Researches Of Plumage Pigmentation Based On Golden Pheasant Genome And Transcriptome Sequencing

Since the initiation of the human genome project, genomics has gradually become the focus of researchers.With the birth of the next generation sequencing technology, high-throughput and high-depth sequencing has been increasingly used into the genome of non model organisms. The completion of the chicken draft genome sequencing marked the advent of the avian genome era. The research on the birds evolutionary characteristics at genome level have also started. Plumage color patterns confer distinct and important advantages to birds in a variety of ways. With its colorful nature and complexity, plumage prefers to be an ideal model to explore the formation of coloration patterns in vertebrates. So it can be of great significance to the investigations of plumage coloration mechanisms in genomics. The golden pheasant (Chrysolophus pictus) is regarded as the famous ornamental pheasant in the world, which mainly inhabit western of China. Based on the analysis of golden pheasant genome, transcriptome sequencing and feather pigment composition, the synthesis and deposition mechanism of melanin and carotenoid in feather were explored. We present a 1.029 Gb de novo assembled genome of the golden pheasant and 16,088 protein-code genes were predicted. We also sequenced the feather follicle transcriptomes with various colors and, obtained a total of 57.66 Gb clean data and 15,118 genes.After further analysis of the whole genome comparison with other species, the expression of the transcriptional groups and the identification of the pigment composition in different colors of feather, we found a series of factors that involved in golden pheasant feather melanin and carotenoid regulations. These results laid the foundation for the investigation to the function of pigment related genes.1. The golden pheasant whole genome comparison with other birdsTo assess the phylogenetic position of the golden pheasant in Galliformes, a maximum-likelihood phylogenetic tree was constructed based on 3,479 single-copy orthologous genes identified from eight candidate birds and three other species. We found that the relationship between the golden pheasant and turkey was closer than that between the golden pheasant and chicken. Then the whole genomes of chicken, turkey, golden pheasant were analyzed with the zebra finch as outgroup. For gene copy number, we found 44 species specific and 200 expaned gene families in the golden pheasant, including lipid-related gene families, POU family and claw keratin genes. It is believed to be related to the synthesis and accumulation of melanins and lipid-soluble pigments. For the gene co linearity, we found 58 scaffolds involved in genomic rearrangement including 15 color genes and 24 Hox genes. These genes directly or indirectly involved in the golden pheasant pigment regulations.2. Pigment identification of golden pheasant and Lady Amherst’s pheasant feathersThe study identified pigment compositions from different colored feathers of pheasants by spectral and chromatographic techniques. Firstly,Raman spectra from red, orange and, yellow feathers and even the rachis of iridescent feather in golden pheasant are consistent with carotenoids previously reported, while Spectral bands from Lady Amherst’s pheasant express the characteristic of pheomelanin. Green iridescent feathers in both birds showed eumelanin bands. In the next, exaction of lipid-souble pigments were performed, which were only found in red, orange and yellow feathers from golden pheasant. Mass spectrometry analysis of these substances showed C40H54O and C40H56O2 existed in these feathers. C40H56O2 should be lutein or zeaxanthin, which are isomers with each other. At last, qualitative and quantitative analyses in further high performance liquid chromatography showed carotenoids in golden pheasant plumage are lutein and zeaxanthin. For melanin analysis, with PTCA and TTCA to be the standards, the content of eumelanin and pheomelanin were confirmed. Both chromatographic and spectra results showed the golden pheasant feather pigment compositions included carotenoids and melanin, while there were only melanins in Lady Amherst’s pheasant plumage.3. Analysis of pigmentation related genes by RNA-seqThis part sequenced the feather follicle transcriptomes from breast, nape, mantle, rump and tail in golden pheasant and Lady Amherst’s pheasant. We carried out screening and enrichment analysis to differentially expressed genes from same parts of the two species and different color feathers. For melanin related genes, TYR, TYRP1, KIT and WNT7 are mainly expressed in mantle, ASIP and members of SLC family are expressed in pheomelanin feathers. In addition, the alternative splicing isoforms, ASIP-IA and MTF-1M may be associated with pheomenlanin synthesis. For carotenoids related genes, we found that a low expression level of BCO2 is associated with carotenoid accumulation in golden pheasant feathers.APOA1 and CYP2J expressed mainly in carotenoid contained feathers.These genes may be key factors affecting carotenoid accumulation in golden pheasant feathers.In addition, GSTA2 and APOD were found to be associated with carotenoid coloration in golden pheasant plumage by real-time PCR.