The Expression Characteristic Of RFRP Gene And Association Of Its Polymorphism And Age At First Egg With Reproduction Traits In Chickens

As an important economic trait of poultry,reproductive traits are regulated by many genes and QTLs.Revealing the genetic effects and regulatory mechanisms of these genes is beneficial to the selection of poultry reproductive traits.This study used RFamide-related peptides(RFRP)as candidate gene,Zhenning yellow chicken as object,direct sequencing was used to detect the single nucleotide polymorphism(SNP)of RFRP.The gene expression characteristics and the association between polymorphisms and reproductive traits of hens were analyzed,and the association between age at first egg with reproductive traits,hormone levels and hypothalamic-pituitary-gonadal axis related genes(GNRH,RFRP,GNRHR,RFRPR,etc.)expression were also analyzed.The results contained two parts.1.The expression characteristic of RFRP gene and association analysis between the SNP of RFRP gene and reproductive traits in chickens.Using RF amide-related peptide gene as the candidate gene for reproductive traits,direct sequencing was used to detect the single nucleotide polymorphism(SNP)of it.A general linear model(GLM)program was performed to analyze the association between the SNP and the reproductive traits,quantity of follicles,hormone levels.Quantitative Real-time PCR was used to measure the RFRP gene expression level in different tissues and to compare RFRP gene expression levels at different sexual maturity stages in hypothalamus.Finally,according to the results of the association analysis,Compare the expression levels of RFRP between individuals based on genotypes at sites that are significantly associated with reproductive traits and reproductive hormones.The experimental results are as follows:(1)According to the results of direct sequencing,a total of 11 mutation sites were detected in RFRP gene.(2)The results of association analysis with reproductive traits showed that 4 loci(A276G,G1396A,G1694A and A2659G)were significantly associated with reproductive traits.The A276G and A2659G were significantly associated with egg production at 300 days of age,G1396A was significantly associated with egg weight at 300 days of age.G1694A is significantly associated with the fertilization rate.(3)The results of the association analysis with reproductive hormones and follicle number showed that three loci were significantly associated with them(A276G,G1396A and G1694A).Among them,A276G was significantly related to the number of pre-hierarchical follicles,and G1396A was significantly associated with LH and PRL levels.G1694A was significantly associated with the LH level.There was no significantly associated between the A2659G site and reproductive hormones and the number of follicles.(4)The result of Quantitative Real-time PCR showed that the RFRP gene is mainly expressed in the hypothalamus,and a small amount is also expressed in the pituitary,ovary and uterus,and little expressed in other tissues.The expression level of RFRP gene in the hypothalamus of sexually mature hens was significantly lower than that in the immature hens(P<0.01).At the three loci A276G,G1396A,and G1694A,the expression levels of RFRP of different genotypes are also different.2.The association between age at first egg with reproductive traits and hypothalamic-pituitary-gonadal axis related gene expression.The age at first egg(AFE)is not only one of the important reproductive traits of hens,but also an important signal of sexual maturity of hens.Generally,animals with precocious puberty or delayed puberty always have disadvantage at reproductive ability.Therefore,400 340-day-old Zhenning yellow chicken were selected and all individuals were grouped based on the age at first egg.Comparing the egg production rate(EPR)at different time points.,reproductive traits,hormone levels and the gene expression levels in hypothalamic-pituitary-gonadal axis among the groups.The results are as follows:(1)All individuals were divided into 6 groups(134-138d,139-143d,144-148d,149-153d,154-158d,>159d).According to the analysis results,the hens in 134-138d group was precocious,hens in>159d group was late-maturing,the hens with normal AFE were in 149-153d group.(2)Using the EPR above 70%as the peak of egg production in the egg production curve of each group,the peak egg production duration of the 6 groups were 54 days,60 days,71 days,70 days,59 days,38 days.In addition,a total of five time points of 220d,260d,300d,340d,245-340d were calculated.The results of the comparative analysis showed that the laying performance of precocious individuals was poor at the early stage,but the egg production performance gradually improved with the age increased,the late-maturing group had well performance at egg production performance at the early stage.(3)The comparison results of reproductive traits showed significant differences in egg production at 340 days,egg production at first 50 days(EPF50)before laying,and fertilization rate.Both the precocious and late maturity groups had lower egg production than the normal group at 340 days of age.The egg production at the first 50 days before the start of laying showed a significant upward trend with the increase of the age at the start of laying.The late maturity group had the highest EPF50 but precocious group had the lowest EPF50.In the comparison of fertilization rate,the fertilization rate of the late maturity group was significantly lower than the other groups.(4)The comparison result of reproductive hormone and follicle number showed that the hens in the normal group had a significant advantage at the number of hierarchical follicles.The level of luteinizing hormone in the normal group was significantly higher than that in the late maturity group,and the prolactin level in the late puberty group was higher than that in the precocious puberty group.(5)Comparison of the expression levels of related genes in hypothalamic-pituitary-gonadal axis among the groups.In the hypothalamus,the GNRH gene expression of the normal group was significantly higher than that of the precocious puberty group and the late maturity group,while was no difference in the expression of RFRP.In the pituitary,the expression of GNRHR gene in late puberty group was lower,and there was no significant difference in RFRPR gene expression.In the ovary,the expression of estrogen receptor gene in the normal group was higher than that in the late maturity group.In follicular granulosa cells,CYP11A1 and KITLG gene expression levels were lower in the late puberty group.