Microsatellite genotyping of single cells of the dinoflagellate species Lingulodinium polyedrum (dinophyceae): a novel approach for marine microbial population genetic studies

Abstract

In recent years, two new approaches have been
introduced in genetic studies of phytoplankton species.
One is the application of highly polymorphic
microsatellite markers, which allow detailed population
genetic studies; the other is the development of
methods that enable the direct genetic characterization
of single cells as an alternative to clonal cultures.
The aim of this study was to combine these
two approaches in a method that would allow microsatellite
genotyping of single phytoplankton cells,
providing a novel tool for high-resolution population
genetic studies. The dinoflagellate species
Lingulodinium polyedrum (F. Stein) J. D. Dodge was
selected as a model organism to develop this novel
approach. The method we describe here is based on
several key developments: (i) a simple and efficient
DNA extraction method for single cells, (ii) the
characterization of microsatellite markers for
L. polyedrum, (iii) a protocol for the species identifi-
cation of single cells through the analysis of partial
rRNA gene sequences, and (iv) a two-step multiplex
PCR protocol for the simultaneous amplification of
microsatellite markers and partial rRNA gene
sequences from single cells. Our protocol allowed
the amplification of up to six microsatellite loci
together with either the complete ITS1-5.8S-ITS2
region or a partial 18S region of the ribosomal gene
of L. polyedrum from single motile cells and resting
cysts. This article describes and evaluates the developed
approach and discusses its significance for
population genetic studies of L. polyedrum and other
phytoplankton species.

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