Polymorphism Detection Of GDF9 Gene And BMP15 Gene And Their Association With Litter Size In Luzhong Mutton Sheep(Ovis Aries)

Litter size is one of the most important economic traits in sheep.Previous studies suggested that the litter size is regulated by multiple genes simultaneously in sheep.Growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15)are two important candidate genes for litter size.They can form heterodimers to participate in the TGF-β pathway and play a regulatory role in cell differentiation,follicle atresia and oocyte maturation.Therefore,there may be synergistic effects between them on litter size.Mutations in these two genes have been identified in European and African sheep breeds,and some mutations are closely related to litter size.However,there are few reports about the mutations of these two genes in local and cultivated Chinese sheep,and synergistic effects of these two genes on litter size have also been rarely reported.The Luzhong mutton sheep,a newly developed sheep breed in China(White Dorper sheep × Hu sheep),may carry mutations in both genes,which make it a good material for exploring candidate genes of litter size and the synergistic effects between GDF9 and BMP15.To investigate the association between GDF9 and BMP15 genes with litter size trait in Luzhong mutton sheep,the entire ORF of GDF9 and BMP15 genes were sequenced in this study to identify all mutant loci in Luzhong ewe population.Then,a general linear model was used to analyze the association between polymorphic loci in GDF9 and BMP15 genes with litter size.Secondary and tertiary structure changes caused by missense mutation sites related to the litter size were analyzed by bioinformatics.The geographical distributions of mutant allele frequencies were mapped to world map using R package ggplot2.The results were as follows:1.A total of eight mutant loci were detected in the ORF of the GDF9 gene in Luzhong mutton sheep,of which the mutation g.41769606 T > G was not annotated in the Ensembl database.Association analysis showed that two closely linked mutations g.41768501 A > G and g.41768485 G > A were significantly associated with litter size in Luzhong mutton ewes.The geographical distribution of mutation frequencies suggests that these two mutations may have originated from southwestern Europe.The missense mutation g.41768485 G > A(Val 332 Ile)is located in the maturation domain of the GDF9 protein and is predicted to lead to significant the tertiary structure alterations around the target region.2.A total of thirteen mutant loci were detected in the ORF of BMP15 gene in Luzhong mutton sheep,of which six mutations(g.50980691 G>T,g.50982388 T>G,g.50983121 T>A,g.50984457 T>C,g.50984616 C>T,g.50985229 A>G)were not annotated in the Ensembl database.Association analysis showed that six mutations g.50986052 C>A,g.50980656 T>C,g.50982538 T>C,g.50985162 T>C,g.50985229 A>G and g.50980691 G>T were significantly associated with litter size in Luzhong mutton sheep.Among them,g.50982538 T>C and g.50986052 C>A mutations had synergistic effects with the g.41768485 G>A mutation of GDF9 gene on litter size(P<0.05).The geographical distribution of mutation frequencies indicated that the frequency of g.50980656(T>C)are higher in Indonesia,Iran and South Africa,while the frequency of g.50982538 and g.50985162(T>C and T>C,respectively)are higher in Europe and Africa and with the highest frequencies exist in Spain and South Africa.The predictive analysis of protein structure shows that the two missense mutations g.50980691 G>T(Gln 240 His)and g.50980656 T>C(Leu 252 Pro)may lead to changes in the tertiary structure of the protein,and the latter also leads to changes in the secondary structure of the protein(from α-helix to random coil).In summary,favorable mutations for high litter size in fecundity genes GDF9 and BMP15 may originated from Europe and Africa.Several mutations in GDF9 and BMP15 are significantly related to the litter size of Luzhong mutton sheep,and some of them have a certain synergistic effect on litter size.Mutations g.41768501 A > G and g.41768485 G > A in GDF9 gene and mutations(g.50986052 C > A,g.50980656 T > C,g.50982538 T > C,g.50985162 T > C,g.50985229 A>G and g.50980691 G>T)in BMP15 gene can be used as potential effective genetic markers for selective breeding of the litter size trait in Luzhong mutton sheep.