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