The Japanese quail expresses polymorphism in plumage colors, including black, yellow, white, wild-type (maroon), and various intermediate colors through hybridization of quail with different plumage colors. The expression levels of MC1R and ASIP play important roles in the regulation of plumage colors in birds. In this study, the eukaryotic expression vector of pcDNA 3.1 + was used to analyze the effects of forced expression of MC1R and ASIP on the plumage colors of Japanese quail embryos. The constructed eukaryotic expression vectors of pcDNA 3.1 (+)-MC1R and pcDNA 3.1(+)-ASIP were transfected into wild-type Japanese quail embryos by Lipofectamine TM 2000 liposome at 6 days of incubation. After 3 days, the embryos were collected to analyze the plumage colors and the expression levels of MC1R, ASIP, and DCT genes in skin tissue. Forced expression of the MC1R gene by transfection of the pcDNA 3.1(+)-MC1R vector led to hyperpigmentation (similar to black plumage), whereas forced expression of the ASIP gene by transfection of the pcDNA 3.1(+)-ASIP vector led to hypopigmentation (similar to white plumage) in wild-type quail embryos. Two kinds of ASIP alternative splicing (ASIP1 and ASIP2) were found in Japanese quail, which did not have a significant effect on the plumage color or the main motifs of the ASIP protein. This study indicated that the black plumage color may be caused by increased production of MC1R and the white plumage color may be caused by increased production of ASIP in Japanese quail.

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MC1R plays a crucial role in controlling the type of melanin synthesized in the melanocytes, which greatly affects plumage color in birds. One g.16796362G/T SNP was found in the MC1R gene coding region, which caused a Met120Ile mutation in the amino acid sequence. The Met120Ile mutation was located in the third transmembrane domain of the MC1R protein and decreased protein stability. The g.16796362G/T locus achieved medium polymorphism and had significant association with feather melanin content in Chinese yellow quails. The contents of total melanin and pheomelanin with AA genotype were significant lower than those with AB or BB genotypes in skin tissues, while the expression levels of MC1R mRNA had no significant difference in feathers with different genotypes. This experiment indicated that the Met120Ile mutation could affect the function of the MC1R protein and change the biosynthesis of melanin in Chinese yellow quails.