Interbreed Distribution of the Myostatin (MSTN) Gene 5′-Flanking Variants and Their Relationship With Horse Biometric Traits

Highlights

• Distribution of the analyzed MSTN gene 5′-flanking SNPs confirms that they potential impact on interbreed variability of the horse morphologic traits.

• The 227 bp SINE insertion is present mainly in Thoroughbreds (THOR) but occurs sporadically also in the other horse breeds (Wielkopolski, Silesian [SIL]). Frequency of the insertion (having an impact on MSTN expression and racing performance) in Polish population of THOR horses is lower than in other countries.

• The g.66495826T>C SNP is significantly associated with chest circumference in Hucul horses, whereas the g.66495696T>C polymorphism reveals the association with height at the withers (SIL) and cannon bone circumference (Polish Coldblood Horse).

Abstract

To assess the involvement of the MSTN gene polymorphisms in the biometric traits interbreed variability, we have examined the distribution of the three known 5′-flanking variants (g.66495696T>C, g.66495826T>C, and 272 bp SINE insertion) within the group of 2,052 horses representing 12 breeds and compared the allele frequencies between breeds assigned to the different morphological types. Additionally, the association of particular genotypes with selected biometric traits intrabreed variability was studied in five horse breeds. Genotyping of the two SNPs (g.66495696T>C and g.66495826T>C) revealed significant (P < .01) differences of minor allele frequencies between horses representing various morphological types. The highest and the lowest minor allele frequency values were recorded for horses representing brachymorphic and dolichomorphic morphological types, respectively. The association study revealed significant (P < .05) differences in height at the withers (Silesian [SIL]) and cannon bone circumference (Polish Coldblood Horse) — the lowest trait means were recorded for horses carrying the CC genotype in the g.66495696T>C polymorphic site. The heterozygous genotype in g.66495826T>C SNP turned out to be associated (P < .05) with the lower chest circumference if compared to the TT genotype carriers in the Hucul horse breed. Similarly to other investigations, the SINE insertion was present predominantly in Thoroughbred and occurred sporadically in other warmblood horse breeds (Wielkopolski [WLKP], SIL). Our study has confirmed the impact of the MSTN gene polymorphisms on the interbreed and intrabreed variability of horse biometric traits.

Introduction

Among domestic animal species, the horse (Equus caballus) is considered as one of the most variable in phenotype. Significant variation can be observed, for example, for body size and composition of horses representing different breeds [1]. Opposite to the human, in which size variability is affected by many (>200) genes with rather small effects, it was recently shown that in the case of the domestic horse, a significant part of body size variation may be explained by very few loci [2]. Among them, the ZFAT and LCORL/NCAPG loci located on chromosomes 9 and 3, respectively, are considered the most promising candidate genes, as it was indicated by two independent GWAS experiments [2], [3]. An important role of the LCORL locus was confirmed also in the functional study by Metzger et al [4], showing an association of gene expression levels with the variability of horse sizes (within and across breeds). However, to date, a majority of studies have been focused on the genetic determination of interbreed differences in morphological traits, and there is a limited number of investigations regarding variability of biometric traits observed within particular horse breeds. As it was reported in many previously published reports, heritability (h²) of morphological traits varies between different studies, but generally, it may be classified as moderate or even high. For example, in the Murgese breed, h² estimated for biometric traits ranged between 0.24 (height at the withers [WH]) and 0.44 (cannon bone circumference [CBC]) [5]. For the same traits investigated in the Wielkopolski (WLKP) horse breed, h² values amounted to 0.57 (WH) and 0.42 (CBC), respectively [6]. In turn, within the Poney Francais de Selle breed, the h² value calculated for height exceeded 0.7 [7]. These examples confirm the important role of the genetic background in the variability of horse morphological traits and indicate why searching for genetic markers associated with this variation is an interesting goal for scientists.

One of the most promising candidate genes for many important traits in horses is the myostatin (MSTN) gene, due to the well-known function of the MSTN protein in the regulation of myogenesis. Extensive studies on the MSTN gene in the domestic horse have resulted, for example, in the discovery of an intronic SNP (g.66493737C>T), which is strongly associated with racing performance of Thoroughbreds (THOR) and has an impact on the myostatin mRNA level in skeletal muscles [8], [9]. As it was shown in an experiment by Petersen et al [10], the 5.5-Mb region of ECA18 harboring the MSTN locus contains visible signatures of strong selection in Paint and Quarter Horses, which is probably related to the association of the MSTN gene polymorphisms with muscle fibers composition. To date, more than 20 SNPs have been described in the equine MSTN gene. A majority of them are located in exon 2 and intron 1 [11], [12], [13]. There are also several polymorphisms located in the MSTN gene 5′-flanking region. Some of them turned out to be putatively associated with morphological traits variability. The two previously discovered SNPs, g.66495826T>C and g.66495696T>C (also known as g.26T>C and g.156T>C), seem to be particularly interesting because the studies have proved that they can affect both, the intrabreed and interbreed variability of the horse biometric traits [12], [14], [15]. Moreover, the 227 bp SINE insertion revealed the relationship with muscle fibers composition and the MSTN gene expression [16], [17] and therefore can be considered as potentially functional in the context of equine biometric traits variation.

The aim of this study was to assess interbreed distribution of selected MSTN gene 5′-flanking variants (g.66495826T>C, g.66495696T>C, and 227 bp SINE insertion) based upon an analysis of a broad spectrum of breeds representing diverse morphological types. Moreover, for several horse breeds (Polish Primitive Horse [PPH], Polish Coldblood Horse [PCH], WLKP, Silesian [SIL], Hucul [HUC]), it was tested whether the genotyped polymorphisms may be associated with intrabreed variability of selected biometric traits.