Variation in salivary cortisol responses in yearling Thoroughbred racehorses during their first year of training.

Thoroughbred horses are bred for competitive racing and undergo intense training regimes. The maintenance of physical soundness and desirable behavioural characteristics are critical to the longevity of a racing career. Horses intended for Flat racing generally enter training as yearlings and undergo introductory training prior to exercise conditioning for racing. This period requires rapid adjustment to a novel environment. As a prey animal, a horse's 'fight-or-flight' response is highly adapted, in which a well-understood component of this response, the hypothalamic-pituitary-axis, is activated in response to a stress stimulus, releasing cortisol. In the Thoroughbred, a significant difference in salivary cortisol concentrations between pre- and post-first time ridden (i.e., first backing) by a jockey have previously been identified. Here, to test the hypothesis that salivary cortisol concentrations may be used to objectively detect individual variations in the acute physiological stress response we investigate individual variation in cortisol response to training milestones. Saliva samples were collected from a cohort of n = 96 yearling Flat racehorses, at the same training yard, across three timepoints at rest: before entering the training yard (n = 66), within three days of entry to the training yard (n = 67) and following 2-3 weeks in the training yard (n = 50). Salivary cortisol concentration was measured using an ELISA. There was no significant difference in cortisol concentration (ANOVA, P > 0.05) across the samples collected at timepoints at rest. Samples were also collected before and 30 minutes after exposure to three novel training events: first time long-reined (n = 6), first time backed by a jockey (n = 34), and first time ridden on the gallops (n = 10). Mean salivary cortisol concentration after all three novel training events was significantly higher than prior to the training event (Paired t-test, P <0.005). The ranges of post-event salivary cortisol concentration across all timepoints suggest individual variation in the measured stress response, reflecting individual differences in stress response to the early training environment. This measure may be used as an objective assessment of the stress response of Thoroughbred racehorses during training.

Integrative genomics analysis highlights functionally relevant genes for equine behaviour.

Behavioural plasticity enables horses entering an exercise training programme to adapt with reduced stress. We characterised SNPs associated with behaviour in yearling Thoroughbred horses using genomics analyses for two phenotypes: (1) handler-assessed coping with early training events [coping] (n = 96); and (2) variation in salivary cortisol concentration at the first backing event [cortisol] (n = 34). Using RNA-seq derived gene expression data for amygdala and hippocampus tissues from n = 2 Thoroughbred stallions, we refined the SNPs to those with functional relevance to behaviour by cross-referencing to the 500 most highly expressed genes in each tissue. The SNPs of high significance (q < 0.01) were in proximity to genes (coping - GABARAP, NDM, OAZ1, RPS15A, SPARCL1, VAMP2; cortisol - CEBPA, COA3, DUSP1, HNRNPH1, RACK1) with biological functions in social behaviour, autism spectrum disorder, suicide, stress-induced anxiety and depression, Alzheimer's disease, neurodevelopmental disorders, neuroinflammatory disease, fear-induced behaviours and alcohol and cocaine addiction. The strongest association (q = 0.0002) was with NDN, a gene previously associated with temperament in cattle. This approach highlights functionally relevant genes in the behavioural adaptation of Thoroughbred horses that will contribute to the development of genetic markers to improve racehorse welfare.

Inbreeding depression and durability in the North American Thoroughbred horse.

The proportion of the genome containing runs of homozygosity (ROH) affects production traits in livestock populations. In European and Australasian Thoroughbreds inbreeding, quantified using ROH (FROH ), is associated with the probability of ever racing. Here, we measured FROH using 333 K SNP genotypes from 768 Thoroughbred horses born in North America to evaluate the effect of inbreeding on racing traits in that region. Among North American horses, FROH was not associated (p = 0.518) with the probability of ever racing but was significantly associated with the number of race starts (p = 0.002). Among raced horses, those with a 10% higher FROH than the mean inbreeding coefficient were predicted to have 3.5 fewer race starts compared to horses with a mean inbreeding coefficient. Considering the trend of increasing inbreeding and a decline in the average number of race starts per runner in North America, mitigating inbreeding in the population could positively influence racing durability.

Selection signatures for local and regional adaptation in Chinese Mongolian horse breeds reveal candidate genes for hoof health.

BACKGROUND: Thousands of years of natural and artificial selection since the domestication of the horse has shaped the distinctive genomes of Chinese Mongolian horse populations. Consequently, genomic signatures of selection can provide insights into the human-mediated selection history of specific traits and evolutionary adaptation to diverse environments. Here, we used genome-wide SNPs from five distinct Chinese Mongolian horse populations to identify genomic regions under selection for the population-specific traits, gait, black coat colour, and hoof quality. Other global breeds were used to identify regional-specific signatures of selection. RESULTS: We first identified the most significant selection peak for the Wushen horse in the region on ECA23 harbouring DMRT3, the major gene for gait. We detected selection signatures encompassing several genes in the Baicha Iron Hoof horse that represent good biological candidates for hoof health, including the CSPG4, PEAK1, EXPH5, WWP2 and HAS3 genes. In addition, an analysis of regional subgroups (Asian compared to European) identified a single locus on ECA3 containing the ZFPM1 gene that is a marker of selection for the major domestication event leading to the DOM2 horse clade. CONCLUSIONS: Genomic variation at these loci in the Baicha Iron Hoof may be leveraged in other horse populations to identify animals with superior hoof health or those at risk of hoof-related pathologies. The overlap between the selection signature in Asian horses with the DOM2 selection peak raises questions about the nature of horse domestication events, which may have involved a prehistoric clade other than DOM2 that has not yet been identified.

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

Background: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype. Results: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. Conclusions: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

Common protein-coding variants influence the racing phenotype in galloping racehorse breeds.

Selection for system-wide morphological, physiological, and metabolic adaptations has led to extreme athletic phenotypes among geographically diverse horse breeds. Here, we identify genes contributing to exercise adaptation in racehorses by applying genomics approaches for racing performance, an end-point athletic phenotype. Using an integrative genomics strategy to first combine population genomics results with skeletal muscle exercise and training transcriptomic data, followed by whole-genome resequencing of Asian horses, we identify protein-coding variants in genes of interest in galloping racehorse breeds (Arabian, Mongolian and Thoroughbred). A core set of genes, G6PC2, HDAC9, KTN1, MYLK2, NTM, SLC16A1 and SYNDIG1, with central roles in muscle, metabolism, and neurobiology, are key drivers of the racing phenotype. Although racing potential is a multifactorial trait, the genomic architecture shaping the common athletic phenotype in horse populations bred for racing provides evidence for the influence of protein-coding variants in fundamental exercise-relevant genes. Variation in these genes may therefore be exploited for genetic improvement of horse populations towards specific types of racing.

Inbreeding depression and the probability of racing in the Thoroughbred horse.

Small effective population sizes and active inbreeding can lead to inbreeding depression due to deleterious recessive mutations exposed in the homozygous state. The Thoroughbred racehorse has low levels of population genetic diversity, but the effects of genomic inbreeding in the population are unknown. Here, we quantified inbreeding based on runs of homozygosity (ROH) using 297 K SNP genotypes from 6128 horses born in Europe and Australia, of which 13.2% were unraced. We show that a 10% increase in inbreeding (FROH) is associated with a 7% lower probability of ever racing. Moreover, a ROH-based genome-wide association study identified a haplotype on ECA14 which, in its homozygous state, is linked to a 32.1% lower predicted probability of ever racing, independent of FROH. The haplotype overlaps a candidate gene, EFNA5, that is highly expressed in cartilage tissue, which when damaged is one of the most common causes of catastrophic musculoskeletal injury in racehorses. Genomics-informed breeding aiming to reduce inbreeding depression and avoid damaging haplotype carrier matings will improve population health and racehorse welfare.

Inspiratory muscle training in young, race-fit Thoroughbred racehorses during a period of detraining.

Although inspiratory muscle training (IMT) is reported to improve inspiratory muscle strength in humans little has been reported for horses. We tested the hypothesis that IMT would maintain and/or improve inspiratory muscle strength variables measured in Thoroughbreds during detraining. Thoroughbreds from one training yard were placed into a control (Con, n = 3 males n = 7 females; median age 2.2±0.4 years) or treatment group (Tr, n = 5 males, n = 5 females; median age 2.1±0.3 years) as they entered a detraining period at the end of the racing/training season. The Tr group underwent eight weeks of IMT twice a day, five days per week using custom-made training masks with resistance valves and an incremental threshold of breath-loading protocol. An inspiratory muscle strength test to fatigue using an incremental threshold of breath-loading was performed in duplicate before (T0) and after four (T1) and eight weeks (T2) of IMT/no IMT using a custom-made testing mask and a commercial testing device. Inspiratory measurements included the total number of breaths achieved during the test, average load, peak power, peak volume, peak flow, energy and the mean peak inspiratory muscle strength index (IMSi). Data were analysed using a linear mixed effects model, P≤0.05 significant. There were no differences for inspiratory measurements between groups at T0. Compared to T0, the total number of breaths achieved (P = 0.02), load (P = 0.003) and IMSi (P = 0.01) at T2 had decreased for the Con group while the total number of breaths achieved (P<0.001), load (P = 0.03), volume (P = 0.004), flow (P = 0.006), energy (P = 0.01) and IMSi (P = 0.002) had increased for the Tr group. At T2 the total number of breaths achieved (P<0.0001), load (P<0.0001), volume (P = 0.02), energy (P = 0.03) and IMSi (P<0.0001) were greater for the Tr than Con group. In conclusion, our results support that IMT can maintain and/or increase aspects of inspiratory muscle strength for horses in a detraining programme.

Selection in Australian Thoroughbred horses acts on a locus associated with early two-year old speed.

Thoroughbred horse racing is a global sport with major hubs in Europe, North America, Australasia and Japan. Regional preferences for certain traits have resulted in phenotypic variation that may result from adaptation to the local racing ecosystem. Here, we test the hypothesis that genes selected for regional phenotypic variation may be identified by analysis of selection signatures in pan-genomic SNP genotype data. Comparing Australian to non-Australian Thoroughbred horses (n = 99), the most highly differentiated loci in a composite selection signals (CSS) analysis were on ECA6 (34.75-34.85 Mb), ECA14 (33.2-33.52 Mb and 35.52-36.94 Mb) and ECA16 (24.28-26.52 Mb) in regions containing candidate genes for exercise adaptations including cardiac function (ARHGAP26, HBEGF, SRA1), synapse development and locomotion (APBB3, ATXN7, CLSTN3), stress response (NR3C1) and the skeletal muscle response to exercise (ARHGAP26, NDUFA2). In a genome-wide association study for field-measured speed in two-year-olds (n = 179) SNPs contained within the single association peak (33.2-35.6 Mb) overlapped with the ECA14 CSS signals and spanned a protocadherin gene cluster. Association tests using higher density SNP genotypes across the ECA14 locus identified a SNP within the PCDHGC5 gene associated with elite racing performance (n = 922). These results indicate that there may be differential selection for racing performance under racing and management conditions that are specific to certain geographic racing regions. In Australia breeders have principally selected horses for favourable genetic variants at loci containing genes that modulate behaviour, locomotion and skeletal muscle physiology that together appear to be contributing to early two-year-old speed.

Genomic inbreeding trends, influential sire lines and selection in the global Thoroughbred horse population.

The Thoroughbred horse is a highly valued domestic animal population under strong selection for athletic phenotypes. Here we present a high resolution genomics-based analysis of inbreeding in the population that may form the basis for evidence-based discussion amid concerns in the breeding industry over the increasing use of small numbers of popular sire lines, which may accelerate a loss of genetic diversity. In the most comprehensive globally representative sample of Thoroughbreds to-date (n = 10,118), including prominent stallions (n = 305) from the major bloodstock regions of the world, we show using pan-genomic SNP genotypes that there has been a highly significant decline in global genetic diversity during the last five decades (FIS R2 = 0.942, P = 2.19 × 10-13; FROH R2 = 0.88, P = 1.81 × 10-10) that has likely been influenced by the use of popular sire lines. Estimates of effective population size in the global and regional populations indicate that there is some level of regional variation that may be exploited to improve global genetic diversity. Inbreeding is often a consequence of selection, which in managed animal populations tends to be driven by preferences for cultural, aesthetic or economically advantageous phenotypes. Using a composite selection signals approach, we show that centuries of selection for favourable athletic traits among Thoroughbreds acts on genes with functions in behaviour, musculoskeletal conformation and metabolism. As well as classical selective sweeps at core loci, polygenic adaptation for functional modalities in cardiovascular signalling, organismal growth and development, cellular stress and injury, metabolic pathways and neurotransmitters and other nervous system signalling has shaped the Thoroughbred athletic phenotype. Our results demonstrate that genomics-based approaches to identify genetic outcrosses will add valuable objectivity to augment traditional methods of stallion selection and that genomics-based methods will be beneficial to actively monitor the population to address the marked inbreeding trend.

Expression Quantitative Trait Loci in Equine Skeletal Muscle Reveals Heritable Variation in Metabolism and the Training Responsive Transcriptome.

While over ten thousand genetic loci have been associated with phenotypic traits and inherited diseases in genome-wide association studies, in most cases only a relatively small proportion of the trait heritability is explained and biological mechanisms underpinning these traits have not been clearly identified. Expression quantitative trait loci (eQTL) are subsets of genomic loci shown experimentally to influence gene expression. Since gene expression is one of the primary determinants of phenotype, the identification of eQTL may reveal biologically relevant loci and provide functional links between genomic variants, gene expression and ultimately phenotype. Skeletal muscle (gluteus medius) gene expression was quantified by RNA-seq for 111 Thoroughbreds (47 male, 64 female) in race training at a single training establishment sampled at two time-points: at rest (n = 92) and four hours after high-intensity exercise (n = 77); n = 60 were sampled at both time points. Genotypes were generated from the Illumina Equine SNP70 BeadChip. Applying a False Discovery Rate (FDR) corrected P-value threshold (P FDR < 0.05), association tests identified 3,583 cis-eQTL associated with expression of 1,456 genes at rest; 4,992 cis-eQTL associated with the expression of 1,922 genes post-exercise; 1,703 trans-eQTL associated with 563 genes at rest; and 1,219 trans-eQTL associated with 425 genes post-exercise. The gene with the highest cis-eQTL association at both time-points was the endosome-associated-trafficking regulator 1 gene (ENTR1; Rest: P FDR = 3.81 × 10-27, Post-exercise: P FDR = 1.66 × 10-24), which has a potential role in the transcriptional regulation of the solute carrier family 2 member 1 glucose transporter protein (SLC2A1). Functional analysis of genes with significant eQTL revealed significant enrichment for cofactor metabolic processes. These results suggest heritable variation in genomic elements such as regulatory sequences (e.g. gene promoters, enhancers, silencers), microRNA and transcription factor genes, which are associated with metabolic function and may have roles in determining end-point muscle and athletic performance phenotypes in Thoroughbred horses. The incorporation of the eQTL identified with genome and transcriptome-wide association may reveal useful biological links between genetic variants and their impact on traits of interest, such as elite racing performance and adaptation to training.

Refinement of Global Domestic Horse Biogeography Using Historic Landrace Chinese Mongolian Populations.

The Mongolian horse is one of the oldest extant horse populations and although domesticated, most animals are free-ranging and experience minimal human intervention. As an ancient population originating in one of the key domestication centers, the Mongolian horse may play a key role in understanding the origins and recent evolutionary history of horses. Here we describe an analysis of high-density genome-wide single-nucleotide polymorphism (SNP) data in 40 globally dispersed horse populations (n = 895). In particular, we have focused on new results from Chinese Mongolian horses (n = 100) that represent 5 distinct populations. These animals were genotyped for 670K SNPs and the data were analyzed in conjunction with 35K SNP data for 35 distinct breeds. Analyses of these integrated SNP data sets demonstrated that the Chinese Mongolian populations were genetically distinct from other modern horse populations. In addition, compared to other domestic horse breeds, the Chinese Mongolian horse populations exhibited relatively high genomic diversity. These results suggest that, in genetic terms, extant Chinese Mongolian horses may be the most similar modern populations to the animals originally domesticated in this region of Asia. Chinese Mongolian horse populations may therefore retain ancestral genetic variants from the earliest domesticates. Further genomic characterization of these populations in conjunction with archaeogenetic sequence data should be prioritized for understanding recent horse evolution and the domestication process that has led to the wealth of diversity observed in modern global horse breeds.

The "speed gene" effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion.

Thoroughbred horses are finely-tuned athletes with a high aerobic capacity relative to skeletal muscle mass, attributable to centuries of genetic selection for speed and stamina. Polymorphisms in the myostatin gene (MSTN), a pronounced inhibitor of skeletal muscle growth, have been shown to almost singularly account for gene-based race distance aptitude in racehorses. In Thoroughbreds, two MSTN polymorphisms, a single nucleotide variation in the first intron (SNP g.66493737C>T) and a non-coding transposable element within the promoter region (a 227 bp SINE insertion) are of particular interest. Until now, it has not been clear which of these variants affect skeletal muscle phenotypes or whether both can impact racing performance. In a large cohort of Thoroughbreds, we observed a complete concordance between the SNP and the SINE insertion. By means of in vitro assays in C2C12 myoblasts, we isolated the SNP variant from the SINE polymorphism and showed the latter is exclusively responsible for adversely affecting transcription initiation and gene expression thereby limiting myostatin protein production. Mapping the MSTN transcription start site in horse skeletal muscle likewise revealed anomalous transcription initiation in the presence of the SINE insertion. Our data provides mechanistic evidence that the SINE insertion uniquely accounts for the MSTN "speed gene" effect on race distance aptitude in the Thoroughbred horse.

Skeletal muscle mitochondrial bioenergetics and associations with myostatin genotypes in the Thoroughbred horse.

Variation in the myostatin (MSTN) gene has been reported to be associated with race distance, body composition and skeletal muscle fibre composition in the horse. The aim of the present study was to test the hypothesis that MSTN variation influences mitochondrial phenotypes in equine skeletal muscle. Mitochondrial abundance and skeletal muscle fibre types were measured in whole muscle biopsies from the gluteus medius of n = 82 untrained (21 ± 3 months) Thoroughbred horses. Skeletal muscle fibre type proportions were significantly (p < 0.01) different among the three MSTN genotypes and mitochondrial content was significantly (p < 0.01) lower in the combined presence of the C-allele of SNP g.66493737C>T (C) and the SINE insertion 227 bp polymorphism (I). Evaluation of mitochondrial complex activities indicated higher combined mitochondrial complex I+III and II+III activities in the presence of the C-allele / I allele (p ≤ 0.05). The restoration of complex I+III and complex II+III activities following addition of exogenous coenzyme Q1 (ubiquinone1) (CoQ1) in vitro in the TT/NN (homozygous T allele/homozygous no insertion) cohort indicated decreased coenzyme Q in these animals. In addition, decreased gene expression in two coenzyme Q (CoQ) biosynthesis pathway genes (COQ4, p ≤ 0.05; ADCK3, p ≤ 0.01) in the TT/NN horses was observed. This study has identified several mitochondrial phenotypes associated with MSTN genotype in untrained Thoroughbred horses and in addition, our findings suggest that nutritional supplementation with CoQ may aid to restore coenzyme Q activity in TT/NN horses.

Equine skeletal muscle adaptations to exercise and training: evidence of differential regulation of autophagosomal and mitochondrial components.

BACKGROUND: A single bout of exercise induces changes in gene expression in skeletal muscle. Regular exercise results in an adaptive response involving changes in muscle architecture and biochemistry, and is an effective way to manage and prevent common human diseases such as obesity, cardiovascular disorders and type II diabetes. However, the biomolecular mechanisms underlying such responses still need to be fully elucidated. Here we performed a transcriptome-wide analysis of skeletal muscle tissue in a large cohort of untrained Thoroughbred horses (n = 51) before and after a bout of high-intensity exercise and again after an extended period of training. We hypothesized that regular high-intensity exercise training primes the transcriptome for the demands of high-intensity exercise. RESULTS: An extensive set of genes was observed to be significantly differentially regulated in response to a single bout of high-intensity exercise in the untrained cohort (3241 genes) and following multiple bouts of high-intensity exercise training over a six-month period (3405 genes). Approximately one-third of these genes (1025) and several biological processes related to energy metabolism were common to both the exercise and training responses. We then developed a novel network-based computational analysis pipeline to test the hypothesis that these transcriptional changes also influence the contextual molecular interactome and its dynamics in response to exercise and training. The contextual network analysis identified several important hub genes, including the autophagosomal-related gene GABARAPL1, and dynamic functional modules, including those enriched for mitochondrial respiratory chain complexes I and V, that were differentially regulated and had their putative interactions 're-wired' in the exercise and/or training responses. CONCLUSION: Here we have generated for the first time, a comprehensive set of genes that are differentially expressed in Thoroughbred skeletal muscle in response to both exercise and training. These data indicate that consecutive bouts of high-intensity exercise result in a priming of the skeletal muscle transcriptome for the demands of the next exercise bout. Furthermore, this may also lead to an extensive 're-wiring' of the molecular interactome in both exercise and training and include key genes and functional modules related to autophagy and the mitochondrion.

Skeletal muscle adaptations and muscle genomics of performance horses.

Skeletal muscles in horses are characterised by specific adaptations, which are the result of the natural evolution of the horse as a grazing animal, centuries of selective breeding and the adaptability of this tissue in response to training. These adaptations include an increased muscle mass relative to body weight, a great locomotor efficiency based upon an admirable muscle-tendon architectural design and an adaptable fibre-type composition with intrinsic shortening velocities greater than would be predicted from an animal of comparable body size. Furthermore, equine skeletal muscles have a high mitochondrial volume that permits a higher whole animal aerobic capacity, as well as large intramuscular stores of energy substrates (glycogen in particular). Finally, high buffer and lactate transport capacities preserve muscles against fatigue during anaerobic exercise. Many of these adaptations can improve with training. The publication of the equine genome sequence in 2009 has provided a major advance towards an improved understanding of equine muscle physiology. Equine muscle genomics studies have revealed a number of genes associated with elite physical performance and have also identified changes in structural and metabolic genes following exercise and training. Genes involved in muscle growth, muscle contraction and specific metabolic pathways have been found to be functionally relevant for the early performance evaluation of elite athletic horses. The candidate genes discussed in this review are important for a healthy individual to improve performance. However, muscle performance limiting conditions are widespread in horses and many of these conditions are also genetically influenced.

Genetic diversity in the modern horse illustrated from genome-wide SNP data.

Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. F(ST) 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.

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.

Horses for courses: a DNA-based test for race distance aptitude in thoroughbred racehorses.

Variation at the myostatin (MSTN) gene locus has been shown to influence racing phenotypes in Thoroughbred horses, and in particular, early skeletal muscle development and the aptitude for racing at short distances. Specifically, a single nucleotide polymorphism (SNP) in the first intron of MSTN (g.66493737C/T) is highly predictive of best race distance among Flat racing Thoroughbreds: homozygous C/C horses are best suited to short distance races, heterozygous C/T horses are best suited to middle distance races, and homozygous T/T horses are best suited to longer distance races. Patent applications for this gene marker association, and other linked markers, have been filed. The information contained within the patent applications is exclusively licensed to the commercial biotechnology company Equinome Ltd, which provides a DNA-based test to the international Thoroughbred horse racing and breeding industry. The application of this information in the industry enables informed decision making in breeding and racing and can be used to assist selection to accelerate the rate of change of genetic types among distinct populations (Case Study 1) and within individual breeding operations (Case Study 2).

MSTN genotypes in Thoroughbred horses influence skeletal muscle gene expression and racetrack performance.

Myostatin, encoded by the MSTN gene, is a member of the TGF-ß superfamily that regulates skeletal muscle development. A MSTN SNP significantly associated with Thoroughbred horse racing phenotypes has recently been identified as well as significant reductions in Thoroughbred skeletal muscle gene expression for three transcripts 400-1500 base pairs downstream of the MSTN gene following a period of training. Together, these findings indicate that MSTN genotypes may influence MSTN gene expression. To investigate this, MSTN mRNA expression was measured in biopsies from the middle gluteal muscle from 60 untrained yearling Thoroughbreds (C/C, n = 15; C/T, n = 28; T/T, n = 17) using two independent real-time qRT-PCR assays. MSTN gene expression was also evaluated in a subset (N = 33) of these animals using samples collected after a ten-month period of training. A significant association was observed between genotype and mRNA abundance for the untrained horses (assay I, P = 0.0237; assay II, P = 0.003559), with the C/C cohort having the highest MSTN mRNA levels, the T/T group the lowest levels and the C/T group intermediate levels. Following training, there was a significant decrease in MSTN mRNA (-3.35-fold; P = 6.9 × 10(-7) ), which was most apparent for the C/C cohort (-5.88-fold, P = 0.001). These data demonstrate the tight relationship between phenotype, genotype and gene expression at the MSTN gene in Thoroughbred racehorses.