Nematode Diversity Of Qingdao Coast Determined By PCR Retrieving And Sequencing Of 18S Ribosomal RNA Gene Fragments

Eukaryotic ribosomal RNA small subunit gene is widely used as an indicator gene in phylogenetic research. It is characterized by slow evolution, high conservation and multiple copies. It is also used for studying the genetic diversity of living things in environmental samples. The 18S rRNA gene fragments are amplified directly from the environmental samples and corresponding clone library is obtained in order to describe the population genetic structure. The method is based on the alignment of multiple DNA sequences. And the total DNA of environmental samples is used as the template for the amplification. This process is not the same way with the amplification from the individual organism avoiding the process of pure culture and microscopic inspection. So this method will greatly help us discover more diversity of small organisms in nature. The aim of our research is to develop molecular methods for classifying nematodes and describing nematode community structures independently or in combination.Based on the lengths and yields of PCR products, eight template DNAs isolated from marine nematode individuals stored respectively in four fixing solutions (acetone, absolute alcohol, alcohol with 0.05mol/L EDTA (pH8.0) and 5% formalin in seawater) by using two different genomic DNA isolation methods (alkaline lysis and protease K treatment) were compared for their performances in the amplification of 18s ribosomal RNA gene fragments. It has been found that absolute alcohol is the best fixing solution for preparing PCR template DNA and 5% formalin in seawater the best for keeping the morphological characters of nematode individuals. The genomic DNA isolated with protease K treatment is better for PCR amplification than that isolated with alkaline lysis. Based on this study, an integrated protocol of morphological and molecular identifications of free-living marine nematode individuals was developed. Marine nematodes individuals were identified morphologically under LM after fixedby formalin and transparented with glycerin. And then its 18S ribosomal RNA gene fragments were retrieved from DNA extracted by alkali-heat treatment with nematode specific primers, cloned and sequenced. The sequences of marker genes widely used in phylogenetic analysis of nematode individuals are very important supplement of morphological identification. They will facilitate also the comparison of data from different sources.Free-living marine nematode 18S ribosomal DNA gene fragments, approximately 1300bp in length, were retrieved from DNA extracted from the inter-tidal silt of Qingdao coast with nematode specific primers, cloned and typed with Rsct I and Hin6 I restriction endonucleases. Among 17 types, type 1, 2 and 6 cover 61.2%, 14.4% and 9.3% of the clones analyzed respectively, while remaining 14 cover only 21 clones, 15.1% of the total. Twenty-four representative clones, at least one each type, were sequenced and phylogenetically analyzed with reference to those currently available in RDP and GenBank databases. Although hard to be assigned to known species or genera due to the shortage of identified marine free-living nematode 18S rRNA gene data and sequence variation ranges within different taxa, these sequences certainly belong to the nematodes of Adenophorea. Comparatively, 12 sequences are close to Pontonema vulgare and Adoncholaimus sp., 4 to Daptonema procerus and 2 (identical each other) to Enoplus brevis. Our results showed that free-living marine nematode diversity could be determined by PCR retrieving and sequencing of their 18S rDNA fragments. It is also clear that the assignment of an 18S rDNA sequence to a species or a genus is waiting for the identification of free-living marine nematode species with their 18S rDNA sequenced.