SNPs Analysis Of BMPR-IB And GDF9 Genes In Sheep And Analysis Of GDF9 MRNA Expression Level

With the development of modern sheep industry,lambing traits become more and more important in the sheep production,but the heritability of lambing traits is low.To provide a theoretical basis for sheep prolificacy marker and breeding,this study took Small Tail Han sheep,Mongolian sheep and Gansu Alpine Merino sheep as research objects.The SNPs of BMPR-IB gene and GDF9 gene in different fertile ewes were detected by PCR-SSCP and direct sequencing,and analysis correlation between SNP and litter size.Using the real-time fluorescence quantitative PCR to detect the mRNA expression of GDF9 gene in different fecundity sheep tissues,in order to provide basic data for finding molecular markers and breeding mechanism of sheep's twin traits.The main results of the study are as follows:1.The polymorphism of the 597,746(FecB gene)and 1113 loci in the BMPR-IB gene coding region of 3 sheep breeds was detected(48 Small Taile Han sheep;188 Mongolian sheep,including 100 Singletons Mongolia sheep and 88 Twins Mongolia sheep;112 Gansu Alpine Merino sheep,including 70 Singletons Gansu Alpine Merino sheep,42 Twins Gansu Alpine Merino sheep;a total of 348).The results showed that:(1)The mutation of G>A was found at the 597 locus of the BMPR-IB gene coding region.Three genotypes were detected in Singletons Gansu Alpine Merino sheep,Twins Gansu Alpine Merino sheep,Singletons Mongolia sheep and Twins Mongolia sheep,which were wild-type CC and heterozygous mutant CD and homozygous mutant DD,the dominant genotypes were CD;heterozygous mutant CD and homozygous mutant DD genotypes were detected in Small Tail Han sheep.With the analysis of polymorphism information content,it was shown that Small Tail Han sheep belonged to low polymorphism(PIC<0.25),the other two breeds of sheep belonged to moderate polymorphism(0.25<PIC<0.5).(2)Only wild type(++ type)was detected in Singletons Gansu Alpine Merino sheep at position 746 of CDS region of BMPR-IB gene;wild type(++ type)and heterozygous mutant type(B+ type)were found in Singletons Mongolia sheep,Twins Mongolia sheep and Twins Gansu Alpine Merino sheep,the dominant genotype was ++ type;the heterozygous mutant(B+)and homozygous mutant(BB)were found in Small Tail Han sheep,and the dominant genotype was BB.PIC analysis of polymorphic information content showed that Singletons Mongolia sheep,Twins Mongolia sheep and Twins Gansu Alpine Merino sheep belonged to low polymorphism(PIC<0.25),and Small Tail Han sheep belonged to moderately polymorphic(0.25<PIC<0.5).(3)There is a mutation of C>A at the 1113 locus of the BMPR-IB gene coding region.In addition,three different genotypes were detected in Mongolian sheep single and twin ewes and Gansu Alpine Merino sheep single and twin ewes,which were wild-type AA,homozygous mutant type BB and heterozygous mutant type AB,the dominant genotype is AB.Small Tail Han sheep appeared two genotypes,including homozygous mutant type BB and heterozygous mutant type AB,and the dominant genotype is BB.The PIC analysis showed that Small Tail Han sheep belonged to low polymorphism(PIC<0.25),Gansu Alpine Merino sheep and Mongolian sheep belonged to moderately polymorphic(0.25<PIC<0.5).2.Whether there is a G>A mutation in the 260 loci of the first exon of GDF9 gene in Small Tail Han sheep(54),Mongolia sheep(190,including 102 single-ewes and 88 twins)and Gansu Alpine Merino sheep(112,including 70 single-ewes and 42 twins).The results showed that the wild type AA and heterozygous mutant type AB were detected in the Gansu Alpine Merino sheep,the Mongolia sheep and the Small Tail Han sheep.The PIC analysis showed that Gansu Alpine Merino sheep,Mongolia sheep and Small Tail Han sheep belonged to low polymorphic(PIC<0.25).3.The expression of GDF9 gene was detected in the Hypothalamus,Ovary,Kidney,Liver,Lung,Pituitary and Heart of different fecundity ewes by real-time fluorescence quantitative technique.It is showed that there was a significant difference in Ovary expression between Small Tail Han sheep and Singletons Mongolian sheep,Twins Mongolian sheep,Dorsett singleton ewes and Dorsett double ewes(P<0.05);there was no significant difference in Ovary expression between Singletons Mongolian sheep and Twins Mongolian sheep(P>0.05);there was significant difference in Ovary expression between Dorsett singleton and Dorsett double ewes(P<0.05).In other tissues,there are different degrees of expression,and there was significant difference(P<0.05)in Heart and Liver expression between Small Tail Han sheep and Singletons Mongolian sheep,Twins Mongolian sheep,Dorsett singleton ewes and Dorsett double ewes.