Identification of single nucleotide polymorphisms in GDF9 gene associated with litter size in Garut sheep

Resti Yuliana Rahmawati(1), Sumadi Sumadi(2), Tety Hartatik(3*)

(1) Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 03, Bulaksumur, Yogyakarta 55281, Indonesia
(2) Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 03, Bulaksumur, Yogyakarta 55281, Indonesia
(3) Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 03, Bulaksumur, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Growth differentiation factor 9 (GDF9) gene has been regarded to have major impacts on ovulation rate and litter size in sheep. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of GDF9 gene and their association with litter size in Garut sheep. For this purpose, a total of 60 ewes of Garut sheep were included in this study. Based on the sheep GDF9 reference sequences (Genbank Acc. No. AF078545.2), one pair of primers (forward: 5’-CTGCTGTTTAACCTGGATCGTG-3 and reverse: 5’-GGAGAGCCATACCGATGTCC-3) was used for PCR amplification. The results of this study revealed that four SNPs (g.54C>T, g.60G>A, g.304G>A and g.333G>A) were found in Garut sheep by direct sequencing. For SNP g.54C>T, Garut sheep exhibited the highest frequency of allele C and genotype CC. On the other hand, SNPs g.60G>A, g.304G>A and g.333G>A showed a higher frequency of allele G than allele A, and the GG genotype was predominant in the population. SNP g.333G>A had a significant effect on litter size (P<0.05), and ewes with GG genotype had a higher litter size than those with GA genotype. Genotype distributions for all identified SNPs were in agreement with Hardy-Weinberg equilibrium. We highlight that SNP g.333G>A may be useful as a genetic marker for litter size in Garut sheep.


Garut sheep; GDF9 gene; prolificacy; single nucleotide polymorphism (SNP)

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Ahmad HI, Liu G, Jiang X, Edallew SG, Wassie T, Tasema B, Yun Y, Pan L, Liu C, Chong Y, Yu ZJ, Jilong H. 2017. Maximum-likelihood approaches reveal signatures of positive selection in BMP15 and GDF9 genes modulating ovarian function in mammalian female fertility. Ecol. Evol. 7: 8895-8902.

Arta PD, Rahayu S. 2013. Analisis polimorfisme gen growth differentiation factor 9 (GDF9) dan hubungannya dnegan keberhasilan inseminasi buatan pada sapi PO. Jurnal Biotropika. 1: 95-100.

Bahrani Y, Bahrani S, Mohammadi HR, Chekani-Azar V, Mousavizadeh SA. 2014. The polymorphism of GDF9 gene in Hisari sheep. Biol. Forum. 6:46-52.

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: 14791485.

Chen SY, Su YH, Wu SF, Sha T, Zhang YP. 2005. Mitochondrial diversity and phylogeographic structure of Chinese domestic goats. Mol. Phylogenetics Evol. 37: 804–814.

Davis GH. 2004. Fecundity genes in sheep. Anim. Reprod. Sci. 82: 247-253.

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.

Elvin JA, Clark AT, Wang P, Wolfman NM, Matsuk MM. 1999. Paracrine action of growth differentiation factor-9 in mammalian ovary. Mol Endocrinol. 13: 1035-1048

Falconer DS, Mackay TFC. 1996. Introduction to Quantitative Genetics. 4th ed. Longman Group Limited. London.

Ghaderi A, Nasiri MTB, Mirzadeh KH, Fayazi J, Sadr AS. 2010. Identification of the GDF9 mutation in two sheep breeds by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Afr. J. Biotechnol. 9: 8020 – 8022.

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.

Gilchrist RB, Lane M, Thompson JG. 2005. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum. Reprod. Update. 14: 159-177.

Goyal S, Anggarwal J, Dubey PK, Mishra BP, Ghalsasi P, Nimbkar C, Joshi BK, Kataria RS. 2017. Expression analysis of genes associated with prolificacy in FecB carrier and noncarrier Indian Sheep. Anim. Biotechnol. 28: 220-227.

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. Biol. Reprod. 70: 900-909.

He Y, Ma X, Liu X, Zhang C, Li J. 2010. Candidate genes polymorphism and its association to prolificacy in Chinese goats. J. Agric. Sci. 2: 88-92.

Javamard A, Azadzadeh N, Esmailizadeh AK. 2011. Mutations in bone morphogenetic protein 15 and growth differentiation factor 9 genes are associated with increased litter size in fat-tailed sheep breeds. Vet. Res. Commun. 35: 157–167.

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.

Kasiriyan, MM, Hafezeyan H, Sayahzadeh H, Jamshidi R, Asghari SR, Irajeyan GH, Buesagh H. 2009. Genetic polymorphism FecB and BMP15 genes and its association with litter size in Sangsari sheep breed of Iran. J. Anim. Vet. Adv. 8:1025-1031.

Khodabakhshzadeh R, Mohammadabadi MR, Esmailizadeh AK, Shahrebabak HM, Bordbar F, Namin SA. 2016. Identification of point mutations in exon 2 of GDF9 gene in Kermani sheep. Pol. J. Vet. Sci. 19:281-289.

Kolosov YU, Getmantseva LV, Shirockova NV, Klimenko A, Bakoev SY, Usatov AV, Kolosov AY, Bakoev NF, Leonova MA. 2015. Polymorphism of the GDF9 gene in Russian Sheep Breeds. J. Cytol. Histol. 6: 1-4.

Lan ZJ, Gu P, Xu X, Jackson KJ, DeMayo FJ, O'Malley BW, Cooney AJ. 2003. Dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility. EMBO J. 22: 4070-4081.

Liao WX, Moore RK, Shimasaki S. 2004. Functional and molecular char-acterization of naturally occurring mutations in the oocyte secreted factors bone morphogenetic protein-15 and growth and differentia-tion factor-9. J Biol Chem. 17:17391–6.

McGrath SA, Esquela AF, Lee SJ. 1995. Oocyte specific expression of growth differentiation factor 9. Mol. Endocrinol. 9: 131-36.

McNatty KP, Smith P, Moore LG, Reader K, Lun S, Hanrahan JP, Groome NP, Laitinen M, Ritvos O, Juengel JL. 2005. Oocyte-expressed genes affecting ovulation rate. Mol. Cell. Endocrinol. 234: 57-66.

Mishra C. 2014. Genetic basis of prolificacy in sheep. Int. J. Livest. Res. 4: 46–57.

Mulen MP, Hanrahan JP. 2014. Direct evidence on the contribution of a missense mutation in GDF9 to variation rate of Finnsheep. PloS One. 9:1-6.

Novitasari DA, Elvyra R, Roslim DI. 2014. Teknik isolasi dan lektroforesis DNA total pada Kryptopterus apogon (Bleeker 1851) dari Sungai Kampar Kiri dan Tapung Hilir Kabupaten Kampar Provinsi Riau. JOM FMIPA 1: 258-261.

Paz E, Diaz JQ, Bravo S, Montaldo H, Sepulveda NK. 2014. Genotyping of BMPR1B, BMP15, and GDF9 genes in Chilean sheep breeds and association with prolificacy. Anim. Genet. 46: 98-109.

Pokharel K, Peippo J, Honkatukia M, Seppala A, Rautianen J, Ghanem N, Hamama TM, Crowe MA, Andersson M, Li MH, Kantanen J. 2018. Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries). BMC Genomics. 1-17.

Polley S, De S, Brahma B, Mukherjee A, Vinesh P, Batabyal S, Arora JS, Pan S, Samanta AK, Datta TK, Goswami SL. 2010. Polymorphism of BMPR1, BMP15 and GDF9 fecundity genes in prolific Garole sheep. Trop. Anim. Health. Prod. 42:985-993.

Souza CJ, McNeilly AS, Benavides MV, Melo EO, Moraes JC. 2014. Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes. Anim. Genet. 45: 732-739.

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. Endocrinol. 141: 3814-3820.

Wang W, La Y, Zhou X, ZhangX, Liu B. 2018. The genetic polymorphisms of TGFβ superfamily genes are associated with litter size in a Chinese indigenous sheep breed (Hu sheep). Anim Reprod Sci 189: 19-29.

Widyastuti DA. 2017. Isolasi DNA Kromosom Salmonella sp. dan visualisasinya pada elektroforesis gel agarosa. Seminar Nasional Pendidikan Biologi dan Saintek. 311-317.

Yuwono T. 2006. Teori dan aplikasi polymerase chain reaction. P. 1-3; 18-21. Penerbit Andi, Yogyakarta.


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