Study On The Genetic Variation And Promoter Methylation Of MITF Gene And Their Associations With Feather Color In Duck

Feather color is not only an important feature of ducks variety,but also is one of the key points in duck breeding.In the use of local ducks for genetic breeding,there must be a problem of feather color changes and stable heredity,which requires the guidance of the basic knowledge of the heredity of feather color.The diversity of feather color lied in the distribution and regulation of melanin,and the formation of melanin was a complex process,which involved a large number of genes and regulatory factors.Among them,microphthalmia associated transcription factor gene(MITF)played an important role in melanin synthesis pathway.In this study,four different feather color ducks(Putian black duck,Putian white duck,Longsheng black duck,Liancheng white duck)were used as the research objects,to explore the regulatory mechanism of MITF gene in duck feather color variation,lay the foundation for further analysis of molecular mechanism of formation of different feather color,providing theoretical support for the stability of molecular technology to solve the strains in the duck feather color genetic breeding.The main results were as followed:1.Fluorescent quantitative PCR method were used to detect the expression level of melanin synthesis pathway related genes(TYR,MITF)in the black and white feather bulbs,The results showed that there were almost no expressions of those genes in white feather bulbs.2.The promoter sequence of duck MITF gene was obtained by homologous comparison and analysis,and the CpG island was predicted online.The results showed that the promoter region of duck MITF gene contained two CpG islands,the size of them were 102bp and 131bp,respectively.3.The methylation of duck MITF gene promoter was detected by the Bisulfite Genomic Sequencing PCR technique(BSP),and two CpG islands were found to stay at a high methylation level.The total methylation level of CpG islands in black feather bulbs was significantly lower than that of white one(P<0.05).Similary,the methylation status of CpG island 2 was same as the total methylation level of CpG islands(P<0.05).The methylation level of CpG island 1 in Liancheng white duck was significantly higher than that in black feather bulbs of Putian black duck and Longsheng black duck,white feather bulbs of Putian black duck,Putian white duck and Longsheng black duck(P<0.05).4.The association between MITF gene expression and methylation of CpG islands promoter was analyzed.It was found that CpG-1 in CpG island 1,CpG-6 and CpG-8 in CpG island 2 were negatively correlated with the relative expression of MITF gene mRNA expression(P<0.05),while the total methylation level of CpG islands also had negatively correlated with the relative expression of the MITF gene mRNA expression(P<0.05).It was presumed that CpG island 2 might be the key region regulating the duck MITF gene expression.5.Four SNPs were found in duck MITF gene by Sanger sequencing(g.312C>T,g.32813G>A,g.39807T>G and g.40862G>A).The g.312C>T mutant was located in 5’UTR,32813G>A was located in exon 4,which was a synonymous mutation,g.39807T>G and g.40862G>A were located in intron 7 and intron 8,respectively.6.According to the PCR-RFLP and association analyses,the distribution of those four mutations in the black and white duck populations were all extremely significant difference(P<0.01).In g.39807T>G,T allele was the dominant one in Putian white duck,while G allele was dominant one in Putian black duck and Longsheng black duck and Liancheng white duck.In g.40862G>A,the dominant allele of Putian white duck was G allele,while that in Putian black duck and Longsheng black duck and Liancheng white duck was A allele.Further analysis of the two mutations in white Pekin duck,we speculated that the muatations of g.39807T>G and g.40862G>A in MITF gene may effect the melanin biosynthesis in duck beak and webs.In conclusion,the differential expression of MITF gene was related to the feather color trait,and the variation of MITF gene may affect the synthesis of melanin in duck beak and webs.