Single-cell PCR amplification of thecate dinoflagellates: a case study of Tripos (Dinophyceae)

Abstract

The relief of amplification inhibition in preserved phytoplankton samples is a major challenge in genetic studies relying on single-cell sequencing. The successful amplification of rDNA genes varies considerably depending on the organisms, fixatives, and time of analysis after collection. Among other fixatives, RNAlater® is found to be a suitable choice for PCR amplification after long-term storage. In this study, we tested the performance of RNAlater® samples applied to single-cell PCR amplification in isolates of the thecate dinoflagellate genus Tripos, with an amplification success over 70%. The single-cell protocol proposed in this study amplified an average of 650 pb of large subunit and small subunit rDNA fragments in RNAlater® preserved cells after 5 months. Furthermore, it was possible to obtain rDNA sequences from samples up to 8 months old. The approach described here could also be useful to amplify a wide range of thecate and non-thecate dinoflagellate taxa, especially those species difficult to maintain in culture.