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.

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.

Summary 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 (hm2 = 0.51, best race distance – elite; hm2 = 0.42, best race distance – nonelite; hm2 = 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.