| Most of the world's sheep are seasonal oestrus animals,most of which mainly produce single lambs.With the production of fat lambs becoming the leading production mode of sheep industry,the molecular marker of lambing number has become a research hotspot.One site of BMP-IB gene has been identified as a molecular marker for lambing number of Australian blue Merino and Chinese Small Tailed Han sheep,but it has not achieved ideal results in other breeds.Xinjiang is the region with the most abundant sheep breed resources in China.Many sheep breeds or genetic resource populations show high fecundity,with an average of 2-4 lambs per litter,which can be used as an ideal sample to study the genetic mechanism of high fecundity of sheep.In this study,Xinjiang local multi lambed sheep population and single lambed breed bashby sheep were selected as the main research objects.The single nucleotide polymorphism(SNP)which may cause the difference of lambing number was screened from the whole genome by SLAF-seq technology.Through genome-wide association study,we found new genes related to lambing number of sheep,and studied the mechanism of character formation,which provided new clues for revealing the regulation mechanism of sheep fertility.The experimental results are as follows:1.Simplified genome association analysis of lambing number traits in Hetan sheepThe genomic DNA of 62 multi lambed and 64 single lambed breeds of Hetian Sheep and Bashibai sheep was extracted.685300 SNPs were identified by SLAF-seq technology,which could be used for subsequent genome-wide analysis.By using general linear model and mixed linear model,155 SNPs were significantly correlated with litter size between high fertility and low fertility in Xinjiang SNPs.Through the analysis of gene function annotation and function prediction,14 candidate genes(COIL,SLK,FSHR,PLXNA3,DDX24,CXCL12,PLA2G7,ATP5FLA,KERA,GUCY1A1,LOC101107541,LOC101107119,LOC101107809,BRAF)related to sheep reproduction were screened out.2.Gene polymorphism and its association with litter sizeAmong the 14 breeding related candidate genes identified by GWAS,based on previous reports and functional prediction analysis,7 candidate genes were selected The 30 loci of genes and candidate genes(CXCL12,FSHR,SLK,GUCY1A1,COIL,LOC101107541,LOC101107119)that regulate ovulation and litter production were tested in kasp population for 5 multi lamb sheep breeds(128 Hetan sheep,65 Altay sheep,16 Duolang sheep,44 Cele black sheep and 240 Turpan black sheep)from Xinjiang.Among them,9 loci of 4 genes were successfully typed.The three loci of FSHR(g.75320741),GUCY1A1(g.43266624)and COIL(g.7321466)were significantly correlated with litter size.These three loci are expected to be the molecular markers of significant litter size in Xinjiang sheep.3.Tissue expression analysis of candidate genesThe expression of 7 genes related to ovulation and lambing identified in different tissues was verified.The results showed that the seven genes were expressed in all tissues,and the mRNA expression of five genes except LOC101107541 in the reproductive site was higher than that in the visceral tissue.The expression of CXCL12,GUCY1A1,SLK and COIL genes in the uterine body was the highest,which was significantly higher than that in the visceral tissue Tissue(P<0.01).The expression of FSHR gene in ovary was the highest,significantly higher than that in other 7 tissues(P<0.01).It is suggested that CXCL12,FSHR,SLK,GUCY1A1,COIL,LOC101107119 genes are highly correlated with sheep reproduction.Indicating that these genes are mainly expressed in the ovaries and the reproductive organs of the uterus and participate in the reproductive effect.4.Cloning,bioinformatics and functional analysis of COIL and GUCY1A1 genesThe total length of the CDS region of goat COIL gene was 1764 bp and that of GUCYIA1 gene was 2124 bp,encoding 571 and 701 amino acids respectively.The biological information of COIL and GUCY1A1 genes was analyzed,and their structures,physical and chemical properties and functions were predicted.It is speculated that the distribution of hydrophilic and hydrophobic amino acids in the whole peptide chain of COIL and GUCY1A1 gene is relatively uniform,but they are all hydrophilic proteins,which can promote protein polymerization and precipitation,and the protein has no transmembrane area,so it belongs to non transmembrane protein.It can promote its binding with the receptor to carry out biochemical activities.The two genes contain different amounts of potential glycosylation sites and phosphorylation sites,which affect the transport and localization of protein translation.So it is more conducive to the regulation of reproductive mechanism5.Overexpression and interference of COIL gene in cellThe expression vector pcDNA3.1-MHY-COIL was constructed successfully,which was validated by double enzyme digestion and sequencing;the interference siRNA sequence was designed,and the sequence with the best interference efficiency was screened out by validation.The overexpression vector and interference sequence were successfully transfected in mouse ovarian fibroblasts and sheep fibroblasts.The results of cell proliferation and activity test showed that COIL gene can significantly improve the proliferation and activity of mouse ovarian fibrocytes and sheep fibrocytes.It is speculated that COIL gene can not only activate the regulation of cell proliferation,but also improve the activity of cells.The effect of COIL gene on the interaction protein was detected by fluorescence quantitative PCR,and the results showed that the expression activities of FBL,ACTN1,WRAP53 and USPL1 genes increased significantly when COIL was over expressed,and SMN7 and GEMIN2 genes increased significantly when COIL gene interfered with the expression,which indicated that they had the opposite effect with COIL gene.Through SLAF-seq technology,the whole genome association analysis of sheep with different lambing numbers was carried out.Through gene typing and specific expression experiments,three genetic markers related to lambing numbers were obtained.According to gene function prediction and cell experiments,the formation mechanism of multiple lambing characters was verified,which provided a new clue for revealing the regulation mechanism of sheep fertility. |
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