Revista Chilena de Historia Natural 81 (4): 515-531, 2008
RESEARCH ARTICLE
Diversity of the marine picocyanobacteria Prochlorococcus and
Synechococcus assessed by terminal restriction fragment length polymorphisms of 16S-23S rRNA internal transcribed
spacer sequences
In order to assess the appropriateness of the use of internal transcribed spacer (ITS)
sequences for the study of population genetics of marine cyanobacteria, we amplified and cloned the 16S rRNA gene plus the
16S–23S ITS regions of six strains of Prochlorococcus and Synechococcus. We analyzed them by
denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphisms (T-RFLP). When using the
standard application of these techniques, we obtained more than one band or terminal restriction fragment (T-RF) per strain or cloned
sequence. Reports in literature have suggested that these anomalies can result from the formation of secondary structures.
Secondary structures of the ITS sequences of Prochlorococcus and Synechococcus strains were
computationally modelled at the different temperatures that were used during the polymerase chain reaction (PCR). Modelling results
predicted the existence of hairpin loops that would still be present at the extension temperature; it is likely that these loops produced
incomplete and single stranded PCR products. We modified the standard T- RFLP procedure by adding the labelled ITS primer in
thelast two cycles of the PCR reaction; this resulted, in most cases, in only one T-RF per ribotype. Application of this technique to a
natural picoplankton community in marine waters off northern Chile, showed that it was possible to identify the presence, and
determine the relative abundance, of several phylogenetic lineages within the genera Prochlorococcus and
Synechococcus inhabiting the euphotic zone. Phylogenetic analysis of ITS sequences obtained by cloning and
sequencing DNA from the same sample confirmed the presence of the different genotypes. With the proposed modification, T-RFLP
profiles should therefore be suitable for studying the diversity of natural populations of cyanobacteria, and should become an important
tool to study the factors influencing the genetic structure and distribution of these organisms.
Key words:
cyanobacteria, DGGE,
T-RFLP, ITS, secondary structure
