The Association Of OCA₂ And MITF Genes With Plumage Color In Korean Quails

Compared with chicken and livestock,molecular biology research in laying quail had just begun,there were few molecular markers in quail.In the long term,it will play crucial roles in identifying breed purity and phylogensis,and prompting the process of molecular breeding through using candidate genes and experiences in pigs and chicken.In this paper,Korean maroon quails and white quails were used as experimental animals.OCA2 and MITF genes were selected as candidate genes of quail plumage color traits,and candidate gene cloning,expression analysis,single nucleotide polymorphism(SNP)testing and correlation analysis were finished.The main research results are as follows:(1)The complete coding region of Korean quails OCA2 gene and MITF gene were successfully cloned by PCR.The complete coding region of Korean quails OCA2 gene encoded protein of 851amino acids.The protein of quail OCA2 had 12 cross structure domains.The molecular weight of Korean quails OCA2 protein was 95.30 KDa and theoretical pI was 6.23,respecially.Moreover,the total number of negatively charged residues was79 and the total number of positively charged residues was 71.The instability index(II)was computed to be 39.16 and Grand average of hydropathicity(GRAVY)was 0.335,so OCA2 protein was identified as stable hydrophobic protein.Furthermore,the amino acid sequence of OCA2 protein has highly homology(more than 90%)with other poultry,while low homology with mammal,this indicate that OCA2 gene is relatively conservative in the species evolution process.The coding region of Korean quails MITF gene encoded a protein of 519 amino acids.Its molecular weight was 57.99 KDa and theoretical pI was 5.84.Moreover,the total number of negatively charged residues was 65 and the total number of positively charged residues was 55.The instability index(II)was computed to be 66.55 and Grand average of hydropathicity(GRAVY)was-0.703,so MITF protein was identified as stable hydrophobic protein.Furthermore,the amino acid sequence of MITF protein has highly homology(more than 90%)with other poultry,while low homology with mammal,this indicate that MITF gene is relatively conservative in the species evolution process.(2)The real-time quantitative PCR results showed that the expression levels of OCA2 mRNA was lower at 6-8 d than that at 10-14 d,then the expression of OCA2gene mRNA reached a peak at 10 d.The expressions in maroon Korean quail and in white Korean quail at 6-8 d increased without significant different.The expression levels of OCA2 mRNA in maroon Korean quail embryos were significantly higher(P<0.05)than in the white Korean quail embryos.The expression levels of MITF mRNA was lower at 6-8 d than that at 10-14 d,then the expression of MITF gene mRNA reached a peak at 10 d.The expressions in maroon Korean quail and in white Korean quail at 14 d increased without significantly different.The expression levels of MITF mRNA in maroon Korean quail embryos were significantly higher(P<0.05)than in the white Korean quail embryos at 6-12 d.(3)The SNPs of the OCA2 gene were searched by cDNA sequencing and ASPCR-SSCP in maroon Korean quail and white Korean quail.A total of 3 SNPs(c.93A>G in exon 2,c.999A>G in exon 9,c.2569C>T in 3?UTR)were detected in OCA2 gene of Korean quail.Genotyping of 300 Korean quails was tested by PCR-SSCP and three kinds of genotypes(AA,AB and BB)were found in this population.The three SNPs had moderate polymorphisms(0.25<PIC<0.5)by population genetic analysis and stayed in Hardy-Weinberg equilibrium(P>0.05)by?~2 tests.The distributions of three genotypes(AA?AB and BB)at c.2569C>T in maroon Korean quail were significantly different with that in white Korean quail,which indicated that this SNP had strong association with plumage color in Korean quail(P<0.01).The SNPs of the MITF gene were searched by cDNA sequencing and ASPCR-SSCP in maroon Korean quail and white Korean quail.A total of 3 SNPs(c.589A>G in exon 4,c.761A>G in exon 5,c.2275C>T in 3?UTR)were detected in MITF gene of Korean quail.The first SNP was a base substitution(A/G)at 589 bp of coding region,which changed a lysine to a glutamic in protein sequence.Genotyping of 300 Korean quails was tested by PCR-SSCP and three kinds of genotypes(AA,AB and BB)were found in this population.The three SNPs had moderate polymorphisms(0.25<PIC<0.5)by population genetic analysis and stayed in Hardy-Weinberg equilibrium(P>0.05)by?~2 tests.The distributions of three genotypes(AA?AB and BB)at c.2275C>T and c.589A>G in maroon Korean quail were significantly different with that in white Korean quail,which indicated that this SNP had strong association with plumage color in Korean quail(P<0.01).In conclusion,OCA2 and MITF genes sequences are highly conserved in either chicken or Korean quails.The low expression level of OCA2 and MITF mRNA in wing tissue may be related to the albinistic feather in white Korean quail.The mutations of OCA2 and MITF genes had strong relationships with feather color phenotype in Korean quail,and this indicated that OCA2 and MITF genes had important effects on feather color traits in Korean quail.