Cloning, Expression For Pigmentation Genes-MC1R And Selection Pressure Analysis For Pigmentation Genes-TYR In Oujiang Color Common Carp

Pigmentation are special phenotypic traits in fish．Pigmentation of fish provideimportant values of biodiversity and genetics theory research. Pigmentation variation offish has been used as a very important research topics in genetics，evolutionary biologyand developmental biology. Oujiang color common carp (Cyprinus carpio var color)display a rich body color types, such as”whole red”,“whole red with big blackpatch”,”whole red with scattered black spots”,“whole white” and “whole white withbig black patch”. It is considered as a good materials or models in studyingpigmentation genetics of fish. In the paper, based on previous studies in our laboratory,gene cloning, expression analysis as well as selective pressure analysis were conductedfor melanocortin1receptor (MC1R) and tyrosinase gene (TYR) which are the two keygenes in melanin synthesis pathway. The detailed contents are presented as follows:1. Cloning and expression analysis of color-related gene (MC1R) in Oujiangcolor common carpMelanocortin1receptor (MC1R), one of the key genes in animal melanin synthesispathway, play an important role in determining animal body color and coat color. In thispaper, MC1R gene was cloned and sequenced, and its expression profile in the fourbody color patterns (“whole red”,“whole red with big black patch”,“whole white” and“whole white with big black patch”) and tissues were also analyzed in Oujiang colorcommon carp. The result showed that the full-length cDNA of MC1R was1914bp,containing a5’ un-translated region (UTR) of637bp, a3’-UTR of311bp and an openreading frame (ORF) of966bp. This gene has only one exon which encoding321amino acids and predicting7transmembrane domains. There were98%nucleotidesimilarity with Carassius auratus and93%similarity with Danio rerio. However, Onlyone nucleotide nonsense mutations (C/G) in the coding region was found among different color patterns, indicating the same amino acid sequence among them. Theresult of qRT-PCR indicated that the expression of MC1R in eyes was significantlyhigher (P<0.05) than that in skin, muscles, gills, kidney, bladder, heart, liversignificantly. It was surprising that there were no significant expression differenceamong the different color patterns (P＞0.05). This founding showed that MC1R geneshould not be directly determine factor in controlling color patterns of Oujiang colorcommon carp, it is necessary to conduct further studies in other color-related genes orfactors.2. Analysis of selection pressure of TYR gene in Oujiang color common carpTyrosinase (TYR) is a key enzyme or rate-limited enzyme in melanin synthesis. Inthis section, based on previous studies in our laboratory, a partial sequences of exon1inthe transcript TYR-1were used to conduct selective pressure analysis for the five colorpatterns of Oujiang color common carp, such as “whole red”,“whole red with big blackpatch”,“whole red with scattered black spots”,“whole white” and “whole white withbig black patch”. The result showed that a tolal of44nucleotide variation sites wasfound in the468bp of length sequence, which resulted in9amino acid mutation sites.These was a significant genetic differentiation (FST=0.0198-0.3717, P <0.01) betweenthe five color patterns. The Z-test of selective pressure showed that the ratios of dN/dSranged from0.070to0.103(all were less than1). The further analysis of selectivepressure using the Codeml model (M2a and M8) showed that there were significantselective pressures in some amino acid sites, including1site in the “whole red withscattered black spots”,2sites in the “whole red”,“whole white” and “whole white withbig black patch”,4sites in the “whole red with big black patch”, and there werepurifying selection in the remaining sites. This result showed that artificial selection ornatural selection pressure would have given small effect on sequence variation oftryosinase gene in oujiang color common carp, and there was a stably genetic pattern inpigmentation trait of this fish.