Tissue Expression,Polymorphism And Its Association With Litter Size In Sheep Reproduction Candidate Gene

The litter size is the main indicator to measure the reproductive performance of sheep.Follicular development directly affects lamb production in sheep.Follicular development and ovulation are complex processes regulated by hormones,proteins,enzymes,and cofactors that requiring endocrine hormones from the hypothalamic-pituitary-ovarian axis and various cytokines from autocrine/paracrine.According to the results of previous studies that ESR1,ESR2,PGR,CYP11A1,CYP17A1,CYP19A1,SMAD1,BMP15,HSD3B1 genes are involved in the synthesis,absorption,and release of ovarian steroid hormones,and play an important role in the growth and ovulation of follicles.To reveal the relationship between the that gene and the litter size of sheep and its mechanism affecting litter size,meanwhile to provide a scientific basis for sheep molecular breeding,we researched the expression pattern of that genes in Sunite sheep(SNT)and Small Tail Han sheep(STH)with different fertility and analyzed the relationship between polymorphism and the litter size in sheep,based on the previous sheep genome resequencing data.First,reverse transcription PCR was used to detect the expression of that 19 gene in STH and SNT(brain,cerebellum,hypothalamus,pituitary,uterus,ovary,oviduct,heart,liver,spleen,lung,kidney,adrenal glands,thyroid,large intestine,small intestine,pancreas,rumen,and kidney fat)tissues with different lambing abilities.Then,Real-time PCR was performed to investigate the expression of that the9 gene in SNT and STH reproduction tissues(hypothalamus,pituitary,ovary)with different lambing abilities.Multiparous and uniparous sheep breeds were selected and Sequenom MassARRAY~?SNP assay was applied to genotype single nucleotide polymorphism sites(SNPs)of that gene.Then the association was analyzed between SNPs and litter size in STH by using SPSS 19.0.The main results were as follows:1.RT-PCR results showed that ESR1 and PGR were mainly expressed in oviduct,uterus,and ovary;ESR2 was mainly expressed in uterus;BMP15 was specifically expressed in ovarian tissue;steroidogenic hormone-related genes(HSD3B1,CYP11A1,CYP17A1,CYP19A1)were mainly expressed in the ovary;the SMAD1 gene was expressed in 19 tissue.The expression characteristics of the above genes are closely related to their functions.The results of real-time PCR showed that 9 genes were mainly expressed in hypothalamus,pituitary,and ovary.The ESR2,PGR,SMAD1,CYP11A1,CYP17A1,and CYP19A1 genes were expressed significant different in STH with different fertility(P<0.05).The ESR1,ESR2,PGR,CYP11A1,CYP17A1,CYP19A1,SMAD1,and BMP15 genes were expressed significant different in STH and SNT(P<0.05).It is suggested that ESR2,PGR,SMAD1,CYP11A1,CYP17A1 and CYP19A1genes may be involved in the regulation of reproduction in Small Tail Han sheep with different fertility.2.Using bioinformatics analysis,it was found that the BMP15 gene g.50971423T>C loci of the STH were missense mutation which changed the 252nd amino acids from Leucine to Proline.The ESR2 gene g.73324006C>T loci were missense mutation which changed the 498th amino acid from Arginine to Histidine.HSD3B1 gene g.96092576G>A locus was a missense mutation which changed the 2nd amino acid from Alanine to Threonine.The ESR1 gene g.75378892A>T loci were missense mutation which changed the 36th amino acid from isoleucine to phenylalanine.The CYP11A1 gene g.33217408C>T loci were missense mutation which changed the41st amino acid from serine to phenylalanine.The PGR gene g.7491179C>T and CYP11A1 gene g.33222725A>G loci were synonymous mutations.The CYP19A1 gene g.56122588G>A locus was 5'UTR mutation and SMAD1 gene g.12485895A>G,g.12487190G>T,g.12487467A>G,g.12487558G>A loci were intron mutations that do not change amino acid.3.Population genetic analysis showed that SNPs which genotyping were at moderate polymorphism(0.25<PIC<0.5)and were under Hardy-Weinberg equilibrium(P>0.05)in most sheep populations.Association analysis indicated that the g.50971423T>C,g.96092576G>A,g.7491179C>T,g.75521224T>Aand g.56122588G>A loci had no significant correlation with the litter size in Small Tail Han sheep(P>0.05).Theg.75378892A>T,g.73324006C>T,g.33222725A>G,g.33217408C>T,g.12487190G>T loci had significant correlation with the litter size in Small Tail Han sheep(P<0.05).The litter size of AA,CC,AA,CT genotype was higher than TT,TC,AG and CC genotype(P<0.05),respectively.The TT genotype was higher than GT genotype and GG genotype(P<0.05).In summary,we researched the expression pattern of the genes of ESR1,ESR2,PGR,CYP11A1,CYP17A1,CYP19A1,SMAD1,BMP15 and HSD3B1 in SNT and STH with different fertility to further reveal the basis of the FecB gene effect.Then,We genotype these genes and several new SNPs information with significant influence on the litter size were obtained,which provided new materials for sheep genetic breeding maker selection.