泥蚶分子系统分化及缢蛏遗传多样性的研究

【作者】 李成华；

【导师】 宋林生； 李太武；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2004， 硕士

【摘要】 泥蚶（Tegillarca granosa Linnaeus）是我国重要的养殖贝类，经济意义十分重大。当前对其研究工作主要集中于生物学特性、增养殖、苗种生产、核型、多倍体育种和组织学等几个方面，对其 DNA 水平上的种质资源分化现状的调查至今为止还未有深入的报道；而其他海洋生物的 DNA 分子水平的研究结果证实了开展物种种质资源调查对物种复壮工作提供有益指导和帮助作用。考虑到我国在泥蚶育苗和养殖的生产实践过程中存在的一系列潜在的问题和其在我国四大海域的广泛分布的特点，以及不同海域环境条件的较大差异等原因都有可能会在一定程度上加速其种质资源的分化和亚群的形成的事实，促使我们开展本次研究工作。而且，在本次研究工作对不同地理群体的泥蚶遗传分化状况了解和掌握的基础上，分析上述分化可能的产生机制，会极大的促进泥蚶管理举措的完善及优良品系的筛选和原种库建立等工作，最终进一步推进泥蚶养殖业的持续、高效和健康发展。 本研究采用采用随机引物扩增多态性 DNA 技术（RAPD）对分布在我国沿 海的山东荣成（R）、浙江奉化（F）、浙江温岭（W）、福建福鼎（D）、广东湛江（Z）、广东汕头（S）和韩国釜山（K）共 7 个地理群体的泥蚶遗传分化进行了分析（其中 K、R、F、W 和 D 群体合称北方类群，Z 和 S 群体合称南方类群）；同时为进一步证实 RAPD 分析结果，作者选取浙江温岭（W）、韩国釜山（K）、广东湛江（Z）和广东汕头（S）四个群体，并以与该种同属的结蚶（Tegillarcanodifera Martens）作为外群分析了进化速率快、适于群体间进化分析的核糖体转录间隔区序列 2 序列（简称 ITS2），进一步确立了当前我国泥蚶的分化程度。 RAPD 分析中，选取的 20 个随机引物，除 S-80 在任何群体中均无扩增外，其他引物在上述所有群体均获得清晰、可辨、适宜于群体分析的 RAPD 标记。20 个随机引物中共检测到 154 个扩增片段，长度在 300bp-2300bp 之间，每个个体扩增条带在 1-11 条不等。利用 Popgen3.2 计算各群体各位点的基因频率和每个群体内 20 个个体间的遗传距离，PHILIP3.5 统计软件分析群体间的各遗传参数，从而确定群体内和群体间的遗传分化状况。结果表明：（1）7 群体泥蚶无论是在多态位点比例还是在平均遗传杂合度上都处于较高水平，说明泥蚶当前种质资源状况良好，遗传多样性水平较高，泥蚶养殖有很好的发展前景； I<WP=7>（2）代表北方类群的 5 泥蚶群体间分化较小，群体间的遗传距离在0.0250-0.0901 之间不等，甚至小于群体内个体间的遗传距离；近交系数（Fst）在 0.05 以下。而南北两大类群泥蚶间却发生了重要的遗传分化。广东两群体与北方类群间的遗传距离在 0.3261-0.4511 之间不等，而两者内的两群体间的遗传距离却仅为 0.0612 和 0.0692；Fst 和有效繁殖亲本数（Nem）两遗传参数的统计结果同样表现出两大类群间的分化远大于类群内。（3）与韩国野生群体相比，其他群体泥蚶在多态位点比例和遗传平均杂合度上都有不同程度的降低，表明人为等因素已开始影响到泥蚶的种质资源状况。在对同为二龄的我国温岭养殖和韩国野生泥蚶群体数量性状测定上，前者壳长和壳宽分别为 24.45±1.84mm和 19.50±1.74mm，而后者则依次为 30.80±2.80mm 与 23.30±2.06mm，且两两差异明显（p〈0.01）。说明养殖环境已开始威胁到泥蚶的资源状况，加强其资源保护势在必行。（4）聚类分析是群体亲缘关系远近的反映，首先聚在一起的群体遗传分化最小，亲缘关系最近。对南北两大类群泥蚶不论用 NJ 还是UPGMA 聚类，总是南方类群的两群体先聚在一起，然后再与北方类群聚在一 起，表明两者间较显著的遗传分化。 ITS2 测序结果证实了我们群体分析时结论——南北两大类群间分化明显，而两大类群内分化较小。在韩国群体和浙江温岭群体比较仅发现韩国 351bp处发生了缺失，354bp 处发生了一个碱基的颠换（G → T ），两群体序列相似性高达 99.60%；汕头群体与湛江群体也仅为 347 处一个碱基的缺失和 412 位置一个碱基的颠换（G →T）；而两大类群间的比较则发生了多处的缺失、替换和颠换，两两群体间的序列相似性在 92.47%-93.08%之间，小于结蚶与两者间的相应值 93.27%-96.82%。聚类分析结果也证实了两大类群间的分化程度大于它们与结蚶间的分化，结蚶或是先与南方类群聚类，然后与北方类群聚类（UPGMA），或者首先与北方类群聚在一起（NJ），提示我们泥蚶两大类群可能已分化到种以上程度。 产生上述分化的可能机制，笔者认为主要有三个方面：一是泥蚶养殖管理规范的缺乏，造成养殖者为追求经济利益而盲目引进苗种和亲本。同时由于操作的偏好性和一些特定的原因，使得引种范围极少发生在两大类群间，一般多存在于两大类群内部。这样做的长期结果会使得这些地区间有广泛的基因交 II<WP=8>流，这些基因流可以在这些地区得到扩散，而不能在南北两大类群间发生，最终产生上述结果。另一方面为生殖隔离的原因，北方泥更多还原

【Abstract】 Li chenghua (marine biology) Directed by professor Song linsheng, Li taiwu and Su xiurong Tegillarca granosa (Linnaeus) belongs to Bivalvia class, Pteriomorphiasub-class, Arcoida order, Arcidae family, Tegillarca genius according to up-to-datataxonomy system. As an important cash shellfish of seawater, it distributes along thewest of Pacific Ocean, the Indian Ocean and the Atlantic Ocean. Concerning ourcountry, it plays an important role in the marine aquaculture industry in theprovinces of Shandong, Zhejiang, Fujian and Guangdong. Nowadays, although much has been done on the research of the species biology,farming technology, breeding methods, karyotype and histology, there are muchmore left and expected to be studied, especially at DNA level which can throw lightto the species recovery. Regarding of T. granosa, the reasons for studying its geneticdifferentiation lie in two aspects. One is the potential problems occur in its breedingand aquaculture; the other is the diversity of its surroundings, spanning from Bohaito the south of China sea (abbreviation for SCS). In the first part of this paper, wefirstly adopted RAPD method to analysis the differential degree of the differentpopulations in China and Korea, then the internal transcribed spacer 2 (abbreviationfor ITS2) was amplified, sequenced to show more proof among the most differentialpopulations. Seven populations were analyzed in our RAPD experiment, there are as follows:Rongcheng in Shandong province(R), Fenghua(F) and Wenling(W) in Zhejiangprovince, Fuding in Fujian province(D), Shantou(S) and Zhanjiang(Z) in Guangdongprovince and Fushan in Korea(K), all samples were collected from China except toK. We preferred to nominated K, R, F, W and D as the north stocks (abbreviation forNP), the others as the south ones (abbreviation for SP). The samples employed toITS2 analysis randomly selected two populations from the NP and SP withTegillarca nodifera (Martens) as an outgroup. Under predetermined optical reaction conditions, all primers showed usinformative message except S-80 without any results in seven populations. 154 IV<WP=10>RAPD sites were detected ranging from 300bp to 2300bp in size. The number ofamplified bands ranged from 1 to 11 in every individual. The experimental data wereanalyzed with the Popgen3.2 and PHILIP3.5 softwares. The results showed that: (1)the high level of mean heterozygosities and polymorphic loci suggested that theresource of T. granosa was in good condition with high genetic variation, whichgave us more confidence to develop its aquaculture; (2) the degree of the geneticdifferentiation was much lower in the NP or SP comparing to that existing betweenthe NP and SP. The genetic distances were ranging from 0.0250 to 0.0901 in the NPand SP, while the corresponding valves were ranging from 0.3261 to 0.4511.betweenthe NP and SP. Other genetic parameters like the inbreeding coefficient (abbreviationfor Fst) also verified the above judgment; (3) the heterozygosities and proportion ofpolymorphic loci of the hatchery populations were lower than that of the wildpopulation (K). What’s more, the quantitative character variability between W and Kwas significant (p<0.01), the shell length and height of the former were24.45±1.84mm and 19.50±1.74mm in turn, the latter were 30.80±2.80mm and23.30±2.06mm respectively. All these deviations could be attributed to artificial orother factors. (4) the nearest phylogenetic relationship occurs between thepopulations with the nearest blood relationship. The NP or SP was firstly cluster nomatter what methods we adopted (NJ and UPGMA), indicating their close sibship. The ITS2 sequences showed the similar trends inferred from RAPD analysis.There are more or less variation existing between each other in our experiment at thedefault parameters with ClustalW software aid, ranging from one base deletion and asingle transversion (G →更多还原