10种海参16SrDNA序列多样性及其亲缘关系分析

16S rDNA sequence diversity and phylogenetic relationship of ten sea cucumbers

运用PCR扩增10种海参的16SrDNA部分序列，并测序。获得大小为566bp的序列，利用DNAsp4．0软件分析比较10种海参的碱基组成、各碱基变异位点数、插入或缺失位点数，并采用MEGA4．0软件分析彼此间的遗传距离。结果发现，10种海参富含AT碱基，A＋T的含量平均为57．0％，不同种海参问发生碱基转换、颠换及发生插入或缺失变异的位点数差异很大，10种海参彼此间的遗传距离在O．007～0．316之间。所得结果与GenBank中10种海参的相应序列进行比对分析，同时用MEGA4．0和PAUP’4．0软件构建进化树，分析其亲缘关系。结果表明，利用16SrDNA序列的差异可以将分属于海参科（Holothuriidae）与剌参科（Stichopodidae）的海参完全分开，瓜参科（Cucumariidae）的2个种与刺参科的亲缘关系较近。对不同海参种间亲缘关系的分析结果还表明，同属于刺参科的仿刺参（Apostichopusjaponicus）与加州拟刺参（Parastichopuscalifornicus）和具疣拟刺参（Parastichopusparvimensis）亲缘关系最近，同属于海参科的格皮氏海参（Pearsonothuriagraeffei）与白尼参属的蛇目白尼参（Bohadschiaargus）和图纹白尼参（B．marmorata）亲缘关系最近，而同样为海参科的墨西哥海参（Holothuriamexicana）则与红腹海参（Holothuriaedulis）关系最近。这些资料为海参的种质资源管理、新种引进和种质改良提供了基础的分子生物学依据。

Holothurian is one kind of invertebrates in the sea, which includes more than 1 400 species, distributing from shallow water to ocean benthal. One hundred and thirty four species of sea cucumbers from China were identified, which mainly distribute in tropic sea areas south of Hainan Island. The sea cucumber species from temperate sea area are few, among which A. japonicus is the one with the highest economic value. A. japonicus distributes in sea areas north of Lianyungang in Jiangsu province, China and has become increasingly popular from 1990s because of its medicinal effect to cancer and unique trophic value. In order to effectively enrich resources of cultured sea cucumber and improve domestic sea cucumber quality, it is essential that Holothurian resource abroad should be introduced, and that the attributes of the exotic sea cucumbers including genetic background, biochemical components, physiological and ecological characteristics should be analyzed. As to the genetic diversity of Holothurian, the sequences including mitochondrial 16S rDNA and CO1 and eukaryotic ribosomal 18S rDNA were amplified and the sequence diversity was analyzed by many researchers, which indicated that the rDNA sequence diversity exists among different Holothurian species. In allusion to a mass of introduction of important Holothurian resources from abroad, 10 species of domestic and overseas sea cucumbers were collected and 16S mitochondrial rDNA sequences from them were amplified and sequenced, and the molecular phylogeny of the 10 species of sea cucumbers were presented. Consequently, the aligned sequences with length of over 566 bp were obtained, the nucleotide composition, invariable/variable sites, insert/deletion （in/ del） sites were calculated by DNAsp 4.0 package.The kimura-two-parameter genetic distance was calculated by MEGA 4.0 package. The results indicate that AT content is rich in the 10 species of sea cucumbers, and the mean content of A＋T was 57.0%, which is similar to that of other invertebrates. A great diversity of the number of transition/transversion and in/del sites among the 10 species of sea cucumbers were observed, and their genetic distance ranges from 0.007 to 0.316. The cluster dendrograms of the 10 collected sea cucumbers and 10 sea cucumbers from GenBank data based on 16s rDNA analyses by means of four methods using MEGA4.0 package and PAUP＊4.0 package reveal the evolutionary relationships： Stichopodidae was separated from Holothuriidae entirely, but two species from Cucumariidae are adjacent to the members of Stichopodidae. This analysis also strongly indicated that in Stichopodidae A. japonicus from china has close affinity with Parastichopus californicus and P parvimensis from North America. On the other hand, Pearsonothuria graeffei, Bohadschia argus and B. marmorata belonging to family of Holothuriidae have close affinity, but the relationship between Holothuria edulis and H. mexicana is much closer. These findings of the study can be used for the management of sea cucumber resources, introduction of exotic species and genetic improvement of commercial species.