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Oceanic Phytoplankton Team Station Biologique de Roscoff

Molecular Analysis of the Genetic Diversity and Physiological Status of Natural Picoplanktonic Populations. RCC 371. Oceanic Phytoplankton Team Station Biologique de Roscoff UMR 7127 CNRS et Université Paris 6, France. Aims for BIOSOPE: 1. Mapping the Picoplankton.

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Oceanic Phytoplankton Team Station Biologique de Roscoff

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  1. Molecular Analysis of the Genetic Diversity and Physiological Status of Natural Picoplanktonic Populations RCC 371 Oceanic Phytoplankton TeamStation Biologique de Roscoff UMR 7127 CNRS et Université Paris 6, France

  2. Aims for BIOSOPE:1. Mapping the Picoplankton • Enumerate picophytoplankton cells (Prochlorococcus, Synechococcus, picoeukaryotes) + bacteria +/- viruses +/- photosynthetic anoxygenic bacteria (to be tested) by flow cytometry => Volume needed: 10 mL all stations / all depths + transect (1 sample / hour)? • Analyze the cell cycle of Prochlorococcus & Synechococcus over L:D cycles (in combination with molecular analyses of stress response) => 4 long-term stations only : Volume needed: 10 mL every 3 h for 2-3 days, 7 depths (to be discussed)

  3. Flow Cytometry FACSAria • Laser source : 488 & 635 nm • High speed cell sorting (> 5 103 cell./sec) • Increased Sensitivity • « Transportable » (180 kg!)

  4. FACSort ARIA

  5. Aims for BIOSOPE :2. Genetic diversity • Isolate new species & organisms representative of non-cultured environmental groups • Assess the genetic diversity of (auto- and heterotrophic) picoeukaryotes in the euphotic hyper-oligotrophic layer (0-200 m) • Assess the diversity & activity of heterotrophic picoeukaryotes over the vertical layer (0-2000 m) • Quantify the abundance of selected taxa and identify the dominant species/taxa • and more (collaborations): Synechococcus, …

  6. Molecular Techniques - Pre-screening of genetic diversity of picoeukaryotes by TTGE (most stations / 6 depths) => Volume : 5L pre-filtered on 3 µm on-line with 0.2µm filter - Cloning & sequencing 18S & 16S rRNA gene from natural populations (8-10selected stations & depths) => same DNA sample as for TTGE - Quantification of the abundance of different taxa • in situ Hybridization (FISH) using 18S & 16S rRNA probes (8-10 selected stations at 6 depths, 1 L) • Quantitative PCR (most stations / 6 depths ?) • - Metagenomics ? • (Long-term stations, 2 depths, >100 L samples, in situ pumps)

  7. in situHybridization (FISH) Natural sample < 3 µm MolecularApproach Filtration 0.2 µm Extraction Cultures DNA Amplification 18S/16S rRNA gene PCR / Quantitative PCR DGGE/TTGE Clone libraries rRNA gene sequencing Data banks Phylogenetical analyses Molecular probes

  8. An example of DGGE analysis D. Marie unpubl.

  9. Example of Clone Library Analysis Moon et al 2001

  10. FISH: Abundance of Prasinophyceae off Roscoff F. Not, unpubl.

  11. Available Probes for FISH (subset) Probes in yellow require development

  12. Quantitative PCR Natural sample Morlaix Bay Advantages/FISH : faster (96/3 = 32 samples at a time) more sensitive (detection : < 0.1 ng d ’ADN) Drawbacks/FISH : more expensive, do not visualize cell morphology measures relative gene abundances, not cell number => 18S rRNA in variable number copies (2-1000 per cell)

  13. Q-PCR: PROSOPE cruise Fei Zhu

  14. Available probes for Q-PCR • Six primer sets (5’ Euk universal, 3’Group specific) • EUK : Eukaryotes • CHL : Chlorophyta • MAM (=PRAS04) : Mamiellales • MIC : Micromonas (not totally specific yet) • BAT : Bathycoccus • OST : Ostreococcus

  15. Aims for BIOSOPE3. Physiological status of natural populations • Expression of specific stress genes (UV stress, nutrient starvation) over a L:D cycle every 3 h for 2-3 days) => at the four long-term stations only, 7 depths (subsurface, 5 m, 10 m, 20m, 30 m, 50 m, DCM (Niskin!) + if bottles : 5 L for DNA, 15 L for RNA + if in situ pumps: 20L for DNA, 80L for RNA • Characterization of the expression of genes of biogeochemical interest in the picoplankton(e.g.: nifH) => same DNA & RNA samples Problems to be solved with the use of in situ pumps - Sampling need to be short (only 100 L necessary) and done as soon as possible after a CTD cast (within an hour) - flow cytometry needed : will be sampled at same depths with the rosette (DCM: 2 x 10 L) - need for RNA-free material (use of gloves to handle filters, RNA-zapping of filter holder,…) - need for pre-tests of RNA extraction from GF/F filters before the cruise !

  16. Q-PCR for measuring Gene Expression Natural sample Filtration 0.2 µm Extraction RNA Reverse transcription cDNA Amplification gene X by quantitative PCR

  17. Analysis of Gene Expression by Q-PCR (Eilat, Sept. 2000) Holtzendorff et al. (2001)

  18. Specific needs Sample storage : - Cruise start (Tahiti): 105-140 L liquid N2 to fill 3 or 4 35-liter containers Note: Samples can be also be stored at -80°C during cruise time but fast freeze is preferable - Cruise end (Chili) : 50-60 kg dry ice to send all frozen samples by express mail in a large cryobox after the cruise Pre-cultures will be either brought back « by hand » in a cryobox or left on board (requires light and air conditioning left on during the transect back) Space : - Beside SBR container lab space, 1.5 m of bench for filtration ramps Flow cytometer: - Needs to be sent by plane!

  19. Sample number (estimation)

  20. Daniel VAULOT DR1 CNRS (leg 1) • Frédéric PARTENSKY DR2 CNRS (leg 2) • Laurence GARCZAREK CR1 CNRS (leg 1) • Laure GUILLOU CR2 CNRS • Nathalie SIMON MdC UPMC • Dominique MARIE IE CNRS (2 legs) • Florence Le GALL AI CNRS • Post-doc X (2003-2004) • PhD student Y on Diversity (2004-2007) (leg 2) • DEA or PhD student Z on Physiology (2004-2007) Personnel SBR Involved in BIOSOPE Collaborations for molecular analyses • Dave Scanlan, Warwick University : diversity Synechococcus (DNA/FISH) +/- John Zehr, USA : nifH gene diversity (DNA/RNA) +/-Colomban de Vargas, USA : Coccolithophorids +/- Alex Worden : Roseobacter diversity (DNA/RNA) ? +/- Carles Pedros-Alio : Archae diversity (DNA) ? • +/- Osvaldo Ulloa, Chili : diversity eukaryotes, anoxygenic Procs • +/-Z. Kolber, USA : anoxygenic bacteria

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