Screening the fertile ewes from national herds to detect the major genes for prolificacy is an effective way to create the fertile flocks. Growth differentiation factor (GDF) 9 is a member of the transforming growth factor β superfamily that is essential for folliculogenesis and female fertility. The aim of this study was to detect single nucleotide polymorphisms (SNPs) in exon 2 of GDF9 gene in Kermani sheep breed using PCR-SSCP. Genomic DNA was extracted from whole blood of collected samples using salting-out method. Whole exon 2 of GDF9 gene was amplified (634 bp and 647 bp fragments) using designed specific primers. The single stranded conformation polymorphism (SSCP) patterns of PCR products were studied using electrophoresis on acrylamide gel and silver-nitrate staining method. Finally, 4 banding patterns for the first primer pair and 4 banding patterns for the second primer pair were obtained. Also, indices of population genetic per SNP were calculated using Gen Alex 6.41 software. The sequencing results showed the presence of 3 mutations (SNP) (443, 477 and 721 positions) in the studied population.
Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, Hovata O, Dale L, Ritvos O (1999) Human growth differentiation factor 9 (GDF9) and its novel homolog GDF9-B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 84: 2744-2750.
Askari N, Mohammadabadi M, Baghizadeh A (2011) ISSR markers for assessing DNA polymorphism and genetic characterization of cattle, goat and sheep populations. Iranian J Biotech 9: 222-229.
Bai J, Shi HC, Liu MJ, Li F (2007) Study on GDF9 and BMP15 as candidate genes for fecundity in Cele Black sheep and Duolang sheep. Grass-Feed Livestock 2: 1-4.
Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196: 80-83.
Bodensteiner KJ, Clay CM, Moeller CL, Sawyer HR (1999) Molecular cloning of the ovine growth/differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries. Biol Reprod 60: 381-386.
Bodensteiner KJ, McNatty KP, Clay CM, Moeller CL, Sawyer HR (2000) Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the inverdale prolificacy gene (FecXI). Biol Reprod 62: 1479-1485.
Bodin L, Elsen JM, Poivey JP, San Cristobal-Gaudy M, Belloc JP, Eychenne F (1998) Hyper-prolificacy in the French Lacaune sheep breed; a possible major gene. In: Proceedings of the Sixth World Congress on Genetics Applied to Livestock Production, Armidale, Australia, 27, pp. 1-14.
Chang JT, Luo YZ, Hu J (2009) Polymorphism of GDF9 as a candidate gene for fecundity in sheep. J Gansu Agric Univ 44: 30-33.
Chen Y, Luo QJ, Zhang YJ, Zhu WY, Yang JQ, Li DZ, Yang FY (2009) Relationships between genetic polymorphisms of BMP15 and GDF9 gene and litter size of sheep. J Xinjiang Agric Univ 32: 10-17.
Chu MX, Sang LH, Wang JY, Fang L, Ye SC (2005) Study on BMPI5 and GDF9 as candidate genes for prolificacy of Small Tail Han sheep. Acta Genetica Sinica (Yi Chuan Xue Bao) 32: 38-45.
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM (1996) Growth differentiation factor-9 is required during early ovarian foliculogenesis. Nature 383: 531-535.
Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM (1998) The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12: 1809-1817.
Eghbalsaied S, Ghaedi K, Shahmoradi S, Pirestani A, Amini H, Saidi T, Nicol L, McNeilly A (2012) Presence of SNPs in GDF9 mRNA of Iranian Afshari sheep. Int J Fertil Steril 5: 225-230.
Gao LX (2007) Polymorphisms of TGF-b1 and GDF9 genes and their relationships with prolificacy in Small Tail Han sheep. Thesis for M.S., Chinese Academy of Agricultural Sciences, pp 32-36.
Ghaffari M, Nejati-Javaremi A, Rahimi-Mianji G (2009) Lack of polymorphism in the oocyte derived growth factor (GDF9) gene in the Shal breed of sheep. S Afr J Anim Sci 39: 355-360
Guan F, Ai JT, Pang XS, Liu SR, Shi GQ, Yang LG (2005) Detection of the polymorphisms of GDF9 and BMP15 genes in sheep. Life Sci Res 9: 184-188.
Hanrahan, JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R, Galloway SM (2004) Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries). Biol Reprod 70: 900-909.
Juengel JL, Bodensteiner KJ, Heath DA, Hudson NL, Moeller CL, Smith P, Galloway SM, Davis GH, Sawyer HR, McNatty KP (2004) Physiology of GDF9 and BMP15 signalling molecules. Anim Reprod Sci 82-83: 447-460.
Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O;Connell AR, Laitinen MP, Cranfield M, Groome NP, Ritvos O, McNatty KP (2002) Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biol Reprod 67: 1777-1789.
Lan ZJ, Gu P, Xu X, Jackson KJ, DeMayo FJ, O’Malley BW, Cooney AJ (2003) GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility. EMBO J 22: 4070-4081.
McGrath SA, Esquela AF, Lee SJ (1995) Oocyte-specific expression of growth/differentiation factor-9. Mol Endocrinol 9: 131-136.
Melo EO, Silva BD, Castro EA, Silva TA, Paiva SR, Sartori R, Franco MM, Souza CJ, Neves JP (2008) A novel mutation in the growth and differentiation factor 9 (GDF9) gene is associated, in homozygosis, with increased ovulation rate in Santa Ines sheep. Biol Reprod 78 (Suppl 1): 141.
Mohammad Abadi MR, Askari N, Baghizadeh A, Esmailizadeh AK (2009) A directed search around caprine candidate loci provided evidence for microsatellites linkage to growth and cashmere yield in Rayini goats. Small Ruminant Res 81: 146-151.
Mousavizadeh A, Mohammad Abadi MR, Torabi A, Nassiry MR, Ghiasi H, Esmailizadeh AK (2009) Genetic polymorphism at the growth hormone locus in Iranian Talli goats by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Iranian J Biotech 7: 51-53.
Nicol L, Bishop SC, Pong-Wong R, Bendixen C, Holm LE, Rhind SM, McNeilly AS (2009) Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep. Reproduction 138: 921-933.
Orisaka M, Orisaka S, Jiang JY, Craig J, Wang Y, Kotsuji F, Tsang BK (2006) Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage. Mol Endocrinol 20: 2456-2468.
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86: 2766-2770.
Orita M, Suzuki Y, Sekiya T, Hayashi K (1989b) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879.
Sadighi M, Bodensteiner KJ, Beattie AE, Galloway SM (2002) Genetic mapping of ovine growth differentiation factor 9 (GDF9) to sheep chromosome 5. Anim Genet 33: 244-245.
Sadighi M, Montgomery W, Bodensteiner KJ, Galloway SM (1998) The growth differentiation factor 9 maps to sheep chromosome 5. Anim Genet 29 (Suppl 1): 36.
Shojaei M, Mohammad Abadi M, Asadi Fozi M, Dayani O, Khezri A, Akhondi M (2010) Association of growth trait and Leptin gene polymorphism in Kermani sheep. J Cell Mol Res 2: 67-73.
Sun H, Tian X, Wang Y (2009) BMPR-IB, BMP15 and GDF9 as candidate genes for prolific trait in Tan sheep. Acta Agricul Boreali-Occident Sin 18: 17-21.
Vacca GM, Dhaouadi A, Rekik M, Carcangiu V, Pazzola M, Dettori ML (2010) Prolificacy genotypes at BMPR1B, BMP15 and GDF9 genes in North African sheep breeds. Small Ruminant Res 88: 67-71
Vitt UA, McGee EA, Hayashi M, Hsueh AJ (2000) In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Endocrinology 141: 3814-3820.
Zamani P, Akhondi M, Mohammadabadi MR, Saki AA, Ershadi A, Banabazi MH, Abdolmohammadi AR (2011) Genetic variation of Mehraban sheep using two intersimple sequence repeat (ISSR) markers. Afr J Biotechnol 10: 1812-1817.