Identification Of Genes Related With The Grey Plumage Coloration In The Anyi Slate-Grey Chicken Through The RNA-Seq

Anyi Slate-Grey Chicken（also known as Slate-Grey Chicken）is an important indigenous chicken breed in Jiangxi Province.It has the characteristics of"three grey”（grey feathers,grey tibia and grey beak）,and is one of the few existing local chicken breeds characterized by"grey feather".At present,many causal genes for plumage coloration have been identified in the chicken one after another,however,there is no report about the causative gene and mutation for the grey feather and the molecular mechanism of grey plumage coloration in Anyi-Slate Grey chicken is still poorly understood.Based on this,we firstly constructed a reciprocal-crossing F₁ population between the Anyi Slate-Grey Chicken and Ningdu Yellow Chicken.Subsequently,three individuals for each plumage coloration including pure black feather,grey feather and yellow feather phenotype were selected from the F₁ population and the RNA isolated from the new feather follicle tissues of nine individuals were analyzed on transcriptome using a Hiseq2500 sequencing platform,after that,we identified the Differentially expressed genes（DEGs）of the three kinds of tissues,which were annotated and enriched by the GO terms and KEGG pathways followed up.Meanwhile,the qRT-PCR analysis were performed to verify the reliability of the results from the RNA-seq.The main results of this study are listed as follows.（1）After quality control,67,773,426,69,693,067 and 68,754,708 clean reads were obtained in each individual sequence library of black,grey and yellow feathers,respectively,of which 92.2%of the reads were mapped to the chicken reference genome（galGal5.0）.（2）A total of 24293 genes expressed in the three different plumage color animals were obtained in this study,including 18165,17552 and 17489 genes expressed in black,grey and yellow feather follicles,respectively.Furthermore,16480 co-expressed genes were found between the chickens with three different feather coloration and there were 720,351 and 333 genes specially expressed in the samples with black,grey and yellow plumage coloration,respectively.Meanwhile,data from RNA-seq in this study were verified as a reliable results by detecting the seven randomly selected genes in virtue of the qRT-PCR approach.（3）Herein,we totally identified 125,290 and 61 significant DEGs（P<0.05）in the three comparative groups including the Black_VS_Grey,Black_VS_Yellow and Grey_VS_Yellow,respectively.Of them,two genes including TYR and CRYAB were co-existed in the three comparison groups.Compared with the black plumage group,68genes were expressed with up-regulated effects and 57 genes with down-regulated effects were found in the grey plumage group.By means of annotation,nine plumage genes were differently expressed between the black and grey plumage animals and may be the candidate genes for grey feather.（4）The Gene Ontology（GO）enrichment analysis were performed in the DEGs and the results showed that 74 DEGs from the Black_VS_Grey group were assigned to twenty one GO terms and 152 genes from the Black_VS_Yellow group were assigned to thirty three GO terms.Nevertheless,there was no any information from the Grey_VS_Yellow group for the limited DEGs found in this group.Furthermore,all the GO terms identified here were mainly involved in the cell components,molecular functions and biological processes.Subsequently,a KEGG analysis for the pathway enrichment of the DEGs indicated that a melanin production pathway dominated by TYR,WNT16,WNT5B,EDNRB2 and other genes was significantly related to the formation of grey plumage coloration.In conclusion,a lots of transcribed information were obtained by the RNA sequencing and many DEGs were identified in the different plumage comparison groups including the Black_VS_Grey,Black_VS_Yellow and Grey_VS_Yellow.Importantly,nine annotated plumage genes were identified here,which may be regarded as the candidate genes for grey plumage,and a melanin production pathway related to the grey plumage was found in this study.These findings will provide transcriptional evidence for further identification of causal gene,mutation and molecular mechanism of"grey feather"in the Anyi Slate-Grey Chicken.