| Silky is favored by consumers for the health products, cultivating high meat yield and fast-growing breeds sllkies are urgent for the demands for the market. With the differences of deposition time and amounts in different organization, conventional breeding methods can not effectively accelerate breeding progress. In this study, Guangxi black-W were the materials, the distribution of melanocytes and melanin content were analyzed, meanwhile, the gene expression and mutation of MCIR, Tyrpl, MIFF and EDN3 were tested, the main purpose was to study the molecular marker-assisted breeding technology for the meat-type silky. And the results are as follows:1. Distribution of melanoma cells and measurement of melanin content.HE staining of the skin and chest muscle shows that melanocytes are widely distributed in skin epidermis and dermis, the melanocytes in the lower dermal are forming dendritic or fibrous. The muscle melanocytes are mainly distributed in the connective tissue, in addition, there are some melanocytes in the perimysium,and the muscle fibers, and the melanocytes in the dark skin and breast muscules are more than that in the light;The skin and breast tissue samples were dissolved in soluene-350 solution, and placed in a spectrophotometer to measurement the contents of melanin, the results showed that the melanin content of skin and breast muscle in dark black-bone chicken were significantly higher than those in the the light chicken (P<0.01); And we also measured the L-value of breast with the colorimeter, the results showed that the L-value had a significant correlation with the melanin content, thus the L value of breast can be used as an indicator to judge the color of blackness.2. The candidate gene expression studies in Guangxi black-bone chicken.Real-time PCR results showed that the relative expression of MC1R gene in dark skin and breast muscles was significantly higher than that in light (P<0.01), the relative expression of MITF in dark skin and breast muscle was significantly higher than those in light chicken (P<0.05), the expression of EDN3 and Tyrpl in dark skin was significantly higher than those in light(P<0.01), and in the breast, the different was significantly higher than the light(P<0.05). The association analysis between relative expression of genes and the melanin content found that mRNA expression of the four candidate genes and melanin content showed a significant positive correlation, the expression levels of genes are consistented with melanin deposition in skin and breast.3. SNPs and genetic diversity of candidate gene.The Sanger sequencing method was used to detect the SNPs in candidate gene, the results showed that there were 40 SNPs in total, for each SNP, the individual genotyping and haplotype were analysised, Tyrpl found two haplotype block, EDN3 found one, MITF found three, the association of the haplotype, haplotype analy and skin color was analyzed, the results showed that there are a certain correlation between them. the genotype frequencies analysis of each SNPs between the dark and light black-bone chicken were tested by independence Chi-square,and we found that the the genotype frequencies of T1183G in MC1R gene, C7704T and C10237G in MITF gene, A20444G, G20643A and T30947C in Tyrpl gene, and C-1368T in EDN3 gene had significant difference between dark and light black-bone chicken.4. Association analysis between SNPs in candidate genes and melanin.The results showed that the melanin in T10184G site in Tyrpl gene and the T30947C site in MITF gene had significant diference in skin(P<0.05), while in the breast, the defference was not significant.The differences of melanin content in other SNP sites were not significant(P>0.05).In conclusion, the above results show that MC1R, MITF, Tyrp1 and EDN3 gene are associated with Guangxi W Silky melanin deposition and have some SNPs related with color of skin, the dissertation provides theoretical basis for the study of black-bone chicken and enriches the conservation Breeding information landraces. |
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