Identification Of Genes Related With Grey Plumage In Anyi Slate-Grey Chicken Via Whole Genome Resequencing

The feather color is one of the most important breeding characteristics of chicken,and the molecular genetics study of plumage color is of great significance for the study of the origin and evolutionary genetics,resource conservation and product quality control of domestic chickens.Anyi Slate-Grey chicken is a high-quality native chicken breed resource in Jiangxi Province,and it was formed under long-term natural selection and artificial selection.It is a rare native chicken breed with grey plumage in China.So far,the molecular genetic mechanism of the grey plumage in Anyi Slate-Grey chicken has not been found.Therefore,in this study,whole-genome resequencing and extracting variation was performed on a total of 72 unrelated individuals,them were 30 individuals form Anyi Slate-Grey chicken and 42 individuals form three Jiangxi yellow feather native chicken breeds（Baier Yellow chicken,Ningdu Yellow chicken and Xiushui Yellow chicken）,and the average sequencing depth of whole genome resequencing was～18 X.Based on the genome-wide SNP dataset,the selection signal analysis and genome-wide association analysis on the grey plumage phenotype in Anyi Slate-Grey chicken were carried out.Combining with the results of the two analyses,candidate functional genes significantly related with the grey plumage phenotype in Anyi Slate-Grey chicken were successfully identified.Subsequently,identification of mutation and the correlation analysis between the identified mutation and the grey plumage phenotype in Anyi Slate-Grey chicken was conducted by using MC1R gene as strong candidate gene.The specific results in the study are as follows:Ⅰ.A total of 1496.04 Gb of Raw data was produced by whole genome resequencing,and 1493.03 Gb of Clean data was obtained after quality control.The Clean data of each sample was in the range of 14.6 G～27 G,and the average sequencing depth was 16.93 X（12.26 X～21.09 X）.Clean Reads mapping to reference genome of chicken（GRCg6a）showed that the mapped rate of each individual was above 97.5%,1 X coverage of each individual was more than 97.86%,and 4 X coverage of each individual was not less than95.25%.A total of 21,745,571 SNPs and 1,158,820 Indels were detected by extracting variation,and high-quality genomic variation data（18,793,039 SNPs and 1,012,978 Indels）were obtained by quality control parameter filtering.Ⅱ.Based on the whole-genome SNP data,the Selection signature analysis using a sliding window strategy was performed to screen the selection signature of the grey plumage phenotype in Anyi Slate-Grey chicken.At the top 0.1%threshold level,105 significant selection window regions were detected for each of the two statistics of F_ST andπratio,and49 and 34 significant selection genes were obtained by the gene annotation analysis,respectively.Seven selection window regions was belong to the overlap regions of the selection regions on F_ST andπratio,and the two regions of GGA1:65.92 M-65.94 Mb and GGA11:18.82 M-18.86 Mb presented the strongest selective signatures in the overlap regions of the selection regions on F_ST andπratio.A total of 7 functional genes were involved in the above regions with strong selection pressure by gene annotation,including MC1R,SOX5,TUBB3,DEF8,TCF25,DBNDD1 and GAS8 genes.Among them,MC1R and SOX5gene were known to be related to melanin formation.Ⅲ.The GWAS of the grey plumage phenotype in Anyi Slate-Grey chicken was conducted by the univariate linear mixed model.The results of GWAS showed a total of 94significant SNPs were screened,among which two missense mutations of c.212T>C and c.274G>A are located in MC1R gene,and 14 functional genes were involved in these above significant sites.Combing the two results of selection signature analysis and GWAS,a total of 7 location candidate genes related with grey plumage in the grey plumage phenotype in Anyi Slate-Grey chicken were identified,including MC1R,SOX5,TUBB3,DEF8,TCF25,DBNDD1 and GAS8 genes.Among them,MC1R and SOX5 gene were known to be related to melanin formation,so MC1R and SOX5 gene can be used as strong candidate genes to affect grey plumage formation of Anyi Slate-Grey chicken.Ⅳ.Identification of mutations and the correlation analysis between the identified mutation and the grey plumage phenotype in Anyi Slate-Grey chicken was conducted by using MC1R gene as strong candidate gene.The results showed the two missense mutations of c.212T>C and c.274G>A in MC1R gene were detected in all individuals form Anyi Slate-Grey chicken,which were both causative mutations for black feathers formation of Chinese indigenous chicken,suggesting that the grey plumage phenotype in Anyi Slate-Grey chicken was formed on the molecular basis of melanin formation.Meanwhile,the c.637T>C missense mutation of MC1R gene was related to melanin dilution but wasn’t completely associated with grey plumage phenotype,indicating that this mutation isn’t the causative mutation for grey plumage formation in Anyi Slate-Grey chicken.In conclusion,there is no structural mutation of SNP in MC1R gene that is completely related to grey plumage formation of Anyi Slate-Grey chicken,but the possibility of regulatory mutations or more complex variants in the MC1R gene cannot be ruled out,which need to be further explored.These above results of this study can provide a foundation for further identifying the causal genes and causal mutation of grey plumage in Anyi Slate-Grey chicken,and expatiating the molecular genetic mechanism of grey plumage phenotype.