Effect of g.46547645T>G Locus of GDF9 Gene on Promoter Activity and Litter Size of Mongolia Sheep (Ovis aries)
CHENG Zi-Xuan1,*, LU Da-Tong1,*, LIU Jia-Seng2, LI Yun-Hua2, ZHANG Xiu-Ying3, CHAO Lu-Meng3, CANG Ming1, WANG Jian-Guo1, YU Hai-Quan1, LI Guang-Peng1,**, TONG Bin1,**
1 State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock/School of Life Sciences, Inner Mongolia University, Hohhot 010071, China;
2 Animal Husbandry Institute, Inner Mongolia Academy of Agricultural & Animal Husbandry, Hohhot 010031, China;
3 Xilinhot Animal Husbandry Station, Xilinhot 026000, China
Abstract：Increasing litter size is the primary aim of breeding in Mongolia sheep (Ovis aries). Growth and differentiation factor 9 (GDF9) is an oocyte-derived growth factors that play an important role in ovarian folliculogenesis in mammals. Many studies have shown that the mutations in GDF9 were associated with litter size and ovulation rate in several sheep breeds. The aims of this study were to detect the association between the g.46547645T>G SNP of the GDF9 gene and litter size in Mongolia sheep, and detect the genetic diversity in Mongolia, Ujimqin, Hulunbuir (big and short tail types), Small-tailed Han and Hu sheep breeds, as well as to validate the effect of the g.46547645T>G SNP on the promoter activity of GDF9. A total of 416 sheep were used in this study including 260 Mongolia sheep, 36 Ujimqin sheep, 30 Hulunbuir sheep (big tail type), 30 Hulunbuir sheep (short tail type), 30 Small-tailed Han sheep and 30 Hu sheep. The g.46547645T>G in GDF9 were genotyped using the PCR-restriction fragment length polymorphism (PCR-RFLP) method, and then performed the association and genetic diversity analyses. The effect of g.46547645T>G on the promoter activity of the GDF9 was detected by the luciferase reporter assay. The χ2 test indicated that the g.46547645T>G SNP is under Hardy-Weinberg equilibrium in the 6 sheep breeds. There were 3 genotypes TT, TG and GG of the g.46547645T>G SNP in Mongolia sheep, 2 genotypes TT and TG in Ujimqin sheep, Hulunbuir sheep (big tail type), Hulunbuir sheep (short tail type), Small-tailed Han sheep and Hu sheep populations. The values of polymorphism information content (PIC) of the g.46547645T>G SNP presented with related low polymorphism in the Mongolia sheep, Ujimqin sheep, Small-tailed Han sheep and Hu sheep breeds, and presented with moderate polymorphism in the Hulunbuir sheep (big tail type), Hulunbuir sheep (short tail type) breeds. The result of association analysis showed that the individuals with TG genotype of g.46547645T>G SNP had extremely significantly more lambs than the individuals with TT genotype in Mongolia sheep (P<0.01). In addition, GDF9 promoter activity analysis showed that the G allele at g.46547645T>G could significantly decrease luciferase activity compared with that of the control (P<0.05), the T allele at g.46547645T>G could significantly increase luciferase activity compared with that of the control (P<0.05), and the G allele at g.46547645T>G could extremely significantly decrease luciferase activity compared with that of the T allele (P<0.01). This study may facilitate effective marker-assisted selection to increase litter size in Mongolia sheep populations, as well as bring new insights into GDF9 expression.
[1] 杜丽, 张利平, 张筱艳, 等. 2017. 三个绵羊品种BMPR-IB基因CDS区1 113位点的多态性与产羔数的相关性分析[J]. 农业生物技术学报, 25(12): 1989-1997.
(Du L, Zhang L P, Zhang X Y, et al.2017. The correlation analysis between polymorphism and fecundity of 1 113 Locus in BMPR-IB gene CDS area of three sheep (Ovis aries) varieties[J]. Journal of Agricultural Biotechnology, 25(12): 1989-1997.)
[2] 国家畜禽遗传资源委员会组编. 2011. 中国畜禽遗传资源志—羊志[M]. 北京: 中国农业出版社, pp. 20-23.
(China national commission of animal genetic resources. 2011. Animal Genetic Resources in China: Sheep[M]. China Agriculture Press, Beijing, China, pp. 20-23.)
[3] 刘荣. 2014. 蒙古羊多羔性状候选基因ADAMTS1的遗传效应分析[J]. 现代农业, (4): 77-79.(Liu R. 2014. Genetic effect of ADAMTS1 gene as the candidate gene for multi-lambs trait of Mongolian sheep[J]. China Academic Journal Electronic, (4): 77-79.)
[4] 马晓明, 张利平, 朱韶华, 等. 2017. 四个绵羊品种BMPR-IB基因CDS区864位点多态性及其产羔数相关性分析[J]. 基因组学与应用生物学, 36(10): 4116-4124.
(Ma X M, Zhang L P, Zhu S H, et al.2017. Analysis of polymorphism at 864 locus of BMPR-IB gene CDS region and its relationship with litter size in four sheep breeds[J]. Genomics and Applied Biology, 36(10): 4116-4124.)
[5] 田秀娥, 孙红霞, 王永军. 2009. 3个绵羊群体BMPR-IB基因的遗传多态性及其对产羔数的影响[J]. 西北农林科技大学学报(自然科学版), 37(11): 31-36.
(Tian X E, Sun H X, Wang Y J.2009. Genet is polymorphism of BMPR-IB gene and effect on litter size in three sheep breeds[J]. Journal of Northwest A & F University (Natural Science Edition), 37(11): 31-36.)
[6] 杨燕燕, 邵凯, 达来, 等. 2010. BMP15基因作为影响蒙古羊双羔性状候选基因的研究[J]. 中国草食动物科学, 25(6): 161-165.
(Yang Y Y, Shao K, Da L, et al.2010. Study on the BMP15 gene as candidate genes for fecundity in Mongolian sheep double-lambs group[J]. China Herbivore Science, 25(6): 161-165.)
[7] Abdoli R, Zamani P, Mirhoseini S Z, et al.2016. A review on prolificacy genes in sheep[J]. Reproduction in Domestic Animals, 51(5): 631-637.
[8] Dong J, Albertini D F, Nishimori K, et al.1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis[J]. Nature, 383(6600): 531-535.
[9] Elvin J A, Clark A T, Wang P, et al.1999. Paracrine actions of growth differentiation factor-9 in the mammalian ovary[J]. Molecular Endocrinology, 13(6): 1035-1048.
[10] Fitzpatrick S L, Sindoni D M, Shughrue P J, et al.1998. Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues[J]. Endocrinology, 139(5): 2571-2578.
[11] Galloway S M, McNatty K P, Cambridge L M, et al.2000. Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[J]. Nature Genetics, 25(3):279-283.
[12] Hreinsson J G, Scott J E, Rasmussen C, et al.2002. Growth differentiation factor-9 promotes the growth, development, and survival of human ovarian follicles in organ culture[J]. The Journal of Clinical Endocrinology and Metabolism, 87(1): 316-321.
[13] Hanrahan J P, Gregan S M, Mulsant P, et al.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)[J]. Biology of Reproduction, 70(4): 900-909.
[14] Kaivo-Oja N, Bondestam J, Kämäräinen M, et al.2003. Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells[J]. The Journal of Clinical Endocrinology and Metabolism, 88(2): 755-762.
[15] McPherron A C, Lee S J.1993. GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines[J]. The Journal of Biological Chemistry, 268(5): 3444-3449.
[16] Mulsant P, Lecerf F, Fabre S, et al.2001. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[J]. Proceedings of the National Academy of Sciences of the United States of America, 98(9): 5104-5109.
[17] McGrath S A, Esquela A F, Lee S J.1995. Oocyte-specific expression of growth/differentiation factor-9[J]. Molecular Endocrinology, 9(1): 131-136.
[18] Nei M, Roychoudhury A K.1974. Sampling variances of heterozygosity and genetic distance[J]. Genetics, 76(2): 379-390.
[19] Pan Z, Wang X, Di R, et al.2018. A 5-methylcytosine site of growth differentiation factor 9 (GDF9) gene affects its tissue-specific expression in sheep[J]. Animal, 8(11): 200.
[20] Roy B, Rajput S, Raghav S, et al.2012. A reporter promoter assay confirmed the role of a distal promoter NOBOX binding element in enhancing expression of GDF9 gene in buffalo oocytes[J]. Animal Reproduction Science, 135(1-4): 18-24.
[21] Sham P, Bader J S, Craig I, et al.2002. DNA Pooling: a tool for large-scale association studies[J]. Nature Reviews and Genetics, 3(11): 862-871.
[22] Souza C J, MacDougall C, Campbell BK,et al.2001. The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1B (BMPR1B) gene[J]. The Journal of Endocrinology, 169(2): R1-R6.
[23] Souza C J, McNeilly A S, Benavides M V, et al.2014. Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes[J]. Animals Genetics, 45(5): 732-739.
[24] Sterneck E, Tessarollo L, Johnson P F.1997. An essential role for C/EBPβ in female reproduction[J]. Genes & Development, 11(17): 2153-2162.
[25] Su J, Hu G, Wang Y, et al.2014. Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos[J]. Cellular Reprogramming, 16(4): 281-289.
[26] Tong B, Wang J, Cheng Z, et al.2020. Novel variants in GDF9 gene affect promoter activity and litter size in Mongolia sheep[J]. Genes, 11(4): E375.
[27] Vinet A, Drouilhet L, Bodin L, et al.2012. Genetic control of multiple births in low ovulating mammalian species[J]. Official Journal of the International Mammalian Genome Society, 23(11-12): 727-740.
[28] Vitt U A, Hayashi M, Klein C, et al.2000b. Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles[J]. Biology of Reproduction, 62(2): 370-377.
[29] Vitt U A, McGee E A, Hayashi M, et al.2000a. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats[J]. Endocrinology, 141(10): 3814-3820.
[30] Wilson T, Wu XY,Juengel JL,et al.2001. Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells[J]. Biology of Reproduction, 64(4): 1225-1235.
[31] Yamamoto N, Christenson L K, McAllister J M, et al.2002. Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells[J]. The Journal of Clinical Endocrinology and Metabolism, 87(6): 2849-2856.
