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18S rRNA clone library of phytoplankton in the Columbia River and its coastal zone By Pete Kahn Mentors: Lydie Herfort and Peter Zuber 1 http://www.mapwatch.com/news-blog/images/phytoplankton.jpg Significance of phytoplankton Major producers: O2: 90% CO2=>C3: 50% Base of aquatic food chain 2 Interactions with other Domains Bacterioplankton Colonization : Competition for NO32- and NH3? POC=>DOC 3 http://www.fossilmuseum.net/Tree_of_Life/Domains_Archaea_Bacteria/Domains_of_life.jpg Significance: Why is CMOP interested in Phytoplankton? Base of aquatic food chain Major producers: O2: 90%; CO2=>C3: 50% Interaction with other two Domains -Crenarchaeota: “competition” -Bacterioplankton: colonization -POC=>DOC Marine Phytoplankton bloom off the coast of Argentina http://www.mapwatch.com/news-blog/images/phytoplankton.jpg 4 Traditional Methods Flow cytometry Microscopy Flow Cytometry Chlorophyll a Photosynthesis: O2 production C14 uptake Microscopy: Pediastrium http://upload.wikimedia.org/wikipedia/commons/c/c7/Pi coplancton_cytometrie.jpg http://www.keweenawalgae.mtu.edu/ALGAL_IMAGES/ chlorophyceans/Pediastrium_n16_dollartow3_402_c.jpg Filtering for chlorophyll a 5 Objectives 18S rRNA clone library for different seasons and different locations: 0 PSU, 30 PSU for spring and summer; compare to winter samples Compare with Bacteria & Archaea clone libraries from Dan Murphy Identify unexpected changes Monitor health of ecosystem Pediastrium http://www.keweenawalgae.mtu.edu/ALGAL_IMAGES/chlorophyceans/Pedia strium_n16_dollartow3_402_c.jpg 6 Understand Phytoplankton community in estuary Methods Overview Phylogenetic analysis Sequence analysis w/ BLAST Yan, Q. Y., Yu, Y. H. & Feng, W. S. (2006). Genetic fingerprinting of plankton community provides new insights into aquatic ecology. Progress in Natural Science 16, 893-898. 7 Methods Overview Water sample => Sterivex on site Total nucleic acid extraction from sterivex in lab Amplification through PCR 8 Variations in nucleic acid in environmental samples Oct 2006 DNA Feb 2007 Between seasons Between locations April Wecoma & Forerunner: Freshwater << Surface Coastal RNA 9