Study On The Mechanisms Of Hair Pigmentation And Skin Transcriptome Annotation In Mink(Neovison Vison) Based On High Throughput RNA-sequencing

Mink（Neovison vison） is a precious fur-bearing animal and coat color is an important economic trait determing quality and value due to use of its fur. Investigation of mechanisms about hair pigmentation will provide important theoretical and practical significance in the process of cultivating new varieties of coat color for mink. In order to analyze the genetic regulatory mechanisms of formation of coat color phenotype in mink, in this study, Jinzhou black mink, Jilin white mink, Silverblue mink, Coffee mink and Pearl mink with five distinct coat color phenotypes were used as samples. The determination of melanin content, histological location of mature melanocyte, screening SNPs of coat color gene, differential expression of mRNA and transcriptome annotation of skin were studied by means of histology, cytology, molecular biology and RNA-seq technology. The main contents and results were as follows:1. The content of total melanin, eumelanin and pheomelanin were measured and analyzed from three kind of hair with different colors during molting and pelting periods. The results revealed that the content of TM, EM and PM in black hair were the highest than that of two others. There was a decreasing tendency in three kind of melanin content with the color gradually becoming light from black to white. In pelting period, the content of TM and PM in black hair were 1.17 and 1.20 times（P<0.01） than that of molting period, and the value of EM was significant higher than that of molting period. The content of TM and PM in white hair were 1.27 and 1.22 times（P<0.05） than that of molting period, and the value of EM was higher than that of molting period（P>0.05）. In addition, the content of TM, EM and PM in gray hair were all the same（P>0.05） in two periods. The formation of gray and white coat color phenotype was related with the content of PM. The EM content may be associated with black hair of mink. In the process of molting, the melanin in the melanosome from skin were transferred into the hair shaft by orientation of hair follicle.2. Toluidine blue, dopa and dopa with toluidine blue staining were conducted to observe the characterization about the distribution of mature melanocytes in hair follicle from mink skin with three different coat colors. The results of toluidine blue staining indicated that the structural feature of skin, hair follicle and accessories were clear because of the blue nuclear in mink with three coat colors. The epidermis was thinner than dermis. There was club hair in the hair root, and hair papilla was not existed. We inferred that the hair follicle was in catagen. Dopa and dopa with toluidine blue staining all showed that the region of tyrosinase-positive was deepened and pigment granules were continuous distribution in dermis of skin with black hair than that of gray, which was discontinuous. There were a large number of clumps-like pigment granule at the top of hair follicle. A small amount of positive staining band in the outer root sheath and hair medulla, and which color gradually fades along the direction of hair shaft. There was less dopa-positive band at the top of hair follicle in mink skin with gray hair, and which was sparse pigment granule. More over, the positive staining of the outer root sheath in mink with gray hair was weaker than that of black hair. Nevertheless, there was not positive staining area in mink skin with white hair. The mature melanocytes at the top of hair follicle were the main cytological basis of hair pigmentation in mink. The coat color differences of mink were determined by the quantity of mature melanocytes and tyrosinase activity in the region of hair follicle. However, the melanocytes in dermis were not involved in the formation of coat color.3. The nucleotide sequence of MC1 R, TYR and Agouti gene in mink were obtained by the method of PCR and clone sequencing. Then, the structural characteristics of nucleotide and amino acid sequence of three genes were predicted and analyzed by the method of comparative genomics and bioinformatics. The results showed that the sequence of American mink MC1 R gene was consisted of one single exon, partial of 5′UTR（188bp） and 3′UTR（182bp） untranslated region, and it contained an open reading frame of 954 bp, which encodes 317 amino acids（GenBank accession No:KJ152766）. Further structural prediction indicated that the MC1 R protein had one simple domain, seven transmembrane domains and no signal peptide at N-terminal, which was located on plasma membrane. The obtained nucleotide sequence of mink TYR gene, which had been deposited in GenBank under the accession number KJ716783, was 2391 bp in length, including 5 complete exons and the full-length open reading frame（ORF） of 1596 bp, which encoded 531 amino acids. The peptide encoded by full ORF was consisted of 18 amino acids signal peptide and 513 amino acids belonging to mature peptide. In addition, the length of obtained Agouti gene of mink was 4231 bp, including intron 1, exon 2, exon 3, intron 2 and partial intron 3, which was 424 bp, 160 bp, 62 bp, 1256 bp and 2329 bp in length respectively.4. The DNA of blood from five mink breeds（Jinzhou black, Jilin white, Silverblue, Coffee and Pearl mink） that possessed significant difference in coat color phenotype were selected as templetes, SNPs of MC1 R, TYR and Agouti gene were screened by PCR and Sanger direct sequencing technology, and then the relationship between mutation sites of three genes and coat color traits were analyzed in 430 minks. The results showed that there was not existed SNPs in the coding region of MC1 R gene and one deletion mutation, g.132₁35delAGTG was detected in the upstream of CDS. The polymorphic information content of the deletion mutation site was 0.3268, which was moderate polymorphism in silverblue mink populations. The mutation might affect gray coat color in silverblue mink, or showed closely linkage with the major gene controlling gray phenotype. One nonsense mutation, g.138T>A was detected at exon 1 only, while no SNPs was discovered in exon 4 and 5. The nonsense mutation leads to a premature stop codon at position 46 of the deduced amino acid sequence（p.Cys46ter）, which deleted a truncated TYR protein lacking of the putative catalytic domains. These results implied that the nonsense mutation in TYR gene exon 1 possibly caused the white coat color phenotype in mink（P<0.0001）. The CT genotype, which generated by g.1189C>T in intron 2 of Agouti gene, was associated with white coat color of mink. Moreover, the other CT genotype from g.252C>T was correlated with the pearl phenotype of mink. Five SNPs（g.290A>C、g.298G>C、g.340A>G、g.343T>C, g.379T>C） and two deletion mutations（g.472₄73delCA, g.974₉77delCTCT） in intron 3, which were closely linked together respectively may be associated with light coat color phenotype in mink.5. The quantitative real-time PCR technology was used to detect the expression level of MC1 R, TYR and Agouti gene in skin of three mink breeds（Jinzhou black, Jilin white and Silverblue mink） with black, white and gray coat color phenotype for investigating differential expression and providing theoretical foundation for elucidating the mechanisms of coat color in American mink. The results showed that the mRNA expression level of three genes were all screened in three mink breeds with different coat colors during pelting period. The mRNA expression level of MC1 R gene in white coat color was very significantly higher than that in black and gray coat colors（P<0.01）. However, there was not different between black and gray coat colors（P>0.05）. The order of relative expression was white>gray>black. The mRNA expression level of TYR gene in black and gray coat colors were 3.2535 and 1.8933 times than that in white coat color, which was very significantly different（P<0.01） between black and white hair. The order of relative expression was black>gray>white. Additionally, The mRNA expression level of Agouti gene in black and gray coat colors were 1.2515 and 0.9501 times than that in white coat color, however, the Agouti gene expression was not associated with different coat color phenotype（P>0.05）. The order of relative expression was black>white>gray.6. Skin transcriptome of mink with black, white and gray coat colors were determined based on the high throughout RNA-sequencing technology. The quantitative real-time PCR was used to detect the expression level of key genes, which might affect hair pigmentation of mink. The results showed that the raw data of transcriptome from skin of mink with three coat colors was about 33.19 G and 403,725 unigenes, which were annotated in seven bioinformatic databases. There were 12,557, 4,334 and 10,643 differential expressed genes（DEGs） in three comparative groups of BLM_S vs WHM_S、BLM_S vs SBM_S and WHM_S vs SBM_S, respectively. And the DGEs shared among three comparative groups were 1,735. There were 20, 10 and 17 DGEs involved in the process of hair pigmentation in mink among three comparative groups. KITLG、LEF1、DCT、TYRP1、PMEL、TYR、Myo5a、Rab27a and SLC7A11 gene might regulate the coat color phenotype in mink by means of four biological processes, which were melanocyte development, components of melanosomes and their precursors, melanosome transport and the swith eumelanin and pheomelanin.