Klinbunga S, Pripue P, Khamnamtong N, Puanglarp N, Tassanakajon A, Jarayabhand P, Hirono I, Aoki T, Menasveta P
Marine Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 113 Paholyothin Rd, Klong 1, Klong Luang, Patthumthani 12120, Thailand.
Mar Biotechnol (NY). 2003 Sep-Oct;5(5):505-17. doi: 10.1007/s10126-002-0108-8. Epub 2003 Aug 14.
Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and FST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P<0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P<0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.
采用聚合酶链反应（PCR）扩增18S和16S核糖体DNA，并结合随机扩增多态性DNA（RAPD）和限制性片段长度多态性（RFLP）技术，分析了泰国的三种鲍鱼，即皱纹盘鲍（Haliotis asinina）、羊鲍（H. ovina）和多变鲍（H. varia）的遗传多样性。在每种鲍鱼中均发现了物种特异性的RAPD标记。用Alu I、Taq I和Hae III对18S（核）核糖体DNA进行限制性分析，以及用Bam HI、Eco RI、Hae III和Alu I对16S（线粒体）核糖体DNA进行限制性分析，分别得到了12种和13种消化模式。共发现了49种复合单倍型。通过对复合单倍型对之间的差异构建的算术平均非加权配对组方法得到的树形图，揭示了这些鲍鱼的生殖隔离基因库，并表明皱纹盘鲍和羊鲍在遗传上比多变鲍更接近。由于样本量较小，在发现多变鲍时，地理异质性分析和FST估计表明，来自安达曼海（西部）的羊鲍和泰国湾（东部）的羊鲍之间存在明显的遗传分化（P<0.0001），而菲律宾的羊鲍与其余皱纹盘鲍样本之间存在部分分化（P<0.0021）。对本研究中发现的代表复合单倍型的个体的扩增16S rDNA进行克隆和测序。根据16S rDNA序列差异构建的邻接树能够根据鲍鱼的物种起源准确地对这些序列进行分类。基于16S rDNA多态性的物种特异性PCR在皱纹盘鲍和多变鲍中成功开发，但在羊鲍中未成功开发。
