Screening And Validation Of Genes Related To Multiplets Traits In Xinji Fine Wool Sheep

Sheep raising industry is an important part of animal husbandry in China.There are about 380 million sheep and goats in China.Among herbivorous livestock,sheep has a high conversion rate of feed,a wide range of forage intake and adaptability,and they can make full use of the resources that cannot be used by other livestock.Improving the comprehensive output rate of agricultural resources in China plays an important role in promoting the development of animal husbandry economy,maintaining industrial balance and healthy and sustainable development.Reproductive characters are important economic characters in sheep industry.The polymorphisms of 8 genes were detected in Xinji fine wool sheep.The expression levels of 8 genes in the ovarian granulosa cells of the hybrid offspring of brumellino sheep and Xinji fine wool sheep were studied.The purpose of this study is to provide theoretical basis for improving the fecundity and litter size of sheep and goats.The main conclusions of this study are as follows:1.BMPR-1B gene polymorphisms of 129 Xinji fine wool sheep were analyzed by PCR-RFLP.The results showed that BMPR-1B gene polymorphisms did not exist in Xinji fine wool sheep.It is speculated that BMPR-1B gene is not the main gene of polyparous trait in Xinji fine wool sheep population.2.Polymorphisms of bone morphogenetic protein 15(BMP15)and growth differentiation factor 9(GDF9)in 118 Xinji fine wool sheep were studied by PCR-SSCP.In the study of BMP15 gene,polymorphism was found in the amplification fragment of P1 primer,presenting three genotypes: AA,AC and CC.The mutation resulted in the deletion of CTT at the position of 58-60 on exon 1 of BMP15 gene.Xinji fine wool sheep ?2 squared value reached significant level,And Hardy Weinberg showed imbalance.There was no significant difference in the average lambing number between different genes(P> 0.05).In the study of GDF9 gene,there was no polymorphism in Xinji fine wool sheep.The T477 C mutation of GDF9 gene could not be used as the genetic marker of multiple embryo trait in Xinji fine wool sheep.3.The Gn RHR,LHR,FSHR,PRLR and ESR polymorphisms of 119 Xinji fine wool sheep were analyzed by ABI sequencing.It was found that there was no mutation site in FSHR gene.There were 13 mutation sites in Gn RHR,LHR,PRLR and ESR genes.Gn RHR gene has mutations of A505 G and G720 C,and LHR gene has mutations of T1262G,A1991 G,C2012T,A2032 T and T2041 A,PRLR gene has mutations of A1263 G and C1544 G,and ESR gene has mutations of A106 T,C181T,G612 A and C735 T.The correlation analysis between four gene polymorphisms and lambing number in Xinji fine wool sheep population showed that only the mutation of ESR gene A106 T had significant effect on lambing number of Xinji fine wool sheep.The average lambing numbers of AA,AT and TT genotypes were 1.391,1.101 and 1.417,respectively;The mean lambing number of AA genotype and TT genotype was 0.290 and 0.316 more than that of AT genotype,respectively(P < 0.05).There was no significant difference in the mean lambing number between AA genotype and TT genotype(P > 0.05).4.The hybrid generation of Booroola Merino Sheep and Xinji fine wool sheep was used as the research animal.After the superovulation of one-month old ewes without Fec B gene(++ type)and Fec B gene heterozygous type(B+ type),the follicular fluid was collected in vivo by surgery,and the ovarian granulosa cells were collected and cultured.The expression of BMPR-1B,BMP15,GDF9,Gn RHR,LHR,FSHR,ESR and AMH in ovarian granulosa cells of ++ type and B+ type were analyzed by real-time fluorescence quantitative PCR.The results showed that there was no significant difference in BMPR-1B,Gn RHR,LHR and FSHR gene expression between ++ and B + ovarian granulosa cells(P > 0.05);The m RNA expression levels of BMP15 and AMH genes in B+ type ovarian granulosa cells were significantly higher than those in the B+ type(P < 0.05);The m RNA expression level of GDF9 and ESR genes was significantly lower than that of the ++ type(P < 0.01).The m RNA expression level of ESR gene was significantly higher than that of the ++ type(P< 0.05).The results showed that BMPR-1B? BMP15? GDF9? LHR? FSHR? PRLR did not have mutations related to the multifetal traits of Xinji fine wool sheep.The A106 T mutation of ESR could be used as a genetic marker for multifetal traits in Xinji fine wool sheep.At the same time,the expression level of genes related to reproductive traits was significantly changed after the introduction of Fec B gene into the Xinji fine wool sheep.The experimental results provide experimental basis for the mechanism of Fec B gene improving the lambing rate of sheep.