Polymorphism at second half of exon 2 of GDF9 gene in Kermani sheep breed using PCR-SSCP and sequencing

Applications of molecular genetics have many important advantages (Mousavizadeh et al. 2009). Using genetic markers in animal selection and breeding may also dramatically expedite genetic improvement (Javanmard et al. 2008). Study of native breeds is necessary for conservation of genetic resource in livestock (Mohammadabadi et al. 2010). There are more than 26 sheep breeds in Iran adapted to different environmental circumstances (Zamani et al. 2015). One of the most important breeds of Iranian sheep is Kermani sheep. This local breed lives in the south-eastern of Iran and is a fat-tail breed and well adapted to a wide range of harsh environmental conditions in Kerman province (Mohammadabadi 2017). Growth differentiation factor (GDF) 9 is a member of the transforming growth factor β superfamily that is secreted from oocytes during folliculogenesis (Aaltonen et al. 1999) and is essential for folliculogenesis and female fertility (Khodabakhshzadeh et al. 2016). Hanrahan et al. (2004) revealed eight single nucleotide polymorphisms across the entire coding region (G1–G8) and these differences correspond to one SNP in exon 1, one SNP in the intron, and five SNPs in exon 2. It is proven that exon 2 is more important than exon 1 and intron.
Material and The blood samples were randomly collected from Kermani sheep (102 animals) from both sexes and with different ages (Kerman, Iran), using vacuum tubes with 0.25% ethylene diamine tetra acetic acid (EDTA). The blood samples were transferred in dry ice to the laboratory and stored at -20 °C pending assays. Blood samples of the animals were used to extract genomic DNA using the salting out procedure described by Abadi et al. (2009). The quality of DNA was checked by spectrophotometry taking ratio of optical density (OD) value at 260 and 280 nm. The sheep GDF9 gene was amplified using the polymerase chain reaction (PCR) with designed specific primers. These primers were used to amplify fragment 647 bp of the exon 2 for the sheep GDF9 gene. The PCR reaction was performed in a 25 μL reaction volume containing 2 μL of genomic DNA (50 ng/μL), 1 μL of MgCl2 (3 mM), 1μL of each forward and reverse primers (10 pmol each), 0.5μL of dNTPs (500 μM each), 0.3 unit of Taq DNA polymerase (Cinna Gene, Iran) and 10X PCR buffer. DNA amplifications were performed using thermo cycler (CLEMENS, Germany) programmed for a preliminary step of 5 min at 94°C, followed by 33 cycles of 30 s at 94°C, 50 s at 62.5°C for the first primer pair and 63.6°C for the second primer pair and 50 s at 72°C, with a final extension of 8 min at 72°C. Amplification was verified by electrophoresis on 1% (w/v) agarose gel in 1 x TBE buffer (2 mM of EDTA, 90 mM of Tris-Borate, pH 8.3), using a 100bp ladder as a molecular weight marker for confirmation of the length of the PCR products. Gels were stained with ethidium bromide (1 μg/mL). The SSCP technique was used to allow the sequence variants to be detected from the migration shift in PCR amplified fragments of the gene. For SSCP analysis, 6 μL of each PCR product was mixed with 12 μL of denaturing loading buffer (19 mL formamide, 0.98 gr NaOH (3% NaOH solution), 0.01 gr xylene cyanol and 0.01gr bromophenol blue). The samples were denatured by heating at 95°C for 10 min, then immediately chilled on ice and loaded onto 8% polyacrylamide gel (37.5:1). Gels were run at 170-180 V for 7-8 hours at 4°C. The electrophoresis was carried out in a vertical unit in 1x TBE buffer (Tris 100 mM, boric acid 9 mM, EDTA 1mM). The gels were stained with silver nitrate to observe the conformational patterns. After revealing the single stranded conformation polymorphism (SSCP) patterns for this locus, from each of the ovine GDF9 variants identified by PCR–SSCP, one sample was sequenced (Mahan Gene, Iran). The raw sequence data were edited using Bioedit 7.0 software. Multiple sequence alignments were performed with Bioedit 7.0 and DNAMAN software to identify single nucleotide polymorphisms (SNPs) in the exon 2 of the GDF9 gene in Kermani sheep. The nucleotide sequence of exon 2 was translated to amino acid sequence for each particular allelic variant. The BLAST algorithm was used to search the NCBI GenBank databases for comparison of the ovine GDF9 sequences with homologous sequences of other animals to determine similarity percentage and detect the novel SNPs in the studied locus. Population genetic parameters were obtained using GenAlex6.41 software.
As expected, PCR amplification of the ovine GDF9 gene for Kermani sheep gave uniform fragment 647 bp by running on 1% agarose gel and the amplified fragment size were consistent with the expected size and subsequently sequencing of the ovine GDF9 amplicons confirmed them to be 647 bp in size (Fig 1). The SSCP analysis revealed four unique banding patterns for the second half of the exon representing different allelic variants (Fig 2). In the studied population, four different genotypes and three haplotypes were observed for the second half of the exon 2 (Table 1). Frequencies of the detected genotypes and haplotypes in the studied population are provided in Table 2. In total, in this population, genotype 2 in the second half of the exon 2 were most common with a frequency of 0.411. The sequencing results were representative of the point mutations at nucleotide positions 994 and 978 in exon 2 of the GDF9. The analysis results of the GenAlex software in the studied position revealed the lack of Hardy-Weinberg equilibrium in single nucleotide polymorphism (SNP) at position 978. The high level of Shannon index in both positions of the identified mutations indicated that the level of Biodiversity in GDF9 gene position associated with the sample population was high. Hanrahan et al. (2004) discovered eight variants (G1 to G8) of GDF9 gene in Cambridge and Belclare sheep breeds using PCR-SSCP and sequencing. However, G8 variant caused serine to phenylalanine substitution at residue 395 which replaced an uncharged polar amino acid with a nonpolar one at residue 77 of the mature coding region and may change the function of GDF9 in sheep (Hanrahan et al. 2004). Nikol et al. (2009) discovered 4 variants (G3, G4, G5 and G6) of GDF9 gene in Icelandic Thoka sheep that is in agreement with the result of the present study. The high level of genetic variability observed in the coding region of the ovine GDF9 gene in this study suggests that this region of the GDF9 gene probably affects folliculogenesis and female fertility in sheep; hence further association studies using appropriate populations are needed to identify genetic variants that can be used as markers related to fertility.
Regarding the estimated criteria and relatively high level of heterozygosity, it can be concluded that the studied population has a relatively high polymorphism in the examined locus. The discovered alleles and genotypes can also be used as markers in marker-assisted selection of sheep for economic traits in future.