228 Journal of Food, Agriculture & Environment, Vol.13 (2), April 2015 www Journal of Food, Agriculture & Environment Vol.13 (2): 228-231. 2015 .world-food.net Meri-Rastilantie 3 B, FI-00980 WFL Publisher Science and Technology Helsinki, Finland e-mail: info@world-food.net Investigation of the polymorphism of the myostatin gene by using PCR-RFLP in two tilapia species (Oreochromis aureus and Oreochromis niloticus) Makbule Baylan 1 *, Gamze Mazi 1 , Numan Ozcan 2 , Bahri Devrim Ozcan 3 and Ali İrfan Güzel 4 1 Çukurova University, Faculty of Fisheries, Department of Basic Sciences, 01330, Adana, Turkey. 2 Çukurova University, Faculty of Agriculture, Department of Animal Science, 01330, Adana, Turkey. 3 Osmaniye Korkut Ata University, Faculty of Arts and Sciences, Department of Biology, 8000, Osmaniye, Turkey. 4 Recep Tayyip Erdoğan University, Faculty of Medicine, Department of Basic Medical Science, 53100, Rize, Turkey. *e-mail: makyan@cu.edu.tr Received 14 January 2015, accepted 30 March 2015. Abstract The aim of the study was to investigate the restriction fragment length polymorphism (RFLP) of the exon 1–3 fragments from the myostatin gene in two tilapia species, Oreochromis aureus and Oreochromis niloticus. Tissue samples were collected from a total of 50 fish, and the total genomic DNA was extracted and used for PCR amplification. Particular regions of exon 1–3 from the myostatin gene (390, 435 and 342 bp, respectively) were amplified with specific primers for each exon region by using PCR. The PCR-amplified products of each exon region were digested with appropriate restriction endonuclease enzymes and analysed using agarose gel electrophoresis. Key words: Tilapia, myostatin, polymorphism, PCR-RFLP. Introduction Myostatin (MSTN) is an important negative regulator gene of the skeletal muscle mass and has a potential application in both agriculture and aquaculture. The myostatin gene, also known as GDF-8 (growth and differentiation factor-8), inhibits muscle growth 1 . It is a member of the TGF-b (transforming growth factor- beta) superfamily, which is comprised of proteins that have important roles in cell growth and signal transduction. Because of its regulator role in muscle development and growth, MSTN has been studied for productivity, growth and performance in livestock, including cattle 2 , pigs 3 , sheep 4 , chickens 5, 6 , rabbits 7 , and some commercial fish species, such as rainbow trout 8 , Mozambique tilapia 9 , white perch 10 , Atlantic salmon 11 , shi drum 12 , gilthead sea bream 13 , European sea bass 14 , white bass 9 , and catfish spp. 15–17 . Several polymorphisms have been identified in the gene, indicating that the MSTN gene is highly variable 8, 10 . Since the discovery of MSTN’sprimary role in skeletal muscle growth and development, a considerable number of MSTN polymorphisms have been investigated. Several researchers showed polymorphisms of this gene in two breeds of cattle, the Piedmontese and the Belgian blue, both of which cause double muscling phenotype naturally occurring mutations (11 bp deletion) of the myostatin gene 1, 2, 18– 20 . These polymorphisms,including an 11-base pair deletion in the third exon of the gene 2, 18 , are directly related to the double muscling phenotype 21–24 . This gene consists of three exons and two introns in fish. Exon 1, responsible for the N-terminal signal sequence for secretion, includes the highest inter-specific variability, while exons 2 and 3 are highly conserved across species and are translated into the pro-peptide and C-terminal bioactive dimer 16, 25 . Sun et al. 26 investigated partial genomic fragments of the MSTN gene in common carp (Cyprinus carpio L.) for single nucleotide polymorphisms (SNPs). They also identified two SNPs in intron 2 and two synonymous SNPs in exon 3. Wang et al. 27 found two mutations by screening 103 cultivated mollusc Chlamys farreri specimens for polymorphisms in the MSTN gene using both DNA sequencing methods and PCR-single strand conformation polymorphisms (PCR-SSCP). The aim of this study was to investigate the variability of three exon fragments located on the myostatin gene of two tilapia species by using polymerase chain reactions (PCR) for the amplification of the region of interest by using appropriate restriction enzymes, followed by the restriction fragment length polymorphism (RFLP) method. Material and Methods Samples and DNA isolation: Tilapia tissue samples (Oreochromis aureus (n = 25), Oreochromis niloticus (n = 25)) were obtained fresh from Çukurova University, Fisheries Faculty, Fresh Water Fishes Research and Production Station in Adana (Turkey). The total genomic DNA was extracted according to Asahida et al. 28 procedures.The purified DNA was electrophoresed on 2% agarose gels and stained with ethidium bromide. PCR: The pairs of primers (Table 1) were designed for each of the exon 1, 2 and 3 regions. The 100 ng of genomic DNA was amplified in the total volume of a 23.875-μl PCRmix. The PCR mix consisted of 16.25 μl Master mix (Thermo, K0171), 2.5 μl forward and reverse primers (20 pmol/μl), 0.125 μl (2.5 U/μl )Taq DNA polymerase and 5-μl ddH 2 O. The amplification conditions are shown in Table 2. Negative controls were used in all experiments. Assays were performed in a thermal cycler (Techne), and the amplicons were analysed with 2% agarose gel electrophoresis. The ethidium bromide stained gels were visualised under ultraviolet light.