Restriction Fragment Length polymorphism (RFLP) and DNA Sequence Analysis of PCR-generated Clones to Assess Diversity of Picoeukaryotic Algae in the Subtropical Central North Pacific Ocean (Station ALOHA)

C. L Moyer, L Campbell, D. M. Karl and J. Wilcox

Abstract

Species composition of the small (<3 µm) eukaryotic phytoplankton is poorly described. Molecular techniques directed at the large subunit (LSU) rRNA genes within plastid genomes were used to limit our analysis exclusively to eukaryotic cells. Specific DNA oligonucleotides were used to PCR amplify rDNA gene fragments (800-850 base pairs) from 5 enrichment cultures from Sta. ALOHA (chrysophytes and chlorophytcs) and Pycnococcus Provasolii (Prasinophyte). After demonstrating the utility of the LSU rRNA PCR primers, picoeukaryotes from the deep chlorophyll maximum layer at Sta. ALOHA were sorted by flow cytometry, bulk picoeukaryotic community DNA extracted and PCR amplified, and a clone library of 102 clones was generated. Preliminary RFLP analysis of the LSU rRNA gene fragment from 6 randomly picked clones revealed 3 different patterns: 4 of one type and one from each of two types. One clone was partially sequenced and found to have an 88% identity with Chlorella ellipsoidea plastid LSU rRNA. The other two clones had an 84% identity with Chlamydomonas eugametos and an 82% identity with C. reinhardtii plastid LSU rRNA.