Association Of FSHR Gene Expression And Sequence Variations With Egg Laying Performance Of Lueyang Black-boned Chicken

Lueyang black-boned chicken(LBC)is a unique poultry breed in Shaanxi Province.Which has excellent egg quality and nutritional value,and is suitable for forest land scattered feed.However,due to the lack of systematic breeding,LBC grew slowly and had poor egg laying performance.The egg laying performance defects of LBC increased the production costs of chicks and commercial eggs,which had a very negative impact on the development of black-boned chicken industry.In this study,FSHR,a follicular development regulatory gene,was used as the object to screen functional mutations affecting FSHR function and egg laying performance of LBC by analyzing the association between FSHR gene expression level and sequence variation and egg laying performance of LBC.In this study,675 Lueyang black-boned chickens were selected from the second generation population of LBC egg line,determination of age at the onset of egg laying(AOE)and the number of eggs(EN)from the onset of egg laying to 30 weeks,40 weeks and 56 weeks,with univariate animal model to estimate the heritability of AOE and EN,and the genetic correlation between AOE and EN was estimated by bivariate animal model.The granulosa cells and pituitary of small white follicles(SWF,1-5 mm),small yellow follicles(SYF,6-8 mm)and large yellow follicles(LYF,9-12 mm)were collected from 8 high-yielding and low-yielding individuals of the third generation population of LBC egg line.The expression levels of FSHR and AMH in follicular granulosa cells and FSHβin pituitary were detected by q PCR.4 individuals with SNP site g.8142094T>C in exon 10 of FSHR were selected as heterozygotes,and the expression imbalance of FSHR between alleles was detected by PCR-RFLP.Genotypes of three non-synonymous mutations Thr46Ile,Lys151Arg and Met335Thr in FSHR gene were detected by mass spectrometry,and the association between these mutations and egg production traits of LBC was analyzed by univariate linear mixed model.The conservation of three non-synonymous mutations was analyzed by sequence alignment,and the 3D structure model of FSHR was constructed by homology modeling method to analyze the influence of three non-synonymous mutations on the 3D structure of FSHR protein.Both the AOE(h~2=0.08)and EN(h~2=0.25～0.32)were low heritability traits,and there was a strong negative genetic correlation between AOE and EN at each stage(r _A=-0.98,95%confidence interval:-1.0～-0.7),but the phenotypic correlation was moderate(r_p=-0.59～-0.21),and there was a strong genetic correlation(r _A=0.99,95%confidence interval:0.84～1.00)and phenotypic correlation(r_p=0.48～0.79)between EN at each stage.In SWF,the expression level of FSHR in the low-yield group was significantly higher than that in the high-yield group.There was no significant difference between high and low yield groups in SYF and LYF.The expression of FSHR was the highest in SWF(P<0.05),and the lowest in SYF(P<0.05).The expression of AMH was the highest in SYF(P<0.05),and there was no significant difference between high and low yield groups.There was no significant difference in FSHβexpression between high and low yield groups.The allele-specific expression test found that FSHR had an unbalanced allele expression,indicating that the differential expression of FSHR gene among individuals with different growth performance was caused by the variation of cis-elements.In order to further identify the functional mutations associated with phenotype,this study analyzed the correlation between the three non-synonymous mutations and the egg production traits of LBC,but no significant mutation associated with egg production traits was found.Among the three FSHR non-synonymous mutations,Thr at 46 and Met at 335 were not conserved,but Lys at 151was completely conserved in birds and mammals.The three mutations caused changes in the hydrogen bonds of the main or side chains of about 60 amino acid residues of FSHR protein and the water solubility and positive phi torsion angles of individual residues.In addition,Lys151Arg caused changes in the cis-peptide bond of amino acid at position 269.Genetic parameter estimation results suggested that directly select from AOE and EN for phenotypic is difficult to achieve the ideal effect,but on the basis of AOE and early egg production for early stage on EN were indirect selection has the feasibility.The results of candidate gene studies showed that the change of FSHR expression level was probably an important genetic factor affecting the egg laying performance of LBC,and related expression regulation mutation might constitute the genetic basis of EN of LBC.This study was the first to estimate the genetic parameters of variety specific egg production traits and analyze the relationship between FSHR gene expression and sequence variation and egg production traits of LBC.These results will lay a theoretical basis for the development of breeding programs for laying traits and identification of functional mutations in LBC.