Association analysis of melanophilin (MLPH) gene expression and polymorphism with plumage color in quail.

We explore the relationship between the melanophilin (MLPH) gene and quail plumage color and provide a reference for subsequent quail plumage color breeding. In this experiment, real-time quantitative PCR (RT-qPCR) technology was used to analyze the relative mRNA expression levels of Korean quail (maroon) and Beijing white quail embryos at different developmental stages. Two single-nucleotide polymorphisms (SNPs) in the MLPH gene were screened based on the RNA-sequencing (RNA-Seq) data of skin tissues of Korean quail and Beijing white quail during the embryonic stage. Kompetitive allele-specific PCR (KASP) technology was used for genotyping in the resource population, and correlation analysis was carried out with the plumage color traits of quail. Finally, bioinformatics was used to predict the effects of these two SNPs on the structure and function of the encoded protein. The results showed that the expression level of the MLPH gene during embryonic development of Beijing white quail was significantly higher than that of Korean quail ( $P<0.01$ ). The frequency distribution of the three genotypes (CC, CA and AA) of the Beijing white quail at the c.1807C  $>$  A mutation site was significantly different from that of the Korean quail ( $P<0.01$ ). The frequency distribution of the three genotypes (GG, GA and AA) of the Beijing white quail at the c.2129G  $>$  A mutation site was significantly different from that of the Korean quail ( $P<0.01$ ). And there was a significant correlation between the c.1807C  $>$  A mutation site and the white plumage phenotype. Bioinformatics showed that SNP1 (c.1807C  $>$  A) was a neutral mutation and that SNP2 (c.2129G  $>$  A) was a deleterious mutation. The prediction of protein conservation showed that the mutation sites of coding proteins R603S and G710D caused by SNP1 (c.1807C  $>$  A) and SNP2 (c.2129G  $>$  A) were highly conserved.