基于ITS2和16S rRNA的西施舌群体遗传差异分析

Stock difference of Coelomactra antiquata based on nuclear (ITS2) and mitochondrial (16S rRNA) DNA sequence and secondary structure

目前,我国西施舌群体分子遗传差异研究结果存在争议。分析我国南北沿海(9个群体)、与广西北海毗邻的越南(1个群体)西施舌核DNA的内转录间隔区2(ITS2)和线粒体DNA的16S rRNA基因(16S)片段核苷酸序列及其二级结构,为解决争议问题提供分子生物学资料。扩增获得西施舌ITS2片段和16S序列,其长度分别为389—402 bp和306 bp,加之下载序列共147条;序列分析显示,74个ITS2序列共有17种基因型,73个16S序列有15种单倍型,其中,长乐(CL)群体独享9种ITS2基因型和5种16S单倍型,非长乐群体(nCL)多数为群体间交叉共享1种或几种基因(单倍)型;基因(单倍)型核苷酸变异位点占5.7%(ITS2)和11.8%(16S);基于ITS2和16S的CL群体和nCL群体间的遗传距离与群体内遗传距离之比分别为2.42和11.08,nCL群体间的平均遗传距离均为0.007;二级结构显示CL群体ITS2的9种基因型和16S的5种单倍型均区别于nCL群体,nCL的ITS2和16S二级结构分别相似;ITS2和16S基因的系统发育分析显示,CL西施舌形成支持率很高(98,96)的单系支,而nCL群体则交叉聚为另一支(98,96)。研究结果揭示,福建西施舌是腔蛤蜊属(Coelomactra)的一个新种。

Coelomactra antiquata （Bivalvia： Mactridae） is distributed widely in the western Pacific Ocean, along the coast of Indo-China Peninsula, Japan, Korea and China. C. antiquata has a wide but erratic distribution, and is one of the most critically endangered species in China. Background genetic data are critical to successful cultivation of C. antiquata, Mitochondrial DNA （mtDNA） is widely used for genetic difference analysis among closely related species, since it accumulates mutations more rapidly than most nuclear regions. The internal transcribed spacer 2 （ITS2） of the nuclear ribosomal repeat unit is one of the most commonly applied phylogenetic markers. It is a fast evolving locus, which makes it appropriate for studies at low taxonomic levels. The present molecular study was conducted on partial sequences of the mitochondrial 16S rRNA genes （16S） and ITS2 sequence from nuclear DNA to assess genetic variations in C. antiquata. Nine wild stocks of C. antiquata were collected from Daliang （Liaonin Prov. DL）, Jimo （Shandong Prov. JM）, Jiaonan （Shandong Prov. JN, JD） and Rizhao （Shandong Prov. RZ）, Lianyungang （Jiangsu Prov. LYG,X3） and Qidong （Jiangsu Prov. QD XM）, Changle （Fujian Prov. CL, X1）, Beihai （Guangxi Prov. BH） and Pingtan （Fujian Prov. X2） in China, and the coast next to China in Vietnam （YN）. We analyzed 147 sequences in total： 74 from ITS2 （389-402bp） and 73 from 16S （306bp）, Seventeen genotypes were detected from ITS2 sequences and 15 haplotypes were detected from 16S sequences. The Changle group （CL, YN,X2） has nine exclusive ITS2 genotypes and five 16S haplotypes. The majority of non-Changle （nCL） groups （DL, JM, JN, JD, RZ, LYG, QD, XM, BH） share one or several genotypes （or haplotypes） among groups. The ITS2 alignment for 17 genotypes contained 401 nucleotide positions （including indels） with 23 variable sites （5.7%）, of which 16 （4.0%） were parsimony informative. The 16S alignment for 15 haplotypes contained 306 bp with 35 （11.4%） variable sites, of which 24 （7.8%） were parsimony informative. The average intergroup genetic distance of nCL group is 0.007. The secondary structures of 9 genotypes and 5 haplotypes of CL group were different from those of nCL group, whereas the secondary structures of ITS2 and 16S are quite similar in nCL group. The phylogenetic analysis of ITS2 and 16S gene showed that the group CL formed a monophyletic clade with a high support （BP=98, 96）, whereas the group nCL formed a second separate clade also with a high support （BP=98, 96）. The results of this study suggests that the C. antiquata from Fujian province may be a new species of genus Coelomactra.