Myostatin (MSTN), a negative regulator of skeletal muscle development in mammals, represents a key target for genetic investigations in meat-producing animals, with mutations responsible for increased skeletal-muscle mass was described in several livestock species. The aim of present study was to investigate myostatin gene polymorphism and its association with body weight records in Marwari sheep using PCR-SSCP methods. Blood samples were collected from randomly selected Marwari sheep ((n=71), and DNA was extracted using spin column method. Polymerase chain reaction was carried out to amplify 311 bp fragment of intron 2 region of myostatin gene. SSCP analysis showed two different conformations (AA and AB) in intron 2 region of MSTN gene in Marwari sheep and the frequencies of “AA” and “AB” genotypes were observed to be 0.86 (n=61) and 0.14 (n=10), respectively. This locus was not found in Hardy-Weinberg equilibrium and no significant effect of intron 2 of MSTN gene on body weights was observed.
Marwari breed of sheep is primarily reared for mutton production in addition to its medium and coarse quality carpet wool. The Marwari sheep serves as lifeline of Raika/Rebari communities (Yadav and Paul, 2009) in the arid and semi-arid regions of Rajasthan. Its importance can be judged from the fact that this numerically largest breed of north-west region of India (Livestock Census, 2012) is well known for its drought tolerance and excellent migrating ability.
Myostatin (MSTN) gene, also called Growth and Differentiation Factor 8 (GDF 8), a TGF-β family member, is a negative regulator of skeletal muscle development and growth in mammals. It consists of three exons and two introns that encode a glycoprotein that is expressed widely in skeletal muscle (Bellige et al., 2005). Loss of function mutations associated with increased skeletal-muscle mass and other pleiotropic effects have been detected in several species, including cattle (Grobet et al., 1997), sheep (Clop et al., 2006), chickens (McFarland et al., 2007) and putatively pigs (Stinckens et al., 2008). Mutations in the MSTN gene can inactivate its expression or produce a non-functional protein, which leads to dramatic muscularity and a “double-muscling” phenomenon in many species (Grisolia et al., 2009). The studies conducted in cattle by several authors (Stinckens et al., 2011) mapped twenty different mutations in MSTNgene in which six mutations were found in intronic regions.
Boman et al. (2009) observed the significant effect of mutations in the MSTN gene in muscular development of sheep. Thus MSTN gene could be considered as primarily responsible for muscle development and could be a potential candidate gene for animal muscle growth. Genetic analysis of myostatin gene in Marwari sheep may open interesting prospects for future selection programs, especially marker-assistant selection for body weight. A number of studies by different workers (Azari et al., 2012; Dimitrova et al., 2016) could not establish the polymorphic status of MSTN gene through restriction fragment length polymorphism (RFLP) as the gene was found to be highly conserved in vertebrates (Farhadian and Hashemi, 2016). Thus, the present study considered the polymerase chain reaction based single strand conformation polymorphism (PCR-SSCP) method for the detection of polymorphism in intron 2 of MSTN gene. The present study also aimed to detect the association of different allelomorphs of intron 2 of myostatin gene with body weight in Marwari sheep to find effective alleles influencing meat quantity and quality traits in sheep.
Materials and Methods
Sample Collection and DNA Extraction
Unrelated Marwari sheep (n=71) blood samples were randomly collected under aseptic conditions in sterile vacutainers from the Regional Station of Central Sheep and Wool Research Institute (CSWRI), Bikaner. Genomic DNA was extracted from 3mL of blood through spin column method as per manufacturer’s protocols. The quality and quantity of DNA extracted was determined by 0.8% agarose gel and NanoDrop spectrophotometer, respectively.
Collection of Body Weight Data
Body weight information from birth to 6 month of age was collected for the respective animal from whose blood samples was withdrawn. The phenotypic information was collected from records of Regional Station of Central Sheep and Wool Research Institute (CSWRI), Bikaner.
Amplification of Intron 2 of MSTN Gene of Marwari Sheep
The primers for the amplification of desired intron 2 segment of MSTN were designed from homologous ovine MSTN gene sequences available at NCBI BLAST (GenBank accession no. JN856480) using primer 3 software (Table 1).
Table 1: Primer Sequences used to Amplify Intron 2 of MSTN Gene
|Intron 2 of MSTN Gene||Forward – 5′- CAC ATT TTT CCC CCA GAA GAG -3′||311 bp|
|Reverse – 5′- AAG ACA GTT CAG AAA ATA GCT GG -3′|
An aliquot of 100 ng genomic DNA was amplified in a total volume of 25 μL PCR mix. The PCR reaction mix consisted of 14.2 μL nuclease free water, 5 μL of 5X PCR assay buffer, 2.5 μL MgCl2 (25mM), 0.5 μL dNTPs (10mM), 0.5 μL forward and reverse primers each (100 pmol/μL), and 0.2 μL Taq DNA polymerase (5U/µl). The optimum number of cycles and annealing temperature were identified through gradient PCR approach. Amplification program adopted for exponential amplification of intron 2 of MSTN region is presented in Table 2.
Table 2: PCR Conditions for Intron 2 of MSTN Gene
|Steps||Temperature||Time||No. of Cycle|
|I. Initial Denaturation||95oC||5 min.||1 cycle|
|III. Final extension||72oC||10 min.||1cycle|
|IV. Hold||4oC||5 min||1 cycle|
Negative controls were used during every batch of amplification to observe any contamination during process. All assays were performed in thermal cycler (Chino Scientific Pvt. Ltd.). The size and integrity of amplicons were analyzed on 1.5% agarose gel with standard molecular marker in horizontal gel electrophoresis. The gels were stained with ethidium bromide dye and visualized under ultraviolet light in gel documentation system.
Detection of Genetic Variation in Intron 2 of MSTN Gene of Marwari Sheep
Genetic analysis and nucleotide variation in intron 2 of MSTN Gene of Marwari sheep was carried by PCR-SSCP method. 5μL PCR amplified products were mixed with 10μL denaturing solution (including 800 μL formamide (99%), 100 μL loading dye, 100 μL glycerol (98%), 3 μL 0.5M EDTA, and 2 μL 10M NaOH), heated for 10 min at 95°C in Thermal cycler, and chilled on ice immediately for 20 min. Polymorphism at studied locus between different samples was detected on 8% nondenaturing polyacrylamide gels at 120 V for 7 h at 4oC. The different polymorphic patterns were visualized using ethidium bromide staining method (Benbouza et al., 2006).
Statistical Analysis of Genotypic Pattern and their Association with Body Weight
The different genotypic patterns were detected on the basis of the conformational changes adopted by the denatured single stranded DNA samples on polyacrylamide gel. The gene and genotypic frequency for intron 2 of MSTN were estimated by standard procedure as suggested by Falconer and Mackey (1998).
Genotypic frequency =
Gene frequency = P+1/2 H
Where, P= frequency of homozygote
H= frequency of heterozygote
The statistical analysis involving association of genotypic patterns of intron 2 of MSTN gene with body weight parameters in Marwari sheep was done using General Linear Model (GLM) procedure of SPSS to observe the effect of genotypic pattern on body weight of Marwari goat by following statistical model:
Yij = µ + Gi + eij
Where, Yij = body weight of the jth animal of ith genotype, µ = overall mean, Gi = fixed effect of ith genotype, eij = random error, NID (0, σ2).
Result and Discussion
The 311 bp fragment of intron 2 of MSTN gene was successfully amplified in all the DNA samples extracted (Fig. 1). The SSCP analysis of amplified fragments on 8% nondenaturing polyacrylamide gel detected two different conformational patterns “AA” and “AB” in Marwari sheep (Fig. 2). The conformation pattern “AA” was characterized with two bands on acrylamide gel and was designated as homozygote. The genotypic pattern “AA” was detected in 86 per cent of the Marwari animals (n=61) studied.
The genotypic pattern “AB” was identified by the presence of three bands of which two bands were in similar position to that of “AA” genotype. Ten animals of Marwari sheep showed the “AB” conformational pattern with genotypic frequency of 14 per cent. The allelic frequency of the allelomorphs “A” and “B” were observed to be 0.93 and 0.07, respectively (Table 3). The association analysis of the two genotypic patterns “AA” and “AB” of intron 2 of MSTN gene with body weight information at three different stages, viz., birth, 3-month and 6-month, in Marwari sheep did not reveal any significant association at any of stage of life indicated above (Table 4).
Table 3: Gene and Genotypic Frequency of Intron 2 of MSTN Gene in Marwari Sheep
|Genotypic Frequency||Gene Frequency|
Very low difference in weight such as 0.06 kg, 0.89 kg and 0.62 kg was observed between genotypes at birth, 3-month and 6-month, respectively.
Table 4: Association of Genotypic Patterns of Intron 2 of MSTN Gene with Body weight in Marwari Sheep
|Body Weight (Kgs) Mean ± SE|
|Genotypic Patterns||Birth Weight NS||3 Month Weight NS||6-Month Weight NS|
The polymorphic nature of intron 2 of MSTN gene in Marwari sheep was established through PCR-SSCP technique in the present study. However, inability to detect any association of body weight with the polymorphic status of MSTN gene in present study might be due to breed specific effect of the locus under study. Similar to these findings, Masoudi et al. (2005) did not report any significant effect of this locus. In a similar study on Iranian Baluchi sheep, Ansary et al. (2008) observed polymorphism in intron 1 of MSTN gene through PCR-SSCP method but the study revealed significant association of SSCP pattern with weaning weight trait however no association was reported for birth, 6-month, 9-month and yearling weights of the studied animals. Hadjipavlou et al. (2008) also detected significant effect of SNP on muscle depth in Charollais sheep.
This inconsistency in results could be attributed to breed difference, sampling size, geographical effect and frequency distribution of genetic variants. Mutations in the intron regions do not have a major role in rank of amino acids and proteins’ instructor (Matthews et al., 1997). It also seems that introns have a role in the expression of gene and age specific effect of intron 2 on expression of quantitative traits may be possible. This may be due to the environmental effects that exist and affect this trait at particular period of time. Low level of heterozygosity and genetic variability observed in the study might masked the effect of intron 2 of MSTN on body weights. The information provides evidence that Indian Marwari sheep have polymorphism in intron 2 for MSTN locus. It can be concluded that intron 2 of MSTN polymorphism did not have effect on body weight in Marwari sheep and may not be useful for developing future selection programs, especially marker assistant selection for improving weight gain in Marwari sheep. Furthermore, results showed PCR-SSCP are appropriate tools for evaluating genetic variability.
Authors are thankful to Rajasthan University of Veterinary and Animal Sciences, Bikaner (Rajasthan) for providing financial support and necessary infrastructure.