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