PCR primers for selective detection of intra-species variations in the bloom-forming cyanobacterium, Microcystis

Takafumi Kataoka, Takamitsu Homma, Shin Ichi Nakano, Yoshikuni Hodoki, Kako Ohbayashi, Ryuji Kondo

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Members of the cyanobacterial genus Microcystis commonly form blooms in eutrophic freshwater systems, and some produce cyclic heptapeptide hepatotoxins called microcystins, thereby often causing serious water management problems. Microcystis species were unified into the single Microcystis aeruginosa classification based on 16S rRNA gene sequences and DNA-DNA re-association experiments; however, the morphological features of the organisms differ in different culturing conditions. Here, we describe a new real-time quantitative PCR (qPCR) method of determining Microcystis intradiversity using the SYBR Green I assay. We analyzed 71 Microcystis 16S-23S rDNA internal transcribed spacer region (16S-23S ITS) sequences, designed three group-specific PCR primers that successfully selected a morphologically M. wesenbergii-like non-toxic group (Group-3), and differentiated between M. viridis-like toxic group (Group-4) and M. aeruginosa-like Group-1 organisms including toxic and non-toxic Microcystis strains. The primers covered 76% of the Microcystis 16S-23S ITS regions from all over the world (six continents) included in GenBank. We constructed a mixed culture with representative Microcystis strains from each group, and estimated their cell densities by qPCR over 7 weeks. Group-1 and Group-3 grew exponentially for 4 weeks; however, the growth of Group-4 declined after 2 weeks, revealing different growth properties for the Microcystis groups in the mixed culture. Finally, we applied this method to natural Microcystis blooms at four freshwater sites, and found the dominance of Group-1 in three blooms and of Group-3 in one bloom, thereby showing the geographically uneven distribution of Microcystis genotypes. The developed qPCR technique targeting the 16S-23S ITS region is both rapid and simple and is useful for selective quantification of group variations among sympatric Microcystis genotypes, such as in mixed cultures and the natural environment.

Original languageEnglish
Pages (from-to)46-54
Number of pages9
JournalHarmful Algae
Volume23
DOIs
Publication statusPublished - 2013 Mar 1

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Keywords

  • 16S-23S rDNA ITS region
  • Intra-species variation
  • Microcystis aeruginosa
  • Quantitative PCR
  • Selective detection

ASJC Scopus subject areas

  • Aquatic Science
  • Plant Science

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