A genome-wide SNP-association study confirms a sequence variant (g.66493737C>T) in the equine myostatin (MSTN) gene as the most powerful predictor of optimum racing distance for Thoroughbred racehorses

Abstract

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) [P(unadj.) = 6.96 x 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 (P(unadj.) = 1.61 x 10⁻⁹; P(Bonf.) = 6.58 x 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 x 10⁻¹⁰; BIEC2-417495, P(unadj.) = 1.61 x 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.

Figure 1

Manhattan plot of P-value for genotype-phenotype GWAS in short (≤ 8 f) and middle-long (> 8 f) distance elite race winners. The y-axis plots -log₁₀(P-values) and the x-axis plots the physical position of the SNPs sorted by chromosome and chromosome position. The most significant SNP was on chromosome 18 (BIEC2-417495). No SNP remained statistically significant following correction for multiple-testing.

Figure 2

Manhattan plot of P-value for quantitative trait GWAS using best race distance as phenotype. The y-axis plots -log₁₀(P-values) and the x-axis plots the physical position of the SNPs sorted by chromosome and chromosome position. A peak of association on chromosome 18 (chr18:65809482-67545806) encompassed a ~1.7 Mb region (shown in Figure 3). Seven of the chromosome 18 SNPs remained significant following correction for multiple testing. The most significant SNP was BIEC2-417495 (P_Bonf. = 6.58 × 10^-5).

Figure 3

A regional plot for the 1.8 Mb peak of association on chromosome 18 containing the MSTN and NAB1 genes. Association plot of the 1.8 Mb region encompassing 40 SNPs (diamonds) and the Ins227bp polymorphism (circle) ranging from one SNP upstream and one SNP downstream of the seven SNPs significantly associated with optimum racing distance following correction for multiple testing. The y-axes plot -log₁₀(P-values) for each SNP (diamonds) and r² (blue line) between g.66493737C>T and all other SNPs. The x-axis plots the physical position of each SNP in the region. The best SNP, g.66493737C>T, is indicated with a blue diamond. Each SNP is color coded according to the strength of LD with g.66493737C>T: r² ≥ 0.8, red; r² ≥ 0.5 < 0.8, orange; r² ≥ 0.2 < 0.5, yellow; r² < 0.2, white.

Figure 4

A visual representation of haplotype blocks across a 1.7 Mb region on chromosome 18. The g.66493737C>T SNP was included in block 3; BIEC2-417495 was included in block 6.