Molecular cloning, sequence characteristics, and polymorphism analyses of the tyrosinase-related protein 2 / DOPAchrome tautomerase gene of black-boned sheep (Ovis aries)

Abstract

Tyrosinase-related protein 2 (TYRP2) plays a pivotal role in the biosynthesis of eumelanin. Black-boned sheep have excessive melanin and eumelanin, resulting in dark (black) muscles and organs. This study was designed to investigate the effects of variants of the TYRP2 gene on black traits and coat colour of black-boned sheep. Melanin traits were measured in three populations of sheep (Nanping black-boned, Nanping normal, and Romney Marsh) and compared in this study. From the TYRP2 cDNA, all 8 exons and their flanking regions were amplified and characterized. Fifteen single nucleotide polymorphisms (SNPs) were identified in the exons and their flanking regions. Five exonic polymorphic sites, including two synonymous (c.93T>G and c.1140C>T) and three non-synonymous mutations (c.163C>T (p.R55W), c.605G>A (p.R202H), and c.1141A>G (p.T381A)), were retrieved. PCR-RFLP analysis of c.605G>A showed that the frequencies of allele G in the Nanping black-boned, Nanping normal, and Romney Marsh sheep were 0.632, 0.603, and 0.886, respectively. Sheep with the GG genotype had significantly (P < 0.05) lower tyrosinase activity, alkali-soluble melanin content, and ratio of eumelanin : total melanin than sheep with GA and AA genotypes when measured across all investigated samples but not when samples within each population of sheep were compared. However, there was no association of TYRP2 genotype at a single SNP position with coat colour across populations. Nonetheless, the two breeds with higher overall tyrosinase activity did produce darker and more varied coat colours than the breed with lower tyrosinase activity.