Candidate Gene Analysis Of Rabbit Agouti Coat Color Variation

The fur color of rabbits is not only a symbol of breed,but also of great economic value.There are many kinds of rabbit’s fur,among which the agouti coat color rabbit’s fur is popular because of its bright color and easy to be processed into wool products.Previous studies have found that agouti signal protein gene（ASIP）,tyrosinase receptor protein 1（TYRP1）,tyrosinase gene（TYR）,melanocorticosteroid gene（MLPH）and melanocorticosteroid receptor 1 gene（MC1R）have effects on rabbit hair color.However,the mechanism of the effects of these five genes on agouti coat color rabbits and how they affect agouti coat color rabbits remain unclear.Therefore,we tried to elucidate the mechanism of the effects of these five genes on agouti coat color rabbits by Sanger sequencing,relative quantitative analysis,methylation degree analysis and gene copy number analysis.In this experiment,three specially cultured colour meat rabbits were selected as the research objects.A total of 820 rabbits were selected to study the effects of these five genes on hemp-coloured rabbits.Firstly,we used Sanger Sequencing to search for SNPs in each exon and part of intron of these five genes.We used DNAMAN,Chromas and other software to genotype SNPs.Finally,we used genotype to establish a pedigree to verify the conclusion.Then,the expression of these five genes was analyzed by relative quantitative method.Subsequently,double luciferase reporting system was used to analyze the active region of ASIP gene promoter,and online software was used to analyze the methylation of ASIP gene promoter region.Finally,the absolute quantitative method was used to analyze the copy number variation of ASIP gene.The main results are as follows:（1）In this study,18 SNPs were found in these five genes,of which 6 SNPs were consistent with previous studies.The 18 SNPs were verified by pedigree analysis,and only 4 SNPs were found to be associated with hair color traits.（2）They were in exon 2 of ASIP gene c.5₆insA,exon 4 of TYR gene c.1118C>A,c.280₂85del 6 of MC1 R gene and c.304₃33ins 30 of ASIP gene,which are related to agouti coat color traits.Moreover,we found that when the second exon c.5₆insA of ASIP gene mutated,the rabbit did not show a agouti coat color trait,and the mutation was recessive by pedigree verification;when the MC1 R gene c.280₂85del 6 and c.304₃33ins 30 mutated,the rabbit showed a black coat trait,and the mutation was recessive by pedigree verification;when the fourth exon c.1118C>A of TYR gene homozygous mutation,the rabbit surface showed a black coat trait.Rabbits showed white color and the mutation was confirmed to be recessive by pedigree analysis.（3）Therefore,the homozygous agouti coat color rabbit genotype AACCEE was determined,and then the family of the homozygous rabbit was established as the parent generation to verify the validity of this conclusion,and the rabbits whose offspring were all agouti coat color trait were obtained.It was found that TYR gene was the epistatic gene of ASIP gene and MC1 R gene,while MC1 R gene was the epistatic gene of ASIP gene.But in the agouti coat color rabbits,we found that the fur color of these rabbits was dark and light.According to the results of hair measurement,the bigger the brown area of rabbit hair,the lighter the color of rabbit hair,the smaller the brown area,and the darker the color of rabbit hair.（4）Relative quantitative results showed that the expression level of ASIP gene was the cause of the influence on the shade of agouti coat color in rabbits,and the higher the expression of ASIP gene,the larger the brown area of rabbits.The active region of ASIP promoter is between – 1001^-912,and the content of CpG islands in the promoter region of ASIP gene is less,so the methylation of ASIP gene CpG islands may have less influence on the expression of ASIP.Absolute quantitative data show that copy number variation of ASIP gene can affect the expression of ASIP gene,so copy number variation of ASIP gene may be one of the reasons that affect the shade of agouti coat color.Generally speaking,genotype determines the formation of agouti coat color rabbits.The expression of ASIP gene and the variation of copy number of gene can affect the color of agouti coat color rabbits.