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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.

Since the first Spanish settlers brought horses to America centuries ago, several local varieties and breeds have been established in the New World. These were generally a consequence of the admixture of the different breeds arriving from Europe. In some instances, local horses have been selectively bred for specific traits, such as appearance, endurance, strength, and gait. We looked at the genetics of two breeds, the Puerto Rican Non-Purebred (PRNPB) (also known as the “Criollo”) horses and the Puerto Rican Paso Fino (PRPF), from the Caribbean Island of Puerto Rico. While it is reasonable to assume that there was a historic connection between the two, the genetic link between them has never been established. In our study, we started by looking at the genetic ancestry and diversity of current Puerto Rican horse populations using a 668 bp fragment of the mitochondrial DNA D-loop (HVR1) in 200 horses from 27 locations on the island. We then genotyped all 200 horses in our sample for the “gait-keeper” DMRT3 mutant allele previously associated with the paso gait especially cherished in this island breed. We also genotyped a subset of 24 samples with the Illumina Neogen Equine Community genome-wide array (65,000 SNPs). This data was further combined with the publicly available PRPF genomes from other studies. Our analysis show an undeniable genetic connection between the two varieties in Puerto Rico, consistent with the hypothesis that PRNPB horses represent the descendants of the original genetic pool, a mix of horses imported from the Iberian Peninsula and elsewhere in Europe. Some of the original founders of PRNRB population must have carried the “gait-keeper” DMRT3 allele upon arrival to the island. From this admixture, the desired traits were selected by the local people over the span of centuries. We propose that the frequency of the mutant “gait-keeper” allele originally increased in the local horses due to the selection for the smooth ride and other characters, long before the PRPF breed was established. To support this hypothesis, we demonstrate that PRNPB horses, and not the purebred PRPF, carry a signature of selection in the genomic region containing the DMRT3 locus to this day. The lack of the detectable signature of selection associated with the DMRT3 in the PRPF would be expected if this native breed was originally derived from the genetic pool of PRNPB horses established earlier and most of the founders already had the mutant allele. Consequently, selection specific to PRPF later focused on allels in other genes (including CHRM5, CYP2E1, MYH7, SRSF1, PAM, PRN and others) that have not been previously associated with the prized paso gait phenotype in Puerto Rico or anywhere else.

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 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.

Osteochondrosis (OC) is an important skeletal disease causing profound welfare concerns in horses. Although numerous studies have explored the genetics underlying OC in various breeds, the Belgian Warmblood (BW) remains unstudied despite having a concerning prevalence of 32.0%. As a result, this study aimed to conduct genome-wide association (GWA) analyses to identify candidate variants associated with OC in BWs. To achieve this, blood samples and radiographs were collected from 407 Belgian Warmbloods registered to one of two BW studbooks (Belgisch Warmbloedpaard and Zangersheide), and genotyping was performed using the 670K Axiom Equine Genotyping Array. GWA analyses using a principle component approach were then performed on OC status (OCS; presence or absence of OC at any joint), hock OC status (HOC) and stifle OC status (SOC). These analyses yielded significantly associated (P < .01) SNPs on Equus caballus chromosome (ECA) 3, ECA 12, and ECA 18 for OCS; however, no single nucleotide polymorphisms (SNPs) reached significance for HOC or SOC. Subsequent analysis of candidate genes within 500 kilobases of the significant SNPs revealed functions broadly related to cell differentiation and chondrocyte development. While this study represents another step forward in uncovering variants and biological pathways associated with OC, additional studies are needed to validate the newly identified candidate SNPs for OC in BWs. Further studies of OC in BWs, as well as other breeds, are critical in our efforts to fully understand the disease's etiopathogenesis and ultimately provide breeding programs better equipped to improve horse health and well-being. ispartof: JOURNAL OF EQUINE VETERINARY SCIENCE vol:111 ispartof: location:United States status: published

BACKGROUND: Dynamic laryngeal collapse (DLC) associated with poll flexion is the most common disorder of the upper respiratory tract (URT) in the Norwegian-Swedish Coldblooded Trotter (NSCT). The disorder, which has also been diagnosed in other breeds of trotters and gaited horses, appears to be related to anatomic phenotypes and only occurs during poll flexion when the horse is exercised 'on the bit'. OBJECTIVES: Identify genomic regions associated with DLC in the NSCT by combining a rigorous phenotyping protocol with genomic data from a high-density equine genotyping array. STUDY DESIGN: Prospective case/control study. METHODS: High-speed treadmill endoscopy was used to phenotype horses (n = 61) for DLC, distinguishing between cases and controls. Genome-wide association (GWA) analysis of DLC status was then performed using a principal component approach (PCA) with haplotype analyses subsequently performed for regions containing single-nucleotide polymorphisms (SNPs) above the suggestive genome-wide significance (GWS) threshold (P

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. ispartof: JOURNAL OF ANIMAL BREEDING AND GENETICS vol:137 issue:2 pages:223-233 ispartof: location:Germany status: published

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.

Analysis of the Y chromosome is the best-established way to reconstruct paternal family history in humans. Here, we applied fine-scaled Y-chromosomal haplotyping in horses with biallelic markers and demonstrate the potential of our approach to address the ancestry of sire lines. We de novo assembled a draft reference of the male-specific region of the Y chromosome from Illumina short reads and then screened 5.8 million basepairs for variants in 130 specimens from intensively selected and rural breeds and nine Przewalski's horses. Among domestic horses we confirmed the predominance of a young'crown haplogroup' in Central European and North American breeds. Within the crown, we distinguished 58 haplotypes based on 211 variants, forming three major haplogroups. In addition to two previously characterised haplogroups, one observed in Arabian/Coldblooded and the other in Turkoman/Thoroughbred horses, we uncovered a third haplogroup containing Iberian lines and a North African Barb Horse. In a genealogical showcase, we distinguished the patrilines of the three English Thoroughbred founder stallions and resolved a historic controversy over the parentage of the horse 'Galopin', born in 1872. We observed two nearly instantaneous radiations in the history of Central and Northern European Y-chromosomal lineages that both occurred after domestication 5,500 years ago. ispartof: SCIENTIFIC REPORTS vol:9 issue:1 ispartof: location:England status: published

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.

Infrared thermography (IRT) is a popular technology used for the detection of thermal changes given its non-invasive nature and lack of direct contact with the individual. Accordingly, the maximal eye temperature (MaxET) measured with IRT has been extensively applied in equine research. However, there is little information available about the potential limitations of the MaxET in field studies. Thus, the aims of this study were to 1) quantify the individual variation of MaxET in field conditions and the effects of individual, breed, body size (height at withers), eye side, sex and age, 2) determine the effects of environment and operator, and 3) explore the relationship between MaxET and rectal temperature (RT) at rest. To accomplish these aims, 791 MaxET measures from 32 horses were collected in Sweden in five different months and five farms over a period of 12 months. There was an effect of individual on IRT (P < .05) and individual MaxET varied from 29.4 to 37.6 °C. IRT was also affected (P < .05) by breed and sex (maximal difference 1.1 °C and 0.3 °C, respectively) but not by eye side, age and height at withers. There were significant effects of month and farm (maximal differences; 2.4 and 2.3 °C, respectively), between outdoor and indoor measurements (0.8 °C) and also between operators (0.2 °C). There were no correlations between MaxET and RT. These results demonstrate that in horses observed at rest in their home environment, MaxET is affected by endogenous (sex and breed) and environmental factors (farm, location and month of the year) and shows no relationship to RT. We strongly suggest that IRT technology should be used with great caution in field studies and only under conditions where these factors can be accurately accounted for. ispartof: PHYSIOLOGY & BEHAVIOR vol:228 ispartof: location:United States status: published

Recessive lethal variants often segregate at low frequencies in animal populations, such that two randomly selected individuals are unlikely to carry the same mutation. However, the likelihood of an individual inheriting two copies of a recessive lethal mutation is dramatically increased by inbreeding events. Such occurrences are particularly common in domestic animal populations, which are often characterised by high rates of inbreeding and low effective population sizes. To date there have been no published investigations into the presence of specific variants at high frequencies in domestic horse populations. This study aimed to identify potential recessive lethal haplotypes in the Thoroughbred horse breed, a closed population that has been selectively bred for racing performance.In this study, we scanned genotype data from Thoroughbred horses(n= 526) for adjacent single nucleotide polymorphisms (SNPs) at high heterozygote frequencies, but with a complete absence of homozygotes. Two SNPs that matched these criteria were mapped to an intronic region in theLY49Bgene, indicating that a closely linked mutation may cause lethality in homozygous state. Despite a complete absence of homozygotes, almost 35% of Thoroughbreds included in these analyses were heterozygous for both SNPs. A similar loss or absence of homozygotes was observed in genotype data from other domestic horse breeds (n= 2030). Variant analysis of whole-genome sequence data (n= 90) identified two SNPs in the 3’UTR region of theLY49Bgene that may result in loss of function. Analysis of transcriptomic data from equine embryonic tissue revealed thatLY49Bis expressed in the trophoblast during placentation stage of development.In this study, a region in theLY49Bgene was identified as a strong candidate for harbouring a variant causing lethality in homozygous state. These findings suggest thatLY49Bmay have an essential, but as yet unknown function in the implantation stage of equine development. Further investigation of this region may allow for the development of a genetic test to improve fertility rates in horse populations. Identification of other lethal variants could assist in improving natural levels of fertility in horse populations.Author SummaryRecessive lethal mutations may reach high frequencies in livestock populations due to selective breeding practices, resulting in reduced fertility rates. In this study, we characterise recessive lethal mutations at high frequencies in the Thoroughbred horse population, a breed with high rates of inbreeding and low genetic diversity. We identified a haplotype in theLY49Bgene that shows strong evidence of being homozygous lethal, despite having high frequencies of heterozygotes in Thoroughbreds and other domestic horse breeds. Two 3’UTR variants were identified as most likely to cause loss of function in theLY49Bgene, resulting in lethality. This finding provides novel insights into the potential importance ofLY49Bin equine development. Additionally, this study may assist with breeding strategies to improve fertility rates in the Thoroughbred and other domestic horse breeds.

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. ispartof: VETERINARY CLINICS OF NORTH AMERICA-EQUINE PRACTICE vol:36 issue:2 pages:323-+ ispartof: location:United States status: published

The roan coat color in horses is characterized by dispersed white hair and dark points. This phenotype segregates in a broad range of horse breeds, while the underlying genetic background is still unknown. Previous studies mapped the roan locus to the KIT gene on equine chromosome 3 (ECA3). However, this association could not be validated across different horse breeds. Performing a genome-wide association analysis (GWAS) in Noriker horses, we identified a single nucleotide polymorphism (SNP) (ECA3:g.79,543.439 A > G) in the intron 17 of the KIT gene. The G -allele of the top associated SNP was present in other roan horses, namely Quarter Horse, Murgese, Slovenian, and Belgian draught horse, while it was absent in a panel of 15 breeds, including 657 non-roan horses. In further 379 gray Lipizzan horses, eight animals exhibited a heterozygous genotype (A/G). Comparative whole-genome sequence analysis of the KIT region revealed two deletions in the downstream region (ECA3:79,533,217_79,533,224delTCGTCTTC; ECA3:79,533,282_79,533,285delTTCT) and a 3 bp deletion combined with 17 bp insertion in intron 20 of KIT (ECA3:79,588,128_79,588,130delinsTTATCTCTATAGTAGTT). Within the Noriker sample, these loci were in complete linkage disequilibrium (LD) with the identified top SNP. Based upon pedigree information and historical records, we were able to trace back the genetic origin of roan coat color to a baroque gene pool. Furthermore, our data suggest allelic heterogeneity and the existence of additional roan alleles in ponies and breeds related to the English Thoroughbred. In order to study the roan phenotype segregating in those breeds, further association and verification studies are required. ispartof: JOURNAL OF EQUINE VETERINARY SCIENCE vol:88 ispartof: location:United States status: published

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7&ndash, 29.9 Mb. The region contains AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in AKR1C genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8&ndash, 9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders.

BACKGROUND Studies of large racing populations have established clear differences in the career profile of stallions, mares and geldings. Multiple studies have also demonstrated positive effects on racing careers for horses that commence racing at a younger age. However, the applicability of these studies to small, native racing populations is unknown and warrants investigation. OBJECTIVES To provide summary statistics for performance outcomes for the Norwegian-Swedish Coldblooded Trotter and to document and provide evidence on the current differences in racing careers across age at first start groups, sexes and country of birth. STUDY DESIGN Cohort study. METHODS Performance data on the population of Norwegian-Swedish Coldblooded Trotters born between 2000 and 2009 (n = 14,548) were acquired and used to describe and compare the racing careers of this native racing breed. Career length, career starts and career earnings were evaluated. Kaplan-Meier survival curves, stratified by sex, age group and country of birth were produced for career length. A Cox proportional hazards model was fitted to assess factors influencing the hazard rate of retirement from racing. RESULTS Log-rank test for equality of career length survival functions showed significant differences (P

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-4877-5) contains supplementary material, which is available to authorized users.

Summary A previous study revealed a strong association between the DMRT3:Ser301STOP mutation in horses and alternate gaits as well as performance in harness racing. Several follow-up studies have confirmed a high frequency of the mutation in gaited horse breeds and an effect on gait quality. The aim of this study was to determine when and where the mutation arose, to identify additional potential causal mutations and to determine the coalescence time for contemporary haplotypes carrying the stop mutation. We utilized sequences from 89 horses representing 26 breeds to identify 102 SNPs encompassing the DMRT3 gene that are in strong linkage disequilibrium with the stop mutation. These 102 SNPs were genotyped in an additional 382 horses representing 72 breeds, and we identified 14 unique haplotypes. The results provided conclusive evidence that DMRT3:Ser301STOP is causal, as no other sequence polymorphisms showed an equally strong association to locomotion traits. The low sequence diversity among mutant chromosomes demonstrated that they must have diverged from a common ancestral sequence within the last 10 000 years. Thus, the mutation occurred either just before domestication or more likely some time after domestication and then spread across the world as a result of selection on locomotion traits.

The Estonian Native Horse (ENH) is a medium-size pony found mainly in the western islands of Estonia and is well-adapted to the harsh northern climate and poor pastures. The ancestry of the ENH is debated, including alleged claims about direct descendance from the extinct Tarpan. Here we conducted a detailed analysis of the genetic makeup and relationships of the ENH based on the genotypes of 15 autosomal short tandem repeats (STRs), 18 Y chromosomal single nucleotide polymorphisms (SNPs), mitochondrial D-loop sequence and lateral gait allele in DMRT3. The study encompassed 2890 horses of 61 breeds, including 33 ENHs. We show that the expected and observed genetic diversities of the ENH are among the highest within 52 global breeds, and the highest among 8 related Northern European ponies. The genetically closest breeds to the ENH are the Finn Horse, and the geographically more distant primitive Hucul and Konik. ENH matrilines are diverse and relate to draught and Pontic-Caspian breeds. ENH patrilines relate to draught breeds, and to a unique haplogroup not described before. None of the 33 ENHs carried the &ldquo, gait&rdquo, mutation, but the mutation was found in 2 Huculs. The study demonstrates that the ENH is a genetically distinct and diverse breed of ancient origin with no notable pressure of selective breeding.

Intensive artificial and natural selection have shaped substantial variation among European horse breeds. Whereas most equine selection signature studies employ divergent genetic population structures in order to derive specific inter-breed targets of selection, we screened a total of 1476 horses originating from 12 breeds for the loss of genetic diversity by runs of homozygosity (ROH) utilizing a 670,000 single nucleotide polymorphism (SNP) genotyping array. Overlapping homozygous regions (ROH islands) indicating signatures of selection were identified by breed and similarities/dissimilarities between populations were evaluated. In the entire dataset, 180 ROH islands were identified, whilst 100 islands were breed specific, all other overlapped in 36 genomic regions with at least one ROH island of another breed. Furthermore, two ROH hot spots were determined at horse chromosome 3 (ECA3) and ECA11. Besides the confirmation of previously documented target genes involved in selection for coat color (MC1R, STX17, ASIP), body size (LCORL/NCAPG, ZFAT, LASP1, HMGA2), racing ability (PPARGC1A), behavioral traits (GRIN2B, NTM/OPCML) and gait patterns (DMRT3), several putative target genes related to embryonic morphogenesis (HOXB), energy metabolism (IGFBP-1, IGFBP-3), hair follicle morphogenesis (KRT25, KRT27, INTU) and autophagy (RALB) were highlighted. Furthermore, genes were pinpointed which might be involved in environmental adaptation of specific habitats (UVSSA, STXBP4, COX11, HLF, MMD).

The Colombian paso horse, the most important horse breed in Colombia, performs specific and particular gaits (paso fino, trocha, and Colombian trot), which display different footfall patterns and stride frequencies. The breed has been selected for gait and conformation for more than 50 years and we hypothesize that this selection has led to kinematic differences of the gaits that can be explained by different genetic variants. Hence, the aims of the study were: 1. To identify if there are any differences in the kinematic and genetic variants between the Colombian paso horse's gaits. 2. To evaluate if and how much the gait differences were explained by the nonsense mutation in the DMRT3 gene and 3. To evaluate these results for selecting and controlling the horses gait performance. To test our hypotheses, kinematic data, microsatellites and DMRT3 genotypes for 187 Colombian paso horses were analyzed. The results indicated that there are significant kinematic and DMRT3 differences between the Colombian paso horse's gaits, and those parameters can be used partially to select and control the horses gait performance. However, the DMRT3 gene does not play a major role in controlling the trocha and the Colombian trot gaits. Therefore, modifying genes likely influence these gaits. This study may serve as a foundation for implementing a genetic selection program in the Colombian paso horse and future gene discovery studies for locomotion pattern in horses.

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 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.

Insect bite hypersensitivity (IBH), which is a cutaneous allergic reaction to antigens from Culicoides spp., is the most prevalent skin disorder in horses. Misdiagnosis is possible, as IBH is usually diagnosed based on clinical signs. Our study is the first to employ IgE levels against several recombinant Culicoides spp. allergens as an objective, independent, and quantitative phenotype to improve the power to detect genetic variants that underlie IBH. Genotypes of 200 Shetland ponies, 127 Icelandic horses, and 223 Belgian Warmblood horses were analyzed while using a mixed model approach. No single-nucleotide polymorphism (SNP) passed the Bonferroni corrected significance threshold, but several regions were identified within and across breeds, which confirmed previously identified regions of interest and, in addition, identifying new regions of interest. Allergen-specific IgE levels are a continuous and objective phenotype that allow for more powerful analyses when compared to a case-control set-up, as more significant associations were obtained. However, the use of a higher density array seems necessary to fully employ the use of IgE levels as a phenotype. While these results still require validation in a large independent dataset, the use of allergen-specific IgE levels showed value as an objective and continuous phenotype that can deepen our understanding of the biology underlying IBH. The research leading to these results has received funding fromthe EuropeanUnion's Seventh Framework Programme managed by REA-Research Executive Agency, http://ec.europa.eu/research/rea (FP7/2007-2013) under grant agreement No. 606142.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5484-9) contains supplementary material, which is available to authorized users.

Diagnostic reports written to assist stud managers in the sale of young Thoroughbreds have not previously been used as a data source for the study of skeletal lesions. However, analyses of these reports may provide efficient and cost-effective insights into the prevalence and distribution of skeletal lesions within a population. Diagnostic reports written by veterinarians were acquired from Thoroughbred stud managers in Australia and New Zealand. The reports were based on approximately 1300 sets of weanling and yearling radiographs taken between 2002 and 2007. The prevalence and anatomical distribution of skeletal lesions in weanlings (299 horses) and yearlings (1004 horses) were determined from these reports. Overall, 69.9% of weanlings and 64.5% of yearlings were reported as having one or more skeletal lesions. Diagnostic reports in weanlings were a strong indication of what was likely to be seen in subsequent yearling reports. These diagnostic reports are typically used by stud managers in the sales process and the potential drawback is that some categories of skeletal lesions may be under-reported. However, there was substantial agreement between the prevalence and distribution of several skeletal lesions reported in this study and those previously reported from direct evaluation of radiographs for Australian and New Zealand Thoroughbred yearlings. Strong agreement was found for osteophytes, enthesiophytes and other modelling in the hocks, and for lesions in the hind fetlocks and stifles. This indicates that written diagnostic reports are a useful and a reliable source of data for the study of some skeletal lesions in young Thoroughbred horses.

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160−180 kb and 60−80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development.

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. Electronic supplementary material The online version of this article (10.1186/s12863-018-0670-3) contains supplementary material, which is available to authorized users.

The Swedish‐Norwegian Coldblooded trotter and the heavier North‐Swedish draught horse both descend from the North‐Swedish horse, but the Coldblooded trotters have been selected for racing performance while the North‐Swedish draught horse is mainly used for agricultural and forestry work. By comparing the genomes of Coldblooded trotters, North‐Swedish draught horses and Standardbreds for a large number of single‐nucleotide polymorphisms (SNPs), the aim of the study was to identify genetic regions that may be under selection for racing performance. We hypothesized that the selection for racing performance, in combination with unauthorized crossbreeding of Coldblooded trotters and Standardbreds, has created regions in the genome where the Coldblooded trotters and Standardbreds are similar, but differ from the North‐Swedish draught horse. A fixation index (Fst) analysis was performed and sliding window Delta Fst values were calculated across the three breeds. Five windows, where the average Fst between Coldblooded trotters and Standardbreds was low and the average Fst between Coldblooded trotters and North‐Swedish draught horses was high, were selected for further investigation. Associations between the most highly ranked SNPs and harness racing performance were analyzed in 400 raced Coldblooded trotters with race records. One SNP showed a significant association with racing performance, with the CC genotype appearing to be negatively associated. The SNP identified was genotyped in 1915 horses of 18 different breeds. The frequency of the TT genotype was high in breeds typically used for racing and show jumping while the frequency of the CC genotype was high in most pony breeds and draught horses. The closest gene in this region was the Endothelin3 gene (EDN3), a gene mainly involved in melanocyte and enteric neuron development. Both functional genetic and physiological studies are needed to fully understand the possible impacts of the gene on racing performance.

Humans have shaped the population history of the horse ever since domestication about 5500 years ago. Comparative analyses of the Y chromosome can illuminate the paternal origin of modern horse breeds. This may also reveal different breeding strategies that led to the formation of extant breeds. Recently, a horse Y-chromosomal phylogeny of modern horses based on 1.46 Mb of the male-specific Y (MSY) was generated. We extended this dataset with 52 samples from five European, two American and seven Asian breeds. As in the previous study, almost all modern European horses fall into a crown group, connected via a few autochthonous Northern European lineages to the outgroup, the Przewalski's Horse. In total, we now distinguish 42 MSY haplotypes determined by 158 variants within domestic horses. Asian horses show much higher diversity than previously found in European breeds. The Asian breeds also introduce a deep split to the phylogeny, preliminarily dated to 5527 ± 872 years. We conclude that the deep splitting Asian Y haplotypes are remnants of a far more diverse ancient horse population, whose haplotypes were lost in other lineages.

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. Electronic supplementary material The online version of this article (10.1186/s12863-018-0657-0) contains supplementary material, which is available to authorized users.

BackgroundTo 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.ResultsUsing whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 millionde novodiscovered 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.ConclusionsHere, 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 Y chromosome directly reflects male genealogies, but the extremely low Y chromosome sequence diversity in horses has prevented the reconstruction of stallion genealogies [1, 2]. Here, we resolve the first Y chromosome genealogy of modern horses by screening 1.46 Mb of the male-specific region of the Y chromosome (MSY) in 52 horses from 21 breeds. Based on highly accurate pedigree data, we estimated the de novo mutation rate of the horse MSY and showed that various modern horse Y chromosome lineages split much later than the domestication of the species. Apart from few private northern European haplotypes, all modern horse breeds clustered together in a roughly 700-year-old haplogroup that was transmitted to Europe by the import of Oriental stallions. The Oriental horse group consisted of two major subclades: the Original Arabian lineage and the Turkoman horse lineage. We show that the English Thoroughbred MSY was derived from the Turkoman lineage and that English Thoroughbred sires are largely responsible for the predominance of this haplotype in modern horses.

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.

While susceptibility to hypersensitive reactions is a common problem amongst humans and animals alike, the population structure of certain animal species and breeds provides a more advantageous route to better understanding the biology underpinning these condi- tions. The current study uses Exmoor ponies, a highly inbred breed of horse known to fre- quently suffer from insect bite hypersensitivity, to identify genomic regions associated with a type I and type IV hypersensitive reaction. A total of 110 cases and 170 controls were gen- otyped on the 670K Axiom Equine Genotyping Array. Quality control resulted in 452,457 SNPs and 268 individuals being tested for association. Genome-wide association analyses were performed using the GenABEL package in R and resulted in the identification of two regions of interest on Chromosome 8. The first region contained the most significant SNP identified, which was located in an intron of the DCC netrin 1 receptor gene. The second region identified contained multiple top SNPs and encompassed the PIGN , KIAA1468 , TNFRSF11A , ZCCHC2 , and PHLPP1 genes. Although additional studies will be needed to validate the importance of these regions in horses and the relevance of these regions in other species, the knowledge gained from the current study has the potential to be a step forward in unraveling the complex nature of hypersensitive reactions ispartof: PLoS One vol:11 issue:4 ispartof: location:United States status: published

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 tolt 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.

Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

The syndrome Multiple Congenital Ocular Anomalies (MCOA) is the collective name ascribed to heritable congenital eye defects in horses. Individuals homozygous for the disease allele (MCOA phenotype) have a wide range of eye anomalies, while heterozygous horses (Cyst phenotype) predominantly have cysts that originate from the temporal ciliary body, iris, and/or peripheral retina. MCOA syndrome is highly prevalent in the Rocky Mountain Horse but the disease is not limited to this breed. Affected horses most often have a Silver coat color; however, a pleiotropic link between these phenotypes is yet to be proven. Locating and possibly isolating these traits would provide invaluable knowledge to scientists and breeders. This would favor maintenance of a desirable coat color while addressing the health concerns of the affected breeds, and would also provide insight into the genetic basis of the disease. Identical-by-descent mapping was used to narrow the previous 4.6-Mb region to a 264-kb interval for the MCOA locus. One haplotype common to four breeds showed complete association to the disease (Cyst phenotype, n = 246; MCOA phenotype, n = 83). Candidate genes from the interval, SMARCC2 and IKZF4, were screened for polymorphisms and genotyped, and segregation analysis allowed the MCOA syndrome region to be shortened to 208 kb. This interval also harbors PMEL17, the gene causative for Silver coat color. However, by shortening the MCOA locus by a factor of 20, 176 other genes have been unlinked from the disease and only 15 genes remain. Electronic supplementary material The online version of this article (doi:10.1007/s00335-011-9325-7) contains supplementary material, which is available to authorized users.

Frequencies of polymorphisms in myostatin vary in Icelandic horses according to the use of the horses

Insect bite hypersensitivity (IBH) is the most common allergic skin disease in horses and is caused by biting midges, mainly of the genus Culicoides. The disease predominantly comprises a type I hypersensitivity reaction, causing severe itching and discomfort that reduce the welfare and commercial value of the horse. It is a multifactorial disorder influenced by both genetic and environmental factors, with heritability ranging from 0.16 to 0.27 in various horse breeds. The worldwide prevalence in different horse breeds ranges from 3% to 60%; it is more than 50% in Icelandic horses exported to the European continent and approximately 8% in Swedish-born Icelandic horses. To minimize the influence of environmental effects, we analyzed Swedish-born Icelandic horses to identify genomic regions that regulate susceptibility to IBH. We performed a genome-wide association (GWA) study on 104 affected and 105 unaffected Icelandic horses genotyped using Illumina® EquineSNP50 Genotyping BeadChip. Quality control and population stratification analyses were performed with the GenABEL package in R (λ = 0.81). The association analysis was performed using the Bayesian variable selection method, Bayes C, implemented in GenSel software. The highest percentage of genetic variance was explained by the windows on X chromosomes (0.51% and 0.36% by 73 and 74 mb), 17 (0.34% by 77 mb), and 18 (0.34% by 26 mb). Overlapping regions with previous GWA studies were observed on chromosomes 7, 9, and 17. The windows identified in our study on chromosomes 7, 10, and 17 harbored immune system genes and are priorities for further investigation.

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.

A nonsense mutation in DMRT3 ('Gait keeper' mutation) has a predominant effect on gaiting ability in horses, being permissive for the ability to perform lateral gaits and having a favourable effect on speed capacity in trot. The DMRT3 mutant allele (A) has been found in high frequency in gaited breeds and breeds bred for harness racing, while other horse breeds were homozygous for the wild-type allele (C). The aim of this study was to evaluate further the effect of the DMRT3 nonsense mutation on the gait quality and speed capacity in the multigaited Icelandic horse and demonstrate how the frequencies of the A- and C- alleles have changed in the Icelandic horse population in recent decades. It was confirmed that homozygosity for the DMRT3 nonsense mutation relates to the ability to pace. It further had a favourable effect on scores in breeding field tests for the lateral gait tolt, demonstrated by better beat quality, speed capacity and suppleness. Horses with the CA genotype had on the other hand significantly higher scores for walk, trot, canter and gallop, and they performed better beat and suspension in trot and gallop. These results indicate that the AA genotype reinforces the coordination of ipsilateral legs, with the subsequent negative effect on the synchronized movement of diagonal legs compared with the CA genotype. The frequency of the A-allele has increased in recent decades with a corresponding decrease in the frequency of the C-allele. The estimated frequency of the A-allele in the Icelandic horse population in 2012 was 0.94. Selective breeding for lateral gaits in the Icelandic horse population has apparently altered the frequency of DMRT3 genotypes with a predicted loss of the C-allele in relatively few years. The results have practical implications for breeding and training of Icelandic horses and other gaited horse breeds.

For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be four-beat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing.

Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F2 generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F2s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome. © 2000 Academic Press

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map.

Several studies based on a variety of genetic markers have attempted to establish the origins of horse domestication. Thus far a discrepancy between the results of mitochondrial DNA analysis, which show high levels of diversity, and results from the Y-chromosome, with almost no genetic variability, has been identified. Most previous work on the horse Y-chromosome has focused on widespread, popular breeds or local Asian breeds. It is possible that these breeds represent a reduced set of the genetic variation present in the species. Additional genetic variation may be present in local breeds and ancient feral populations, such as the Retuertas horse in Spain. In this study we analyzed the Y-chromosome of the Retuertas horse, a feral horse population on the Iberian Peninsula that is at least several hundred years old, and whose genetic diversity and morphology suggests that it has been reproductively isolated for a long time. Data from the Retuertas horse was compared to another 11 breeds from the region (Portugal, Spain and France) or likely of Iberian origin, and then to data from 15 more breeds from around the globe. We sequenced 31 introns, Zinc finger Y-chromosomal protein (ZFY) and anonymous Y-linked fragments and genotyped 6 microsatellite loci found on the Y-chromosome. We found no sequence variation among all individuals and all breeds studied. However, fifteen differences were discovered between our data set and reference sequences in GenBank. We show that these likely represent errors within the deposited sequences, and suggest that they should not be used as comparative data for future projects.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:16Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:16Z : No. of bitstreams: 1 2-s2.0-84873867256.pdf: 796974 bytes, checksum: 64f172274307a2abd3e5977743eb1671 (MD5) Made available in DSpace on 2014-05-27T11:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-30 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. © 2013 Petersen et al. University of Minnesota College of Veterinary Medicine, St Paul, MN Texas AandM University College of Veterinary Medicine and Biomedical Science, College Station, TX Swedish University of Agricultural Sciences Department of Animal Breeding and Genetics, Uppsala University of Kentucky Department of Veterinary Science, Lexington, KY University of California Davis School of Veterinary Medicine, Davis, CA Equine Analysis, Midway, KY University Estadual Paulista Department of Veterinary Clinical Science, Botucatu-SP University College Dublin School of Veterinary Medicine, Dublin University of Azores Institute for Biotechnology and Bioengineering Biotechnology Centre of Azores, Angra do Heroísmo University of Veterinary Medicine Hannover Institute for Animal Breeding and Genetics, Hannover University of Perugia Faculty of Veterinary Medicine, Perugia Animal Health Trust, Lanwades Park, Newmarket, Suffolk French National Institute for Agricultural Research-Animal Genetics and Integrative Biology Unit, Jouy en Josas University of Sydney Veterinary Science, New South Wales Nihon Bioresource College, Koga, Ibaraki University of Helsinki Faculty of Veterinary Medicine, Helsinki University College Dublin College of Agriculture, Food Science and Veterinary Medicine, Belfield, Dublin University of Bern Institute of Genetics, Bern Institute of Cancer Research Breakthrough Breast Cancer Research Centre, London Royal Veterinary College Comparative Neuromuscular Diseases Laboratory, London Swiss National Stud Farm Agroscope Liebefeld-Posieux Research Station, Avenches Norwegian School of Veterinary Science Department of Basic Sciences and Aquatic Medicine, Oslo Animal DNA Diagnostics Ltd, Cambridge Laboratory of Racing Chemistry Department of Molecular Genetics, Utsunomiya, Tochigi

A premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses, and the Dmrt3 transcription factor is critical in the development of a coordinated locomotor network in mice, suggesting that it has an important role in configuring the spinal circuits that control stride. Some horses — notably the harness-racing American Standardbred and the all-terrain Icelandic breed — have the ability to perform extra gaits. All horses can walk, trot, canter and gallop, but some can also 'pace' — moving the two legs on the same side of the body in unison — and/or perform other novel ambling gaits. A genome-wide association analysis of Icelandic horses has identified linkage between a premature stop codon in the DMRT3 gene and the ability to perform alternative gaits. Functional studies in mice show that Dmrt3 is expressed in a subset of spinal cord neurons that are crucial for the normal development of a coordinated locomotor network that controlling limb movements. Dmrt3 may therefore have a key role in configuring the spinal circuits that control stride in vertebrates. In the domestic horses, the DMRT3 mutation has had a major impact on the creatures' diversification, because the altered gait characteristics of a number of breeds apparently require this mutation. Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement1. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles2. 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.

Selective breeding for speed in the racehorse has resulted in an unusually high frequency of the C-variant (g.66493737C/T) at the myostatin gene (MSTN) in cohorts of the Thoroughbred horse population that are best suited to sprint racing. Here we show using a combination of molecular- and pedigree-based approaches in 593 horses from 22 Eurasian and North-American horse populations, museum specimens from 12 historically important Thoroughbred stallions (b.1764-1930), 330 elite-performing modern Thoroughbreds and 42 samples from three other equid species that the T-allele was ancestral and there was a single introduction of the C-allele at the foundation stages of the Thoroughbred from a British-native mare. Furthermore, we show that although the C-allele was rare among the celebrated racehorses of the 18th and 19th centuries, it has proliferated recently in the population via the stallion Nearctic (b.1954), the sire of the most influential stallion of modern time, Northern Dancer (b.1961).

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 Przewalski's 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., Author Summary We utilized the previously generated horse genome sequence and a large SNP database to design an ∼54,000 SNP assay for use in the domestic horse and related species. The utility of this SNP array was demonstrated through genome-wide linkage disequilibrium, inbreeding and genetic distance measurements within breeds, as well as multidimensional scaling and parsimony analysis. Association mapping confirmed a large conserved segment containing the chestnut coat color locus in domestic horses. We also assess the utility of the SNP array in related species, including the Przewalski's Horse, zebras, asses, tapirs, and rhinoceros. This SNP genotyping tool will facilitate many genetics applications in equids, including identification of genes for health and performance traits, and compelling studies of the origins of the domestic horse, diversity within breeds, and evolutionary relationships among related species.

Insect bite hypersensitivity (IBH) is a chronic allergic dermatitis common in horses. Affected horses mainly react against antigens present in the saliva from the biting midges, Culicoides ssp, and occasionally black flies, Simulium ssp. Because of this insect dependency, the disease is clearly seasonal and prevalence varies between geographical locations. For two distinct horse breeds, we genotyped four microsatellite markers positioned within the MHC class II region and sequenced the highly polymorphic exons two from DRA and DRB3, respectively. Initially, 94 IBH-affected and 93 unaffected Swedish born Icelandic horses were tested for genetic association. These horses had previously been genotyped on the Illumina Equine SNP50 BeadChip, which made it possible to ensure that our study did not suffer from the effects of stratification. The second population consisted of 106 unaffected and 80 IBH-affected Exmoor ponies. We show that variants in the MHC class II region are associated with disease susceptibility (praw = 2.34 × 10−5), with the same allele (COR112:274) associated in two separate populations. In addition, we combined microsatellite and sequencing data in order to investigate the pattern of homozygosity and show that homozygosity across the entire MHC class II region is associated with a higher risk of developing IBH (p = 0.0013). To our knowledge this is the first time in any atopic dermatitis suffering species, including man, where the same risk allele has been identified in two distinct populations. Electronic supplementary material The online version of this article (doi:10.1007/s00251-011-0573-1) contains supplementary material, which is available to authorized users.