Genetic Variation Of Litter Size Candidate Genes In Erhualian Pigs And Their Correlation With Litter Size

Litter size is one of the most important traits in the breeding of commercial pigs.However,the heritability of litter size is low and traditional breeding methods displayed limited improvement.It’s essential to optimize porcine fertility using molecular markers.Erhualian pig is a native breed with high fertility but the underlying genetic mechanism remains unclear.In the previous study,we found a large litter size variation within Erhualian pig breed.QTLs for litter size were found by genome-wide association analysis(GWAS)and genetic differentiation coefficient(Fst)analysis.But the key loci affecting litter size remained unknown.Based on our previous study,the key loci affecting the variation of litter size in Erhualian pigs were studied through candidate gene method.First,we selected ESR and FSHβ genes as two candidate genes.At the same time RBR1,CLTC and RPS6KB1 were selected according to the results of the SSC12 and SSC13 QTL loci.The candidate genes were studied for polymorphisms and their association with litter size in Erhualian population.Meanwhile,Meishan pig,Shawutou pig and Yorkshire pig population were used to verify the results.The correlation analysis of ESR and FSHβ genotypes with litter size in Erhualian breed showed that the genotypes of each gene and their combined genotypes were not correlated with TNB and NBA.Indicating that the ESR and FSHβ genes could not be certified to be the main genes affecting litter size in Erhualian pigs.The population of Suhuai pigs was analyzed for further verify.And the results showed that the TNB,NBA and birth weight of Suhuai pigs were not related to each gene and their combined genotypes.Indicating that the ESR and FSHβ genes could not be certified to be the main genes of litter size in Suhuai pigs.In the study of RBP],CLTC and RPS6KB1 genes,SNP loci were screened and verified.According to the results of re-sequencing,all the SNPs of the candidate genes were screened.The critical area affecting litter size was determined by RNAseq results.The SNPs of the functional regions were selected and verified by genotyping in extremely high and low prolific Erhualian sows.After analysis the g.88075242 locus of RBP1 gene and g.37384779,g.37385057 and g.37386712 loci in promotor region and g.37431651,g.37433196,g.37433262 and g.37433718 loci in 3’UTR region of RPS6KB1 gene were selected.No SNP was selected in CLTC gene.The correlation analysis showed that there was no significant association between SNP in RBP1 gene with litter size.Loci g.37433196 and g.37433718 in the 3’UTR region of RPS6KB1 gene were highly linked and significantly related to litter size while other loci were not.The two loci were significantly correlated with TNB(P<0.05)and highly significantly correlated with NBA(P<0.01).The same correlation analysis was also carried out in Meishan pig,Shawutou pig and Yorkshire pig population.The results showed that the two loci were completely linked in Meishan pig and Shawutou pig population.In the Yorkshire pig population only g.37433196 locus found polymorphisms.In the Meishan pig population,the two loci were significantly correlated with TNB and NBA(P<0.05).In the Shawutou pig population the two loci had relevant trends with litter size.In the Yorkshire pig population g.37433196 wasn’t associated with litter size.Indicating that there was group heterogeneity in the effects of the two loci on litter size.To study the mechanism of g.37433196 and g.37433718 loci in RPS6KB1 gene affecting litter size,we first predicted the function of two loci online.The g.37433 196 locus was located at the binding site of RPS6KB1 gene and ssc-miR-7135-5p or ssc-miR-23b.The g.37433718 locus was located at the binding site of RPS6KB1 gene and ssc-miR-10a-3p.It means different genotypes may lead to different gene expression.The mRNA of RPS6KB1 gene expressed in many tissues and organs of sows.And the expression of RPS6KB1 was active in ovary.The expression level of the ovaries of 10 days old sows was significantly higher than that of 182 days(P<0.05);The expression level of 96 days old sows was significantly higher than that of 182 days(P<0.01);The results suggest that RPS6KB1 gene may be involved in ovarian development.The expression of ovarian and endometrial mRNA in high and low fertility groups showed that the low fertility group had significantly higher expression than that in high fertility group(P<0.05).There was no significant difference in the expression of endometrium between high and low fertility groups.Indicating that the RPS6KB1 gene may affect litter size through ovary.