长江草鱼群体MHC Class ⅡB的多态性和进化分析

克隆及测序草鱼(Ctenopharyngodon idella)长江3个群体的主要组织相容性复合体(MHC)Class II B基因编码β1和β2区的第2和第3个外显子及两个外显子之间的内含子,分析了草鱼MHC的进化模式和种群遗传结构。结果显示:实验共定义了34个等位基因,每条序列包括长为130~136 bp的第2个外显子,长为218 bp的第3个外显子以及长81~371 bp的内含子。序列分析揭示,第2个外显子有106个核苷酸变异位点(78%)和40个氨基酸变异位点(88%),而第3个外显子有100个核苷酸变异位点(45%)和41个氨基酸变异位点(56%),β1变异要大于β2区。用β1和β2区序列分别构建的邻接(NJ)系统树均显示5个具有高支持率的谱系,结合序列变异特点和内含子长度,推测草鱼至少存在5个MHC Class II座位。分别计算β1的肽结合位点(PBR)、非肽结合位点及β2的非同义替换率(dN)和同义替换率(dS),PBR的dN/dS为2.03(P<0.05),非肽结合位点和β2则小于1,表明草鱼MHC受到歧化选择作用。根据等位基因在群体中的分布频率作分子方差分析(AMOVA),得出FST为0.37%,提示长江草鱼MHC没有遗传分化。

Polymorphism and evolution of MHC Class II B grass carp (Ctenopharyngodon idella) populations of Yangtze River

To investigate the evolutionary patterns and population genetic variation of grass carp(Ctenopharyngodon idella) major histocompatibility complex(MHC),the DNA fragments of exon 2 and exon 3,encoding β1 domain and β2 domain of MHC Class II β,and the intron between them were amplified from three populations of carp collected from Yangtze River,and then cloned and sequenced.A total of 34 alleles was defined,and each included an exon 2 of 130~136 bp,an exon3 of 218 bp and an intron of 81~371 bp in length.Analysis of alignments of nucleotide acid and putative amino acid sequences revealed that there were 106 variable nucleotide acid(78%) and 40 amino acid(88%) sites in exon 2,and 100 variable nucleotide acid(45%) and 41 amino acid(56%) sites in exon 3,suggesting more variability in β1 than in β2.Two phylogenetic trees constructed separately from sequences of β1 and β2 domains showed 5 distinct lineages with high bootstrap values.Considering the different introns of alleles in lineages,it indicated that it should harbor at least 5 loci in Grass carp.The numbers of synonymous substitutions per synonymous site(dS) and nonsynonymous substitutions per nonsynonymous site(dN) were calculated for peptide binding residues(PBR) and non-PBR of the β1 domain and β2 domain.The results showed the ratio of dN to dS in PBR was 2.03(P<0.05),whereas ratios in non-PBR and β2 domain were lower than 1,implying divergent selection impacted at MHC of carp.Analysis of molecular variance(AMOVA) based on allele frequency indicated FST=0.37%,which revealed little genetic differentiation among populations of carp in Yangtze River.