Location Of The Major Gene Of Sub-Columbian Feather In H Line Of Yufen No.1 Laying Hens And Functional Verification

Yu Pink No.1 Laying Hen H line is a nationally recognized laying hen line bred by Henan Agricultural University,whose feather color is characterized by white feathers with black horizontal stripes on the neck,wings and tail.The feather color is similar to Columbia feather,except that Columbia feather has black vertical stripes on the neck,wings and tail,while H line has black horizontal stripes,so we call the feather color of H line "sub-Columbian feather".In production,when a sub-Columbian hen is crossed with a jute-feathered rooster,the offspring can distinguish the male from the female by the color of their feathers.However,the primary gene for sub-Columbian plumage has not yet been identified.In this study,we performed BSA-seq on the F3 generation of the feather color segregation resource population formed by crossing H line hens(sub-Columbian feather)and Goodyear roosters(jute feather)to locate the loci affecting sub-Columbian feather to a certain interval of the chromosome and to screen for genetic variation loci associated with sub-Columbian feather.Genetic variant loci associated with gene expression were mined and validated by performing RNA-seq on hair follicle tissues of different plumage colors.Then BSA-seq and RNAseq were jointly analyzed,followed by one-generation sequencing and KASP assay to validate the primary effector genes in sub-Columbian plumage.Finally,the mined candidate genes were functionally validated at the cellular level.The main results of this study are as follows:1.BSA-seq-based screening of candidate genes for sub-Columbian feather in H line of Yu Fan 1 laying hensIn this study,based on the F3 generation feather color segregation resource group formed by crossing H line hens(sub-Columbian feather)and Goodyear roosters(jute feather)in the pre-laboratory period,we used the jute-feathered roosters and subColumbian-feathered hens from the F0 generation as the parents,and the jute-feathered hens and sub-Columbian-feathered hens from the F2 generation as the offspring mixing pools for the BSA-seq analysis.In this study,we screened for genetic variant loci controlling sub-Columbian feather,including 1,118,029 Single Nucleotide Polymorphism(SNP)and 181,074 Insertion and Deletion(InDel)mutations.The Euclidean Distance(ED)algorithm and SNP Index algorithm(SNP-Index)were then used to identify markers with significant differences,and the candidate region where the candidate genes were located was localized on the Z chromosome,with a total size of 10.43 Mb,containing a total of 400 genes,which included ADAMTS12,RXFP3,SLC45A2,AMACR,PRLR,LMBRD2,OXCT1,ITGA1 and SLC38A9.By GO and KEGG enrichment analysis of the candidate genes,we found that the genes CREB3,FGF10,MAPKK,PDE4 D and FST within the interval were enriched in the pathway of melanin deposition.The genetic variant loci in all of the above genes can be used as candidate variant loci affecting sub-Columbian plumage.2.RNA-seq-based screening of candidate genes for sub-Columbian plumage in the H line of Yu Fan 1 laying hensIn this study,7,934 differentially expressed genes(DEGs)were detected by transcriptome sequencing of wing(W)and neck(N)follicle tissues of 5-week-old F2 generation H-line hens with sub-Columbian plumage(H),F2 generation Jute-feathered Gusi hens(Y),and Silver-feathered Hyland Brown hens(S).Among them,differentially expressed genes such as SLC45A2 and GPNMB were found to have the same expression trend in the YN vs.HN group and YW vs.HW group.Functional annotation and enrichment analysis of DEGs by Gene ontology(GO)and KEGG Kyoto Encyclopedia of Genes and Genomes(KEGG)revealed that DEGs were enriched in melanin-related pathways,such as the melanogenic pathway.PPI analysis revealed that SLC45A2 and GPNMB interact with melanin deposition marker proteins such as MLPH,TYR and KIT to co-regulate melanin deposition.In addition,the transcriptome was screened for a total of 81,306 SNPs from 5,970 DEGs and 4,912 InDel from 2,685 DEGs,resulting in the final screening of DEGs that both carry genetic variant loci and are associated with melanin,such as TYR,OCA2,MC1 R,GPR143,CDKN2 A,SLC45A2 and SLC24A5.The genetic variant loci in the above genes may affect gene expression and provide a basis for further screening of candidate genes affecting sub-Columbian plumage.3.Combined BSA-Seq and RNA-Seq to localize the main genes of subColumbian feather in the H line of Yu Fan 1 laying hens.In this study,BSA-Seq and RNA-Seq were jointly analyzed,aiming to explore some genetic variation loci that cause changes in gene expression and thus lead to the phenotype of sub-Columbian plumage.As a result,it was found that a mutation L347 M in the CDS region of SLC45A2 and a mutation(A10331272T)in the 3’UTR region were simultaneously measured in both RNA-Seq and BSA-Seq intervals,and moreover,SLC45A2 was a differentially expressed gene in RNA-Seq(P<0.05),and then the two loci were analyzed by one-generation sequencing and KASP technology.The genotypes of these two loci were found to be mutant in all H lines(sub-Columbian plumage)and light-flowered Sussex chickens(Colombian plumage);mutant heterozygous in all Hyland’s chickens(silver plumage);and mutant heterozygous in all Wenshang’s ruffed grouse(transverse mottled plumage),Goodyear’s chickens(jute plumage),Changshun’s chickens(jute plumage),Romain’s chickens(jute feather)and Luzhi chicken(jute feather)were all wild-type phenotypes.It indicates that both SNP loci L347 M and A10331272 T of SLC45A2 are causal mutations in sub-Columbian plumage and the two loci are completely interlocked.In addition,a mutation R10 C in the CDS region of CDKN2 A was measured only in RNA-Seq,and in addition CDKN2 A was a differentially expressed gene in RNA-Seq(P<0.05).In response to the black transverse spots on the neck,wings and tail of the sub-Columbian plumage,we validated the reported CDKN2 A locus controlling chicken transverse spot feather type B2 by typing it in different plumage color groups of chickens.The results revealed that the SNPs in the CDKN2 A promoter region,CDS region and intron region of the H line(sub-Columbian plumage)were all mutant;the SNPs in the CDKN2 A promoter region,CDS region and intron region of the light-flowered Sussex chickens(Colombian plumage)were wild-type;and the SNPs in the CDKN2 A promoter region and intron region of the Wenshuang ruffed grouse(transverse-spotted plumage)were mutant and the SNPs in the CDS region were wild-type.SNPs in the CDS region were wild-type.The SNPs in CDKN2 A CDS region and intron region of Hailan chicken(silver feather)were mutant,while the SNPs in the promoter region were wild-type.It indicates that all three SNP loci in CDKN2 A are one of the causal mutations in sub-Columbian feather and the three loci are completely interlocked.In this study,we found and verified that both SLC45A2 and CDKN2 A are the main effector genes of subColumbian feather,which lays the foundation for the subsequent verification of the functions of the main effector genes.4.Effects of SLC45A2 genetic variation on sub-Columbian feather in H line of Yu Fan 1 laying hensIn this study,the function of SLC45A2 genetic mutation on melanin deposition was verified on melanocytes.Firstly,chicken primary melanocytes were successfully isolated,and also SLC45A2 wild-type and mutant vectors were successfully constructed and overexpressed on the cells.It was found by q-PCR,WB,transmission electron microscopy and ELISA that SLC45A2 wild type promoted melanin deposition in primary melanocytes,and mutation decreased melanin deposition in melanocytes(P<0.05).In addition,it was found by q-PCR,WB,CCK8 and flow cytometry that SLC45A2 wild type promotes the proliferation of primary melanocytes,and the mutation decreases the proliferation of melanocytes(P<0.05).The H line,due to the decrease in gene expression after the mutation of SLC45A2,leads to a decrease in the deposition of melanin in the follicle and inhibits the proliferation of melanocytes,which may be in sub Columbia plumage may play an important role in the formation of white plumage.5、Effects of GPNMB on sub-Columbian feathers of H line of Yu Pink No.1 laying hensIn this study,the function of GPNMB on melanin deposition was verified on melanocytes.By q-PCR,WB,transmission electron microscopy and ELISA,it was found that GPNMB significantly promoted melanin deposition in chicken primary melanocytes(P<0.05).In addition,GPNMB was found to significantly promote the proliferation of primary melanocytes by q-PCR,WB,CCK8 and flow cytometry(P<0.05).The H line,due to the high expression of GPNMB,may play an important role in melanin deposition and melanocyte proliferation in sub-Columbian plumage by promoting melanin deposition in the follicle and also promoting melanocyte proliferation.To summarize,in this study,by combining BSA-seq and RNA-seq for analysis,and also by one-generation sequencing and KASP test,the primary effector genes of sub-Columbian plumage of the H line of Yu Pink No.1 laying hens were localized to SLC45A2 and CDKN2 A on the Z chromosome.cellular level validation revealed that SLC45A2 wild type could promote melanin deposition in chicken melanocytes,promote melanocyte proliferation;the mutation decreased melanin deposition in melanocytes and melanocyte proliferation.Meanwhile RNA-seq screening of GPNMB was empirically validated and found to promote melanin deposition in chicken melanocytes and proliferation of chicken melanocytes.Taken together,SLC45A2,CDKN2 A and GPNMB are involved in the formation of H-lineage sub-Columbian plumage.This study provides new insights for resolving and localizing the candidate genes regulating the sub-Columbian plumage,and also provides a theoretical basis for further research on the formation mechanism of chicken plumage color.