A protocol for a single-cell PCR of diatoms from fixed samples: method validation using Ditylum brightwellii (T. West) Grunow

Abstract

Many major advances in macro-organismal biology, population genetics and evolutionary biology may be attributed to the development of rapid and inexpensive molecular methodology. Here, a simple protocol is presented for a polymerase chain reaction (PCR) of alcohol-preserved single diatom cells that facilitate studies requiring a large number of individuals, inclusive of archived and uncultivable specimens. Our approach integrates cell isolation and PCR amplification of two molecular markers, as well as post-PCR identification by scanning electron microscopy, thereby bridging the gap between classical and molecular diatom biology. The amplification of two genes was performed in a two-step protocol. By applying multiple sets of PCR primers to a single diatom cell, an internal fragment of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (RuBisCO, rbcL) was successfully amplified and sequenced, as well as the internal transcribed spacer (ITS) region in 20% of the same, preserved cells derived from natural phytoplankton. Multiple-gene recovery from a single-cell source requires further optimization, likely specific for the taxa of interest. Single marker recovery (from first-step amplification to sequencing) from natural, fixed single-cell samples was more successful with overall success rate of ∼31% for ITS and 67% for rbcL, respectively. Admittedly it is 50–70% lower than rates for a culture-pelleted DNA source (68% for ITS and 100% for rbcL) using current standard methods of sequence recovery in diatom studies, but our method is much less labor- and cost-intensive. This approach was applied to the diatom Ditylum brightwellii to assess the quality of recovered sequences. The phred quality scores of our sequences derived from single-cell material were similar to phred scores of our own, published sequences obtained from cultures grown in our laboratory.

Keywords:

• diatom
• multiple gene single-cell PCR
• fixed samples

Acknowledgements

We thank the sampling team of the Canadian Aquatic Invasive Species Research Network for sample collection in Vancouver Harbor and J.L. Martin in the Bay of Fundy, J.M. Ehrman for SEM and M.B.J. Moniz for four Ditylum brightwellii ITS sequences. Two anonymous reviewers provided much appreciated helpful comments. We acknowledge funding from the Natural Science and Engineering Research Council of Canada (NSERC) and from Canadian Aquatic Invasive Species Network (CAISN).