Roscoff clone libraries (2000-2001)
Picoeukaryote diversity and their temporal changes, in the well mixed water of the English
Channel, was pre-analysed by comparative RFLP patterns. From each clone library a total of
100 re-amplified 18S rDNA fragments (size 1800 - 2000 bp) were digested with tetrametric
endonuclease HaeIII. Identical patterns were grouped into common RFLP type. The summary
of 700 amplified rDNA restriction analysis is listed in Table 1.
Table 1. Summary of amplified rDNA restriction analysis
Clone library
No. of clones
No. of RFLP type
Coverage value (%)
RA000412
100
66
34
RA000609
100
54
46
RA000907
100
68
32
RA001219
100
60
40
RA010412
100
54
46
RA010516
100
65
35
RA010613
100
41
59
To determine whether the clone sampling carried out covered the total diversity of the
picoeukaryotes 18S rDNA in the libraries, the cumulative number of OTUs was plotted as a
function of their detection during the sampling of clones, generating thus the rarefaction
curve. (Fig. 1). The shape of the curves obtained shows that there is no real saturation and this
in spite of the analysis of 100 clones per library, indicating that analysis of an increasing
number of clones would have revealed further diversity.
At least one clone that was representative of each RFLP type and from each clone library was
partially sequenced. BLAST analysis of all sequences obtained showed that all clones are
eukaryotes, indicating the high specificity of the eukaryotic Moon's primers used for clone
libraries construction. Overall, the greatest numbers of cloned partial sequences had more
than 88% similarity to 18S rDNA sequences in the DNA database. Sequences having a
similarity upper than 99,5 % are gathered in the same OTU. These clones, which were found
approximately in all libraries, were associated with Prasinophyceae (27 OTUs);
Prymnesiophycea (5 OTUs); Cryptophyceae (20 OTUs); Stramenopiles (38 OTUs); Alveolate
(52 OTUs); Rhizopoda (24); Ciliophora (30 OTUs) and 2 unknown groups LKM74 and
Helgoland groups represented respectively by 9 clones (8 OTUs) and 14 clones (10 OTUs).
The Metazoan clones and one fungal sequence were exclueded from the analysis. The
distribution of all sequences (384) found in the 7 clone libraries is shown in Figures below
Plans : During the next month, I am planning to analyse full sequences obtained from New
red group and Telonema (LKM74); to continue the screening of cultures obtained by Florence
and finalize the draft of picoeukaryote diversity paper.
Rarefaction curves
70
RA000412
RA000609
60
RA000907
50
RA001219
RA010412
40
RA010516
RA010613
30
20
10
0
0
20
40
60
80
100
Number of clones
Fig.1: Rarefaction curves for the different RFLP type of 18S rDNA clones in 7 clone
libraries (2000-2001).