| The litter size is a quantitative trait controlled by minor polygenes,which is difficult to improve through traditional breeding methods.Therefore,the screening of genes and molecular markers associated with prolific traits in sheep has always been the focus of breeders.In recent years,with the development of biotechnology,various omics technologies such as transcriptomics,genomics,proteomics,and metabolomics had become important tools for screening prolific genes in sheep.Published researches on prolific traits had focused mainly in pituitary,ovary and testis,but only a few reported studies in the uterus of sheep.In this study,we selected the FecB++ genotype Small Tail Han sheep ewes as the research object.Firstly,uterine RNA-Seq and uterine proteomics analyses were used to screen the differentially expressed mRNAs and proteins associated with prolific trait of sheep.Secondly,MassARRAY assay was applied to genotype on the candidate genes and the association was analyzed between the SNPs and litter size in Small Tail Han sheep,which provide a theoretical basis for researching the molecular mechanism of sheep prolificacy.The results are as follows: 1.Transcriptomic studies of uterine in sheepRNA-Seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous(PG)and monotocous(MG)sheep in follicular and luteal phases.In the follicular phase,166 mRNA,473 lncRNA and 147 circRNA candidates were found to be differentially expressed.Of them,33 mRNAs,242 lncRNAs and 80 circRNAs were upregulated in the PG,while 133 mRNAs,231 lncRNAs and 67 circRNAs were downregulated.GO and KEGG enrichment analyses showed that the differentially expressed transcripts were enriched in reproduction-related signaling pathways,such as ovarian steroidogenesis,oxytocin signaling pathway,TGF-? signaling pathway and estrogen signaling pathway.In the luteal phase,505 mRNA,967 lncRNA and 364 circRNA candidates were found to be differentially expressed.Of them,359 mRNAs,330 lncRNAs and 178 circRNAs were upregulated in the PG,while 146 mRNAs,637 lncRNAs and 186 circRNAs were downregulated.GO and KEGG enrichment analyses showed that the differentially expressed transcripts were also enriched in ovarian steroidogenesis,oxytocin signaling pathway,TGF-? signaling pathway,estrogen signaling pathway and VEGF signaling pathway.2.Proteomics studies of uterine in sheepProteomics analysis of polytocous and monotocous Small Tail Han sheep uterine tissue using TMT technology identified 41 and 43 differentially expressed proteins in the follicular phase and luteal phase,respectively.Transcriptome and proteomics correlation analysis found that the spearman correlation coefficients of the follicular phase and luteal phase were 0.106 and 0.216,respectively,and ACSS3,SDSL and ENSOARP00000002929 reached significant differences in mRNA and protein levels.GO and KEGG enrichment analyses showed that compared with MG,mRNA and protein significantly up-regulated in PG were involved in metabolic pathways,such as sphingolipid metabolism and amino acid metabolism,among which the differentially expressed genes SDS,SDSL,ACSS1,ACSS3,MGST1 and MGST3 provide energy for early embryo development,embryo implantation and uterine morphological changes by regulating metabolic-related pathways.3.Polymorphism analysis of litter size related genesIn this study,14 genes(SLC5A1,CCNA1,ABCC1,CYP24A1,MGST1,MGST3,ACSS1,ACSS3,SDS,SDSL,SMPD1,SMPD4,SMPD2 and HADHB)related to reproduction were screened based on uterine transcriptome and proteomics.Then,based on previous data from the whole-genome sequencing(WGS)previously performed,all significant SNPs loci of the above genes were screened.Finally,the 32 SNPs screened were genotyped,and the association with litter size in Small Tail Han sheep was conducted.The results indicated that the g.70067210 T>C locus of SLC5A1 gene,the mean litter sizes of the first,second,and third parities for TT and TC were significantly higher those of the CC genotype(P < 0.05).However,a comparison of litter size between TT and TC showed no significant difference(P > 0.05).Furthermore,a comparison of average litter sizes among TT,TC,and CC showed a significant difference(P < 0.05).In the g.198329390 T>C locus of MGST1 gene,the mean litter sizes of the first parity,second parity and average for CC was significantly higher those of the TT genotype(P < 0.05).In the g.41750020 C>T locus of ACSS1 gene,the mean litter sizes of the first,second,and third parities for TT was significantly higher those of the TC and CC genotype(P < 0.05).However,a comparison of litter size between TC and CC showed no significant difference(P > 0.05).In the g.117028798 G>A and g.117068514 A>G loci of ACSS3 gene,the mean litter sizes of the first,second,and third parities for GG was significantly higher those of the AA genotype(P < 0.05).In the g.117049071 G>A locus of ACSS3 gene,the mean litter sizes of the first parity for GA was significantly higher those of the GG genotype(P < 0.05).In the g.60663271 C>T locus of SDSL gene,the mean litter sizes of the first,second,and third parities for CC was significantly higher those of the TT genotype(P < 0.05).In the g.46061191 C>T locus of SMPD1 gene,the mean litter sizes of the first and second parities for TT was significantly higher those of the CT genotype(P < 0.05).In the g.71660757 C>T locus of SMPD4 gene,the mean litter sizes of the second and third parities for TT was significantly higher those of the CT and CC genotype(P < 0.05).However,a comparison of litter size between CC and CT showed no significant difference(P > 0.05).In conclusion,the above nine SNPs were significantly associated with litter size,indicating that the above SNPs were useful as genetic markers for litter size,and SLC5A1,MGST1,ACSS1,ACSS3,SMPD1,SDSL and SMPD4 genes were useful as candidate genes for prolific traits in sheep. |
PDF Full Text Request