Association Analysis Of GDF9 Gene In Sonid Sheep And The Effect Of Its Mutation On Promoter Activity

Mongolia sheep populations are mainly distributed in grassland,desert and agricultural areas in northern China,Mongolia Plateau and Central Asia.They are fattailed,carpet wool,produce quality meat,and have sound body conformation,strong walking ability and admirable adaptation to various different ecological conditions.However,due to low prolificacy,the reproduction of Mongolia sheep was limited.In the current study,total of 416 sheep samples,including 260 Sonid sheep ewes(77 ewes with twin lambs and 183 ewes with single lamb),thirty Hulunbuir sheep(big tail type)ewes,thirty Hulunbuir ewes(short tail type),thirty-six Ujimqin sheep ewes,thirty Small Tailed Han sheep ewes and 30 Hu sheep ewes,were collected for association and genetic diversity analysis.The PCR-RFLP and DNA sequencing techniques were used to identify SNPs(single nucleotide polymorphisms)in the GDF9(Growth differentiation factor 9)in Sonid sheep.The results showed that:(1)There were 11 variations found in the Sonid sheep,in which two groups of SNPs had strong linage disequilibrium(LD),namely LD1 of g.41770932C>T,g.41771134A>G and g.41771399C>T SNPs,and LD2 of g.41771007T>G,g.41771305A>G and g.41771380C>T SNPs.Furthermore,the g.41770718T>G and the LD1 were linked in the Sonid sheep with high prolificacy.Association analysis showed that six variations were significantly related with high prolificacy,including g.41770718T>G(P<0.01),LD1(P<0.001),p.Ser347Phe(P<0.05)and c.1525A>G SNP(P<0.01);(2)Analyses of the GDF9 promoter activities by dual-luciferase showed that g.41770932C>T SNP played a leading role in LD1,and-460 to-177 bp region was the core area in GDF9 promoter;the G at-118 position(g.41770718T>G SNP)reduced luciferase activity compared with that of the T allele(P<0.01);the T allele at-332 position(g.41770932C>T SNP)increased luciferase activity compared with that of the C allele(P<0.01);co-existence of G and T alleles in-118 and-332 positions,the luciferase activity also increased.These results indicated that the g.41770932C>T SNP significantly promoted the expression of GDF9 gene and significantly correlated with the litter size of Sonid sheep.