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

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. Key words: black-boned sheep, TYRP2 gene, black traits, association analyses. La proteine 2 apparentee a la tyrosinase (TYRP2) joue un role important dans la biosynthese de l'eumelanine. Les moutons a chair noire ont des quantites excessives de melanine et d'eumelanine ce qui leur confere des muscles et des organes fonces (noirs). Cette etude a ete concue pour investiguer les effets de differents variants du gene TYRP2 sur les caracteres noirs et la couleur du pelage chez les moutons a chair noire. Dans ce travail, les caracteres affectes par la melanine ont ete mesures et compares chez trats populations du mouton (Nanping black-boned, Nanping normal et Romney Marsh). L'ADNc, les huit exons et les regions bordantes du gene TYRP2 ont ete amplifies et caracterises. Quinze SNP ont ete identifies au sein des exons et des regions bordantes. Cinq sites polymorphes dans les exons, deux mutations synonymes (c.93T>G et c.1140C>T) et trais non-synonymes (c.163C>T (p.R55W), c.605G>A (p.R202H) et c.1141A>G (p.T381A)), ont ete releves. Par analyse PCR-RFLP du polymorphisme c.605G>A, il a ete montre que les frequences de l'allele G au sein des populations Nanping black-boned, Nanping normal et Romney Marsh etaient respectivement 0,632, 0,603 et 0,886. Les moutons de genotype GG affichaient une activite tyrosinase, une teneur en melanine soluble en conditions alcalines et un ratio eumelanine : melanine totale significativement (P < 0,05) plus faibles que chez les moutons de genotype GA ou AA lorsque les mesures compaees etaient prises sur l'ensemble des animaux mais pas au sein de chaque population. Cependant, aucune association n'a ete observee entre le genotype de TYRP2 a une seine position SNP et la couleur du pelage chez ces populations. Neanmoins, les deux races avec une activite tyrosinase globalement plus elevee presentaient des pelages plus fonces et plus varies que la race a faible activite tyrosinase. Mots-cles : moutons a chair noire, gene TYRP2, caracteres fonces, analyses d'association. [Traduit par la Redaction]
Introduction Melanin is found naturally in a wide range of species from microbes to humans and is present as a mixture of the black-brown pigment eumelanin and the yellow-red pigment [...]