Study Of Model Of Gene Regulation Of Shank Color Conversion

Shank color is an important quality trait of chickens,which is mainly regulated by inhibitor of dermal pigmentation(Id).The Id is located at the distal of the long arm of Z chromosome,and some research found SNPs that significantly linked to Id locus in this position.But Id has not been accurately located so far.In order to explore sequence variants related to Id locus,this study used the phenotype data of the self-built Id family and chicken 600 K Affymetrix Axiom HD chip to perform genome-wide association analysis on black shank.GWAS results were verified and the candidate regions were sequenced to discover sequence variants.These results can provide molecular markers for dark shank trait and have great significance for promoting the mapping of Id.The dark shank controlled by Id is a hereditary trait,and it is feasible to establish an auto-sexing system by using Id.In the hybrid offspring of dark shank roosters and light shank hens,the hens showed dark shank and the rooster showed light shank.After 23 days of age,the sexuality of chicks can be distinguished by shank color,with an accuracy rate of 100%.However,the accuracy rate of this method at birth is not high,because some hens are born with light-colored shank,and gradually converted to dark shank as the age increases.This phenomenon is called shank color conversion.There is currently no research on the conversion of shank color.In order to study the gene regulation pattern of shank color conversion,we used the cross between the dark shank rooster and the White leghorn hen in the resource family to produce 12 offspring hens,which were divided into dark shank group and the converted group.Transcriptome sequencing analysis was used to study the differences in gene expression levels between the two groups at the age of 1 day,10 days and 20 days.Also,we construct a coexpression network to screen the core genes regulating shank color conversion.Our results will provide a theoretical basis for the establishment of auto-sexing systems of shank color.The main results of this study are as follows:1.Using chip data from 362 chickens,GWAS analysis was performed on the black shank,and it was found that there was a T / C mutation at 78,732,319 bp in the Z chromosome of the chicken(gal Gal5.0),which was closely linked to the black shank(P value = 9.54E-29).Randomly selecting 86 individuals within the Id family to verify the SNP by enzyme typing and found that this site was significantly associated with black shank.2.It was found that the yellow shank individuals had a deletion.PCR and gel electrophoresis were used to verify the result and it was found that the deletion was 100% linked to the yellow shank in 86 family individuals.3.Turpan fighting cock is crossed with white Leghorn hens and the phenotype of the offspring verify the law of sex-linked dark shank.All of the hens were converted ones.4.Differential expression gene analysis and functional analysis revealed that DEGs are connected to the melanocyte differentiation function,and 9 genes that were significantly differentially expressed at various ages were identified,including OCA2,RASGRP3,CRP,SLC24A5,TRPM1,PAX3,EDNRB2,MLPH,ENSGALG00000037596.5.By constructing a co-expression network,the modules associated with the traits were predicted,and the genes in the modules were significantly related to the melanocyte differentiation function.The core genes were screened out,including TYR,TYRP1,MLPH,MLANA,OCA2,EDNRB2,PMEL.In summary,through research,we identified two sequence variants in the Id family that are significantly associated with shank color,which proves that Id is near the 72Mb-78 Mb on the Z chromosome.Few candidate genes of Id within this region have been found mutations linked to shank color,suggesting that they may regulate the shank color by other means.The rest genes need more research to study their effects for shank color.Meanwhile,we speculate that Id may be an unknown gene.Through the Turpan fighting cock hybridization experiment,it is proved that the dark shank is a sex-linked trait,and the shank color conversion is the normal manifestation of the dark shank in hens.Through transcriptome analysis,it was found that the melanocyte differentiation function in the early stage of the dark shank group was more active,and there was a gene EDNRB2 that regulates this function,which was both included in the differentially expressed genes and core genes.This gene can combine with EDN3 to control melanocyte development.Fm was reported to promote the large expression of EDN3 in embryonic and juvenile stages,accelerate melanin synthesis and deposition.Therefore,we believe that EDN3 is a candidate gene for dark shank at 1-day-old in hens.