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PCR-RFLP anaLysis oF MsTn gene in KaRaKaChan sheeP bReedM. BOZHILOVA-SAKOVA1, I. DIMITROVA1, A. TENEVA1 and N. PETROV2

1University of Forestry, Department of Genetics and Selection, Agronomy Faculty, 10, Kl. Ohridski bl.,1756 Sofia, Bulgaria

2Institute of Soil Science “Nikola Poushkarov” 7, Shousse Bankya Str., 1080 Sofia, Bulgaria

bozhilova-sakova, M., i. dimitrova, a. Teneva and n. Petrov, 2016. PCR-RFLP analysis of MSTN gene in Karakachan sheep breed. Bulg. J. Agric. Sci. 22 (Suppl. 1): 115–117

abstract

The present study was conducted in order to investigate and identify the allelic variants of myostatin (MSTN) gene asso-ciated with meat traits in sheep. Blood samples were taken from a population of 25 animals of Bulgarian breed Karakachan sheep – 22 ewes and 3 rams. Genomic DNA was extracted and myostatin genotypes were estimated by means of PCR am-plification and PCR-RFLP method. The PCR products were digested with HaeIII restriction enzyme. In the total population of sheep polymorphism was not found. The MSTN locus was found to be monomorphic – only the allele m and the genotype mm were detected.

Key words: Karakachan sheep breed, MSTN gene, PCR-RFLP method

Bulgarian Journal of Agricultural Science, 22 (Supplement 1) 2016, 115–117Anniversary scientific conference „Animal Science - Challenges and Innovations”, November 4-6, 2015, SofiaAgricultural Academy, Institute оf Animal Science – Kostinbrod

E-mail: bojilova_milena@abv.bg

introduction

Nowadays the consumers of sheep meat demand not only quantitative traits but qualitative traits of meat. The candidate genes for meat traits in sheep and gene assisted selection are in great help of breeders. Molecular genetic researches have shown a discovery of several genes which affect some quanti-tative traits, such as meat production (Tambasco et al., 2003). There are many published articles on different genes associ-ated with meat traits in sheep and one of them is myostatin gene (Shafey et al., 2014).

Growth and differentiation factor 8 (GDF8) or myostatin is known to influence muscle hypertrophy and meat tender-ness in sheep (Kijas et al., 2007). MSTN gene is located in chromosome 2, locus 2q32.2 from the sheep genome (Ovis aries L.). The gene contains 3 exons and 2 introns in all spe-cies studied (Bellinge et al., 2005; Nakev et al., 2013). Myo-statin is part of the mammalian growth transforming family (TGF-beta superfamily) (Sontegard et al., 1998). This gene is considered to be an inhibitor of skeletal muscle growth therefore a mutation in this region causes mutation leading

to increased muscling (McPherron et al., 1997; Georgieva et al., 2015).

Different polymorphisms have been detected in MSTN gene, related with growth and carcass traits and these poly-morphisms are used as efficient markers in genetic assisted selection in order to increase the quality and quantity of meat (Mirhoseini and Zare, 2012).

Materials and Methods

The present investigation was carried out in the DNA lab-oratory part of Faculty of Agronomy at the University of For-estry. The material in this study includes animals from one indigenous Bulgarian sheep breed, Karakachan - 22 ewes and three rams which are part of one private herd in town Sa-pareva banya, placed in Southwest Bulgaria. Approximately 5 mL of peripheral blood was collected from V. jugularis in vacuum tubes, containing EDTA. DNA was extracted by manual commercial kit for DNA purification according to the manufacturer’s instruction (QIAamp DNA Blood Mini

M. Bozhilova-Sakova, I. Dimitrova, A. Teneva and N. Petrov116

Kit Qiagen). The DNA concentration of each sample was de-termined by spectrophotometer Biodrop.

PCR amplification reaction was carried out in total vol-ume of 10 µl consisting of 4 µl DNA, 5 µl Red TaqPolymerase Master Mix (VWR) and 0.4 µl of each primer – forward and reverse (Bioneer) (Table 1). One pair of primers was used for amplifying exon 3 of the MSTN gene suggested by Dehnavi et al. (2012).

F: 5’- CCG GAG AGA CTT TGG GCT TGA -3’R: 5’- TCA TGA GCA CCC ACA GCG GTC -3’After PCR amplification it was obtained a PCR product

with length 337 bp. The PCR conditions are presented in Ta-ble 2.

The digestion reaction was carried out in 10 µl final vol-ume, containing 6 µl PCR product and 4 µl HaeIII restriction enzyme with buffer (Bioneer). PCR products were incubated at 37oC for overnight (up to 15 h). The restriction enzyme cuts in following specific region:

5’G↑GCC 3’ CCG↑GThe fragment sizes were determined using GeneRuler.

Ladder, 50 bp (Sigma) supplied with 1 mL 6xDNA Loading dye. The obtained restriction products were tested on 2% aga-rose (Healthcare) gel in 1xTBE buffer, stained with GelRed (Biotium) and visualized under UV - transilluminator UVP.

Results and discussion

The quality of extracted DNA samples was tested on 1% agarose gel and it was visualized under UV - transillumina-tor (Figure 1). After PCR amplification of exon 3 of MSTN sheep gene it was obtained a fragment with size 337 bp (Fig-ure 2). After digestion with specific endonuclease HaeIII in present study was detected only genotype mm (Figure 3) and only one allele m. Allele M and genotypes Mm and MM were not detected in this herd. Exon 3 of MSTN locus was found to be monomorphic in this population.

After digestion with HaeIII restriction enzyme the other researchers detected two alleles - M and m and three geno-types respectively: genotype MM - one fragment with length 337 bp, genotype Mm - four fragments: 337 bp, 131 bp, 123 bp and 83 bp and genotype mm – three fragments: 131 bp, 123 bp and 83 bp (Dehnavi et al., 2012; Jamshidi et al., 2014; Soufy et al., 2009).

The results in the present study are in agreement with other studies. In our previous investigations on two Bulgar-ian sheep breeds – 121 adult sheep of Synthetic Population Bulgarian Milk and 32 rams of Northeast Bulgarian Meri-no we found similar results and all studied sheep carried the genotype mm (Georgieva et al., 2015; Dimitrova et al., 2016). In Zel sheep Dehnavi et al., (2012) reported polymorphism in intron 2, but intron 1 and exon 3 were monomorphic. In Egypt Elkorshy et al. (2013) studied five Egyptian and Saudi sheep breeds and in all of them the exon 3 of MSTN locus was reported as monomorphic. In Iran, Azzari et al. (2012) reported similar results in 110 native Dalagh sheep.

On the contrary, in Iranian Mehraban’s sheep Jamshidi et al. (2014) detected two genotypes of exon 3 in MSTN gene – genotype Mm and genotype mm with frequencies 0.053 and 0.947, respectively. Soufy et al., (2009) observed polymor-phism for exon 3 in Sanjabi sheep in Iran. They determined all three possible genotypes – MM, Mm and mm with fre-quencies 2.00 %, 1.33 % and 96.70 %, respectively and the estimated allele frequencies were: for allele M 3.00% and for allele m 97.0%.

Table 1Reaction mix for PCR amplificationProduct VolumeddH2O 0.2μlRed Taq Master mix 5.0μlForward primer 0.4μlReverse primer 0.4μlDNA 4.0μlTotal volume 10μl

Table 2Conditions of PCR amplificationStage Temperature TimePrimary denaturation 95 oC 5 min30 cyclesDenaturation 95 oC 30 sAnnealing 58oC 45 sElongation 72 oC 1 minFinal extension 72 oC 10 minStore 10 oC

Fig. 1. Testing of extracted dna samples on 1% agarose gel with agarose electrophoresis

PCR-RFLP Analysis of MSTN Gene in Karakachan Sheep Breed 117

Conclusion

In present investigation it may be concluded that exon 3 of MSTN sheep gene is found to be monomorphic for this herd of indigenous Karakachan breed kept in Southwest Bulgaria. It was detected only the allele m and the genotype mm, re-spectively with frequency 1.00.

Referencesazari, M. a., e. dehnavi, s. yousseli and L. shahmohamadi,

2012. Polymorphism of calpastatin, calpain and myostatin genes in native Dalaghsheep in Iran. Slovak J. Anim. Sci., 45 (1): 1-6.

bellinge, R. h. s., d. a. Liberles, s. P. iaschi, P. a. o’brien and g. K. Tay, 2005. Myostatin And Its Implications On Animal Breeding: A Review. Anim. Genet., 36: 1–6.

dehnavi, e., M. azari, s. hasani, M. R. nassiry, M. Mohajer, A. Ahmadi, L. Shahmohamadi and S. Yousefi, 2012. Poly-morphism 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. Biotechnology Research International, Volume 2012, Article ID 472307, 5 pages, DOI: 10.1155/2012/472307.

dimitrova, i., M. bozhilova-sakova, n. stancheva and T. Tzonev, 2016. Molecular Analysis of Ovine Myostatin Gene (MSTN) in Northeast Bulgarian Merino Sheep Breed Using PCR-RFLP Method. Bulg. J. Agric. Sci. (in print).

elkorshy, n., K. Mahrous and L. salem, 2013. Genetic poly-morphism detection in four genes in Egyptian and Saudi sheep breeds. World Applied Science Journal, 27 (1): 33-43.

georgieva, s., d. hristova, i. dimitrova, n. stancheva and M. bozhilova-sakova, 2015. Molecular analysis of ovine calpasta-tin (CAST) and myostatin (MSTN) genes in Synthetic Popula-tion Bulgarian Milk sheep using PCR-RFLP. Journal of BioSci-ence and Biotechnology, 4 (1): 95-99.

Jamshidi, s., s. Karani and M. goudarzi, 2014. Study polypor-phism myostatin gene in Mehraban’s sheep using PCR-RFLP method. Sci,Int (Lahore), 26(3): 1129-1135.

Kijas, J.W., R. McCulloch, J. e. h. edwards, V. h. oddy, s. h. Lee, and J. van der Werf, 2007. Evidence for multiple allele effecting muscling and fatness at the Ovine GDF8 locus.BMC Genetics. Doi:10.1186/1471-2156-8-80.

McPherron, a. C. and s. J. Lee, 1997. Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. USA 94(23): 12457-12461.

Mirhoseini, s. Z. and J. Zare, 2012. The role of Myostatin on growth and carcass raits and its application in animal breeding, Life Sci J, 9 (3); 2353-2357.

nakev, g., P. stamberov, i. dimitrova, n. stancheva, g. geor-gieva, d. hristova, g. angelov, T. Mehmedov, K. genova and a. Teneva, 2013. Growth and development of skeletal muscle in connection with the expression of the myostatin gene (MSTN). In: Proceedings of the 10th International Symposium Modern Trends in Livestock Production, October 2-4, 2013, pp. 640-647.

shafey, h. i., K. F. Mahrous, M. s. hassanane, M. abdel Mordy and h. e. Rushdi, 2014. Genetic polymorphism of myostatin and insulin-like growth factor binding protein-3 genes in Egyp-tian sheep breeds, Global Veterinaria, 13 (3): 419-424, DOI: 10.5829.

sontegard, T. s., g. a. Rohrer and T. P. L. smith, 1998. Myo-statin maps to porcine chromosome 15 by linkage and physical analysis, Anim. Genet., 29 (1): 19-22.

soufy, b., M. R. Mohammad abadi, K. shojaeian, a. baghiza-deh, s. Ferasaty, n. askari and o. dayani, 2009. Evaluation of Myostatin gene polymorphism in Sanjabi sheep by PCR-RFLP method. Animal Science Reserches Tabriz Univ., 19 (1): 81-89.

Tambasco, d. d., C. C. P. Paz, M. Tambasco-studart, a. P. Pereira, M. M. alencar, a. R. Freitas, L. L. Coutinho, L. U. Packer and L. C. a. Regitano, 2003. Candidate genes for growth traits in beef cattle cross Bostaurus × Bosindicus. Jour-nal of Animal Breeding and Genetics, 120: 51-56.

Fig. 3. Restriction analysis of amplified product of MsTn gene with HaeIII restriction enzyme on 2% agarose gel electrophoresis in Karakachan breed.

Fig. 2. Testing of obtained PCR fragment after PCR amplification on 2% agarose gel