Preliminary Study On The 18S RDNA Sequence Diversity Of Jiaozhou Bay Planktonic Copepods

Eukaryotic ribosomal RNA small subunit gere (18S rDNA) 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 rDNA 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 biodiversity researches about marine picoplankton based on 18S rRNA gene have been published at home and abroad. But the study on planktonic copepods using similar method has not been reported yet. The aim of our research is to analyze the 18S rRNA gene genetic diversity and develop a molecular method to describe the community structure of copepods of Jiaozhou Bay.The total DNA was extracted directed fron the mixed zooplanktons in Jiaozhou Bay using alkaline-boiling. The copepod-specific primers (247F and 1701R) were designed to amplify the 18S rRNA gene from the total DNA selectively. The length of these fragments was 1.5 kb. It is expensive to get the whole sequences of 1.5 kb.The copepod-specific primer (247F) and eukaryote -specific primer (EK1269R) were used to amplify about 1 kb 18S rDNA fragments from total DNA. Then the correspondingplasmid libraries were made respectively. The OTU types were detected by using RFLP method and one or two clones of every OTU type were chosen randomly and sequenced. So three sequences of 1.35kb and eight sequences of 1kb were obtained. The systemic analysis on different subclass of Crustacean based on 18S rRNA gene was done for describing copepod genetic diversity.The analysis proves that the primers (247F and 1701R) amplified selectively copepod 18S rRNA genes from marine environmental samples. And more copepod species were amplified and more OTU types were found using the primers (247F and EK1269R). Two high and one low frequency OTU types were detected in two experiments. The results of the nucleotide alignments show that the Calanoida organisms predominated the zooplankton community in Jiaozhou Bay. The values of genetic diversity index are respectively 0.95 and 0.96 on the basis of OTU frequency. So we can conclude that the 1.35kb sequences and 1 kb sequences have the similar or equal genetic information. This point can also be received from the analysis of conserved and variable sections of 18S rRNA gene. The analysis of genetic distance matrix, online BLAST searching and phylogenetic tree prove that all of the sequences got in the experiments are derived from the Copepoda. The nucleotides between any two sequences got in this experiment are so different and the value of diversity is from 0.03% to 13%. Three sequences (AY437862, AY491388, AY491390) are so far similar with that of Calanus paciftcus (the value of identity is 98%). And the bootstrap value on the node is above 99%, which is calculated 500 times. So it is confirmed that they belong to the Calanus genus. The remnant sequences are derived from the Calanoida order excluding AY437863 and AY491391.