Genetic diversity of eukaryotic marine picoplankton studied by DGGE Beatriz Díez, Ramon Massana and Carlos Pedrós-Alió Department of Marine Biology, Institut de Ciències del Mar, Barcelona, Spain

DGGE because... Fingerprinting analysis. Easy and quick method to compare many samples. Phylogenetic capacity. Sequencing of bands.

Steps in the DGGE method Collection of samples DNA extraction PCR amplification DGGE Image analysis

Collection of microbial biomass Prefiltration unit Polycarbonate 5 m, 47 mm 0.2 m Sterivex filter (Durapore, Millipore) 5-20 liters seawater Peristaltic pump

DNA extraction protocol Digestion with lysozyme, proteinase K and SDS Extraction of DNA with phenol: chloroform: isoamyl alcohol Concentration and purification of DNA with a Centricon Quantification of DNA extract

Annealing at 65°C with touchdown to 55°C PCR conditions Choice of primers for PCR Tested with cultures Tested with natural samples 1209 F 516 R 1392 R Two sets of primers used Euk1209f-GC and Un1392r Euk1f and Euk516r-GC EUK1F Annealing at 65°C with touchdown to 55°C

DGGE conditions 0.75 mm-thick 6% polyacrylamide gel 600-800 ng PCR product Staining with SybrGold and visualized with a FluorS MultiImager Primers Denaturing Voltage Time Temperature gradient Euk1209f-GC and Un1392r 40-80% 200V 5h 60°C Euk1f and Euk516r-GC 45-65% 100V 16h 60°C

Euk1209f-GC and Un1392r Euk1f and Euk516r-GC Nannochloropsis Pelagomonas Thalassiosira Olisthodiscus Pelagomonas Thalassiosira Olisthodiscus Dunaliella Platymonas Heterocapsa Dunaliella Platymonas Heterocapsa

DGGE studies applied to marine picoeukaryotes Study 1 Estimation of genetic diversity (by sequencing some bands) and comparison with genetic libraries. e.g. ME1 sample Study 2 Estimation of temporal succession by fingerprinting analysis. e.g. Blanes samples

Study 1 ME1 ME1 Euk1f and Euk516r-GC Euk1209f-GC and Un1392r Mantoniella squamata Euk1f and Euk516r-GC Euk1209f-GC and Un1392r Pfiesteria sp. Unidentified prymnesiophyte Ostreococcus tauri Ostreococcus tauri Unidentified prymnesiophyte Mantoniella squamata Mantoniella squamata Aureococcus anophagefferens Ostreococcus tauri Oikopleura sp. Oikopleura sp.

ME1 Oikopleura 28 25 37 Prasinophytes 20 30 16 Prymnesiophyceae 3 3 1 % band intensity with Euk1209f-GC and Un1392r % band intensity with Euk1f and Euk516r-GC % Clones ME1 Oikopleura 28 25 37 Prasinophytes 20 30 16 Prymnesiophyceae 3 3 1 Pelagophyceae 8 _ 1 Dinophyta 3 _ 13 Others 37 43 32 Number of bands/patterns RFLP 18 28 32

Study 1 Conclusions DGGE is appropriate to study genetic diversity Similar phylogenetic groups appear by cloning and DGGE (using different sets of primers) Similar dominant species contribution by either approach (cloning and DGGE)

Study 2. Fingerprinting comparison Temporal variability of eukaryotic picoplankton communities (5-0.2m), along an annual sampling at surface during 1998 Does picoeukaryotes species succession exist?

Picoeukaryotic succession in Blanes´98 DGGE (primers Euk1f and 516r-GC) Jan February March Apr Jun Jul Sep Oct Nov Dec ANT12 27 5 25 3 11 18 26 29 3 2 29 3 9 4 1 31 27 28 26 20 15 32 25 34 28 28 27 32 33 40

Matrix of normalized Euclidean distances 26 MARCH ---- +--------- 11 MARCH ---- | +----------------------------------- 26 MARCH ------- | | +-- | | 29 APRIL ------+ | | | | | | | 3 MARCH --- | | | | +-- | | | 25 FEBRUARY --- | | | | | +-- | | | 5 FEBRUARY ----- | | | +---- | 27 JANUARY --------- | +------- 1 DECEMBER ----------- | +------- | 3 SEPTEMBER --------- | | | +-- | | 4 NOVEMBER ------ | | | +--- | | 3 JUNE ------ | | +------------------------------ 2 JULY --------- | +----- | 29 JULY --------- | | +---- 9 OCTOBER -------------- DISTANCE METRIC IS EUCLIDEAN DISTANCE WARD MINIMUM VARIANCE METHOD Winter and Spring Summer and Autumn

Bacterial Succession in Blanes

Blanes samples Summer Autumn Spring Winter

Study 2 Conclusions DGGE fingerprinting is useful to compare assemblages Temporal succession (possible species succession). Summer/Autumn and Winter/Spring periods Similar temporal succession of bacterioplankton and eukaryotic picoplankton communities