Eukaryotic Algal-Biased PCR And Its Application

In this article, the problem concerning large amount of heterotrophic organismsaffecting the investigation of the sedimentary eukaryotic algae diversity is to besolved. We used a series of bioinformatics tools to analyze the nucleic acid database,and successfully obtained a pair of eukaryotic algal-biased PCR primers amplifyingthe small subunit nuclear ribosomal DNA (SSU nrDNA). High sensitivity and highspecificity for eukaryotic algae of the primers was validated by examining theamplification of genomic and sedimentary DNA. Then we use this newly designedprimer to investigate sedimentary eukaryotic algae diversity of the Yangtze Riverestuary and its adjacent area, where diverse sedimentary eukaryotic algae was found.By constructing the eukaryotic algae-biased PCR, we provide a new method toinvestigate the sedimentary eukaryotic algal diversity, avoiding the large-scale libraryto be established and screening process to be launched using universal primers PCRamplification as well as reducing costs, making large-scale sampling and analysispossible, and ultimately providing a methodological basis for investigating the spatialand temporal distribution of sedimentary eukaryotic algae community. The mainfindings are as follows:We constructed the the SSU nrDNA database for primer design and evaluation,used Primrose to generate alternative primers and TestProbe to analyze the primer-template mismatch sites. Combined with the the previous research ofprimers-template mismatching on amplification efficiency, we designed a pair ofeukaryotic algae18S rDNA specific primers968F-1643R. Database results shows,except for some protozoan taxa, this pair of primers enjoy a high sensitivity andspecificity for eukaryotic algae.In order to verify the database research conclusion, we carried otu genomic DNAand sedimentary PCR. Genomic DNA from seven major eukaryotic algae phyla, twofungi species, one crustacean, and one ciliate was employed for primer PCR amplification verification. Electrophoresis results after PCR optimization showed theeukaryotic algae genomic DNA is amplified, while the non-algae group was not. Onthe contrary, PCR using the universal primers results showed that the11genomicDNA were amplified. Similarly, clone library of universal primers amplicon failed todetect eukaryotic algae. These findings preliminarily confirmed the reliability ofdatabase research results.By employing Primer pair968F-1643R to amplify the surface sediment DNA inYangtze Estuary and constructing clone library, we sequenced250clones and found atotal of70operational taxonomic units using the criterion of of97%similarity.17diatom species,24dinoflagellates species,24species of other algae phyla, and5non-algae eukaryotes were identified. This result shows a unique and diverse surfacesedimentary eukaryotic algal community of the border of Yellow Sea and East ChinaSea, differed from the diatom-dominated sedimentary eukaryotic algal community inthe Yangtze estuary area.