作 者 ：孟学平*,高如承,申欣,王帅,程汉良,董志国,阎斌伦
期 刊 ：生态学报 2010年 30卷 20期 页码：5555~5561
Keywords:Coelomactra antiquata, ITS1, sequence analysis,
摘 要 ：利用核糖体rRNA基因第一内转录间隔区（ITS1）序列，对我国山东日照（RZ）、江苏连云港（LYG）和启东（QD）、福建长乐（CL）、广西北海（BH）沿海西施舌5个野生群体的遗传结构进行了分析。经PCR扩增、克隆、非克隆测序，获得长度为816—843bp的ITS1核苷酸序列,其平均G+C含量（60.8%）显著高于A+T含量。在西施舌5个群体93个个体118个序列中共检测到153个变异位点，多态位点比例为18.3%，其中，简约信息位点为63个，共有63种基因型。群体间遗传距离在0.74%—338%之间，群体内遗传距离为0.72%—1.06% 之间。CL群体与其他4个群体间的遗传距离明显高（3.17%—3.38%）。AMOVA分析结果显示长乐群体有明显的遗传分化（FST = 0.670—0.702，P﹤0.001）。以中国蛤蜊为外群构建的NJ树和ML树显示CL群体聚为一支，非长乐群体互相重叠聚为另一支，研究结果显示，长乐群体已经形成了具有明显遗传结构的地理种群。
Abstract:Coelomactra antiquata Spengler, 1802, called Xishishe in Chinese, is a bivalve, which belongs to the phylum Mollusca, the class Bivalvia, the order Veneroida, the family Mactridae and the genus Coelomactra. The species is distributed widely in the western Pacific Ocean, along the coast of Indochina, Japan, Korea and China. C. antiquata has a wide but erratic distribution in China from Taiwan, Guangdong and Hainan Provinces in the south northward to the Shandong Peninsula. C. antiquata, a commercially important clam in aquaculture, is one of the most critically endangered species in China. It has experienced severe population declines due to habitat destruction and overfishing during the last two decades. The depletion of natural resources encouraged development of culture techniques for this species, in order to satisfy the demand of the market and maintain the wild population. However, cultivation may result in reduction of genetic diversity unless we know the genetic background of the species. Therefore it is necessary to investigate the genetic structure of this species. Molecular markers have been very useful for analysis of genetic structure. Among many types of molecular markers, we selected the ribosomal rRNA gene first internal transcribed spacer (ITS1). It is widely used in genetic variation and phylogenetic studies of animals and plants because of its high rate of molecular evolution. In recent years, the usage of ITS1 to assess genetic variations in bivalves has increased. To study the genetic variability, five wild stocks of C. antiquata were collected from Shandong Rizhao (RZ), Jiangsu Lianyungang (LYG) and Qidong (QD), Fujian Changle (CL), Guangxi Beihai (BH) in China, and the genetic variation was analyzed based on ITS1 nucleotide sequences of rDNA. PCR amplification with the primers ITS1-F and ITS1-R was performed and the PCR products were cloned and sequenced. DNA sequences were analyzed using DNAStar, and were aligned in ClustalX. The phylogenetic trees were constructed using Neighbor-Joining method (on MEGA 4.1) and Maximum likelihood method (online: http://www.atgc-montpellier.fr/phyml/). Analysis of molecular variance (AMOVA) was performed by Arlequin3.11. The results showed that the ITS1 region ranged in size from 816 to 843 bp. 118 sequences were obtained from 93 samples of five stocks of C. antiquata, and 153 polymorphic sites were detected. The proportion of polymorphic sites was 18.3%, in which parsimony informative sites were up to 63. Intrastock genetic distance was 0.72%-1.06% whereas interstock genetic distance was 074%-3.38%. Significantly, higher genetic variation was observed between the CL stock and the other four stocks (309%-3.34%). Further, AMOVA analysis revealed a substantial genetic differentiation in the CL stock (FST =0.670-0.702, P<0.001). Phylogenetic relationships of these individuals were analyzed using NJ and ML methods with Mactra chinensis Philippi as an out-group. Phylogenetic analysis showed that CL species formed a monophyletic clade whereas the other non-CL species formed a separate clade. Combined with previous findings, this evidence strongly supports CL C. antiquata as a distinct geographical population. In this study, the analysis of ITS1 nucleotide sequences of C. antiquata geographical stocks revealed the genetic structure and phylogenetic relationship of five wild stocks of C. antiquata in typical coastal regions, which provides the molecular basis for genetic breeding and resource protection of the species.
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