Study On The Expression Of Seven Candidate Genes Related With Reproductive Performance Of Xiang Pig

Pig is one of the important agricultural animals,and it is restricted by the litter size of sow in pig industry,which is an indicator for reproductive performance of sow.Xiang pig is miniature breed with a large range of fertility,but the underlying genetic mechanism remains unclear.The present paper aimed to explore the molecular mechanism of litter size from the transcriptome level and epigenetics manner taking two groups of Xiang pig with high and low litter sizes as samples.The expression level of candidate genes was detected by real-time fluorescent qPCR method and the methylation status of RARRES1 gene was tested by bisulfite PCR sequencing method?BSP?.The main results were as follows:1.The expression patterns of transcriptome were detected to the ovaries of Xiang pig with high and low litter sizes by using Illumina HiseqTM 2000 platform.A total of 92 diferent genes were found out between two groups of Xiang pig.Of those 92 genes,seven candidate genes?DMBT1,SERPINE2,RARRES1,LRP8,CDK1,CDC25 C and PKA?were chosen to investigate further and three of them,CDK1,CDC25 C and PKA were enriched in the pathway of oocyte meiosis.2.The expression levels of seven candidate genes deduced from transcriptome sequencing data were tested by real-time PCR method.The results of six genes were confirmed by qPCR method except CDC25 C gene.The expression levels of RARRES1 gene and SERPINE2 were significantly higher in Xiang pig with higher litter size than that with low litter size group?P<0.05?without difference for the other five genes.3.The 5'-flanking region of five genes,SERPINE2,RARRES1,PKA,CDK1 and CDC25 C genes were cloned and sequenced from the genome of the two groups respectively.The amplificated lengths were 736 bp,828 bp,791 bp,929 bp and 991 bp respectively with identities from 92 to 99 percentages.The core promoter region was present in four fragments based on bioinformatics analysis except PKA,and there were many transcription factor binding sites in the four 5?-flanking sequences.For example,it was found that the A/C polymorphisms at 307 bp of 5`-end of SERPINE2 gene and G/C changes at-781 bp in the 5'-flanking region of RARRES1 gene might change the numbers of transcription factor binding sites,which might be the reason for the high expression levels of SERPINE2 and RARRES1 gene in ovary of Xiang pig group with high litter size.4.It was predicted that the first exon of RARRES1 gene,downstream of ATG start codon is riched in CpG islands based on bioinformatics analysis.Designing two pairs of primers in this region and using the same samples,the methylation status of RARRES1 gene was detected by bisulfite PCR sequencing method?BSP?and its relationship with the gene expression level was analyzed.The results showed that the methylation level for the group of high litter size was higher,and in both of the two groups,there were four CG sites keeping unmethylated states,while the other three sites were all modified by methylation.There were four CG sites were differently modified between the two groups with sites CG₄,CG₉,CG₁6 to be higher and site CG6 to be lower in fecundity pig group.A strong positive correlationship between the methylation ratio of CG₉ and CG₁6 and the expression level of RARRES1 gene?r=0.896?were estimated by Spearman correlation analysis method.It suggested that the methylation of the first exon region might have a role on the promotion for the expression of RARRES1 gene in Xiang pig ovary.In summary,seven candidate genes deduced from transcriptome sequencing data were investigated for the genw expression and SNP sites.Of those genes,two genes,RARRES1 and SERPINE2,might have an effect on the litter size of Xiang pig while the other five genes might be less impact on the fecundity of Xiang pig breed.