated with body height in humans. C20orf85 and ANKRD60 are potentially linked to adolescent idiopathic scoliosis in humans. CONCLUSIONS: Our results show that the detected QTL for conformation of back and croup is of importance for quality of lateral gaits in Icelandic horses. These findings could result in a genetic test to aid in the selection of breeding horses, thus they are of major interest for horse breeders. The results may also offer a gateway to comparative functional genomics by potentially linking both motor laterality and back inclination in horses with scoliosis in humans.

Equine vitiligo-like depigmentation in grey horses is related to genes involved in immune response and tumor metastasis.

BACKGROUND: In horses, the autoimmune disease vitiligo is characterized by the loss of melanocytes and results in patchy depigmentation of the skin around the eyes, muzzle and the perianal region. Vitiligo-like depigmentation occurs predominantly in horses displaying the grey coat colour and is observed at a prevalence level of 26.0-67.0% in grey horses compared with only 0.8-3.5% in non-grey horses. While the polygenetic background of this complex disease is well documented in humans, the underlying candidate genes for this skin disorder in horses remain unknown. In this study we aim to perform a genome-wide association study (GWAS) for identifying putative candidate loci for vitiligo-like depigmentation in horses. METHODS: In the current study, we performed a GWAS analysis using high-density 670 k single nucleotide polymorphism (SNP) data from 152 Lipizzan and 104 Noriker horses, which were phenotyped for vitiligo-like depigmentation by visual inspection. After quality control 376,219 SNPs remained for analyses, the genome-wide Bonferroni corrected significance level was p < 1.33e-7. RESULTS: We identified seven candidate genes on four chromosomes (ECA1, ECA13, ECA17, ECA20) putatively involved in vitiligo pathogenesis in grey horses. The highlighted genes PHF11, SETDB2, CARHSP1 and LITAFD, are associated with the innate immune system, while the genes RCBTB1, LITAFD, NUBPL, PTP4A1, play a role in tumor suppression and metastasis. The antagonistic pathogenesis of vitiligo in relation to cancer specific enhanced cell motility and/or metastasis on typical melanoma predilection sites underlines a plausible involvement of RCBTB1, LITAFD, NUBPL, and PTP4A1. CONCLUSIONS: The proposed candidate genes for equine vitiligo-like depigmentation, indicate an antagonistic relation between vitiligo and tumor metastasis in a horse population with higher incidence of melanoma. Further replication and expression studies should lead to a better understanding of this skin disorder in horses.

Genome-wide association study for insect bite hypersensitivity susceptibility in horses revealed novel associated loci on chromosome 1.

Equine insect bite hypersensitivity (IBH) is a pruritic skin allergy caused primarily by biting midges, Culicoides spp. IBH susceptibility has polygenic inheritance and occurs at high frequencies in several horse breeds worldwide, causing increased costs and reduced welfare of affected horses. The aim of this study was to identify and validate single nucleotide polymorphisms (SNPs) associated with equine IBH susceptibility. After quality control, 33,523 SNPs were included in a Bayesian genome-wide association study on 177 affected and 178 unaffected Icelandic horses. We report associated regions in E. caballus (ECA) 1, 3, 15 and 18, overlapping with known IBH QTLs in horses, and novel regions containing several genes, together explaining 11.46% of the total genetic variance. For validation, three SNPs on ECA 1 and ECA X (explaining the largest percentage of genetic variance) within 1-mb genomic windows for IBH were genotyped in an independent population of 280 Exmoor ponies. The associated genomic region (152-153 mb) on ECA 1 was confirmed in Exmoor ponies and contains the AQR gene involved in splicing processes and a long non-coding RNA. This study confirms the polygenic nature of IBH susceptibility and suggests a role of transcriptional regulatory mechanisms (e.g., alternative splicing) for IBH predisposition in these horse breeds.

Genetics of Skin Disease in Horses.

Equine skin diseases are common, causing increased costs and reduced welfare of affected horses.Genetic testing, if available, can complement early detection, disease diagnosis, and clinical treatment and offers horse breeders the possibility to rule out carrier status. The mechanisms of complex disease can be investigated by using the latest state-of-the-art genomic technologies. Genome-based strategies may also serve as an efficient and cost-effective strategy for the management of the disease severity levels, with particular interest in complex traits such as insect bite hypersensitivity, chronic progressive lymphedema, and melanoma.

Signatures of selection in the genome of Swedish warmblood horses selected for sport performance.

BACKGROUND: A growing demand for improved physical skills and mental attitude in modern sport horses has led to strong selection for performance in many warmblood studbooks. The aim of this study was to detect genomic regions with low diversity, and therefore potentially under selection, in Swedish Warmblood horses (SWB) by analysing high-density SNP data. To investigate if such signatures could be the result of selection for equestrian sport performance, we compared our SWB SNP data with those from Exmoor ponies, a horse breed not selected for sport performance traits. RESULTS: The genomic scan for homozygous regions identified long runs of homozygosity (ROH) shared by more than 85% of the genotyped SWB individuals. Such ROH were located on ECA4, ECA6, ECA7, ECA10 and ECA17. Long ROH were instead distributed evenly across the genome of Exmoor ponies in 77% of the chromosomes. Two population differentiation tests (FST and XP-EHH) revealed signatures of selection on ECA1, ECA4, and ECA6 in SWB horses. CONCLUSIONS: Genes related to behaviour, physical abilities and fertility, appear to be targets of selection in the SWB breed. This study provides a genome-wide map of selection signatures in SWB horses, and ground for further functional studies to unravel the biological mechanisms behind complex traits in horses.

Exploring the genetics of trotting racing ability in horses using a unique Nordic horse model.

BACKGROUND: Horses have been strongly selected for speed, strength, and endurance-exercise traits since the onset of domestication. As a result, highly specialized horse breeds have developed with many modern horse breeds often representing closed populations with high phenotypic and genetic uniformity. However, a great deal of variation still exists between breeds, making the horse particularly well suited for genetic studies of athleticism. To identify genomic regions associated with athleticism as it pertains to trotting racing ability in the horse, the current study applies a pooled sequence analysis approach using a unique Nordic horse model. RESULTS: Pooled sequence data from three Nordic horse populations were used for FST analysis. After strict filtering, FST analysis yielded 580 differentiated regions for trotting racing ability. Candidate regions on equine chromosomes 7 and 11 contained the largest number of SNPs (n = 214 and 147, respectively). GO analyses identified multiple genes related to intelligence, energy metabolism, and skeletal development as potential candidate genes. However, only one candidate region for trotting racing ability overlapped a known racing ability QTL. CONCLUSIONS: Not unexpected for genomic investigations of complex traits, the current study identified hundreds of candidate regions contributing to trotting racing ability in the horse. Likely resulting from the cumulative effects of many variants across the genome, racing ability continues to demonstrate its polygenic nature with candidate regions implicating genes influencing both musculature and neurological development.

Inter- and intra-breed genome-wide copy number diversity in a large cohort of European equine breeds.

BACKGROUND: Copy Number Variation (CNV) is a common form of genetic variation underlying animal evolution and phenotypic diversity across a wide range of species. In the mammalian genome, high frequency of CNV differentiation between breeds may be candidates for population-specific selection. However, CNV differentiation, selection and its population genetics have been poorly explored in horses. RESULTS: We investigated the patterns, population variation and gene annotation of CNV using the Axiom® Equine Genotyping Array (670,796 SNPs) from a large cohort of individuals (N = 1755) belonging to eight European horse breeds, varying from draught horses to several warmblood populations. After quality control, 152,640 SNP CNVs (individual markers), 18,800 segment CNVs (consecutive SNP CNVs of same gain/loss state or both) and 939 CNV regions (CNVRs; overlapping segment CNVs by at least 1 bp) compared to the average signal of the reference (Belgian draught horse) were identified. Our analyses showed that Equus caballus chromosome 12 (ECA12) was the most enriched in segment CNV gains and losses (~ 3% average proportion of the genome covered), but the highest number of segment CNVs were detected on ECA1 and ECA20 (regardless of size). The Friesian horses showed private SNP CNV gains (> 20% of the samples) on ECA1 and Exmoor ponies displayed private SNP CNV losses on ECA25 (> 20% of the samples). The Warmblood cluster showed private SNP CNV gains located in ECA9 and Draught cluster showed private SNP CNV losses located in ECA7. The length of the CNVRs ranged from 1 kb to 21.3 Mb. A total of 10,612 genes were annotated within the CNVRs. The PANTHER annotation of these genes showed significantly under- and overrepresented gene ontology biological terms related to cellular processes and immunity (Bonferroni P-value < 0.05). We identified 80 CNVRs overlapping with known QTL for fertility, coat colour, conformation and temperament. We also report 67 novel CNVRs. CONCLUSIONS: This work revealed that CNV patterns, in the genome of some European horse breeds, occurred in specific genomic regions. The results provide support to the hypothesis that high frequency private CNVs residing in genes may potentially be responsible for the diverse phenotypes seen between horse breeds.

Genomic measures of inbreeding in the Norwegian-Swedish Coldblooded Trotter and their associations with known QTL for reproduction and health traits.

BACKGROUND: Since the 1950s, the Norwegian-Swedish Coldblooded trotter (NSCT) has been intensively selected for harness racing performance. As a result, the racing performance of the NSCT has improved remarkably; however, this improved racing performance has also been accompanied by a gradual increase in inbreeding level. Inbreeding in NSCT has historically been monitored by using traditional methods that are based on pedigree analysis, but with recent advancements in genomics, the NSCT industry has shown interest in adopting molecular approaches for the selection and maintenance of this breed. Consequently, the aims of the current study were to estimate genomic-based inbreeding coefficients, i.e. the proportion of runs of homozygosity (ROH), for a sample of NSCT individuals using high-density genotyping array data, and subsequently to compare the resulting rate of genomic-based F (FROH) to that of pedigree-based F (FPED) coefficients within the breed. RESULTS: A total of 566 raced NSCT were available for analyses. Average FROH ranged from 1.78 to 13.95%. Correlations between FROH and FPED were significant (P < 0.001) and ranged from 0.27 to 0.56, with FPED and FROH from 2000 to 2009 increasing by 1.48 and 3.15%, respectively. Comparisons of ROH between individuals yielded 1403 regions that were present in at least 95% of the sampled horses. The average percentage of a single chromosome covered in ROH ranged from 9.84 to 18.82% with chromosome 31 and 18 showing, respectively, the largest and smallest amount of homozygosity. CONCLUSIONS: Genomic inbreeding coefficients were higher than pedigree inbreeding coefficients with both methods showing a gradual increase in inbreeding level in the NSCT breed between 2000 and 2009. Opportunities exist for the NSCT industry to develop programs that provide breeders with easily interpretable feedback on regions of the genome that are suboptimal from the perspective of genetic merit or that are sensitive to inbreeding within the population. The use of molecular data to identify genomic regions that may contribute to inbreeding depression in the NSCT will likely prove to be a valuable tool for the preservation of its genetic diversity in the long term.

Genome data uncover four synergistic key regulators for extremely small body size in horses.

BACKGROUND: Miniature size in horses represents an extreme reduction of withers height that originated after domestication. In some breeds, it is a highly desired trait representing a breed- or subtype-specific feature. The genomic changes that emerged due to strong-targeted selection towards this distinct type remain unclear. RESULTS: Comparisons of whole-genome sequencing data from two Miniature Shetland ponies and one standard-sized Shetland pony, performed to elucidate genetic determinants for miniature size, revealed four synergistic variants, limiting withers height to 34.25 in. (87 cm). Runs of homozygosity regions were detected spanning these four variants in both the Miniature Shetland ponies and the standard-sized Shetland pony. They were shown to be characteristic of the Shetland pony breed, resulting in a miniature type under specific genotypic combinations. These four genetic variants explained 72% of the size variation among Shetland ponies and related breeds. The length of the homozygous regions indicate that they arose over 1000 years ago. In addition, a copy number variant was identified in DIAPH3 harboring a loss exclusively in ponies and donkeys and thus representing a potential height-associated variant. CONCLUSION: This study reveals main drivers for miniature size in horses identified in whole genome data and thus provides relevant candidate genes for extremely short stature in mammals.

A genome-wide association study for harness racing success in the Norwegian-Swedish coldblooded trotter reveals genes for learning and energy metabolism.

BACKGROUND: Although harness racing is of high economic importance to the global equine industry, significant genomic resources have yet to be applied to mapping harness racing success. To identify genomic regions associated with harness racing success, the current study performs genome-wide association analyses with three racing performance traits in the Norwegian-Swedish Coldblooded Trotter using the 670 K Axiom Equine Genotyping Array. RESULTS: Following quality control, 613 horses and 359,635 SNPs were retained for further analysis. After strict Bonferroni correction, nine genome-wide significant SNPs were identified for career earnings. No genome-wide significant SNPs were identified for number of gallops or best km time. However, four suggestive genome-wide significant SNPs were identified for number of gallops, while 19 were identified for best km time. Multiple genes related to intelligence, energy metabolism, and immune function were identified as potential candidate genes for harness racing success. CONCLUSIONS: Apart from the physiological requirements needed for a harness racing horse to be successful, the results of the current study also advocate learning ability and memory as important elements for harness racing success. Further exploration into the mental capacity required for a horse to achieve racing success is likely warranted.

Copy number variations in Friesian horses and genetic risk factors for insect bite hypersensitivity.

BACKGROUND: Many common and relevant diseases affecting equine welfare have yet to be tested regarding structural variants such as copy number variations (CNVs). CNVs make up a substantial proportion of total genetic variability in populations of many species, resulting in more sequence differences between individuals than SNPs. Associations between CNVs and disease phenotypes have been established in several species, but equine CNV studies have been limited. Aim of this study was to identify CNVs and to perform a genome-wide association (GWA) study in Friesian horses to identify genomic loci associated with insect bite hypersensitivity (IBH), a common seasonal allergic dermatitis observed in many horse breeds worldwide. RESULTS: Genotypes were obtained using the Axiom® Equine Genotyping Array containing 670,796 SNPs. After quality control of genotypes, 15,041 CNVs and 5350 CNV regions (CNVRs) were identified in 222 Friesian horses. Coverage of the total genome by CNVRs was 11.2% with 49.2% of CNVRs containing genes. 58.0% of CNVRs were novel (i.e. so far only identified in Friesian horses). A SNP- and CNV-based GWA analysis was performed, where about half of the horses were affected by IBH. The SNP-based analysis showed a highly significant association between the MHC region on ECA20 and IBH in Friesian horses. Associations between the MHC region on ECA20 and IBH were also detected based on the CNV-based analysis. However, CNVs associated with IBH in Friesian horses were not often in close proximity to SNPs identified to be associated with IBH. CONCLUSIONS: CNVs were identified in a large sample of the Friesian horse population, thereby contributing to our knowledge on CNVs in horses and facilitating our understanding of the equine genome and its phenotypic expression. A clear association was identified between the MHC region on ECA20 and IBH in Friesian horses based on both SNP- and CNV-based GWA studies. These results imply that MHC contributes to IBH sensitivity in Friesian horses. Although subsequent analyses are needed for verification, nucleotide differences, as well as more complex structural variations like CNVs, seem to contribute to IBH sensitivity. IBH should be considered as a common disease with a complex genomic architecture.

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice.

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

The refractive state of the eye in Icelandic horses with the Silver mutation.

BACKGROUND: The syndrome Multiple Congenital Ocular Anomalies (MCOA) is a congenital eye disorder in horses. Both the MCOA syndrome and the Silver coat colour in horses are caused by the same missense mutation in the premelanosome protein (PMEL) gene. Horses homozygous for the Silver mutation (TT) are affected by multiple ocular defects causing visual impairment or blindness. Horses heterozygous for the Silver mutation (CT) have less severe clinical signs, usually cysts arising from the ciliary body iris or retina temporally. It is still unknown if the vision is impaired in horses heterozygous for the Silver mutation. A recent study reported that Comtois horses carrying the Silver mutation had significantly deeper anterior chambers of the eye compared to wild-type horses. This could potentially cause refractive errors. The purpose of the present study was to investigate if Icelandic horses with the Silver mutation have refractive errors compared to wild-type horses. One hundred and fifty-two Icelandic horses were included in the study, 71 CT horses and five TT horses. All horses were genotyped for the missense mutation in PMEL. Each CT and TT horse was matched by a wild-type (CC) horse of the same age ± 1 year. Skiascopy and a brief ophthalmic examination were performed in all horses. Association between refraction and age, eye, genotype and sex was tested by linear mixed-effect model analysis. TT horses with controls were not included in the statistical analyses as they were too few. RESULTS: The interaction between age and genotype had a significant impact on the refractive state (P = 0.0001). CT horses older than 16 years were on average more myopic than wild-type horses of the same age. No difference in the refractive state could be observed between genotypes (CT and CC) in horses younger than 16 years. TT horses were myopic (-2 D or more) in one or both eyes regardless of age. CONCLUSION: Our results indicate that an elderly Icelandic horse (older than 16 years) carrying the Silver mutation is more likely to be myopic than a wild-type horse of the same age.

Conformation Traits and Gaits in the Icelandic Horse are Associated with Genetic Variants in Myostatin (MSTN).

Many genes are known to have an influence on conformation and performance traits; however, the role of one gene, Myostatin (MSTN), has been highlighted in recent studies on horses. Myostatin acts as a repressor in the development and regulation of differentiation and proliferative growth of skeletal muscle. Several studies have examined the link between MSTN, conformation, and performance in racing breeds, but no studies have investigated the relationship in Icelandic horses. Icelandic horses, a highly unique breed, are known both for their robust and compact conformation as well as their additional gaits tölt and pace. Three SNPs (g.65868604G>T [PR8604], g.66493737C>T [PR3737], and g.66495826A>G [PR5826]) flanking or within equine MSTN were genotyped in 195 Icelandic horses. The SNPs and haplotypes were analyzed for association with official estimated breeding values (EBV) for conformation traits (n = 11) and gaits (n = 5). The EBV for neck, withers, and shoulders was significantly associated with both PR8604 and PR3737 (P < 0.05). PR8604 was also associated with EBV for total conformation (P = 0.05). These associations were all supported by the haplotype analysis. However, while SNP PR5826 showed a significant association with EBVs for leg stance and hooves (P < 0.05), haplotype analyses for these traits failed to fully support these associations. This study demonstrates the possible role of MSTN on both the form and function of horses from non-racing breeds. Further analysis of Icelandic horses as well as other non-racing breeds would be beneficial and likely help to completely understand the influence of MSTN on conformation and performance in horses.

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 F(ST)-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.