Gene 538 (2014) 150–154

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Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses Ran Li a,1, Dong-Hua Liu b,1, Chun-Na Cao c, Shao-Qiang Wang a, Rui-Hua Dang a, Xian-Yong Lan a, Hong Chen a, Tao Zhang d, Wu-Jun Liu e, Chu-Zhao Lei a,⁎ a

Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China Institute of Hexi Ecology and Oasis Agriculture, Hexi University, Zhangye, Gansu 734000, China Animal Husbandry and Veterinary Bureau of Yining, Yili, Xinjiang 835100, China d Shaanxi University of Technology, Hanzhong, Shaanxi 723001, China e Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China b c

a r t i c l e

i n f o

Article history: Accepted 11 December 2013 Available online 22 December 2013 Keywords: Chinese horse MSTN SNP Genetic diversity

a b s t r a c t The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26 T N C, g.156 T N C, g.587A N G, g.598C N T, g.1485C N T, g.2115A N G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A N G and g.598C N T residing in the 5′UTR region were novel SNPs identified by this study. The g.2115A N G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The myostatin gene (MSTN) belonging to the TGF-β superfamily is widely known for its genetic effect on double muscling trait as a negative regulator of skeletal muscle growth (Grobet et al., 1998). It controls the proliferation of muscle precursor cells by inhibiting cell cycle progression mediated by the p21 gene (Thomas et al., 2000) and participates in bone formation and regeneration by inhibiting the recruitment and proliferation of progenitor cells in the fracture blastema (Hamrick et al., 2010; Kellum et al., 2009). Mutations in the MSTN gene have been found to be strongly associated with growth, reproduction and carcass quality traits in many mammalian species. In cattle, six different

Abbreviations: MSTN, myostatin gene; SNP, single nucleotide polymorphism; RFLP, restriction fragment length polymorphism; TGF-β, transforming growth factor beta; p21, cyclin-dependent kinase inhibitor 1 encoded by the CDKN1A gene; 3′-UTR, 3′ untranslated region; 5′-UTR, 5′ untranslated region; ACD, acid citrate dextrose; Hd, haplotype diversity; AMOVA, analysis of molecular variance; HWE, Hardy–Weinberg equilibrium. ⁎ Corresponding author at: College of Animal Science and Technology, Northwest A & F University, Yangling, Shaanxi 712100, China. Tel.: + 86 29 87091373; fax: + 86 29 87092164. E-mail address: [email protected] (C.-Z. Lei). 1 The two authors contributed equally to this article. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.027

loss-of-function mutations in the MSTN gene caused an increase in the number of muscle mass resulting in enlarged muscles (Kambadur et al., 1997; Marchitelli et al., 2003). In sheep, mutations in coding region were shown to affect carcass conformation and fatness (Boman and Våge, 2009; Boman et al., 2009, 2010). In dogs, a mutation in the third exon increased muscle mass and enhances racing performance (Mosher et al., 2007). The equine MSTN gene mapped at the distal end of the equine chromosome 18 (ECA18) consists of three exons and two introns (GQ183900.1). In total, 23 equine MSTN gene SNPs have been reported by far, namely two in the promoter region, eight in intron-1, one in intron-2, 10 in exon-2 and two in 3′-UTR (Boman et al., 2010; Dall'Olio et al., 2010; Hill et al., 2010). A mutation in intron-1 of the MSTN gene can serve as a powerful marker for prediction of race distance aptitude in Thoroughbreds (Hill et al., 2010). The association between MSTN gene and horse racing performances was further evidenced by Binns et al. (2010) and Tozaki et al. (2010). In addition, two mutations located in the promoter of the MSTN gene are associated with breeds of different morphological types (Dall'Olio et al., 2010) while eight mutations in exon-2 caused alterations of protein function (Baron et al., 2012). In Chinese horse breeds, sequence variants of MSTN gene have been poorly characterized with only limited SNPs identified in Mongolian

R. Li et al. / Gene 538 (2014) 150–154 Table 1 Sampling information of the Chinese domestic horse breeds in this study. Distribution

Breed

Abbreviation

Sample size

Source region

Northwestern China

Kazakh Yanqi Balikun Chakouyi Datong Yushu Hequ Chaidamu Ningqiang Guanzhong Mongolian

HSK YQ BLK CKY DT YS HQ CD NQ GU MG

20 42 24 50 32 56 30 42 29 28 16

Changji, Yili county, Xinjiang Hejing county, Xinjiang Balikun country, Xinjiang Tianzhu county, Gansu Qilian county,Qinghai Yushu county,Qinghai Maqu county, Gansu Geermu city, Qinhai Ningqiang county, Shaanxi Fufeng county, Shaanxi Inner Mongolia

Lichuan Guizhou Debao Baise

LC GZ DB BS

21 49 39 36

Lichuan city, Hubei Guiyang city, Guizhou Debao county, Guangxi Baise county, Guangxi

Northeastern China Central China Southwestern China

horse (Wang, 2005). The significance of MSTN SNPs on horse racing performance urges the need for a thorough characterization of MSTN polymorphisms among Chinese domestic horses. Here the partial sequence of the MSTN gene including 5′-UTR region (671 bp), intron-1 (1829 bp), exon-1 (373 bp) and exon-2 (374 bp) in 15 Chinese horse breeds was screened. Our aim is to screen the sequence polymorphisms of the MSTN gene and analyze their genetic diversities in Chinese horse breeds. Considering the strong association between MSTN gene and racing performance, this study will provide instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds and helps to select the best Chinese domestic breed for further cultivation towards optimum racing traits.

151

(Northwestern, Northeastern, Central and Southern China). The Guanzhong horse (GU) belongs to the cultivated breed. Details of the horse breeds involved in the analysis are presented in Table 1. All the samples were collected randomly from local villages based on the pedigree information from the herdsmen to prevent the related individuals and ensure the coverage of native tract and purity of each population. 20 ml of jugular blood was collected for each sample, immediately treated by 4 ml of ACD anti-coagulation and stored at − 4 °C for transportation. Genomic DNA was extracted from blood using Genomic DNA isolation kit (Sangon, Shanghai, China) according to the manufacturer's instructions. 2.2. PCR amplification In total, 3267 bp of DNA fragments (GQ183900.1) was amplified including partial 5′-UTR (671 bp), exon-1 (373 bp), intron-1 (1829 bp), and exon-2 (374 bp) of the equine MSTN gene. Primers (Table 2) were designed using Primer Premier 5.0 software (PREMIER Biosoft International, CA, USA) (Table 2). PCR amplification was conducted in a 50 μl volume containing 5 μl of 10 × buffer, 1.5 mM MgCl2, 0.25 mM dNTPs, 0.2 μM each primer, 1.5 U Taq DNA polymerase (TaKaRa Biosystems) and 10 ng pooled genomic DNA which consist of DNA from ten individuals (Sham et al. 2002). The PCR conditions were as follows: an initial step at 95 °C for 5 min, 35 cycles for 35 s at 94 °C, 35 s at a specific annealing temperature for each primer pair (Table 2), and 35 s at 72 °C, followed by a final extension for 10 min at 72 °C. PCR products from the pooled DNA samples were sequenced directly by an ABI PRIZM 377 DNA sequencer (Perkin-Elmer). DNA sequences were edited using the DNASTAR 5.0 package (DNAstar, Madison, Wis., USA) and aligned by ClustalX version 2.0 (Larkin et al., 2007). 2.3. Genotyping by PCR-RFLP

2. Methods and materials 2.1. Specimen collection and DNA extraction A total of 514 blood samples from 15 Chinese native horse breeds were divided into four groups based on their geographical distributions

After identifying all the SNPs from the horse genomic DNA pools, PCR-RFLP protocols were designed to identify and genotype SNPs in the 514 samples. Restriction enzymes including RsaΙ, SspI, TasΙ, BsmΙ, AccΙ and ScaΙ were chosen to screen the SNPs. The RFLP condition was as follows: 5 μl of PCR products was digested with 3U of restriction

Table 2 Primers, PCR conditions, amplified regions, SNP location and restriction enzyme information. Primer pair

Primers (5′ → 3′)

Amplified region

Products length (bp)

Annealing temperature

SNP

Restriction enzymes

P1

1 F:TCAGGGAAACAAGTTTCTCAAAT 1R: TGCTCCACAATGAATCTCG 2 F: GACTTGTGACAGACAGGGTT 2R: CGCAGTTTACTGAGGATTT 3 F: TGCTGATTCTTGCTGGTCC 3R: GGCATGGTAATGATTGTTTC 4 F: CGACGACGGAAACAATCAT 4R: TTAGGCAACCAAACGCAAT 5 F: CATAATTGCGTTTGGTTGC 5R: CCTCCCTCCCAAGAAGAATA 6 F: AGGCAGGCACATTGCTTAAT 6R: GAATGTTATATTCAGGCTATCTCAA 7 F: CTAACTTTTGAGATAGCCTG 7R: CCAGAAAACTGTGAACTAAG 8 F: ATGTTCCTCCACGGTGTCT 8R: GGGCCTTTACTACTTTATTG 9 F: GCAAGTGGAAGGAAAACCCA 9R: TATTTTCATTTATCACTTACC 1 F: TCAGGGAAACAAGTTTCTCAAAT 10R: ACTTCCTCAGAAATTAAGATTTAAT 2 F: GACTTGTGACAGACAGGGTT 11R: GGACCAGCAAGAATCAGCAC 2 F: GACTTGTGACAGACAGGGTT 12R: TACTTTTCTTTTGCTTTTGAGGAAT 6 F: AGGCAGGCACATTGCTTAAT 13R: GCAGAGTCATAAAGGAAAAGTA

Promoter Promoter Promoter Exon 1 Exon 1 Exon 1 Exon 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Exon 2 Exon 2 Exon 2 Promoter Promoter 5′-UTR 5′-UTR 5′-UTR 5′-UTR Intron 1 Intron 1

484

63.2 °C

g.26 T N C

Rsa I

457

56.6 °C

–

–

325

55.6 °C

–

–

415

56.6 °C

–

–

467

57.8 °C

g.1485C N T

Acc I

480

56.6 °C

-

-

366

59 °C

-

-

446

56.6 °C

–

–

381

65 °C

-

-

204

50.7 °C

g.156 T N C

Ssp (CRS)

322

60 °C

g.587 A N G

Tas I

215

51.6 °C

g.598C N T

Bsm I (CRS)

330

55.4 °C

g.2115 A N G

Sca I (CRS)

P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13

Note: Primer pairs 1, 7 and 9 were from references (Baron et al., 2012; Dall'Olio et al., 2010); Primer pairs 2–6 and 8 were designed based on horse MSTN sequence (GenBank No. GQ183900.1). CRS: created restriction site.

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enzymes (MBI Fermentas) at a specific incubation temperature overnight for different primers, following the supplier's instructions. The digested products were detected in 3.0% agarose gel. 2.4. Statistical analysis Based on the genotyping information among the analyzed breeds, genotypic frequencies were directly calculated. Exact tests for deviations from Hardy–Weinberg equilibrium (HWE) for all locus–population combinations were assessed by SHEsis (http://analysis2.bio-x.cn/ myAnalysis.php) (Yong and Lin, 2005). Haplotype diversity (Hd), nucleotide diversity (π) and average number of nucleotide differences (K) were analyzed by DnaSP (version 5.0) (Librado and Rozas, 2009). Genetic variation among the four geographical groups was compared using analysis of molecular variance (AMOVA) in ARLEQUIN ver 3.1 (Excoffier et al., 2005). Significance levels of variance were evaluated by 1000 permutations for the analysis. Median-joining networks were constructed with NETWORK v. 4.6.0.0 (http://www. fluxus-engineering.com/) based on haplotype information (Bandelt et al., 1999).

Table 4 Genetic diversities of horse MSTN gene among 15 horse breeds. Breed

No.

h

hD

π (%)

K

HSK YQ BLK CKY DT YS HQ CD NQ GU MG LC GZ DB BS Total

20 42 24 50 32 56 30 42 29 28 16 21 49 39 36 514

4 8 5 9 8 10 7 8 6 3 6 7 11 6 4 13

0.460 0.416 0.235 0.520 0.714 0.528 0.406 0.378 0.580 0.288 0.480 0.417 0.713 0.447 0.535 0.503

0.019 0.017 0.008 0.021 0.039 0.021 0.019 0.014 0.022 0.009 0.019 0.017 0.039 0.017 0.023 0.022

0.6231 0.5422 0.2465 0.6935 1.1692 0.6709 0.6288 0.4515 0.7181 0.3007 0.6270 0.5412 1.2756 0.5658 0.7473 0.7112

Note: h: number of haplotypes; hD: haplotype diversity (standard deviation); π: nucleotide diversity (standard deviation); K: average number of nucleotide differences.

g.2115A N G locus, GG genotype only existed in DT, GZ, GU and YS breeds. Two loci (g.156 T N C and g.2115A N G) deviated from the Hardy–Weinberg equilibrium (HWE) in all breeds (P b 0.01) whereas other are in HWE.

3. Results 3.1. SNP polymorphism and genotypes Comparison of the sequences of the MSTN gene including promoter region, 5′-UTR, intron-1, exon -1 and exon-2 with reference sequence (GQ183900.1) revealed six SNPs (GQ183900.1:g.26 T N C,g.156 T N C, g.587A N G, g.598C N T, g.1485C N T, g.2115A N G) in 15 Chinese breeds. Of these, two SNPs (g.26 T N C and g.156 T N C) were located in the promoter region, two (g.587A N G and g.598C N T) in the 5′-UTR region, and two (g.1485C N T, g.2115A N G) in intron-1 of the equine MSTN gene, respectively. g.587A N G and g.598C N T were novel SNPs whereas the others were previously reported (Binns et al., 2010). Among the six detected SNPs, g.26 T N C, g.587A N G and g.1485C N T can be identified by their natural endonuclease restriction sites, while g.156 T N C, g.598C N T and g.2115A N G were genotyped by the introduction of artificial restriction sites. The frequencies of each genotype in 15 Chinese horse breeds were shown in Table 3. Homozygous genotypes were dominant at each locus (Table 3). The g.26 T N C and g.587A N G only produced two different genotypes (TT, TC and AA, AG), while the remaining loci each demonstrated three different genotypes respectively. At g.598C N T site, individuals with the TT genotype only existed in CKY, CD and BS breeds. At g.1485C N T site, TT genotype was only present in CKY, DT, HSK and YS breeds. At

3.2. Genetic diversity Genetic diversity was evaluated using the DnaSP software (version 5.0) (Table 4). The average nucleotide diversity was 0.022%, ranging from 0.008% to 0.039% among all the analyzed Chinese horse breeds. The GZ breed had the highest haplotype numbers of 11, while GU breed had the lowest haplotype number of three (Table 5). The average haplotype diversity was 0.503 varying from 0.235 to 0.714. The DT breed possessed both the largest haplotype diversity (0.714) and nucleotide diversity (π = 0.039%), whereas BLK breed showed the lowest haplotype diversity (0.235) and the lowest nucleotide diversity (π = 0.008%). Median-joining networks were constructed based on haplotype information. Among all of these haplotypes, Haplotype H1, accounting for 69.46% of the total haplotypes was shared among all breeds. Haplotype H3 was distributed in 13 horse breeds except for the HSK and HQ breeds. The Haplotype H12 and H13 only existed in the GZ breed (Fig. 1).

Table 3 Genotype frequencies of horse MSTN gene at each SNP locus. Breed

HSK YQ BLK CKY DT YS HQ CD NQ GU MG LC GZ DB BS Total

Sample size

20 42 24 50 32 56 30 42 29 28 16 21 49 39 36 514

g.26 T N C

g.156 T N C

g.587 A N G

g.598C N T

TT

TC

TT

TC

CC

AA

AG

CC

CT

TT

CC

g.1485C N T CT

TT

AA

g.2115 A N G AG

GG

0.900 0.905 0.917 0.960 0.844 0.893 1.000 0.929 0.931 1.000 1.000 1.000 1.000 0.949 1.000 0.946

0.100 0.095 0.083 0.040 0.156 0.107 0.000 0.071 0.069 0.000 0.000 0.000 0.000 0.051 0.000 0.054

0.850 0.905 0.958 0.840 0.656 0.929 0.867 0.857 0.897 0.893 0.875 0.905 0.633 0.744 0.833 0.835

0.050 0.095 0.000 0.060 0.250 0.071 0.133 0.095 0.103 0.036 0.000 0.000 0.204 0.154 0.167 0.105

0.100 0.000 0.042 0.100 0.094 0.000 0.000 0.048 0.000 0.071 0.125 0.095 0.163 0.103 0.000 0.061

0.900 1.000 0.917 0.880 1.000 0.821 1.000 0.952 1.000 1.000 1.000 0.857 0.714 0.949 0.750 0.905

0.100 0.000 0.083 0.120 0.000 0.179 0.000 0.000 0.000 0.048 0.000 0.143 0.286 0.051 0.250 0.095

0.950 0.857 0.875 0.620 0.563 0.857 0.800 0.517 0.893 0.810 0.750 0.762 0.796 0.641 0.500 0.735

0.050 0.143 0.125 0.360 0.438 0.143 0.200 0.414 0.107 0.190 0.250 0.238 0.204 0.359 0.417 0.251

0.000 0.000 0.000 0.020 0.000 0.000 0.000 0.069 0.000 0.000 0.000 0.000 0.000 0.000 0.083 0.014

0.850 0.810 0.833 0.780 0.750 0.857 0.933 0.897 0.929 0.905 0.813 0.762 0.755 0.897 1.000 0.856

0.100 0.190 0.167 0.200 0.188 0.107 0.067 0.103 0.071 0.095 0.188 0.238 0.245 0.103 0.000 0.132

0.050 0.000 0.000 0.020 0.063 0.036 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.012

1.000 0.905 0.875 0.940 0.875 0.821 0.867 0.897 0.893 0.905 1.000 0.905 0.796 1.000 1.000 0.907

0.000 0.095 0.125 0.060 0.063 0.143 0.133 0.103 0.107 0.048 0.000 0.095 0.163 0.000 0.000 0.083

0.000 0.000 0.000 0.000 0.063 0.036 0.000 0.000 0.000 0.048 0.000 0.000 0.041 0.000 0.000 0.013

R. Li et al. / Gene 538 (2014) 150–154 Table 5 The 13 haplotypes of horse MSTN gene derived from the six SNPs. 1

2

1

5

5

4

1

153

Table 6 AMOVA performed by grouping Chinese horse breeds according to their geographical distribution.

2

5

8

9

8

1

Haplotype

6

6

7

8

5

5

Percentage

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13

T T T T T T T T C T T T T

T T T T T C T T T C C C C

A A A A G A A A A A A G G

C C T C C C C T C T C C C

C C C T C C T T C C T T C

A G A G A A A A A A A A A

69.46% 2.33% 8.46% 2.04% 1.85% 6.81% 3.31% 0.97% 0.78% 1.65% 1.07% 0.88% 0.39%

3.3. AMOVA analysis AMOVA was performed based on RFLP data by partitioning the 15 breeds into four groups based on their geographical distributions (Table 1). The results revealed that the highest source of variation was derived from within individuals (about 87%), whereas about 13% of the total variation was attributable to differences among groups (Table 6). The results showed that the difference was all significant except among groups (P b 0.001). 4. Discussion In this study, six mutations were observed and genotyped among 15 Chinese horse breeds. The two novel polymorphism sites (g.587A N G and g.598C N T) were identified in the 5′-UTR of the horse MSTN

Source of variation

d.f.

Sum of squares

Variance components

Percentage of variation

Among groups (geographical distribution) Among populations within groups Among individuals within populations Within individuals

3

3.622

0.00108

0.26

11 499 514

13.677 221.667 186

0.01109* 0.04120* 0.36187*

2.67 9.92 87.15

Note: *shows the difference is extremely significant (P b 0.01).

gene. Polymorphisms in 5′UTR of the goat MSTN gene have been shown to have a significant effect on body weight and size (Li et al., 2008). The potential effect of these two novel SNPs on horse body weight and size needs to be further studied. Two SNPs (g.26 T N C and g.156 T N C) are located in the promoter region which is a rather conservative area closely associated with gene expression level, birth weight, growth traits, and muscle growth (Han et al., 2012; Hu et al., 2013; Liu et al., 2011). These two mutations might be associated with different morphological types (Dall'Olio et al., 2010), but this hypothesis couldn't be tested in our study due to the lack of morphological information. The CC genotype at g.26 T N C site was not found in Chinese horse breeds, but present in American and European horse breeds (Dall'Olio et al., 2010). In addition, ten mutations which are found in exon-2 of the MSTN gene in American and European horse breeds (Baron et al., 2012) are absent in Chinese horse breeds. These differences could be mostly explained by the loss of genetic diversity of Chinese horse breeds due to their decreasing importance in economic and agriculture in past decades. Based on the g.2115A N G locus, horses with the GG genotype were best suited for fast and short-distance races in thoroughbred horses; GA genotype horses were most competitive in middle-distance races; and AA genotype horses had greater stamina (Hill et al., 2010). In this study, g.2115A N G was found genotyped among Chinese horse breeds.

Fig. 1. Median-joining network of 13 haplotype sequences from MSTN gene of 15 Chinese horse breeds. Coloured circles represent haplotypes; Size of the circle is proportional to the haplotype frequency except for H1. Branch length is proportional to number of mutations. Different colors represent different horse breed.

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The result showed that the individuals with AA genotype were much more frequent than those of the GG genotype, which is in concordance with the fact that the majority of Chinese horses have great stamina. Chinese horses have been mainly used in transportation, agriculture and war over history, resulting in the favorite of horses with better stamina. Therefore, we suppose that most of the Chinese horse breeds would be potential candidates for cultivation for long-distance races. Meanwhile DT, GZ, GU and YS breeds were dominated by GG genotype, indicating that they could be the candidates for shorter-distance races. The average haplotype diversity in Chinese horses was abundant. Most of the haplotypes were present in more than one breed, and no haplotype was restricted to a certain region or a single horse breed. This could be explained by the no strict and systematic breeding and the constant intermixing among breeds in the history, which is also evidenced by the AMOVA analysis where genetic difference among the Chinese horse breeds was insignificant (Table 6). The BLK breed had the lowest genetic diversity indicating that the BLK breed has undergone a recent bottleneck. The GU breed had lower nucleotide diversity and less number of nucleotide differences in average than others except for the BLK breed, which could be the results of directional selection in this cultivated breed which is consistent with mtDNA study in Chinese horse breeds (Lei et al., 2009). Based on haplotype analysis and network, there was only one nucleotide difference between H1 and H2, H3, H5, H6, H7and H9 haplotypes. Haplotype H1 was most frequent and centered in the Median-joining network, implying it could be the original haplotype. AMOVA analysis indicated that there is no obvious genetic difference among the Chinese horse breeds and most of the genetic variance was attributable to differences among individuals. China's Silk Road which is the Ancient Tea Route as well as the widely use of horses in wars throughout Chinese history drove the gene flow and mixture of horses from different regions, resulting in reduced genetic diversity among horse breeds in different regions. 5. Conclusion In this study, the sequence polymorphisms of the MSTN gene were analyzed in 15 Chinese horse breeds. Six SNPs including two novel SNPs (g.587A N G and g.598C N T) were identified in the 15 breeds investigated. The SNP locus (g.2115A N G) which was associated with racing performances could serve as a potential marker in predicting the racing performance of Chinese horse breeds. Further studies are required to evaluate their associations with racing performance in Chinese horses and apply them to breeding practices. Conflict of interest All the authors stated no conflict of interest. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 31072001) and the National Science and Technology Support Program (2012BAD44B01).

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