Application of pyrosequencing method for investigating the diversity of synechococcus subcluster 5.1 in open ocean

Abstract

Synechococcus are distributed throughout the world's oceans and are composed of diverse genetic lineages. However, as they are much less abundant than Prochlorococcus in oligotrophic open oceans, their in-depth genetic diversity cannot be investigated using commonly used primers targeting both Prochlorococcus and Synechococcus. Thus, in this study, we designed a primer specific to the 16S-23S rRNA internal transcribed spacer (ITS) of the Synechococcus subcluster 5.1. Using the primer, we could selectively amplify Synechococcus sequences in oligotrophic seawater samples. Further, we showed that a barcoded amplicon pyrosequencing method could be applicable to investigate Synechococcus diversity using sequences retrieved in GenBank and obtained from environmental samples. Allowing sequence analyses of a large number of samples, this high-throughput method would be useful to study global biodiversity and biogeographic patterns of Synechococcus in marine environments.

Fig. 1

Neighbor-joining (NJ) tree based on 899 positions of the 16S–23S rDNA ITS sequences. A maximum-likelihood (ML) tree was also constructed using the PhyML program (ver. 3.0) with the HKY+i+g model (not shown). Numbers at nodes represent bootstrap values where bootstrap support is >60% in at least one method. Clade names are shown on the right of the vertical lines. Numbers in parentheses represent the number of sequences belonging to each clade. Previously designated clades CB2, CB3, XVIII, XV, XIX and WPC2 were grouped together with other clades (see text). In these cases, the earliest published clade names were selected. Four sequences belonging to Synechococcus 5.3 were used as an outgroup (not shown).