Assessment Of Marine Microalgae DNA Barcode Genes And Development Of New Gene For The Diversity Analysis And Quantitation Of Environmental Samples

Marine microalgae played a very important role in the marine ecological system, and their compositions and secretions was huge the exploitable marine resource, While diatoms and dinoflagellates were the domination of the marine microalgae. Thus, the classification of Marine microalgae was the foundation of all other related works.DNA barcoding was a very important assistant for the traditional morphological taxonomy. DNA barcoding of the diatoms and dinoflagellates developed lately and slowly. Researchers have not reached consensus of opinion, though they have done many analysis for SSU, ITS, LSU, COI, cob, rbcL and UPA focused on some special tax instead of the whole diatom group. In addition, there were two mainly methods for researching the marine microalgae communities, one is microscopic examination which was subjected to the limitation of the taxonomy and the other one is the method of rDNA amplicons sequencing which was often used at present by researchers to study the marine microalgae community structure. However, rDNA copy number was different within different eukaryote; Thus, there might be bias in evaluating the species abundance of the eukaryote microbial communities by using the method of rDNA amplicons sequencing. Some nuclear codon protein gene owing fewer copies have been used for the phylogenetic analysis, community structure probably be more precise if they were analyzed by these genes.In this study, degenerate primers were designed for 18S rDNA, ITS rDNA, COI and rbcL, and with sequences download from NCBI the amplified sequences were used to evaluate whether these regions are effective on different levels of diatom clustering analysis to assess the potential of these regions on barcoding some taxa. The mocked diatom community was made to build the rDNA clone library to evaluate the analysis accuracy of diatom community structures by rDNA amplicons sequencing. Then some nuclear codon protein genes possessing fewer copies were detected in the discrimination of dinoflagellates, after that the chosen gene was amplified from the mocked dinoflagellates community to access its effectiveness in analysis of the community structure. Finally 18S v9 and actin gene were sequenced by Miseq sequencer from different mocked diatom and dinoflagellates communities and try to associate the amplified sequences with their cells percent.Results of the barcoding evaluation part showed that all the designed primers got tested in 37 diatoms. The blast result showed that rbcL has the highest blast hitting (88.9%),18S was second and COI has the highest mismatching (25%). Then sequences for these genes amplified by these primers were used to calculate the divergence and make Bayesian inference tree with the sequences download from NCBI, the results indicated that the divergence of 18S rDNA was moderate,18S could cluster the diatoms at the phylum level and also in lower tax level such as Melosira, Licmophora, Skeletonema and Amphipleuraceae. While the divergence of ITS and COI sequences was very large and was not suitable for the clustering analysis of higher tax level. However, ITS showed a large potential in barcoding species of the Thlassiosirales and analyzing their phylogenetic. And COI was more suitable for the clustering and phylogenetic analyses of some pennate diatoms. Although rbcL was more conserved than 18S, it could not do the phylogenetic analyses at diatom higher tax level.The fragment of rDNA (5.8S rDNA+ITS-2) was amplified from DNA and cell samples derived from the same mock diatom community. This result displayed that species in DD was two more than CD, but species in both datasets were much fewer in the mixed sample, and showed the same amplification biases. Meanwhile the species quantification in both datasets differed with their cell percent; it revealed that rDNA copy number varied in different diatoms and rDNA clone library made a large bias in species quantification of the eukaryotic microbial communities. There were ITS variations (p<0.013) in different diatom cultivations.Nuclear coded protein genes HSP90, elf2 and actin were used to make trying in species quantification of the dinoflagellates communities. The degenerate primers were designed for them, and the amplification and sequencing result showed that only the actin gene showed a potential in amplification, sequence availability and discriminating dinoflagellates, actin mainly could cluster the dinoflagellates at the generic level. The actin clone library for 8 dinoflagellates mixed sample showed a result that 8 species were all detected and the bias in species quantification was lower than rDNA especially in generic level.The 18S v9 and the actin gene were sequenced by Miseq sequencer for 7 mocked samples mixed with diatoms and dinoflagellates. Result of this portion showed that rDNA amplicons sequencing made a larger bias when analyze the community structure of the mocked samples and the amplification of 18S rDNA easily affected by other contamination (such as fungi). The actin gene was amplified without any contamination, and it could more similarly show the real community structure and the sample clustering relationship. Thus, it was considered that the actin gene was more suitable than rDNA as ecologic molecular marker in analysis of the eukaryotic microbial communities.