Polymorphism Analysis And Expression In Different Tissues Of ESR2 Gene In Leizhou Black Duck

Leizhou black duck is an egg-type duck population commonly allocated in the Leizhou Peninsula in China with good egg-laying performance.A previous study on the ovarian transcriptomic analysis in Leizhou black duck revealed that the ESR2 gene was involved in hormone regulation in reproduction and the estrogen signaling pathway related to reproductive performance was enriched.This suggested that ESR2 may have a functional role in the reproductive performance of the Leizhou black duck.ESR2 gene belongs to the nuclear receptor family mainly expressed in the ovaries and granulosa cells to regulate ovarian follicle development and egg production.The ovary and oviduct are important organs responsible for egg-laying and their development significantly affects the egg-laying traits in poultry.This study therefore aimed at evaluating the polymorphism of the ESR2 gene and its association with egglaying traits,the distribution pattern of ESR2 m RNA in the HPG axis,oviduct and nonreproductive organs,and the localization of ESR2 in different cells of the ovarian follicles to investigate the functional role of ESR2 in Leizhou black ducks.(1)In this study,genomic DNA was extracted from blood samples of 101 Leizhou black ducks to identify single nucleotide polymorphisms(SNPs)of the ESR2 gene to elucidate molecular markers highly associated with egg-laying traits in Leizhou black duck.From the sequencing results of the 10 PCR amplification products,obvious sets of peaks were found in primers 2,5,6,and 7.After searching,23 SNP sites were identified from the four(4)different primers.Association analysis of single SNP sites showed that SNPs g.56805646 T>C and exon3-20G>A were significantly(P<0.05)associated with egg weight.Ducks with CT and AG genotypes had significantly higher egg weight than those with other genotypes.Haplotype association analysis of g.56805646 T>C and exon 3-20G>A showed that the haplotypes were significantly associated with egg weight where higher egg weight was seen in individuals with H3H4 haplotypes.Haplotype association analysis of SNPs found on primer 6 showed that the haplotypes were significantly associated with egg weight where higher egg weight was seen in individuals with H1H2 haplotypes.Again,haplotype association analysis of SNPs found on primer 7 revealed that the haplotypes were significantly associated with first egg age and egg weight.H2H3 haplotype individuals had significantly(P<0.05)higher first egg age and egg weight than the other individuals.(2)To compare and explore the differential distribution pattern of ESR2 m RNA in laying and non-laying Leizhou black ducks,tissues from the Hypothalamus-pituitary-ovarian axis,oviduct(Infundibulum,magnum,isthmus,and uterus),and non-reproductive tissues(Heart,liver,spleen,lung,kidney,breast muscle,and leg muscle)were collected.In the HPG axis,the results of qRT/PCR showed that ESR2 m RNA was significantly(P<0.05)expressed in the ovaries of both duck groups than in the hypothalamus and pituitary.Comparatively,laying ducks had significantly(P<0.05)higher expression of ESR2 in the HPG axis than non-laying ducks.In the oviduct,the qRT/PCR results showed that ESR2 was significantly(P<0.05)higher in the infundibulum and magnum of laying and non-laying ducks respectively.Comparatively,ESR2 m RNA expression was significantly(P<0.01,0.05)higher in the oviduct of laying ducks than non-laying ducks.In the various non-reproductive tissues,qRT/PCR results showed that the ESR2 m RNA was significantly(P<0.05)higher in the spleen of both laying and non-laying ducks.Comparatively,ESR2 m RNA was highly expressed(P<0.01,0.05)in all the nonreproductive tissues of laying ducks than non-laying ducks.(3)To elucidate the expression pattern and the cell-specific localization of ESR2 of the ovarian follicles,different grade-sized follicles were collected from laying Leizhou black ducks.The follicles were grouped into seven(7)as small white follicles(SWF),large white follicles(LWF),small yellow follicles(SYF),large yellow follicles(LYF),follicle 5(F5),follicle 2(F2),and follicle 1(F1).The qRT/PCR results displayed that ESR2 m RNA was expressed in all follicles with the highest(P<0.05)level of expression found in F1 compared to other follicles.Immunohistochemistry analysis of the cell-specific localization of ESR2 protein revealed that ESR2 was distributed in both granulosa and theca cells region in all the follicles examined.There was a significantly higher localization of ESR2 protein in the granulosa cells than the theca cells of SWF,SYF,LYF,F2,and F1.Comparatively,ESR2 was highly expressed in the granulosa cells of LYF than in all the other follicles.In summary,single-SNP association analysis of SNPs,g.56805646 T>C and exon 3-20G>A had a significant association with egg weight.Haplotype-SNP association analysis revealed that the haplotypes were significantly associated with first egg age(FEA)and egg weight(EW).ESR2 was highly expressed in the ovaries and highly localized in the granulosa cells of Leizhou black ducks.These results provide theoretical knowledge for the in-depth study of the related biological functions of the ESR2 gene and its application at the cellular level.Also,this study demonstrates a strong association between the ESR2 gene and egg-laying traits and can be used as a novel molecular marker for selecting Leizhou black ducks for egg production.