- Email: [email protected]
Polymorphism in exon 3 of myostatin (MSTN) gene and its association with growth traits in Indian sheep breeds
Small Ruminant Research 149 (2017) 81–84
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Research Paper
Polymorphism in exon 3 of myostatin (MSTN) gene and its association with growth traits in Indian sheep breeds Amiya Ranjan Sahu a,∗ , V. Jeichitra b , R. Rajendran c , A. Raja d a
Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai, India Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Orathanadu, Thanjavur, India Directorate of Research, TANUVAS, Madhavaram Milk Colony, Chennai, India d Department of Animal Biotechnology, Madras Veterinary College, Chennai, India b c
a r t i c l e
i n f o
Article history: Received 28 March 2016 Received in revised form 12 January 2017 Accepted 23 January 2017 Available online 31 January 2017 Keywords: Myostatin gene PCR-RFLP Polymorphism Sheep
a b s t r a c t The polymorphism of myostatin (MSTN) gene was analysed as a genetic marker for growth traits in Madras Red and Mecheri sheep breeds of Tamil Nadu. PCR-RFLP revealed a SNP G5622C in exon 3 and was confirmed by sequencing. The breed-wise genotypic and allelic frequencies observed for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively. The estimated allelic frequencies for M and m in Madras Red and Mecheri breeds were 0.7085 and 0.2915; and 0.7430 and 0.2570 respectively. Madras Red sheep with homozygous (MM) genotypes were significantly (P < 0.05) heavier than heterozygous (Mm) genotype by 1.162 kg and 1.227 kg at nine and 12 months weight respectively. However, no significant effect was observed for birth, weaning (three months) and six months weight in the same breed. There was no significant difference in mean birth, weaning, six, nine and 12 months weight between MM and Mm genotypes in Mecheri sheep. © 2017 Elsevier B.V. All rights reserved.
1. Introduction India is endowed with biologically diverse sheep genetic resources (40 breeds) possessing 65.06 million sheep (Livestock Census Report, 2012) which constitute 12.7% of the total livestock population of India and ranks third in the world sheep population. Sheep meat is a major source of animal protein for which sale of surplus animals with male body weight at the market age forms a major component of income in sheep husbandry. Therefore, faster growth and early maturity are important components of profitable sheep production. Growth indicated as the live body weight measured at various ages is one of the most important traits in the genetic improvement for meat sheep. Single nucleotide polymorphism of gene sequence which affects body growth starting from pre-weaning daily gain till 11 months of age acts as a basis for breeding of the economically important growth traits through marker assisted selection (Hua et al., 2009). Myostatin, the protein whose expression is under the control of myostatin (MSTN) gene acts as a negative regulator of muscle cell growth, where the loss of functional myostatin is known to cause the “double-muscled” phenotype in cattle (Kambadur et al., 1997;
∗ Corresponding author. E-mail address: [email protected] (A.R. Sahu). http://dx.doi.org/10.1016/j.smallrumres.2017.01.009 0921-4488/© 2017 Elsevier B.V. All rights reserved.
McPherron et al., 1997 McPherron and Lee, 1997; Grobet et al., 1998), in sheep (Broad et al., 2000) and in goats (Li et al., 2006). MSTN also called as growth differentiation factor 8 (GDF 8) gene is located on chromosome 2 of sheep (Archibald et al., 2010). Mutation in MSTN acts in various ways like myostatin null mice causes hypertrophy and hyperplasia of skeletal muscle mass (McPherron et al., 1997) whereas deletion, missense or nonsense mutations leads double muscling in Belgian Blue and Piedmontese breeds of cattle (McPherron et al., 1997 McPherron and Lee, 1997) and hypertrophy in child due to splice site mutation in human being (Schuelke et al., 2004). However, there is no report regarding the variation in the exons of MSTN in Indian sheep breeds till date. Hence, the identification of the variation in coding region of ovine MSTN gene using PCR-RFLP followed by sequencing and the association analyses with growth traits are described in this study.
2. Material and methods 2.1. Experimental sheep flocks and PCR amplification Madras Red and Mecheri are two large sized hairy meat breeds of Tamil Nadu, India considered for the study. The genomic DNA was isolated from Madras Red (127) and Mecheri (105) sheep using standard Phenol-Chloroform extraction procedure
82
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
(Sambrook et al., 1989) with slight modifications by using DNAzol reagent, instead of SDS and proteinase K. The exon 3 (380 bp) of MSTN, with part of intron 2 and 3 UTR on either side was amplified as a 615 bp fragment, using the primer sequence designed by Fast PCR Primer designing (FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis) software (Kalendar et al., 2014). The primer was synthesised at M/s. Eurofins Genomics, Bangalore. The PCR was performed with volume of 20 l using 10 l master mix (Ampliqon), 0.6 l each of forward primer (CGG TAG GAG AGT GTT TGG GGA T) and reverse primer (GAT GGT TAA ATG CCA ACC ATT GC), 1.3 l template DNA (50 ng/l) and 7.5 l of NFW. The amplification was carried out with a program of 5 min initial denaturation at 95 ◦ C followed by 32 cycles of 95 ◦ C for 35 s, annealing at 61.5 ◦ C for 30 s and extension at 72 ◦ C for 35 s, with a final extension of 5 min at 72 ◦ C. The product was checked by electrophoresis (2 per cent agarose) and the gel was stained with ethidium bromide, visualized under UV light. 2.2. PCR- RFLP and analysis of sequence variation The PCR product (615 bp) was digested with MspI restriction enzyme at 37 ◦ C for 6 h in water bath with the restriction digestion mix of 10.0 l of PCR product, 1.5 l of 1X cut smart buffer, 0.1 l of MspI enzyme (3 units/l) and 3.4 l of NFW. The digested products were checked in 2% agarose gel using 50 bp DNA ladder. Amplicons of eight samples from each breed (Madras Red and Mecheri) were sequenced for 615 bp fragment of MSTN gene. The amplicon was sequenced in both forward and reverse directions at M/s. Eurofins Genomics India Pvt. Ltd., Bangalore. The instrument used for sequencing was ABI PRISM 3730XL Genetic analyzer (Applied Biosystems, USA). Sequence data were analysed using the SeqMan program of LASERGENE software version 7.1.0 (44) (DNASTAR Inc., USA). 2.3. Statistical analysis The genotypes were assigned on the basis of band patterns of the PCR products. The genotype and gene frequencies were estimated and the locus was tested for Hardy-Weinberg equilibrium. The polymorphism observed at the G5622C locus of MSTN gene in Madras Red and Mecheri sheep breeds was analysed separately for their association with body weights at various ages viz., birth, weaning (three month), six, nine and 12 months using least-squares procedures (Harvey, 1990). The data on number of animals used for association analysis was lower than the number of animals genotyped due to the non-availability of complete set of data on body weights and deletion of outliers. Data were classified into two periods of birth viz., period 1 (2007–2010) and period 2 (2011–2014) in Madras Red; and period 1 (2008–2010) and period 2 (2011–2013) in Mecheri breed of sheep; sex as males and females; and different genotypes at G5622C locus of MSTN gene. Weight of dam at lambing was also included as a covariable in the model as below: Y ijkl = + Pi + Sj + Gk + b(WMijk − WM) + eijkl
Fig. 1. MspI-RFLP of PCR amplicon (615 bp) in exon 3 of MSTN gene. Lane M: 50 bp DNA ladder, Lanes 1–5: Madras Red, and Lanes 6–9: Mecheri sheep. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Gk = fixed effect of kth genotype(k = 1and2) ¯ = regression of Y on dam s weight at lambing b (WMijk − WM) eijkl = residual random error, NID (0, 2 ) 3. Results and discussion The restriction enzyme, MspI had cleavage site (C/CGG) at nucleotide position 314 of the amplified region (615 bp) and showed polymorphism in both sheep breeds studied. Restriction digestion revealed the existence of two genotypes viz., MM (301 bp and 314 bp) and Mm (301 bp, 314 bp and 615 bp) and the locus was characterized by the presence of two alleles M and m (Fig. 1). The mm genotype was absent in both breeds. The breed-wise genotypic and allelic frequencies observed (Table 1) for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively. The estimated allelic frequencies for M and m in Madras Red and Mecheri breeds were 0.7085 and 0.2915; and 0.7430 and 0.2570 respectively. The frequency of MM genotype was higher in Mecheri than Madras Red and vice versa for Mm genotype. The sequences were assembled and screened for single nucleotide polymorphisms (SNPs). On analysing the amplicon (615 bp), one SNP was found in the exon 3 of MSTN at locus G5622C, characterized by transversion of G > C, but no change in amino acid was observed in both the sheep breeds. Two genotypes viz., GG and GC were observed as shown in chromatogram (Fig. 2). The 5622G > C transversion observed in the present study is contrary to the reports in the New Zealand Romney sheep (Zhou et al., 2008) and in Chinese breeds of sheep (Gan et al., 2008), where exon 3 was monomorphic. The estimated allelic frequency for M was higher than m in both the breeds of sheep studied. The locus 5622G > C has significantly (P < 0.01) deviated from genetic equilibrium in both sheep breeds. The effect of 5622 G > C mutation in exon 3 of MSTN gene on nine
Where, Y ijkl = is the body weight of the lth animal of the kth genotype of the jth sex born in the ith period of lambing = overallmean Pi = fixed effect of ith period of lambing (i = 1and2) Sj = fixed effect of jth sex of the lamb (j = 1and2)
and twelve months weights was significant (P < 0.05) in Madras Red sheep. Animals with homozygous (MM) genotype were heavier than heterozygous (Mm) genotype by 1.162 kg and 1.227 kg at nine and 12 months weight respectively. However, a non-significant effect of this mutation was observed for birth, weaning (three months) and six months weight in the same breed. There was no
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
83
Table 1 Breed-wise genotypic and allelic frequencies at G5622C locus in exon 3 of MSTN gene. Locus
Breed
Genotype
Genotypic frequency
Allelic frequency
2 value
G5622C
Madras Red (127)
MM (53) Mm (74) mm (0) MM (51) Mm (54) mm (0)
0.417 0.583 0 0.486 0.514 0
M = 0.7085 m = 0.2915
21.45*
M = 0.7430 m = 0.2570
12.59*
Mecheri (105)
Figures in parentheses indicate number of observations. * highly significant (P < 0.01).
Fig. 2. Assembly of the chromatograms of Exon 3 of MSTN gene to determine the polymorphism across the Madras Red and Mecheri breeds of sheep. Chromatograms of the genotypes in Exon 3: Madras Red MM and Mm; and Mecheri MM at position 5622 locus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Least-squares means ± S.E. (kg) for the effect of 5622G > C in MSTN gene on growth traits. Body weight at various ages
Breeds Madras Red MM (42)
Birth weight
NS 2.737 ± 0.062
3 months weight
NS 9.528 ± 0.402
6 months weight
NS 13.602 ± 0.405
9 months weight
* 17.518 ± 0.512b
12 months weight
* 19.656 ± 0.537b
Mecheri Mm (68) 2.729 ± 0.063 8.842 ± 0.412 13.217 ± 0.415 16.356 ± 0.524a 18.429 ± 0.550a
Prob
MM (42)
Mm (43)
Prob
0.9030
NS 2.600 ± 0.066
2.462 ± 0.082
0.0959
0.1263
NS 12.325 ± 0.439
12.920 ± 0.545
0.2787
0.3924
NS 14.865 ± 0.483
14.717 ± 0.600
0.8054
0.0432
NS 17.799 ± 0.552
17.022 ± 0.686
0.2614
0.0419
NS 20.121 ± 0.623
19.220 ± 0.774
0.2482
Figures in parentheses indicate number of records used for analysis. NS = not significant, * = significant (P < 0.05). Means with at least one common superscript within classes do not differ significantly (P ≥ 0.05).
significant difference in body weights at different ages and genotypes MM and Mm at locus 5622 G > C in Mecheri sheep (Table 2). Simillar findings for the non-significant effect of genetic variants in exon 3 of MSTN gene with growth traits were reported in Zel sheep (Dehnavi et al., 2012) and Baluchi sheep (Masoudi et al., 2005).
4. Conclusion The exon 3 of MSTN gene is polymorphic in Madras Red and Mecheri sheep breeds of India. SNP 5622 G > C is novel and first report in Indian sheep breeds. This mutation had significant association with nine and 12 months body weight, where the genotype
84
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
MM is heavier than Mm in Madras Red sheep. The genotypic frequencies for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively and there is complete absence of mm. This suggests the possible use of M allele at G5622C locus as a potential marker for selection in Madras Red sheep breed. The locus 5622G > C has significantly (P < 0.01) deviated from genetic equilibrium in both the sheep breeds would possibly be due to small sample size and selection. Further studies are suggested on a larger sample size to confirm the effect of this mutation G5622C on growth traits in Madras Red sheep, so as to use this as a potential marker. Absence of significant association of this mutation with body weights at various ages in Mecheri sheep could possibly due to founder effect and inadequate pasture availability. It is also concluded that the absence of association for growth traits in Madras Red and Mecheri might be due to the high genetic similarity, forces of evolution and distribution in the similar agro-climatic conditions of these two breeds. Being this as the first report of MSTN polymorphism in Indian sheep breeds needs further research to throw light on the significant effect of MSTN mutations on growth traits. Conflict of interest The authors declare no conflict of interest regarding this article. Acknowledgements Authors are thankful to the Professor and Heads, Post-Graduate Research Institute in Animal Sciences (PGRIAS), Kattupakkam and Mecheri Sheep Research Station (MSRS), Pottaneri for their necessary support to carry out the research work. References Archibald, A.L., Cockett, N.E., Dalrymple, B.P., Faraut, T., Kijas, J.W., Maddox, J.F., McEwan, J.C., Hutton Oddy, V., Raadsma, H.W., Wade, C., Wang, J., Wang, W., Xun, X., 2010. The sheep genome reference sequence: a work in progress. Anim. Genet. 41 (5), 449–453. Broad, T.E., Glass, B.C., Greer, G.J., Robertson, T.M., Bain, W.E., Lord, E.A., McEwan, J.C., 2000. Search for a locus near to myostatin that increases muscling in Texel sheep in New Zealand. Proc. N. Z. Soc. An. 60, 110–112.
Dehnavi, E., Azari, M.A., Hasani, S., Nassiry, M.R., Mohajer, M., Ahmadi, A.K., Shahmohamadi, L., Yousefi, S., 2012. Polymorphism of myostatin gene in Intron 1 and 2 and Exon 3, and their associations with yearling weight, using PCR-RFLP and PCR-SSCP techniques in Zel sheep. Biotechnol. Res. Int., http:// dx.doi.org/10.1155/2012/472307, Article ID 472307, 5 pages. Gan, S.Q., Du, Z., Liu, S.R., Yang, Y.L., Shen, M., Wang, X.H., Yin, J.L., Hu, X.X., Fei, J., Fan, J.J., Wang, J.H., He, Q.H., Zhang, Y.S., Li, N., 2008. Association of SNP haplotypes at the myostatin gene with muscular hypertrophy in sheep. Asian-Aust. J. Anim. Sci. 21, 928–935. Grobet, L., Pncelet, D., Royo, L.J., Brouwers, B., Pirottin, D., Michau, C., Menissier, F., Zanotti, M., Dunner, S., George, M., 1998. Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling. Mamm. Genome 9, 210–213. Harvey, W.R., 1990. Mixed Model Least-squares and Maximum Likelihood Computer Programme. PC-2 Version. Ohio State University, Colombus. Hua, G.H., Chen, S.L., Jn, Y.U., Cai, K.L., Wu, C.J., li, Q.L., Zhang, C.Y., Liang, A.X., Han, L., Geng, L.Y., Shen, Z., Xu, D.Q., Yang, L.G., 2009. Polymorphism of the growth hormone gene and its association with growth traits in Boer goat bucks. Meat Sci. 81, 391–395. Kalendar, R., Lee, D., Schulman, A.H., 2014. Fast PCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. In: Valla, Svein, Lale, Rahmi (Eds.), DNA Cloning and Assembly Methods, Methods in Molecular Biology. Humana Press, 1116, pp. 271–302. Kambadur, R., Sharma, M., Smith, T.P.L., Bass, J.J., 1997. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res. 7, 910–916. Li, X.L., Wu, Z.L., Gong, Y.F., Liu, Y.Q., Liu, Z.Z., Wang, X.J., Xin, T.R., Ji, Q., 2006. Single nucleotide polymorphism identification in the caprine myostatin gene. J. Anim. Breed. Genet. 123, 141–144. Livestock Census Report, 2012. 19th Livestock Census. Department of Animal Husbandry and Dairying, Ministry of Agriculture, Government of India, New Delhi. Masoudi, A., Hemrani, H., Abbasi, A., 2005. Using PCR-SSCP techniques to study polymorphism of myostatin gene and its association with production traits in Baluchi sheep. In: Proc. 4th National Biotechnol. Congr, Iran, Kerman, Iran. McPherron, A.C., Lee, S.J., 1997. Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. U. S. A. 94, 12457–12461. McPherron, A.C., Lawler, A.M., Lee, S.J., 1997. Regulation of skeletal muscle mass in mice by a new TGF- superfamily member. Nature 387, 83–90. Sambrook, J.E., Fritsch, F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold spring Harbor Laboratory Press, New York, USA. Schuelke, M., Wagner, K.R., Stolz, L.E., 2004. Myostatin mutation associated with gross muscle hypertrophy in a child. N. Engl. J. Med. 350 (26), 2682–2688. Zhou, H., Hickford, J.G.H., Fang, Q., 2008. Variation in the coding region of the myostatin (GDF8) gene in sheep. Mol. Cell. Probes 22, 67–68.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Research Paper
Polymorphism in exon 3 of myostatin (MSTN) gene and its association with growth traits in Indian sheep breeds Amiya Ranjan Sahu a,∗ , V. Jeichitra b , R. Rajendran c , A. Raja d a
Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai, India Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Orathanadu, Thanjavur, India Directorate of Research, TANUVAS, Madhavaram Milk Colony, Chennai, India d Department of Animal Biotechnology, Madras Veterinary College, Chennai, India b c
a r t i c l e
i n f o
Article history: Received 28 March 2016 Received in revised form 12 January 2017 Accepted 23 January 2017 Available online 31 January 2017 Keywords: Myostatin gene PCR-RFLP Polymorphism Sheep
a b s t r a c t The polymorphism of myostatin (MSTN) gene was analysed as a genetic marker for growth traits in Madras Red and Mecheri sheep breeds of Tamil Nadu. PCR-RFLP revealed a SNP G5622C in exon 3 and was confirmed by sequencing. The breed-wise genotypic and allelic frequencies observed for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively. The estimated allelic frequencies for M and m in Madras Red and Mecheri breeds were 0.7085 and 0.2915; and 0.7430 and 0.2570 respectively. Madras Red sheep with homozygous (MM) genotypes were significantly (P < 0.05) heavier than heterozygous (Mm) genotype by 1.162 kg and 1.227 kg at nine and 12 months weight respectively. However, no significant effect was observed for birth, weaning (three months) and six months weight in the same breed. There was no significant difference in mean birth, weaning, six, nine and 12 months weight between MM and Mm genotypes in Mecheri sheep. © 2017 Elsevier B.V. All rights reserved.
1. Introduction India is endowed with biologically diverse sheep genetic resources (40 breeds) possessing 65.06 million sheep (Livestock Census Report, 2012) which constitute 12.7% of the total livestock population of India and ranks third in the world sheep population. Sheep meat is a major source of animal protein for which sale of surplus animals with male body weight at the market age forms a major component of income in sheep husbandry. Therefore, faster growth and early maturity are important components of profitable sheep production. Growth indicated as the live body weight measured at various ages is one of the most important traits in the genetic improvement for meat sheep. Single nucleotide polymorphism of gene sequence which affects body growth starting from pre-weaning daily gain till 11 months of age acts as a basis for breeding of the economically important growth traits through marker assisted selection (Hua et al., 2009). Myostatin, the protein whose expression is under the control of myostatin (MSTN) gene acts as a negative regulator of muscle cell growth, where the loss of functional myostatin is known to cause the “double-muscled” phenotype in cattle (Kambadur et al., 1997;
∗ Corresponding author. E-mail address: [email protected] (A.R. Sahu). http://dx.doi.org/10.1016/j.smallrumres.2017.01.009 0921-4488/© 2017 Elsevier B.V. All rights reserved.
McPherron et al., 1997 McPherron and Lee, 1997; Grobet et al., 1998), in sheep (Broad et al., 2000) and in goats (Li et al., 2006). MSTN also called as growth differentiation factor 8 (GDF 8) gene is located on chromosome 2 of sheep (Archibald et al., 2010). Mutation in MSTN acts in various ways like myostatin null mice causes hypertrophy and hyperplasia of skeletal muscle mass (McPherron et al., 1997) whereas deletion, missense or nonsense mutations leads double muscling in Belgian Blue and Piedmontese breeds of cattle (McPherron et al., 1997 McPherron and Lee, 1997) and hypertrophy in child due to splice site mutation in human being (Schuelke et al., 2004). However, there is no report regarding the variation in the exons of MSTN in Indian sheep breeds till date. Hence, the identification of the variation in coding region of ovine MSTN gene using PCR-RFLP followed by sequencing and the association analyses with growth traits are described in this study.
2. Material and methods 2.1. Experimental sheep flocks and PCR amplification Madras Red and Mecheri are two large sized hairy meat breeds of Tamil Nadu, India considered for the study. The genomic DNA was isolated from Madras Red (127) and Mecheri (105) sheep using standard Phenol-Chloroform extraction procedure
82
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
(Sambrook et al., 1989) with slight modifications by using DNAzol reagent, instead of SDS and proteinase K. The exon 3 (380 bp) of MSTN, with part of intron 2 and 3 UTR on either side was amplified as a 615 bp fragment, using the primer sequence designed by Fast PCR Primer designing (FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis) software (Kalendar et al., 2014). The primer was synthesised at M/s. Eurofins Genomics, Bangalore. The PCR was performed with volume of 20 l using 10 l master mix (Ampliqon), 0.6 l each of forward primer (CGG TAG GAG AGT GTT TGG GGA T) and reverse primer (GAT GGT TAA ATG CCA ACC ATT GC), 1.3 l template DNA (50 ng/l) and 7.5 l of NFW. The amplification was carried out with a program of 5 min initial denaturation at 95 ◦ C followed by 32 cycles of 95 ◦ C for 35 s, annealing at 61.5 ◦ C for 30 s and extension at 72 ◦ C for 35 s, with a final extension of 5 min at 72 ◦ C. The product was checked by electrophoresis (2 per cent agarose) and the gel was stained with ethidium bromide, visualized under UV light. 2.2. PCR- RFLP and analysis of sequence variation The PCR product (615 bp) was digested with MspI restriction enzyme at 37 ◦ C for 6 h in water bath with the restriction digestion mix of 10.0 l of PCR product, 1.5 l of 1X cut smart buffer, 0.1 l of MspI enzyme (3 units/l) and 3.4 l of NFW. The digested products were checked in 2% agarose gel using 50 bp DNA ladder. Amplicons of eight samples from each breed (Madras Red and Mecheri) were sequenced for 615 bp fragment of MSTN gene. The amplicon was sequenced in both forward and reverse directions at M/s. Eurofins Genomics India Pvt. Ltd., Bangalore. The instrument used for sequencing was ABI PRISM 3730XL Genetic analyzer (Applied Biosystems, USA). Sequence data were analysed using the SeqMan program of LASERGENE software version 7.1.0 (44) (DNASTAR Inc., USA). 2.3. Statistical analysis The genotypes were assigned on the basis of band patterns of the PCR products. The genotype and gene frequencies were estimated and the locus was tested for Hardy-Weinberg equilibrium. The polymorphism observed at the G5622C locus of MSTN gene in Madras Red and Mecheri sheep breeds was analysed separately for their association with body weights at various ages viz., birth, weaning (three month), six, nine and 12 months using least-squares procedures (Harvey, 1990). The data on number of animals used for association analysis was lower than the number of animals genotyped due to the non-availability of complete set of data on body weights and deletion of outliers. Data were classified into two periods of birth viz., period 1 (2007–2010) and period 2 (2011–2014) in Madras Red; and period 1 (2008–2010) and period 2 (2011–2013) in Mecheri breed of sheep; sex as males and females; and different genotypes at G5622C locus of MSTN gene. Weight of dam at lambing was also included as a covariable in the model as below: Y ijkl = + Pi + Sj + Gk + b(WMijk − WM) + eijkl
Fig. 1. MspI-RFLP of PCR amplicon (615 bp) in exon 3 of MSTN gene. Lane M: 50 bp DNA ladder, Lanes 1–5: Madras Red, and Lanes 6–9: Mecheri sheep. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Gk = fixed effect of kth genotype(k = 1and2) ¯ = regression of Y on dam s weight at lambing b (WMijk − WM) eijkl = residual random error, NID (0, 2 ) 3. Results and discussion The restriction enzyme, MspI had cleavage site (C/CGG) at nucleotide position 314 of the amplified region (615 bp) and showed polymorphism in both sheep breeds studied. Restriction digestion revealed the existence of two genotypes viz., MM (301 bp and 314 bp) and Mm (301 bp, 314 bp and 615 bp) and the locus was characterized by the presence of two alleles M and m (Fig. 1). The mm genotype was absent in both breeds. The breed-wise genotypic and allelic frequencies observed (Table 1) for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively. The estimated allelic frequencies for M and m in Madras Red and Mecheri breeds were 0.7085 and 0.2915; and 0.7430 and 0.2570 respectively. The frequency of MM genotype was higher in Mecheri than Madras Red and vice versa for Mm genotype. The sequences were assembled and screened for single nucleotide polymorphisms (SNPs). On analysing the amplicon (615 bp), one SNP was found in the exon 3 of MSTN at locus G5622C, characterized by transversion of G > C, but no change in amino acid was observed in both the sheep breeds. Two genotypes viz., GG and GC were observed as shown in chromatogram (Fig. 2). The 5622G > C transversion observed in the present study is contrary to the reports in the New Zealand Romney sheep (Zhou et al., 2008) and in Chinese breeds of sheep (Gan et al., 2008), where exon 3 was monomorphic. The estimated allelic frequency for M was higher than m in both the breeds of sheep studied. The locus 5622G > C has significantly (P < 0.01) deviated from genetic equilibrium in both sheep breeds. The effect of 5622 G > C mutation in exon 3 of MSTN gene on nine
Where, Y ijkl = is the body weight of the lth animal of the kth genotype of the jth sex born in the ith period of lambing = overallmean Pi = fixed effect of ith period of lambing (i = 1and2) Sj = fixed effect of jth sex of the lamb (j = 1and2)
and twelve months weights was significant (P < 0.05) in Madras Red sheep. Animals with homozygous (MM) genotype were heavier than heterozygous (Mm) genotype by 1.162 kg and 1.227 kg at nine and 12 months weight respectively. However, a non-significant effect of this mutation was observed for birth, weaning (three months) and six months weight in the same breed. There was no
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
83
Table 1 Breed-wise genotypic and allelic frequencies at G5622C locus in exon 3 of MSTN gene. Locus
Breed
Genotype
Genotypic frequency
Allelic frequency
2 value
G5622C
Madras Red (127)
MM (53) Mm (74) mm (0) MM (51) Mm (54) mm (0)
0.417 0.583 0 0.486 0.514 0
M = 0.7085 m = 0.2915
21.45*
M = 0.7430 m = 0.2570
12.59*
Mecheri (105)
Figures in parentheses indicate number of observations. * highly significant (P < 0.01).
Fig. 2. Assembly of the chromatograms of Exon 3 of MSTN gene to determine the polymorphism across the Madras Red and Mecheri breeds of sheep. Chromatograms of the genotypes in Exon 3: Madras Red MM and Mm; and Mecheri MM at position 5622 locus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Least-squares means ± S.E. (kg) for the effect of 5622G > C in MSTN gene on growth traits. Body weight at various ages
Breeds Madras Red MM (42)
Birth weight
NS 2.737 ± 0.062
3 months weight
NS 9.528 ± 0.402
6 months weight
NS 13.602 ± 0.405
9 months weight
* 17.518 ± 0.512b
12 months weight
* 19.656 ± 0.537b
Mecheri Mm (68) 2.729 ± 0.063 8.842 ± 0.412 13.217 ± 0.415 16.356 ± 0.524a 18.429 ± 0.550a
Prob
MM (42)
Mm (43)
Prob
0.9030
NS 2.600 ± 0.066
2.462 ± 0.082
0.0959
0.1263
NS 12.325 ± 0.439
12.920 ± 0.545
0.2787
0.3924
NS 14.865 ± 0.483
14.717 ± 0.600
0.8054
0.0432
NS 17.799 ± 0.552
17.022 ± 0.686
0.2614
0.0419
NS 20.121 ± 0.623
19.220 ± 0.774
0.2482
Figures in parentheses indicate number of records used for analysis. NS = not significant, * = significant (P < 0.05). Means with at least one common superscript within classes do not differ significantly (P ≥ 0.05).
significant difference in body weights at different ages and genotypes MM and Mm at locus 5622 G > C in Mecheri sheep (Table 2). Simillar findings for the non-significant effect of genetic variants in exon 3 of MSTN gene with growth traits were reported in Zel sheep (Dehnavi et al., 2012) and Baluchi sheep (Masoudi et al., 2005).
4. Conclusion The exon 3 of MSTN gene is polymorphic in Madras Red and Mecheri sheep breeds of India. SNP 5622 G > C is novel and first report in Indian sheep breeds. This mutation had significant association with nine and 12 months body weight, where the genotype
84
A.R. Sahu et al. / Small Ruminant Research 149 (2017) 81–84
MM is heavier than Mm in Madras Red sheep. The genotypic frequencies for MM genotype were 0.417 and 0.486; and Mm were 0.583 and 0.514 in Madras Red and Mecheri breeds respectively and there is complete absence of mm. This suggests the possible use of M allele at G5622C locus as a potential marker for selection in Madras Red sheep breed. The locus 5622G > C has significantly (P < 0.01) deviated from genetic equilibrium in both the sheep breeds would possibly be due to small sample size and selection. Further studies are suggested on a larger sample size to confirm the effect of this mutation G5622C on growth traits in Madras Red sheep, so as to use this as a potential marker. Absence of significant association of this mutation with body weights at various ages in Mecheri sheep could possibly due to founder effect and inadequate pasture availability. It is also concluded that the absence of association for growth traits in Madras Red and Mecheri might be due to the high genetic similarity, forces of evolution and distribution in the similar agro-climatic conditions of these two breeds. Being this as the first report of MSTN polymorphism in Indian sheep breeds needs further research to throw light on the significant effect of MSTN mutations on growth traits. Conflict of interest The authors declare no conflict of interest regarding this article. Acknowledgements Authors are thankful to the Professor and Heads, Post-Graduate Research Institute in Animal Sciences (PGRIAS), Kattupakkam and Mecheri Sheep Research Station (MSRS), Pottaneri for their necessary support to carry out the research work. References Archibald, A.L., Cockett, N.E., Dalrymple, B.P., Faraut, T., Kijas, J.W., Maddox, J.F., McEwan, J.C., Hutton Oddy, V., Raadsma, H.W., Wade, C., Wang, J., Wang, W., Xun, X., 2010. The sheep genome reference sequence: a work in progress. Anim. Genet. 41 (5), 449–453. Broad, T.E., Glass, B.C., Greer, G.J., Robertson, T.M., Bain, W.E., Lord, E.A., McEwan, J.C., 2000. Search for a locus near to myostatin that increases muscling in Texel sheep in New Zealand. Proc. N. Z. Soc. An. 60, 110–112.
Dehnavi, E., Azari, M.A., Hasani, S., Nassiry, M.R., Mohajer, M., Ahmadi, A.K., Shahmohamadi, L., Yousefi, S., 2012. Polymorphism of myostatin gene in Intron 1 and 2 and Exon 3, and their associations with yearling weight, using PCR-RFLP and PCR-SSCP techniques in Zel sheep. Biotechnol. Res. Int., http:// dx.doi.org/10.1155/2012/472307, Article ID 472307, 5 pages. Gan, S.Q., Du, Z., Liu, S.R., Yang, Y.L., Shen, M., Wang, X.H., Yin, J.L., Hu, X.X., Fei, J., Fan, J.J., Wang, J.H., He, Q.H., Zhang, Y.S., Li, N., 2008. Association of SNP haplotypes at the myostatin gene with muscular hypertrophy in sheep. Asian-Aust. J. Anim. Sci. 21, 928–935. Grobet, L., Pncelet, D., Royo, L.J., Brouwers, B., Pirottin, D., Michau, C., Menissier, F., Zanotti, M., Dunner, S., George, M., 1998. Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling. Mamm. Genome 9, 210–213. Harvey, W.R., 1990. Mixed Model Least-squares and Maximum Likelihood Computer Programme. PC-2 Version. Ohio State University, Colombus. Hua, G.H., Chen, S.L., Jn, Y.U., Cai, K.L., Wu, C.J., li, Q.L., Zhang, C.Y., Liang, A.X., Han, L., Geng, L.Y., Shen, Z., Xu, D.Q., Yang, L.G., 2009. Polymorphism of the growth hormone gene and its association with growth traits in Boer goat bucks. Meat Sci. 81, 391–395. Kalendar, R., Lee, D., Schulman, A.H., 2014. Fast PCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. In: Valla, Svein, Lale, Rahmi (Eds.), DNA Cloning and Assembly Methods, Methods in Molecular Biology. Humana Press, 1116, pp. 271–302. Kambadur, R., Sharma, M., Smith, T.P.L., Bass, J.J., 1997. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res. 7, 910–916. Li, X.L., Wu, Z.L., Gong, Y.F., Liu, Y.Q., Liu, Z.Z., Wang, X.J., Xin, T.R., Ji, Q., 2006. Single nucleotide polymorphism identification in the caprine myostatin gene. J. Anim. Breed. Genet. 123, 141–144. Livestock Census Report, 2012. 19th Livestock Census. Department of Animal Husbandry and Dairying, Ministry of Agriculture, Government of India, New Delhi. Masoudi, A., Hemrani, H., Abbasi, A., 2005. Using PCR-SSCP techniques to study polymorphism of myostatin gene and its association with production traits in Baluchi sheep. In: Proc. 4th National Biotechnol. Congr, Iran, Kerman, Iran. McPherron, A.C., Lee, S.J., 1997. Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. U. S. A. 94, 12457–12461. McPherron, A.C., Lawler, A.M., Lee, S.J., 1997. Regulation of skeletal muscle mass in mice by a new TGF- superfamily member. Nature 387, 83–90. Sambrook, J.E., Fritsch, F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold spring Harbor Laboratory Press, New York, USA. Schuelke, M., Wagner, K.R., Stolz, L.E., 2004. Myostatin mutation associated with gross muscle hypertrophy in a child. N. Engl. J. Med. 350 (26), 2682–2688. Zhou, H., Hickford, J.G.H., Fang, Q., 2008. Variation in the coding region of the myostatin (GDF8) gene in sheep. Mol. Cell. Probes 22, 67–68.