6种帘蛤科贝类18S rRNA基因全序列比较分析

Sequence analysis of 18S rRNA gene of six Veneridae clams (Mollusca:Bivalvia)

对文蛤（Meretrix meretrix）、青蛤（Cyclina sinensis）、江户布目蛤（Protothaca jedoensis）、薄片镜蛤（Dosinia corrugata）、紫石房蛤（Saxidomus purpuraus）和菲律宾蛤仔（Ruditapes philippinarum）6种帘蛤科（Veneridae）贝类的18S rRNA基因序列进行了PCR扩增并测序,以期获得这一序列的基本特征,评估其种间变异程度,探讨这一序列在种类鉴定和分子系统发育等研究中的应用价值。测序结果表明,文蛤、青蛤、江户布目蛤、薄片镜蛤、紫石房蛤和菲律宾蛤仔18S rRNA基因序列全长分别为1900bp、1838bp、1831bp、1831bp、1829bp和1833bp。序列中A、T、C和G碱基的平均含量分别为24.0%、24.0%、24.2%和27.8%。用MEGA软件对6种帘蛤18S rRNA基因全序列进行了分析,对位排列后的总长度1 906 bp,其中变异位点210个,简约信息位点28个,si/sv=1.4（46/32）。从GenBank下载了7种帘蛤科贝类18S rDNA全序列,与本研究实测的6种帘蛤一起用MegAlign软件对其18S rDNA序列进行了比对,物种间序列相似百分比为88.7%-99.7%。文蛤与其他12物种间序列差异较大,序列差异百分比均超过了10%,其他各物种间序列差异百分比不超过3%。以异韧带亚纲（Anomalodesmata）笋螂目（Pholadomyoida）的Lyonsia floridana和Cardiomya costellata为外群,采用相邻连接法（NJ）和最大简约法（MP）构建了帘蛤科贝类的系统发育树,其拓扑结构显示雪蛤亚科（Chioninae）、帘蛤亚科（Venerinae）和镜蛤亚科（Dosiniinae）的种类首先聚在一起,形成一个聚类簇;缀锦蛤亚科（Tapetinae）、卵蛤亚科（Pitarinae）、仙女蛤亚科（Callistinae）、青蛤亚科（Cyclininae）和文蛤亚科（Meretricinae）的种类先后分别单独聚成一枝;最后所有帘蛤科物种聚为一枝,与外群相区别,说明18S rDNA序列适合作为帘蛤科系统发育研究的分子标记。

A nucleotide sequence analysis of 18S rRNA gene was conducted among six species in Veneridae, Meretrix meretrix L. 1758, Cyclina sinensis G. 1791, Protothacajedoensis L. 1874, Dosinia corrugata R. 1850, Ruditapes philippinarum A. 1850 and Ruditapes philippinarum A., 1850 to determine the utility of this sequence in identifying species and phylogenetic structure. The result showed that 18S rDNA of six species were 1 900 bp, 1 838 bp, 1 831 bp, 1 831 bp, 1 829 bp and 1 833 bp in length, respectively. The average contents of A, T, C and G in these 18S rRNA gene were 24.0%, 24.0%, 24.2% and 27.8%. Analysis of sequence variation across six species revealed 210 variable sites, among which 28 were parsim-informative sites; the transition to transversion substitution ratio （si/sv） was 1.4 （46/32）. The 18S rDNA sequences of 6 Veneridae species in this study together with 7 Veneridae species downloaded from GenBank were aligned using program MegAlign of DNAStar package by Clustal W method. The percentage of sequence identity across 13 species were from 88.7% （M. meretrix and Periglypta listeri） to 99.7% （D. corrugata and Venus verrucosa）. The percentage of sequence divergence between M. meretrix and other 12 species was more than 10%, meanwhile this value was not more than 3% between other 12 species. The phylogenetic tree of Veneridae clams was reconstructed using 18S rRNA gene complete sequences as markers, using MP and NJ method with Lyonsia floridana and Cardiomya costellata as outgroups. Tree topologies indicated that the species belonging to subfamilies of Chioninae, Venerinae and Dosiniinae formed a clade firstly, then the species belonging to Tapetinae, Pitarinae, Callistinae, Cyclininae and Meretricinae formed a monophyletic group respectively, all Veneridae species formed a clade finally. The results reveal that the 18S rRNA gene is an appropriate marker for studies of phylogeny of the Veneridae clams at subfamily level.