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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.

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 ( F ROH ) 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 F ROH . 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.

BACKGROUND: Conflicting results have been reported for risk factors for recurrent laryngeal neuropathy (RLN) based on resting endoscopic evaluation and comparison of single conformation traits, with many traits correlated to one another. OBJECTIVES: To simplify identification of signalment and conformation traits (i.e. variables) associated with RLN cases and controls diagnosed with exercising overground endoscopy (OGE) using exploratory factor analysis (EFA). STUDY DESIGN: Prospective cohort. METHODS: Pearson's rank correlation was used to establish significance and association between variables collected from n = 188 Thoroughbreds from one stable by observers blinded to OGE results. Exploratory factor analysis was conducted on nine variables for cases and controls; common elements between variables developed a factor, with variables grouped into three factors for cases and controls respectively. Correlation (loading) between each variable and factor was calculated to rank relationships between variables and cases/controls, with factors retrospectively named based on their underlying correlations with variables. RESULTS: Numerous inter‐correlations were present between variables. Most strongly correlated in cases were wither height with body weight (r = 0.70) and ventral neck length (r = 0.68) and in controls body weight with rostral neck circumference (r = 0.58). Wither height (r = 0.61) significantly loaded the top‐ranked factor for cases (‘heightRLN’), explaining 25% of conformational variance. Ventral neck length (r = 0.69) and age (r = 0.57) significantly loaded the second‐ranked factor for cases (‘neck lengthRLN’), explaining 16% of conformational variance. Rostral neck circumference (r = 0.86) and body weight (r = 0.6) significantly loaded the top‐ranked factor for controls (‘body sizeCON’), explaining 19% of the variance. Wither height (r = 0.84) significantly loaded the second‐ranked factor for controls (‘heightCON’), explaining 13% of the variance. MAIN LIMITATIONS: Horses had not reached skeletal maturity. CONCLUSIONS: Exploratory factor analysis allowed weightings to be determined for each variable. Wither height was the predominant conformational feature associated with RLN. Exploratory factor analysis confirms aggregated conformational differences exist between RLN cases and controls, suitable for future evaluations.

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high-density SNP array data, additive SNP heritability (hSNP2) was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome-wide association studies (GWAS) for age at first race and age at best race, a 1.98-Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10-12 and P = 1.89 × 10-14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10-5 ). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4007-9) contains supplementary material, which is available to authorized users.

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 (FISR2 = 0.942, P = 2.19 × 10−13; FROHR2 = 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.

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 (FISR2= 0.942,P= 2.19 × 10−13;FROHR2= 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.Author SummaryIn the highly valuable global Thoroughbred horse industry, there is no systematic industry-mediated genetic population management. Purposeful inbreeding is common practice and there is an increasing use of popular sires. Inbreeding can lead to population health and fertility decline, but there is little objective genomics-based data for the Thoroughbred to catalyse action and support changes in breeding practices. Here, we describe the most comprehensive genetic analysis in the population among 10,000 Thoroughbreds from the major bloodstock regions of the world and reveal a highly significant increase in inbreeding during the last five decades. The main drivers of genetic diversity are the most influential ‘breed-shaping’ sire lines,Sadler’s Wells, DanehillandA.P. Indy. We identified genomic regions subject to positive selection containing genes for athletic traits. Our results highlight the need for population-wide efforts to proactively avert the potential increase of deleterious alleles that may impact on animal health in order to safeguard the future of a breed that is admired for its athleticism and enjoyed for sport worldwide.

Despite strong selection for athletic traits in Thoroughbred horses, there is marked variation in speed and aptitude for racing performance within the breed. Using global positioning system monitoring during exercise training, we measured speed variables and temporal changes in speed with age to derive phenotypes for GWAS. The aim of the study was to test the hypothesis that genetic variation contributes to variation in end‐point physiological traits, in this case galloping speed measured during field exercise tests. Standardisation of field‐measured phenotypes was attempted by assessing horses exercised on the same gallop track and managed under similar conditions by a single trainer. PCA of six key speed indices captured 73.9% of the variation with principal component 1 (PC1). Verifying the utility of the phenotype, we observed that PC1 (median) in 2‐year‐old horses was significantly different among elite, non‐elite and unraced horses (P < 0.001) and the temporal change with age in PC1 varied among horses with different myostatin (MSTN) g.66493737C>T SNP genotypes. A GWAS for PC1 in 2‐year‐old horses (n = 122) identified four SNPs reaching the suggestive threshold for association (P < 4.80 × 10−5), defining a 1.09 Mb candidate region on ECA8 containing the myosin XVIIIB (MYO18B) gene. In a GWAS for temporal change in PC1 with age (n = 168), five SNPs reached the suggestive threshold for association and defined candidate regions on ECA2 and ECA11. Both regions contained genes that are significantly differentially expressed in equine skeletal muscle in response to acute exercise and training stimuli, including MYO18A. As MYO18A plays a regulatory role in the skeletal muscle response to exercise, the identified genomic variation proximal to the myosin family genes may be important for the regulation of the response to exercise and training.

Durability traits in Thoroughbred horses are heritable, economically valuable and may affect horse welfare. The aims of this study were to test the hypotheses that (i) durability traits are heritable and (ii) genetic data may be used to predict a horse's potential to have a racecourse start. Heritability for the phenotype ‘number of 2‐ and 3‐year‐old starts’ was estimated to be urn:x-wiley:02689146:media:age12798:age12798-math-0001 = 0.11 ± 0.02 (n = 4499). A genome‐wide association study identified SNP contributions to the trait. The neurotrimin (NTM), opioid‐binding protein/cell adhesion molecule like (OPCML) and prolylcarboxypeptidase (PRCP) genes were identified as candidate genes associated with the trait. NTM functions in brain development and has been shown to have been selected during the domestication of the horse. PRCP is an established expression quantitative trait locus involved in the interaction between voluntary exercise and body composition in mice. We hypothesise that variation at these loci contributes to the motivation of the horse to exercise, which may influence its response to the demands of the training and racing environment. A random forest with mixed effects (RFME) model identified a set of SNPs that contributed to 24.7% of the heritable variation in the trait. In an independent validation set (n = 528 horses), the cohort with high genetic potential for a racecourse start had significantly fewer unraced horses (16% unraced) than did low (27% unraced) potential horses and had more favourable race outcomes among those that raced. Therefore, the information from SNPs included in the model may be used to predict horses with a greater chance of a racecourse start.

Background: Race distance aptitude in Thoroughbred horses is highly heritable and is influenced largely by variation at the myostatin gene (MSTN). Objectives: In addition to MSTN, we hypothesised that other modifying loci contribute to best race distance. Study design: Using 3006 Thoroughbreds, including 835 ‘elite’ horses, which were >3 years old, had race records and were sampled from Europe/ Middle-East, Australia/New Zealand, North America and South Africa, we performed genome-wide association (GWA) tests and separately developed a genomic prediction algorithm to comprehensively catalogue additive genetic variation contributing to best race distance. Methods: 48,896 single‐nucleotide polymorphism (SNP) genotypes were generated from high‐density SNP genotyping arrays. Heritability estimates, tests of GWA and genomic prediction models were derived for the phenotypes: average race distance, best race distance for elite, nonelite and all winning horses. Results: Heritability estimates were high (urn:x-wiley:04251644:media:evj13058:evj13058-math-0001 = 0.51, best race distance – elite; urn:x-wiley:04251644:media:evj13058:evj13058-math-0002 = 0.42, best race distance – nonelite; urn:x-wiley:04251644:media:evj13058:evj13058-math-0003 = 0.40, best race distance – all) and most of the variation was attributed to the MSTN gene. MSTN locus SNPs were the most strongly associated with the trait and included BIEC2‐438999 (ECA18:66913090; P = 4.51 × 10−110, average race distance; P = 2.33 × 10−42, best race distance – elite). The genomic prediction algorithm enabled the inclusion of variation from all SNPs in a model that partitioned horses into short and long cohorts following assignment of MSTN genotype. Additional genes with minor contributions to best race distance were identified. Main limitations: The nongenetic influence of owner/trainer decisions on placement of horses in suitable races could not be controlled. Conclusions: MSTN is the single most important genetic contributor to best race distance in the Thoroughbred. Employment of genetic prediction models will lead to more accurate placing of horses in races that are best suited to their inherited genetic potential for distance aptitude.

Exercise in horses induces neutrophil degranulation and subsequent increases in plasma myeloperoxidase concentrations (MPO). It is not known whether this response is affected by training or the sampling time in relation to exercise. Our objective was to evaluate plasma MPO concentration at different time points in response to exercise in Thoroughbreds before and following high-intensity training and to evaluate relationships between plasma MPO concentration, physiological measurements and performance. Throughbred racehorses in active training and racing (n=26) performed an exercise test on a high-speed treadmill at least once at the beginning (first three months), middle (second three months) or end (last three months) of a nine month training period with training intensity (number of fast work sessions) collated for each period. Heart rate, speed and distance were recorded and venous blood collected before (T0), during and up to maximal speed (TVmax), 5 min (T5min) and 4 h (T4h) following exercise for measurement of plasma lactate, serum creatine kinase concentration, plasma MPO concentration, white blood count (WBC), neutrophil count (Neut), lymphocyte count and neutrophil percentage (%Neut). WBC increased above T0 values at all sampling points with no training effect. Neut and %Neut increased above T0 values at T4h, while values at TVmax, T5min and T4h increased as training duration increased. Plasma MPO concentration increased above T0 values at all sampling points in response to exercise with all values increasing as training duration increased. Training intensity did not affect any of the measurements, correlations were not identified between any of the biological markers including MPO and physiological and training measurements and no differences were identified between elite versus non-elite horses. It was concluded that plasma MPO concentration is affected by exercise and training duration, and that further investigation of plasma MPO as an assessor of fitness and readiness for competition is warranted.

Sequence variation at the equine myostatin gene ( MSTN) locus has previously been shown to have a singular genomic influence on optimum race distance in Thoroughbred racehorses. Myostatin, encoded by the MSTN gene, is a member of the TGF-β superfamily that regulates skeletal muscle development in a range of mammalian species including the horse. In the Thoroughbred, the C-allele at the g.66493737C/T SNP has been found at significantly higher frequency in subgroups of the population that are suited to fast, short distance, sprint races and also influences body composition phenotypes. We investigated the influence of the g.66493737C/T SNP on speed indexes measured in a cohort of n = 85 Thoroughbred horses-in-training. We found significant associations between genotypes at the g.66493737C/T SNP and all measured speed variables: Dist6 [distance travelled during 6 s before and after maximal velocity (Vmax); P = 0.0040], Vmaxt (duration at Vmax; P = 0.0249), Vmax ( P = 0.0265), Dist6b (distance travelled during 6 s before Vmax; P = 0.0032), and Dist6a(distance travelled during 6 s after Vmax; P = 0.0317). For each measure, horses with the C/C and C/T genotypes outperformed T/T horses, indicating the requirement for at least one C-allele to improve speed. For the most significantly associated variables (Dist6 and Dist6b) the C/C cohort performed better than the T/T cohort with the heterozygotes intermediate, indicating a dose-dependent manifestation. These findings clearly indicate that variation at the MSTN gene influences speed in Thoroughbred horses.

Athletic performance is influenced by a complex interplay among the environment and a suite of genes, which contributes to system-wide structure and function. In a panel of elite and non-elite Thoroughbred horses (n=148), we genotyped 68 SNPs in 17 putative exercise-relevant genes chosen from a genome scan for selection. We performed a series of case-control and quantitative association tests for relationships with racecourse performance. Thirteen SNPs in nine genes were significantly (P

Summary Reasons for performing study: The role of molecular signalling pathways in the phenotypic adaptation of skeletal muscle to different exercise stimuli in the Thoroughbred horse has not been reported previously. Objective: To examine CKM, COX4I1, COX4I2 and PDK4 gene expression following high intensity sprint and moderate intensity treadmill exercise stimuli in skeletal muscle of Thoroughbred horses. Materials and methods: Two groups of trained 3-year-old Thoroughbred horses participated. Group A (n = 6 females, n = 3 males) participated in an incremental step test (moderate intensity) to fatigue or HRmax on a Sato high speed treadmill (distance = 5418.67m 343.21). Group B (n = 8 females) participated in routine 'work' (sprint) on an all- weather gallop (distance = 812.83 m 12.53). Biopsy samples were obtained from the gluteus medius pre-exercise (T0), immediately post exercise (T1) and 4 h post exercise (T2). For physiological relevance venous blood samples were collected to measure plasma lactate and creatine kinase concentrations. Changes in mRNA expression were determined by quantitative real-time RT-PCR for creatine kinase muscle (CKM), cytochrome c oxidase subunit IV isoform 1 (COX4I1), cytochrome c oxidase subunit IV isoform 2 (COX4I2) and pyruvate dehydrogenase kinase, isozyme 4 (PDK4) genes. Statistical significance (a

Summary Reasons for performing study: The wild progenitors of the domestic horse were subject to natural selection for speed and stamina for millennia. Uniquely, this process has been augmented in Thoroughbreds, which have undergone at least 3 centuries of intense artificial selection for athletic phenotypes. While the phenotypic adaptations to exercise are well described, only a small number of the underlying genetic variants contributing to these phenotypes have been reported. Objectives: A panel of candidate performance-related genes was examined for DNA sequence variation in Thoroughbreds and the association with racecourse performance investigated. Materials and methods: Eighteen candidate genes were chosen for their putative roles in exercise. Re-sequencing in Thoroughbred samples was successful for primer sets in 13 of these genes. SNPs identified in this study and from the EquCab2.0 SNP database were genotyped in 2 sets of Thoroughbred samples (n = 150 and 148) and a series of population-based case-control investigations were performed by separating the samples into discrete cohorts on the basis of retrospective racecourse performance. Results: Twenty novel SNPs were detected in 3 genes: ACTN3, CKM and COX4I2. Genotype frequency distributions for 3 SNPs in CKM and COX4I2 were significantly (P T was confirmed (P

Circadian rhythms are innate 24-h cycles in behavioral and biochemical processes that permit physiological anticipation of daily environmental changes. Elucidating the relationship between activity rhythms and circadian patterns of gene expression may contribute to improved human and equine athletic performance. Six healthy, untrained mares were studied to determine whether locomotor activity behavior and skeletal muscle gene expression reflect endogenous circadian regulation. Activity was recorded for three consecutive 48-h periods: as a group at pasture (P), and individually stabled under a light-dark (LD) cycle and in constant darkness (DD). Halter-mounted Actiwatch-L data-loggers recorded light exposure and motor activity. Analysis of mean activity (average counts/min, activity bouts/day, average bout length) and cosinor parameters (acrophase, amplitude, mesor, goodness of fit) revealed a predominantly ultradian (8.9 ± 0.7 bouts/24 h) and weakly circadian pattern of activity in all three conditions (P, LD, DD). A more robust circadian pattern was observed during LD and DD. Muscle biopsies were obtained from the middle gluteal muscles every 4 h for 24 h under DD. One-way qRT-PCR results confirmed the circadian expression ( P < 0.05) of six core clock genes ( Arntl, Per1, Per2, Nr1d1, Nr1d2, Dbp) and the muscle-specific transcript, Myf6. Additional genes, Ucp3, Nrip1, and Vegfa, demonstrated P values approaching significance. These findings demonstrate circadian regulation of muscle function and imply that human management regimes may strengthen, or unmask, equine circadian behavioral outputs. As exercise synchronizes circadian rhythms, our findings provide a basis for future work determining peak times for training and competing horses, to reduce injury and to achieve optimal performance.

Myostatin, encoded by the MSTN gene, is a member of the TGF-β 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.

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

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).

Campylobacter jejuni colonises the caecum of more than 90% of commercial chickens. Even though colonisation is asymptomatic, we hypothesised that it is mediated by activation of several biological pathways. We therefore used chicken-specific 20K oligonucleotide microarrays to examine global gene expression in C. jejuni-challenged birds. Microarray results demonstrate small but significant fold-changes in expression of 270 genes 20 h post-challenge, corresponding to a wide range of biological processes including cell growth, nutrient metabolism and immunological activity. Expression of NOX1 (2.3-fold) and VCAM1 (1.5-fold) were significantly increased in colonised birds (P

Variants of the MSTN gene encoding myostatin are associated with muscle hypertrophy phenotypes in a range of mammalian species, most notably cattle, dogs, mice, and humans. Using a sample of registered Thoroughbred horses (n = 148), we have identified a novel MSTN sequence polymorphism that is strongly associated (g.66493737C>T, P = 4.85x10(-8)) with best race distance among elite racehorses (n = 79). This observation was independently validated (P = 1.91x10(-6)) in a resampled group of Thoroughbreds (n = 62) and in a cohort of Thoroughbreds (n = 37, P = 0.0047) produced by the same trainer. We observed that C/C horses are suited to fast, short-distance races; C/T horses compete favorably in middle-distance races; and T/T horses have greater stamina. Evaluation of retrospective racecourse performance (n = 142) and stallion progeny performance predict that C/C and C/T horses are more likely to be successful two-year-old racehorses than T/T animals. Here we describe for the first time the identification of a gene variant in Thoroughbred racehorses that is predictive of genetic potential for an athletic phenotype.

Background Selection for exercise-adapted phenotypes in the Thoroughbred racehorse has provided a valuable model system to understand molecular responses to exercise in skeletal muscle. Exercise stimulates immediate early molecular responses as well as delayed responses during recovery, resulting in a return to homeostasis and enabling long term adaptation. Global mRNA expression during the immediate-response period has not previously been reported in skeletal muscle following exercise in any species. Also, global gene expression changes in equine skeletal muscle following exercise have not been reported. Therefore, to identify novel genes and key regulatory pathways responsible for exercise adaptation we have used equine-specific cDNA microarrays to examine global mRNA expression in skeletal muscle from a cohort of Thoroughbred horses (n = 8) at three time points (before exercise, immediately post-exercise, and four hours post-exercise) following a single bout of treadmill exercise. Results Skeletal muscle biopsies were taken from the gluteus medius before (T0), immediately after (T1) and four hours after (T2) exercise. Statistically significant differences in mRNA abundance between time points (T0 vs T1 and T0 vs T2) were determined using the empirical Bayes moderated t-test in the Bioconductor package Linear Models for Microarray Data (LIMMA) and the expression of a select panel of genes was validated using real time quantitative reverse transcription PCR (qRT-PCR). While only two genes had increased expression at T1 (P < 0.05), by T2 932 genes had increased (P < 0.05) and 562 genes had decreased expression (P < 0.05). Functional analysis of genes differentially expressed during the recovery phase (T2) revealed an over-representation of genes localized to the actin cytoskeleton and with functions in the MAPK signalling, focal adhesion, insulin signalling, mTOR signaling, p53 signaling and Type II diabetes mellitus pathways. At T1, using a less stringent statistical approach, we observed an over-representation of genes involved in the stress response, metabolism and intracellular signaling. These findings suggest that protein synthesis, mechanosensation and muscle remodeling contribute to skeletal muscle adaptation towards improved integrity and hypertrophy. Conclusions This is the first study to characterize global mRNA expression profiles in equine skeletal muscle using an equine-specific microarray platform. Here we reveal novel genes and mechanisms that are temporally expressed following exercise providing new knowledge about the early and late molecular responses to exercise in the equine skeletal muscle transcriptome.

Intense selection for elite racing performance in the Thoroughbred horse ( Equus caballus) has resulted in a number of adaptive physiological phenotypes relevant to exercise; however, the underlying molecular mechanisms responsible for these characteristics are not well understood. Adaptive changes in mRNA expression in equine skeletal muscle were investigated by real-time qRT-PCR for a panel of candidate exercise-response genes following a standardized incremental-step treadmill exercise test in eight untrained Thoroughbred horses. Biopsy samples were obtained from the gluteus medius before, immediately after, and 4 h after exercise. Significant ( P < 0.05) differences in gene expression were detected for six genes ( CKM, COX4I1, COX4I2, PDK4, PPARGC1A, and SLC2A4) 4 h after exercise. Investigation of relationships between mRNA and velocity at maximum heart rate (VHRmax) and peak postexercise plasma lactate concentration ([La]T1) revealed significant ( P < 0.05) associations with postexercise COX4I1 and PPARCG1A expression and between [La]T1and basal COX4I1 expression. Gene expression changes were investigated in a second cohort of horses after a 10 mo period of training. In resting samples, COX4I1 gene expression had significantly increased following training, and, after exercise, significant differences were identified for COX4I2, PDK4, and PPARGC1A. Significant relationships with VHRmaxand [La]T1were detected for PPARGC1A and COX4I1. These data highlight the roles of genes responsible for the regulation of oxygen-dependent metabolism, glucose metabolism, and fatty acid utilization in equine skeletal muscle adaptation to exercise.

Introduction In Thoroughbreds, genetic variation in myostatin (MSTN g.66493737C/T) is highly predictive of optimum race distance and influences mRNA expression, precocity, skeletal muscle development, two-year-old earnings, speed variables and skeletal muscle fibre type. The aim of this study was to investigate MSTN variation associations with VO2peak in untrained yearling Thoroughbreds. Methods Peak oxygen consumption (VO2peak) was measured in 41 untrained Thoroughbred yearlings (16 colts, 25 fillies, 19.8 ± 1.4 months) from one flat racing yard (November-December). Data was collected as horses freely travelled up an all-weather gallop (2% incline) with no rider. VO2 was measured using an ergospirometry and oximetry mask and associated customized software validated for field testing. Horses were instrumented with a GPS-HR monitor, light-weight mask and data-capture equipment. HRpeak, maximal velocity (Vmax) and exercise distance were recorded. MSTN genotypes were determined using a customised Taqman SNP genotyping assay. Tests of association between genotype and phenotype were performed using PLINK software. Results Average exercise distance was 1700.5 ± 33.4 m, Vmax 15.0 ± 1.4 m/s, and HRpeak 221.2 ± 9.6 bpm. MSTN genotypes and VO2peak based on 6 breaths were n = 16 CC, VO2peak = 119.49 ± 21.8; n = 22 CT, VO2peak = 116.40 ± -27.2; n = 3 TT, 115.08 ± 20.2.There was no association (P>0.05) between genotype and VO2peak. Conclusions In yearling Thoroughbreds no association between MSTN genotype and untrained peak oxygen consumption was observed. This may be a function of the small number of TT genotype individuals evaluated or a reflection of a requirement for MSTN-associated skeletal muscle entrainment in VO2peak changes resulting from a period of exercise training. Such changes may be associated with MSTN genotype and this requires further investigation. Ethical Animal Research Institutional Animal Research Ethics Committee approval was obtained. Explicit owner informed consent for participation in this study is not stated. Sources of funding: Science Foundation Ireland under Grant Number 11/PI/1166. Competing interests: Dr Hill is a co-founder and Chairman of Equinome Ltd.

Introduction Recurrent laryngeal neuropathy (RLN) is considered height-linked in Thoroughbreds. A SNP (BIEC2-808543) proximal to the LCORL/NCAPG gene significantly influences height variation in horses. It has been suggested that the C-allele of this SNP is associated with RLN risk. Our objective was to investigate the association between the BIEC2-808543 C-allele, RLN and height in a cohort of Thoroughbreds. Methods Resting and exercising upper respiratory tract endoscopic videos, laryngeal ultrasonograms, wither height, age and sex were recorded in 79 Thoroughbreds (age 34 ± 11 months). The Havemeyer grading scale was used to characterise RLN cases (Grade B or C at exercise) and controls (Grade 1.1 or 2.1 at rest, Grade A at exercise, normal laryngeal ultrasound). All horses were genotyped for the BIEC2-808543 SNP using a custom TaqMan SNP Genotyping assay. Logistic regression was performed measuring additive effects of allele dosage with sex, age and height as covariates (P≤0.05). Horses were subcategorised into tall (T,≥162 cm) and short (S

Introduction Body composition including percentage of fat, fat mass and fat-free mass (FFM) is an important consideration for athletes. An evaluation of FFM in racehorses concluded that racing performance is positively correlated with FFM both pre- and post-training. Athletic performance is influenced by interactions among the environment and genes that influence anatomical, metabolic and physiological traits relevant to exercise. The aim of this study was to determine the effects of genetic variation in six candidate genes with functions in growth and metabolism (IL1RA, PTPN1, MC3R, IGF1, LEPR and MSTN) on FFM in Thoroughbreds. Methods FFM was calculated from rump fat thickness measured ultrasonographically in 110 two-year-old Thoroughbred horses-in-training in the same yard after two (T2) and eight (T8) months of training. Twenty-one SNPs in six genes were selected from the EquCab2.0 SNP database and were genotyped for all horses using custom Sequenom SNP genotyping assays. Quantitative tests of association were performed between SNPs and FFM at T2 and T8, and the FFM ratio between the two time points (T2 : T8) using PLINK software. Results Two SNPs in both the IGF1 (Insulin-Like Growth Factor 1) and PTPN1 (Protein Tyrosine Phosphatase, Non-Receptor Type 1) genes were significantly associated with FFM (T2) (P

Introduction Measurement of maximal oxygen consumption (VO2max) is the gold standard for assessing aerobic capacity, but in horses requires use of stationary equipment and a high-speed treadmill. Study objectives were to evaluate a portable system designed to measure VO2 in a field setting and to describe these measurements in a cohort of untrained Thoroughbreds. Methods Ventilatory parameters and VO2 were measured in 41 untrained Thoroughbreds (16 colts, 25 fillies, 19.8 ± 1.4 months) using an ergospirometry and oximetry mask with customized software validated for horses galloping on a treadmill with an open-circuit flow-through gas collection system. Horses were instrumented with a GPS-HR monitor, light-weight mask and data-capture equipment. Data was collected as horses travelled riderless up a 2% inclined woodchip track. Pre- and 5 minute post-exercise plasma lactate ([La5]), HRpeak, maximal velocity (Vmax) and exercise distance were recorded and analysed by unpaired t-test and Pearson's correlation (P ≤ 0.05). Results All horses tolerated the mask and saddle pad. Exercise distance was 1700.5 ± 33.4 m, Vmax 15.0 ± 1.4 m/s, HRpeak 221.2 ± 9.6 bpm and [La5] 22.7 ± 5.5 mmol/l. Average VO2peak, based on 6 breaths, was 117.7 ± 24.1 ml/kg/min, breathing frequency 125.8 ± 7.1 bpm, inspiratory volume 13.2 ± 1.7 L, expiratory volume 13.4 ± 1.9 L and minute ventilation 1669.3 ± 193.3 L/min. VO2peak was not significantly different between colts (112.2 ± 27.7 ml/kg/min) and fillies (121.4 ± 21.3 ml/kg/min). There were no correlations between VO2peak and other measured parameters. There was a positive linear correlation between VO2peak and HRpeak (P = 0.046, R2 = 0.16). Conclusions VO2peak values were similar to published reports for yearling Thoroughbreds. The high exercise intensity and linear relationship between VO2peak and HRpeak provides confidence in the system for field studies of aerobic capacity in untrained, unridden horses. Ethical Animal Research Institutional Animal Research Ethics Committee approval was obtained. Sources of funding: Science Foundation Ireland (11-P1- 1166). Competing interests: None.

Introduction PGC-1α and irisin, encoded by the PPARGC1A and FNDC5 genes, play key roles in mediating the beneficial effects of exercise. PPARGC1A gene expression is induced by exercise and has been shown to upregulate FNDC5 expression. Higher levels of FNDC5 have been observed in young male human athletes compared to sedentary controls. FNDC5 gene expression has also been shown in some species to be modulated by myostatin (MSTN), a growth factor that limits muscle hypertrophy. In horses MSTN variation (g.66493737C>T) affects gene expression and racing performance. Thoroughbreds homozygous for the C-allele have significantly higher levels of MSTN mRNA than homozygous T : T horses. The aim of this study was to investigate the responses of PPARGC1A and FNDC5 in skeletal muscle to exercise and investigate the relationship with MSTN genotype. Methods Skeletal muscle (gluteus medius) biopsies were taken from untrained yearling Thoroughbreds (n = 27) at rest and after a ten month period of training at two-years-old. Skeletal muscle gene expression was measured using real-time qRT-PCR assays and data was analysed using qBase software. Results A significant increase in both PPARGC1A (P = 0.0001, FC = 1.82) and FNDC5 (P = 0.039, FC = 1.72) was observed post-training. A significant positive correlation was found between FNDC5 and PPARGC1A expression levels post training (P = 0.01, R2 = 0.22). There was no relationship between PPARGC1A or FNDC5 gene expression and MSTN genotype. Conclusions These data indicate a coordinated role for PGC-1α and irisin in the skeletal muscle response to exercise in Thoroughbred horses. Their known roles in mitochondrial biogenesis and brown fat metabolism suggest important roles in the adaptive response to exercise, which is independent of MSTN-determined muscle type. Ethical Animal Research Institutional Animal Research Ethics Committee approval was obtained. Explicit owner informed consent for participation in this study is not stated. Sources of funding: Science Foundation Ireland under Grant Number 11/PI/1166. Competing interests: Dr Hill is a co-founder and Chairman of Equinome Ltd.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse., Author Summary A breed of the horse typically consists of individuals sharing very similar aesthetic and performance traits. However, a great deal of variation in traits exists between breeds. The range of variation observed among breeds can be illustrated by the size difference between the Miniature horse (0.74 m and 100 kg) and draft horse (1.8 m and 900 kg), or by comparing the optimum racing distance of the Quarter Horse (1/4 mile) to that of the Arabian (100 miles or more). In this study, we exploited the breed structure of the horse to identify regions of the genome that are significantly different between breeds and therefore may harbor genes and genetic variants targeted by selective breeding. This work resulted in the identification of variants in the Paint and Quarter Horse significantly associated with altered muscle fiber type proportions favorable for increased sprinting ability. A strong signature of selection was also identified in breeds that perform alternative gaits, and several genomic regions identified are hypothesized to be involved in the determination of size. This study has demonstrated the utility of this approach for studying the equine genome and is the first to show a functional consequence of selective breeding in the horse.

Background Thoroughbred horses have been selected for traits contributing to speed and stamina for centuries. It is widely recognized that inherited variation in physical and physiological characteristics is responsible for variation in individual aptitude for race distance, and that muscle phenotypes in particular are important. Results A genome-wide SNP-association study for optimum racing distance was performed using the EquineSNP50 Bead Chip genotyping array in a cohort of n = 118 elite Thoroughbred racehorses divergent for race distance aptitude. In a cohort-based association test we evaluated genotypic variation at 40,977 SNPs between horses suited to short distance (≤ 8 f) and middle-long distance (> 8 f) races. The most significant SNP was located on chromosome 18: BIEC2-417495 ~690 kb from the gene encoding myostatin (MSTN) [Punadj. = 6.96 × 10-6]. Considering best race distance as a quantitative phenotype, a peak of association on chromosome 18 (chr18:65809482-67545806) comprising eight SNPs encompassing a 1.7 Mb region was observed. Again, similar to the cohort-based analysis, the most significant SNP was BIEC2-417495 (Punadj. = 1.61 × 10-9; PBonf. = 6.58 × 10-5). In a candidate gene study we have previously reported a SNP (g.66493737C>T) in MSTN associated with best race distance in Thoroughbreds; however, its functional and genome-wide relevance were uncertain. Additional re-sequencing in the flanking regions of the MSTN gene revealed four novel 3' UTR SNPs and a 227 bp SINE insertion polymorphism in the 5' UTR promoter sequence. Linkage disequilibrium was highest between g.66493737C>T and BIEC2-417495 (r2 = 0.86). Conclusions Comparative association tests consistently demonstrated the g.66493737C>T SNP as the superior variant in the prediction of distance aptitude in racehorses (g.66493737C>T, P = 1.02 × 10-10; BIEC2-417495, Punadj. = 1.61 × 10-9). Functional investigations will be required to determine whether this polymorphism affects putative transcription-factor binding and gives rise to variation in gene and protein expression. Nonetheless, this study demonstrates that the g.66493737C>T SNP provides the most powerful genetic marker for prediction of race distance aptitude in Thoroughbreds.

Background Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Skeletal muscle biopsies were collected at rest from the gluteus medius at two time points: T1 - untrained, (9 ± 0.5 months old) and T2 - trained (20 ± 0.7 months old). Results The most abundant mRNA transcripts in the muscle transcriptome were those involved in muscle contraction, aerobic respiration and mitochondrial function. A previously unreported over-representation of genes related to RNA processing, the stress response and proteolysis was observed. Following training 92 tags were differentially expressed of which 74 were annotated. Sixteen genes showed increased expression, including the mitochondrial genes ACADVL, MRPS21 and SLC25A29 encoded by the nuclear genome. Among the 58 genes with decreased expression, MSTN, a negative regulator of muscle growth, had the greatest decrease. Functional analysis of all expressed genes using FatiScan revealed an asymmetric distribution of 482 Gene Ontology (GO) groups and 18 KEGG pathways. Functional groups displaying highly significant (P < 0.0001) increased expression included mitochondrion, oxidative phosphorylation and fatty acid metabolism while functional groups with decreased expression were mainly associated with structural genes and included the sarcoplasm, laminin complex and cytoskeleton. Conclusion Exercise training in Thoroughbred racehorses results in coordinate changes in the gene expression of functional groups of genes related to metabolism, oxidative phosphorylation and muscle structure.