Screening Of Genes Related To Multiple Birth Traits And Their Functions In Qira Black Sheep Based On Resequencing Technology

Qira Black sheep have excellent reproductive characteristics such as perennial estrus,high reproductive rate and early sexual maturity.Fully exploiting the excellent genes of this sheep breed and improving the multiple birth traits is an important way to protect and improve sheep germplasm resources in South Xinjiang.In this study,we screened candidate genes related to excellent multi-lambing traits in Qira black sheep based on genome resequencing technology,and performed polymorphism analysis and association analysis between different genotypes and lambing number on the obtained genes,and the genes initially validated were BMP2,POU1F1,BMPR1B,IGFBP7,ESR1;Further,the expression of IGFBP7,BMPR1B and ESR1 were analyzed at the transcriptome level in different physiological periods of the ovary（luteal phase,follicular phase and 30 d of gestation）to provide a reference for the conservation of germplasm resources and the improvement of reproductive performance of Qira Black sheep.Experiment 1 Genome resequencing and Fst selection signal analysis to screen candidate genes for multi-trait traitsThis chapter sequencing selected local sheep breeds in Xinjiang:28 Altay sheep（Samples from sheep breeding farm in Fuyun County,Xinjiang）;20 Bayinbuluke sheep（Samples from Hejing breeding sheep farm）;20 multi-born Qira black sheep（The test samples were obtained from Xinjiang Kunlun lv Yuan Sheep farm,Cele country）;The extracted qualified sheep genomic DNA was sent to the company for genomic resequencing at a depth of 10×.Variant detection and padding were performed separately for all samples,and 100,998 significant SNP loci were obtained.Plink（V1.09）software was used for quality control of SNP data,and the quality control criteria were:removal of individual samples with less than95%detection rate,less than 90%detection rate of SNPs,less than 5%minimum allele frequency,and less than 1×10^-6P value of Hardy-Weinberg equilibrium test.Qira black sheep were subjected to genomic selection signal analysis with Altay and Bayinbuluke sheep,respectively,and after obtaining the top 1%of loci,gene annotation was performed.The intersection of the two was taken and 450 differentially significant genes were obtained（P<0.05）,which were subjected to GO and KEGG enrichment analysis,among which nine candidate genes associated with reproductive traits were IGFBP7,BMP2,BMPR1B,BMP5,ESR1,OXTR,POU1F1,MAP3K13,MSI2,and an attempt was made to identify among these candidates polymorphic loci associated with multiple fetus traits.Experiment 2:Validation of SNP loci of candidate genes and analysis of their genotypes in relation to lambing numberA total of 138 healthy adult Qira black sheep ewes were randomly selected,of which 64 were single-born and 74 were multi-born.The test samples were obtained from Xinjiang Kunlun lv Yuan Sheep farm,Cele country.Primer design,PCR amplification and detection of SNP loci for the candidate genes BMPR1B,BMP2,POU1F1,IGFBP7 and ESR1 obtained by resequencing in Chapter 1.The results revealed that the BMPR1B gene g.431965 A>G,the Fec B locus,and three genotypes,B+,++and BB,were detected.The BMP2 gene g.2367 C>T,locus detected three genotypes TC type,TT type and CC type;g.2611 G>A locus detected two genotypes GG type and AG type 2.POU1F1 gene g.11043 G>A,3 genotypes of AG type,GG type and AA type were detected at the locus;IGFBP7 gene g.35468 G>A with three genotypes detected at the locus,AG type,AA type and GG type.ESR1 gene was detected at g.188958 A>G locus with three genotypes,AG type,AA type and GG type.Experiment 3:m RNA expression of BMPR1B,ESR1 and IGFBP7 in different physiological periods in the ovaries of Qira black sheepOn the basis of experiment 2,two genes with significant differences in polymorphic locus genotype and lambing number and one gene with insignificant differences:BMPR1B,ESR1 and IGFBP7 were selected and their m RNA expression at 30 d of follicular phase,luteal phase and gestation was investigated by fluorescence quantitative PCR.The results showed that the expression of BMPR1B gene m RNA was highly significant higher in the 30 d of gestation than in the luteal phase（P<0.01）and in the follicular phase than in the luteal phase（P<0.05）.IGFBP7 gene m RNA expression was highly significant higher in the follicular phase than in the luteal phase and 30 d of gestation（P<0.01）and significantly higher in the30 d of gestation than in the luteal phase（P<0.05）.ESR1 gene m RNA expression was significantly higher in the 30 d of gestation than in the luteal phase（P<0.05）and did not differ significantly from follicular phase expression（P>0.05）,nor did it differ significantly from luteal phase expression（P>0.05）.In summary,a total of 450 differentially significantly related genes were obtained by genome resequencing technology in this experiment,and nine reproduction-related candidate genes were screened by enrichment analysis.Five of these genes（BMPR1B,ESR1,POU1F1,BMP2,IGFBP7）were selected for SNP locus population validation,and a total of three differentially significantly related lambing loci were found.Then,verified the expression of BMPR1B,ESR1 and IGFBP7 genes m RNA in the pre-pregnancy（follicular and luteal phases）and late gestation（30 d of gestation）of the ovary,and analyzed the effects on the different physiological periods of follicles in order to analyze the excellent characteristics of multiple births in Qira black sheep.