1. 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

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

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

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

5. 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

6. 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

7. DGGE conditions 0.75 mm-thick 6% polyacrylamide gel
ng PCR product
Staining with SybrGold and visualized with a FluorS MultiImager
Primers Denaturing Voltage Time Temperature
gradient
Euk1209f-GC and Un1392r % V h °C
Euk1f and Euk516r-GC % V h °C

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

9. 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

10. 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.

11. 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
Prasinophytes
Prymnesiophyceae
Pelagophyceae _
Dinophyta _
Others
Number of
bands/patterns RFLP

12. 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)

13. 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?

14. 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

15. Matrix of normalized Euclidean distances
26 MARCH ----
11 MARCH |
26 MARCH | |
+-- | |
29 APRIL | | |
| | | |
3 MARCH | | | |
+-- | | | 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

16. Bacterial Succession in Blanes

17. Blanes samples
Summer
Autumn
Spring
Winter

18. 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