Background: A single nucleotide polymorphism (SNP) in the first intron of the myostatin gene (MSTN) is associated with aptness of elite Thoroughbreds to race over sprint, middle or long distances. This intronic marker (g.66493737 T ≻ C), a short interspersed nuclear element (SINE) of 227 bp (Ins227bp) insertion polymorphism in the MSTN promoter, and the adjacent SNP BIEC2-417495 have not been studied for their association with racing aptness of the average Thoroughbreds raced in countries with lower status of the racing industry. This study investigated these markers regarding their prevalence and association with performance in common race horses. Markers were genotyped by amplification refractory mutation system-quantitative PCR (ARMS-qPCR) or amplicon melting. Furthermore, we asked whether the Ins227bp marker might theoretically regulate the expression of myostatin by generating a novel target for DNA methylation or by changing binding sites for transcription factors. Putative sites for DNA methylation or binding of transcription factors were predicted by MethPrimer and by the softwares JASPAR, MatInspector and UniPROBE, respectively.
Results: Pairwise linkage disequilibrium between g.66493737 T ≻ C and Ins227bp was high (r (2) = 0.93). A lower linkage was determined for g.66493737 T ≻ C and BIEC2-417495 (r (2) = 0.69) as well as for BIEC2-417495 and Ins227bp (r (2) = 0.76). The estimated frequencies for the presence of Ins227bp (I) indel and the C alleles at g.66493737 T ≻ C and BIEC2-417495 were 0.46, 0.47 and 0.43, respectively. Heterozygotes represented the most abundant genotype at each locus. The best racing distance (BRD) was significantly different between the homozygotes of each SNP (p = 0.01 to 0.03). C allele homozygotes at BIEC2-417495 or g.66493737 T ≻ C, as well as Ins227bp homozygotes earned most money on a mean distance ranging from 1211 to 1230 m. Heterozygotes earned most money on races over 1690 to 1709 m. The BRD for the T/T carriers at both SNP loci and for the SINE-free genotype was 1812 to 1854 m. Other performance parameters were not significantly different between the genotypes, except of the relative success score (RSS). The RSS was significantly slightly better on a distance of ≤ 1300 m for all carriers of the C allele and the Ins227bp compared to homozygous T genotypes and SINE-negative horses (p = 0.037 to 0.046). For distances of more than 1300 m the RSS was not significantly different between genotypes. In silico assessment indicated that the Ins227bp promoter insertion might have generated a CpG island and a few novel putative binding sites for transcription factors.
Conclusions: All three target polymorphisms (Ins227bp, g.66493737 T ≻ C, BIEC2-417495) are suitable markers to assess the ability of non-elite Thoroughbreds to race at short or longer distances. The CpG island generated by Ins227bp may cause training-induced silencing of MSTN expression.