MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES¹

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 identification 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.

Microsatellite markers for the dinoflagellate Gambierdiscus caribaeus from high-throughput sequencing data

“…Annealing temperatures ranged from 52 to 60°C in the different PCR reactions. Similar numbers of microsatellites were developed for other dinoflagellate species in different laboratories (Frommlet and Iglesias-Rodriguez 2008;Lundholm et al 2014;Minter et al 2015) with the exception of higher numbers of markers for the genus Alexandrium (Nagai et al 2004(Nagai et al , 2006(Nagai et al , 2014Sehein et al 2016). Fur- thermore, the ratio of applicable microsatellites to the amount of tested markers in our study (partial genome 7.8%, transcriptome 23%) is comparable to the success rate of other studies that use high-throughput sequencing (Nagai et al 2014;Minter et al 2015).…”

Assessing Symbiodinium diversity in scleractinian corals via next‐generation sequencing‐based genotyping of the ITS2 rDNA region

“…To conduct single-cell PCRs on Symbiodinium , we developed an approach modified from Frommlet & Iglesias-Rodríguez (2008) . Cells from cultures CCMP2467, KB8, rt-064 and rt-147 were harvested with the following changes: 1 mL of each culture was filtered through a 40-μm cell strainer (BD Biosciences, San Jose, CA), spun down at 1934 g for 10 min and washed 3 times with 1 mL of 1× TE buffer.…”

Bloom formation potential in the harmful dinoflagellate Akashiwo sanguinea: Clues from movement behaviors and growth characteristics

“…For example, microsatellite markers have revealed considerable genetic variability in field populations of phytoplankton (e.g. Rynearson and Armbrust, 2005;Frommlet and Iglesias-Rodriguez, 2008) and this clonal diversity has been linked to differences in traits such as genome size (Whittaker et al, 2012), morphology (Saravanan and Godhe, 2010), and growth rate (Rynearson and Armbrust, 2004;Campbell et al, 2003). While trait based approaches typically emerge over order of magnitude ranges (e.g.…”