Genetic diversity of eukaryotic marine picoplankton studied by DGGE Beatriz Díez, Ramon Massana and Carlos Pedrós-Alió Department of Marine Biology, Institut.

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

Prefiltration unitPolycarbonate 5 m, 47 mm

0.2 m Sterivex filter(Durapore, Millipore)

Peristaltic pump5-20 liters seawater

Collection of microbial biomass

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

EUK1F

1209 F

1392 R

516 R

Two sets of primers used

• Euk1209f-GC and Un1392r

• Euk1f and Euk516r-GC

PCR conditions

Choice of primers for PCR

Tested with cultures

Tested with natural samples

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

Het

eroc

apsa

Olis

thod

iscu

s

Pla

tym

onas

Dun

alie

lla

Tha

lass

iosi

ra

Nan

noch

loro

psis

Pel

agom

onas

Het

eroc

apsa

Olis

thod

iscu

s

Pla

tym

onas

Dun

alie

lla

Tha

lass

iosi

ra

Nan

noch

loro

psis

Pel

agom

onas

DGGE studies applied to marine picoeukaryotes

Study 1Estimation of genetic diversity (by sequencing some bands) and

comparison with genetic libraries. e.g. ME1 sample

Study 2Estimation of temporal succession by fingerprinting analysis. e.g.

Blanes samples

Euk1f and Euk516r-GC

Euk1209f-GCand Un1392r

ME1 ME1

Mantoniella squamata

Pfiesteria sp.

Ostreococcus tauri

Unidentified prymnesiophyte

Ostreococcus tauri Aureococcus anophagefferens

Oikopleura sp.

Oikopleura sp.

Ostreococcus tauriMantoniella squamata

Mantoniella squamata

Unidentified prymnesiophyte

Study 1

% band intensity with Euk1209f-GC and Un1392r

% band intensity with Euk1f and Euk516r-GC

% Clones

Oikopleura 28 25 37

Prasinophytes 20 30 16

Prymnesiophyceae 3 3 1

Pelagophyceae 8 _ 1

Dinophyta 3 _ 13

Others 37 43 32

Number ofbands/patterns RFLP 18 28 32

ME1

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

(5-0.2m), along an annual sampling at surface during 1998

Does picoeukaryotes species succession exist?

Picoeukaryotic succession in Blanes´98 DGGE (primers Euk1f and 516r-GC)

AN

T12 Jan February March Apr Jun Jul Sep Oct Nov Dec

403332272828342532152028 262731

5 25 3 11 18 2627 29 3 2 29 3 9 4 1

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 --------------

Summer and Autumn

Winter and Spring

Matrix of normalized Euclidean distances

DISTANCE METRIC IS EUCLIDEAN DISTANCEWARD MINIMUM VARIANCE METHOD

-10 27 36 43 50 56 62 70 77 85 119 154 183 210 246 282 308 335

Dec97 Jan Feb Apr Jun Jul Aug Sep Oct Nov Dec Mar

Bacterial Succession in Blanes

Blanes samplesBlanes samples

3 September

2July

29 July

9 October

4 November

1 Desember

26 March

29 April

3 June

27 January

22 Desember 97

25 February

5 February

12 February

19 February

3 March

18 March

11 March

1.0

Verano Otoño

Primavera

Invierno

SummerAutumn

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