Abstract:

To investigate the allele polymorphism of MHC-DAA in Japanese flounder (Paralichthys olivaceus), specific primers were designed based on the published DAA-cDNA sequence of P. olivaceus to amplify the entire introns from flounder. Gene DAA consisted of four exons and three introns. The exon-intron structure was similar as that of Atlantic salmon. A polymorphic microsatellite was detected in intron 2. Subsequently, specific primer was designed based on the DAA genomic sequences. The genetic polymorphism within exon 2 of MHC-DAA was investigated by means of sequence analysis. Forty-five individuals were selected to amplify exon 2 and intron 2. One hundred and twenty-nine fragments revealed 30 novel alleles belonging to 18 allele major type. The frequency of alleles and the numbers of subtypes were not distributed equally. There were 55 variable sites in exon 2. The diversity of nucleotide within the sequence of exon 2 was 0.0887. The nucleotide sequence was corresponded with a putative peptide. Among the 83 amino acids in this peptide, 37.80% were variable, and 30 parsimony informative sites were observed. The rates of non-synonymous substitution (dN) were 3.30 and 2.43 times higher than the rate of synonymous substitution (dS) in the peptide-binding regions (PBR) and non-PBR, respectively. In this study, the high ratio of dN/dS value and high allelic diversity observed in flounder suggested a balancing selection in the exon2 of the MHC class IIA gene, which explained the high polymorphism of MHC-DAA gene in Paralichthys olivaceus.