Genetic Variations of MSTN and Callipyge in Tibetan Sheep: Implications for Early Growth Traits

Simple Summary Tibetan sheep are integral to the ecosystems and livelihoods of the Tibetan Plateau; however, their breeding practices limit their production and growth. Candidate genes myostatin (MSTN)and Callipyge have been recognized as important for improving growth traits in livestock. This research examined the polymorphic loci of these genes in Tibetan sheep and their association with growth traits. The results indicated that SNP loci of MSTN could potentially be utilized as a molecular indicator for early growth traits in Tibetan sheep. Abstract Tibetan sheep are vital to the ecosystem and livelihood of the Tibetan Plateau; however, traditional breeding methods limit their production and growth. Modern molecular breeding techniques are required to improve these traits. This study identified a single nucleotide polymorphism (SNP) in myostatin (MSTN) and Callipyge in Tibetan sheep. The findings indicated notable associations between MSTN genotypes and growth traits including birth weight (BW), body length (BL), chest width (ChW), and chest circumference (ChC), as well as a particularly strong association with cannon circumference (CaC) at 2 months of age. Conversely, Callipyge polymorphisms did not have a significant impact on Tibetan sheep. Moreover, the analyses revealed a significant association between sex and BW or hip width (HW) at 2 months of age and ChW, ChC, and CaC at 4 months of age. Furthermore, the study’s results suggested that the genotype of MSTN as a GA was associated with a notable sex effect on BW, while the genotype of Callipyge (CC) showed a significant impact of sex on CaC at 2 months of age. These results indicated that the SNP of MSTN could potentially serve as a molecular marker for early growth traits in Tibetan sheep.


Introduction
The Qinghai-Tibetan Plateau is distinguished by its distinctive ecosystem and extreme environmental factors, including high altitude, low temperature, and intense ultraviolet radiation [1].Tibetan sheep (Ovis aries) hold a prominent ecological position as the predominant and extensively distributed livestock species on the Tibetan Plateau and serve as a vital resource for the indigenous population [2].Additionally, they play a crucial role in supporting the livelihoods of the Tibetan Plateau inhabitants by offering a dependable source of meat, wool, and leather [3].The morphology of Tibetan sheep is characterized by a rectangular body shape, with an average body height of 68.19 cm for rams and 65.11 cm for ewes, and body lengths of 76.56 cm and 65.79 cm, respectively.The predominant color of their hair is white, with variegated patterns on the head and limbs [4].In terms of growth performance, Tibetan sheep exhibit lower weights compared to high-quality meat sheep breeds, with adult rams weighing around 51.0 kg and ewes around 43.6 kg [5].In contrast, Hu sheep have adult weights of 53 kg for ewes and 85 kg for rams [6], while Dubo sheep reach weights of approximately 120 kg and 85 kg for adult rams and ewes [7].Furthermore, Tibetan sheep, being a traditional meat sheep breed, exhibit problems such as constrained productivity, sluggish growth, and diminished slaughter rates attributed to the absence of sophisticated breeding techniques.Numerous research studies have identified a wide array of candidate genes implicated in the control of metabolism and modulation of growth rates in domestic animals.[8].Variations in these genes have been extensively employed as molecular markers to facilitate breeding and enhance productivity [9].However, studies on Tibetan sheep are limited.
Myostatin (MSTN), also known as GDF-8, is a growth factor belonging to the TGF-β superfamily, located on the long arm of chromosome 2 in sheep.It is composed of three exons and two introns [10].Predominantly found in muscle tissues, it plays a critical role in the control of development and growth through its inhibition of cell cycle progression [11].Extensive research has demonstrated that the suppression of MSTN expression leads to increased meat production in animals, commonly called double-muscle animals [12,13].A notable augmentation (2-3-fold) in skeletal muscle mass has been documented in MSTNnull mice [14].Multiple investigations have recognized MSTN as a pivotal candidate gene that impacts productivity, growth, and performance in diverse domestic animal species, including pigs, sheep, and chickens [15][16][17].Specifically, investigations have demonstrated that MSTN-KO sheep exhibit a propensity for accelerated growth compared with wild-type (WT) sheep [18].Additionally, studies have demonstrated that mutations in MSTN in sheep led to notable gains in body weight and muscle mass [19].
The Callipyge mutation site, situated in the proximal telomere region of chromosome 18, known as the DLK1-GTL2 imprinted domain, is characterized by a remarkably preserved A-G mutation [20].This domain spans approximately 20 kb [21].Furthermore, mice and humans share similar homology regions on chromosomes 12 and 14 [22,23].This area of study centers on a significant quantity of imprinted genes that have been characterized.The gene products of these imprinted genes primarily encompass growth hormones, cell cycle regulators, nutrient transport proteins, and diverse non-coding RNA molecules, all of which are essential for governing growth and development [24].The genetic mechanism underlying the Callipyge gene is distinguished by its polar predominance.This suggests that in the heterozygous state, only the C allele is paternally transmitted, resulting in the manifestation of the Callipyge phenotype in sheep.Conversely, if heterozygous offspring inherit the C allele maternally or form a CC homozygous genotype, the sheep display a normal phenotype, precluding the manifestation of the Callipyge phenotype [25,26].Multiple studies have documented the capacity of the Callipyge gene to not only induce hypertrophy in the rump muscles of sheep but also enhance the efficiency of slaughter, increase carcass weight, and augment the proportion of lean meat [27][28][29].
Because of their vital role, the polymorphisms of these genes were explored in the Tibetan sheep population and their impact on pre-weaning growth traits was examined.This study contributes to the evaluation of MSTN and Callipyge genes as potential markers for growth marker-assisted selection (MAS) in Tibetan sheep breeding, laying the groundwork for the incorporation of molecular markers in breeding programs.

Experimental Animals
The experimental group comprised 313 purebred, single-parity Tibetan sheep (male = 180, female = 133) born between January and February 2022 in Gangcha County, Haibei Tibetan Autonomous Prefecture.The sheep were raised on the Qinghai Provincial Sheep Breeding and Promotion Center ranch, where they were allowed to graze freely and had unlimited access to water and grass.Growth traits such as body weight (BW), body length (BL), body height (BH), chest circumference (ChC), chest depth (ChD), chest width (ChW), hip width (HW), and cannon circumference (CaC) were observed from birth until weaning at four months of age.The animal experiments adhered to the regulations outlined in the approved "Guidelines for animal care and use" manual by the Animal Care and Use Committee.

Sample Collection and Primer Design
Ear tissues were collected and preserved in a 75% alcohol solution, followed by DNA extraction using a DNA extraction kit from TIANGEN, Beijing, China.Custom-designed primers were utilized for all exons of the MSTN and Callipyge genes with the assistance of Primer3 v0.4.0 [30].The MSTN gene consists of three exons, and the entire region of each exon was targeted by specific primers.Conversely, the Callipyge gene sequence was obtained from GenBank (AF401294), and a set of primers was developed to target this region.Additional details regarding the primers employed can be found in Table 1.

SNP Identification and Sequencing
We performed PCR reactions in a reaction volume of 30 µL, comprising 1.0 µL of DNA, 15 µL of 2×Taq PCR Master Mix, 1.0 µL of each primer, and double-distilled water (ddH 2 O) to reach the intended volume.We used a Bio-Rad S1000 thermal cycler (Bio-Rad, Hercules, CA, USA) for amplification, which consisted of an initial denaturation step at 94 • C for 2 min, 35 cycles of denaturation for 10 s at 94 • C, followed by annealing at 60 • C for 30 s, and elongation at 72 • C for 60 s.In the final step, PCR products were visualized on 1.0% agarose gel electrophoresis to determine the quality and quantity of the amplicons.Mutations were identified through Sanger sequencing using the Agilent 3730 system (Santa Clara, CA, USA).DNAMAN version 5.2.10 (Lynnon BioSoft, Vaudreuil, QC, Canada) was used for sequence analysis.

Population Genetic Index Calculation
Genetic parameters including allele frequency, heterozygosity (He), observed heterozygosity (Ho), effective allele numbers (Ne), and polymorphism information content (PIC) were evaluated utilizing Nei's method [31].The genotypes of the SNPs were examined for adherence to Hardy-Weinberg equilibrium (HW) in accordance with the Hardy-Weinberg law [32].

Statistical Analysis
The data analysis in this study was conducted using SPSS Statistics (Version 19, IBM SPSS Statistics, New York, NY, USA), with results presented as mean ± standard error.Associations between genotypes and individual growth traits were examined using general linear mixed models (GLMMs), with statistical significance defined as p < 0.05.The specific statistical model utilized in this research is detailed below: µ is the mean for the growth traits.
Genotypes represent the effects of the genotype.Sex represents the effects of sex.The interaction represents the interaction effect of sex and genotype.ε is a random error, and it is assumed to be independent, with N (0, σ2) distribution.

Polymorphism in Genes
An SNP locus was discovered within the MSTN and Callipyge, labeled A592G (rs410961001) in the MSTN and C232T in the Callipyge (AF401294).The sequencing peak maps for these genes illustrating the mutated sites are shown in Figure 1.
specific statistical model utilized in this research is detailed below: where Y represents the trait measured for each animal, including BW, BL, BH, ChW ChW, HW, and CaC.µ is the mean for the growth traits.
Genotypes represent the effects of the genotype.Sex represents the effects of sex.The interaction represents the interaction effect of sex and genotype.ε is a random error, and it is assumed to be independent, with N (0, σ2) distri

Polymorphism in Genes
An SNP locus was discovered within the MSTN and Callipyge, labeled (rs410961001) in the MSTN and C232T in the Callipyge (AF401294).The sequencin maps for these genes illustrating the mutated sites are shown in Figure 1.

Population Genetic Analysis
Ne was calculated for each SNP and varied between 1 and 2. The allele freque the SNPs were found to be in Hardy-Weinberg equilibrium.The PIC revealed t SNP in MSTN was categorized as a locus with low-grade polymorphism (PIC whereas the SNP in Callipyge was classified as a locus with moderate polymorphis < PIC ≤ 0.5) (Table 2).

Population Genetic Analysis
Ne was calculated for each SNP and varied between 1 and 2. The allele frequencies of the SNPs were found to be in Hardy-Weinberg equilibrium.The PIC revealed that the SNP in MSTN was categorized as a locus with low-grade polymorphism (PIC ≤ 0.25), whereas the SNP in Callipyge was classified as a locus with moderate polymorphism (0.25 < PIC ≤ 0.5) (Table 2).

Association Analyses of Sex with Growth Traits
The results revealed a statistically significant relationship between sex and various growth traits at different ages, including HW at 2 months, BW and ChW at 4 months (p < 0.05), and ChC and CaC at 4 months (p < 0.01).Nevertheless, no significant associations were found between sex and other growth traits at birth, 2 months, or 4 months of age (p > 0.05; Figure 2).
1 A represents the wild-type genotype, B represents the mutant genotype, N represents group Ho for homozygosity, He for heterozygosity, PIC for polymorphism information content, Ne effective allele numbers, HW for Hardy-Weinberg equilibrium, and ns for non-significance.

Association Analyses of Sex with Growth Traits
The results revealed a statistically significant relationship between sex and vari growth traits at different ages, including HW at 2 months, BW and ChW at 4 months 0.05), and ChC and CaC at 4 months (p < 0.01).Nevertheless, no significant associat were found between sex and other growth traits at birth, 2 months, or 4 months of ag > 0.05; Figure 2).

Analyses of SNPs Associated with Growth Traits
The analyses demonstrated statistically significant associations between the ge types of MSTN and BL, ChW, and ChC (p < 0.05), and there was a highly significant a ciation with CaC at 2 months of age (p < 0.01; Figure 3).However, all other associat between SNP in MSTN and Callipyge were not significant (p > 0.05; Figures 3 and 4).

Analyses of SNPs Associated with Growth Traits
The analyses demonstrated statistically significant associations between the genotypes of MSTN and BL, ChW, and ChC (p < 0.05), and there was a highly significant association with CaC at 2 months of age (p < 0.01; Figure 3).However, all other associations between SNP in MSTN and Callipyge were not significant (p > 0.05; Figures 3 and 4).
effective allele numbers, HW for Hardy-Weinberg equilibrium, and ns for non-significance.

Association Analyses of Sex with Growth Traits
The results revealed a statistically significant relationship between sex and va growth traits at different ages, including HW at 2 months, BW and ChW at 4 month 0.05), and ChC and CaC at 4 months (p < 0.01).Nevertheless, no significant associa were found between sex and other growth traits at birth, 2 months, or 4 months of a > 0.05; Figure 2).

Analyses of SNPs Associated with Growth Traits
The analyses demonstrated statistically significant associations between the g types of MSTN and BL, ChW, and ChC (p < 0.05), and there was a highly significant ciation with CaC at 2 months of age (p < 0.01; Figure 3).However, all other associa between SNP in MSTN and Callipyge were not significant (p > 0.05; Figures 3 and 4).

Analysis of Interaction Associations between Sex and SNPs with Growth Traits
The results revealed that there was a significant effect of sex on BW when the genotype of the MSTN gene was GA (p < 0.05).Additionally, when the genotype of Callipyge was CC, the effect of sex on CaC was highly significant at 2 months of age (p < 0.01, Table 3).

Discussion
Notably, MSTN and Callipyge are widely recognized as the most extensively studied genes that influence muscle cell movement and growth [33,34].This study aimed to identify SNPs in two specific genes and establish their association with pre-weaning growth traits in Tibetan sheep.Furthermore, we aimed to identify effective loci that could enhance the growth performance of Tibetan sheep.The findings of this study will contribute to establishing a molecular foundation for improving the growth performance of Tibetan sheep.
Studies have indicated that sex is a significant determinant of the growth traits of domestic animals, with male animals typically exhibiting superior growth traits to their female counterparts [35].In this study, it was found that the HW of ewes at 2 months of age and BW, ChC, and ChW at 4 months of age were significantly higher compared to rams.Furthermore, the CaC of rams at 4 months of age was observed to be greater than that of ewes.CaC is an indicator of somatic bone development, suggesting that the skeleton of rams is superior to that of ewes, specifically in terms of body size.It is crucial to acknowledge that the timeframe analyzed in this research aligns with the initial phases of sheep growth and maturation, potentially obscuring the observable reproductive benefits of rams [36].
The MSTN gene has been recognized as a suppressor of skeletal muscle growth and suggested as a potential gene for enhancing muscle production in sheep [37].Importantly, mutations in MSTN that interfere with its expression have been associated with the phenomenon of "double-muscling" observed in various species.Prior research has demonstrated the identification of approximately 8-10 SNPs in this gene of sheep, as indicated in Table 4 [38][39][40][41][42][43][44], and these mutations lead to the production of nonfunctional proteins, ultimately promoting substantial muscle growth and increased muscularity [45].Considering the significant importance of MSTN in animal breeding, using CRISPR/Cas9 technology to generate MSTN-edited brown sheep, the results revealed a substantial enhancement in myofiber diameter, mean daily gain, and overall body weight in MSTN-edited sheep [46,47].In contrast, an analysis of variations within intron 1 of MSTN revealed no significant association with the average BW, weaning weight, or pre-weaning growth rate [48].Additionally, g.6223G exhibited no discernible effect on the growth or weight of Australian white Suffolk, Bordeaux Set, or Lincoln sheep [44].Our study found that the mutation site significantly affected the BH, ChC, Chw, and CaC in Tibetan sheep at 2 months of age.This observation indicated that the mutation site plays a crucial role in the early growth and development of Tibetan sheep, highlighting its potential as a molecular marker for evaluating early growth traits.Conversely, only 2 SNPs have been identified in the Callipyge gene of sheep as shown in Table 4 [49][50][51].The Callipyge phenotype has been documented in sheep populations, with subsequent research establishing that the A→G mutation situated 32.8 kb upstream of GTL2 serves as the principal factor influencing this phenotype.However, it is important to acknowledge that the mutation frequency at this locus is notably low [49].Notably, no instances of A→G mutations were detected in the Callipyge gene analysis conducted on Chinese and foreign sheep breeds [52].This study was consistent with the results that identified a C→T mutation located 35 base pairs upstream of the mutation site responsible for the manifestation of the Callipyge phenotype.The findings of the study indicate a significant association between the C→T mutation located upstream of the mutation site of the double muscle hip gene and the occurrence of hind breech [50,51].Moreover, a study indicated that the mutation in question did not have a substantial influence on the growth characteristics of Hornless Tawset, Suffolk, and Texel breeds.Nonetheless, significant discrepancies in growth traits were noted in the Tan sheep [50].Studies analyzed the association between genotype and multiple growth traits in a cohort of 83 mature Oula sheep.Their findings suggested that the genotype of the locus under study did not yield statistically significant effects on BW, ChC, or ChD (p > 0.05).Nevertheless, heterozygous individuals tended to display higher values than homozygous individuals.In the present study, the genotype of the locus under investigation had no significant impact on growth performance at birth, 2 months of age, and 4 months of age [23].This finding was consistent with previous studies where they observed that the growth rate of mutant Callipyge sheep before weaning was comparable to that of normal sheep.Furthermore, the phenotypic outcomes of the three genotypes did not exhibit any discernible trend, except for a significant difference in the CaC of rams when the genotype was heterozygous for CC, which was higher than that of ewes (p < 0.01) [53].In summary, the SNP of the Callipyge gene did not exhibit any discernible effect on the pre-weaning growth performance of Tibetan sheep.However, additional studies are required to determine their potential effects on muscle development.

Conclusions
In conclusion, this study identified polymorphisms in two significant candidate genes in Tibetan sheep and assessed their impact on growth traits at birth and 2 and 4 months of age.Our findings reveal an association between the SNP locus of MSTN and growth and development at 2 months of age, suggesting its potential utility as a molecular marker for early growth traits in Tibetan sheep.Data Availability Statement: The data will be made available on request.

Figure 1 .
Figure 1.The peak map of mutated SNPs in MSTN and Callipyge of Tibetan sheep 1 . 1 The se were subsequently analyzed utilizing the DNAMAN software.The identified location of arrow is the mutation site.(A) MSTN mutations marked by the red arrows are located in e MSTN A592G (rs410961001).(B) SNP mutation C232T was identified in Callipyge (AF40129 sites marked with the red arrow.

Figure 1 .
Figure 1.The peak map of mutated SNPs in MSTN and Callipyge of Tibetan sheep 1 . 1 The sequences were subsequently analyzed utilizing the DNAMAN software.The identified location of the red arrow is the mutation site.(A) MSTN mutations marked by the red arrows are located in exon 2 of MSTN A592G (rs410961001).(B) SNP mutation C232T was identified in Callipyge (AF401294) at the sites marked with the red arrow.

Figure 2 .
Figure 2. The association between sex and growth traits in Tibetan sheep 1 . 1 (A) Analyses of associations between sex and growth traits in Tibetan sheep at birth.(B) Analyses of the associat between sex and growth traits in Tibetan sheep at 2 months of age.(C) Analyses of the associat between sex and growth traits in Tibetan sheep at 4 months of age.BW stands for body weight stands for body length; BH stands for body height; ChC stands for chest circumference; ChD sta for chest depth; ChW stands for chest width; HWstands for hip width; CaC stands for cannon cumference.Different superscript letters indicate significant differences at different levels of sig icance, with * representing p < 0.05, ** representing p < 0.01, and *** representing p < 0.001.

Figure 3 .
Figure 3.The association of SNPs in MSTN with Tibetan sheep's growth traits 1 . 1 (A) Associa analysis of SNPs in MSTN and growth traits in Tibetan sheep at birth.(B) Association analys SNPs in MSTN and growth traits in Tibetan sheep at 2 months of age.(C) Association analys SNPs in MSTN and growth traits in Tibetan sheep at 4 months of age.AB represents the heter gous mutant genotype; BB represents the homozygous mutant genotype; BW stands for b weight; BL stands for body length; BH stands for body height; ChC stands for chest circumfere ChD stands for chest depth; ChW stands for chest width; HWstands for hip width; CaC stand cannon circumference.Different superscript letters indicate significant differences at different le of significance, with * representing p < 0.05, ** representing p < 0.01.

Figure 2 .
Figure 2. The association between sex and growth traits in Tibetan sheep 1 . 1 (A) Analyses of the associations between sex and growth traits in Tibetan sheep at birth.(B) Analyses of the associations between sex and growth traits in Tibetan sheep at 2 months of age.(C) Analyses of the associations between sex and growth traits in Tibetan sheep at 4 months of age.BW stands for body weight; BL stands for body length; BH stands for body height; ChC stands for chest circumference; ChD stands for chest depth; ChW stands for chest width; HWstands for hip width; CaC stands for cannon circumference.Different superscript letters indicate significant differences at different levels of significance, with * representing p < 0.05, ** representing p < 0.01, and *** representing p < 0.001.

Figure 2 .
Figure 2. The association between sex and growth traits in Tibetan sheep 1 . 1 (A) Analyses associations between sex and growth traits in Tibetan sheep at birth.(B) Analyses of the associa between sex and growth traits in Tibetan sheep at 2 months of age.(C) Analyses of the associa between sex and growth traits in Tibetan sheep at 4 months of age.BW stands for body weigh stands for body length; BH stands for body height; ChC stands for chest circumference; ChD s for chest depth; ChW stands for chest width; HWstands for hip width; CaC stands for canno cumference.Different superscript letters indicate significant differences at different levels of s icance, with * representing p < 0.05, ** representing p < 0.01, and *** representing p < 0.001.

Figure 3 .
Figure 3.The association of SNPs in MSTN with Tibetan sheep's growth traits 1 . 1 (A) Assoc analysis of SNPs in MSTN and growth traits in Tibetan sheep at birth.(B) Association analy SNPs in MSTN and growth traits in Tibetan sheep at 2 months of age.(C) Association analy SNPs in MSTN and growth traits in Tibetan sheep at 4 months of age.AB represents the hete gous mutant genotype; BB represents the homozygous mutant genotype; BW stands for weight; BL stands for body length; BH stands for body height; ChC stands for chest circumfer ChD stands for chest depth; ChW stands for chest width; HWstands for hip width; CaC stan cannon circumference.Different superscript letters indicate significant differences at different of significance, with * representing p < 0.05, ** representing p < 0.01.

Figure 3 .
Figure 3.The association of SNPs in MSTN with Tibetan sheep's growth traits 1 . 1 (A) Association analysis of SNPs in MSTN and growth traits in Tibetan sheep at birth.(B) Association analysis of SNPs in MSTN and growth traits in Tibetan sheep at 2 months of age.(C) Association analysis of SNPs in MSTN and growth traits in Tibetan sheep at 4 months of age.AB represents the heterozygous mutant genotype; BB represents the homozygous mutant genotype; BW stands for body weight; BL stands for body length; BH stands for body height; ChC stands for chest circumference; ChD stands for chest depth; ChW stands for chest width; HWstands for hip width; CaC stands for cannon circumference.Different superscript letters indicate significant differences at different levels of significance, with * representing p < 0.05, ** representing p < 0.01.

Figure 4 .
Figure 4.The association of SNPs in Callipyge with Tibetan sheep's growth traits 1 . 1 (A) Association analysis of SNPs in Callipyge and growth traits in Tibetan sheep at birth.(B) Association analysis of SNPs in Callipyge and growth traits in Tibetan sheep at 2 months of age.(C) Association analysis of SNPs in Callipyge and growth traits in Tibetan sheep at 4 months of age.AA represents the wild-type genotype; AB represents the heterozygous mutant genotype; BB represents the homozygous mutant genotype; BW stands for body weight; BL stands for body length; BH stands for body height; ChC stands for chest circumference; ChD stands for chest depth; ChW stands for chest width; HWstands for hip width; CaC stands for cannon circumference.

Author
Contributions: K.Z. and X.L. wrote and revised the manuscript; D.L., L.W. and Q.P. provided the study concept and design; B.H. (Buying Han), Z.Z. and D.T. analyzed the data; S.W., J.Z., B.H. (Bin Huang) and F.Z. collected data and samples.All authors have read and agreed to the published version of the manuscript.Funding: This study was supported by the Natural Science Foundation of the Qinghai Province (2022-ZJ-901) and the Tibetan Sheep Germplasm Innovation Joint National Breeding Project.Institutional Review Board Statement: All animal experiments adhered to the protocols outlined in the "Guidelines for Animal Care and Use manual (Approval No. NWIPB2023015, Date: 12 July 2023), which were approved by the Animal Care and Use Committee of the Northwest Institute of Plateau Biology, Chinese Academy of Sciences.Informed Consent Statement: Not applicable.

Table 1 .
Primer information of MSTN and Callipyge genes in Tibetan sheep.

Table 2 .
Population genetic analyses of MSTN and Callipyge in Tibetan sheep 1 .

Table 2 .
Population genetic analyses of MSTN and Callipyge in Tibetan sheep 1 .

Table 3 .
Interaction association analyses between sex and SNPs in MSTN and Callipyge with growth traits in Tibetan sheep 1 .

Table 4 .
SNPs have been identified in the sheep MSTN and Callipyge genes.