Polymorphism Analysis Of MC1R,TYR And MITF Genes And Their Tissue Expression In Chishui Black-bone Chickens

Chishui Black-bone Chickens is an excellent local chicken breed in Guizhou Province.It has two main characteristics of black feather and black bone.However,it is found that the degree of variability of Wudu in the individual is large.Therefore,this study took Chishui Black-bone Chickens as the research object and Taihe Silky Fowl chicken as the control.The SNPs of promoter regions and exon regions of MC1R,TYR and MITF genes in Chishui Black-bone Chickens were screened by PCR direct sequencing technology,and the correlation of melanin-related substances in different tissues was analyzed.At the same time,the expression of MC1R,TYR and MITF genes in different tissues and tissues were analyzed by real-time PCR.The main results are as follows:1.Screening of SNPs for MC1R,TYR and MITF genesA total of 24 pairs of primers were designed for the promoter region and exon region of MC1R,TYR and MITF genes.Sixteen SNPs of MC1R gene in Chishui Black-bone Chickens were found by PCR amplification and sequencing(including 12 promoter regions and exons).District 4);There are 23 SNPs in the MC1R gene of Taihe Silky Fowl(15 promoters and 8 exons);There are 7 SNPs of TYR gene in Chishui Black-bone Chickens(including 2 promoter regions and 5 introns);There are 6 SNPs of TYR gene in Taihe Silky Fowl(2 promoter regions,2 exon regions and 2 introns);There are 18 SNPs of MITF gene in Chishui black-bone chicken(including 2 promoter regions,1 exon region and 15 introns);There are 21 SNPs in the MITF gene of Taihe Silky Fowl(including 2 promoter regions,1 exon region and 18 introns).2.The law of melanin deposition in Chishui Black-bone Chickens and Taihe Silky FowlContent of 5-hydroxyindole,melanin,5-hydroxyindole-2-carboxylic acid,melanin and demelanin in the pectoral muscle,leg muscles and skin tissues,Taihe Silky Fowl hens > Taihe Silky Fowl cock > Chishui black-bone chicken cock > Chishui black-bone chicken hen;The content of 5-hydroxyindole,5-hydroxyindole-2-carboxylic acid and demelanin in the liver is Taihe Silky Fowl cock>Chishui black-bone chicken cock> Taihe Silky Fowl hen> Chishui black-bone chicken hen;The content of melanin and true melanin in the liver is Taihe Silky Fowl cock> Taihe Silky Fowl hen> Chishui black-bone chicken cock> Chishui black-bone chicken hen,but 5-hydroxyindole,melanin,5-hydroxyl in the chest muscle,liver and skin.The content of 5-hydroxyindole-2-carboxylic acid,true melanin and demelanin was not significantly different between varieties and sex;In the leg muscles,the contents of 5-hydroxyindole,melanin,5-hydroxyindole-2-carboxylic acid,true melanin and demelanin were significantly higher in Taihe Silky Fowl hens than in Chishui black-bone chicken hens,and in Taihe Silky Fowl.The difference between Chishui black-bone Chicken is not significant;In addition,there is no significant difference between Taihe Silky Fowl cock,hen and Chishui black-bone chicken cock.3.Association analysis between MC1R,TYR and MITF gene polymorphisms and melaninThe results showed that the CT genotype of the promoter region of MC1R gene was significantly higher than that of CC in the chest muscle of Chishui black-bone chickens.At the Exon1-G23 A locus,the content of melanin-related substances in the chest muscles of Chishui black-bone chicken hens was significantly higher than that of AG type;At the Exon1-T398 C locus,the TC type of the melanin-related substance in the legs of Taihe Silky Fowl cock was significantly higher than that of the TT type.At the Exon1-T212 C locus,the content of melanin-related substances in the leg muscles of Taihe Silky Fowl hens was significantly higher than that of CC type,while the TT type Taihe Silky Fowl hens were significantly higher than CC-type Chishui black-bone hens,and the TT type was in Chishui.Molecular breeding of Chishui black-bone chicken hens can be used as a supplementary reference.In addition,in the MITF gene Exon7-T72 C locus,the content of melanin-related substances in the leg muscles of Taihe Silky Fowl hens was significantly higher than that of TC type Taihe Silky Fowl e hens.4.Tissue expression of MC1R,TYR and MITF genes in two varietiesThe expression of MC1R gene in the liver of Taihe Silky Fowl was significantly higher than that in Chishui Black-bone Chickens(p<0.01).The TYR gene and MITF gene in Taihe Silky Fowl cock skin were significantly higher than those in Chishui Black-bone Chickens(p<0.01).Combined with the determination of the content of melanin-related substances in the two varieties of chickens determined above,it is further speculated that the expression of these three genes can promote the expression of melanin-related substances.In summary,four sites associated with melanin-related substances were found in the MC1R gene,namely C-2175 T and Exon1-G23 A,which can be used as genetic markers for the selection of red-brown chicken and hens.The Exon1-T398 C locus can be used as a genetic marker for the breeding of Taewu and Wuji Rooster.Exon1-T212 C can be used as a supplementary reference for the breeding of Chisui black-bone chicken hens.In addition,the MTF gene Exon7-T72 C locus may be a key site affecting the difference in muscle length of the legs of Taihe Silky Fowl hen and Chishui black-bone chicken.