TECHNICAL NOTE Twelve microsatellite markers in the invasive tunicate, Didemnum vexillum, isolated from low genome coverage 454 pyrosequencing reads Cathryn L. Abbott • Daniel Ebert • Amy Tabata • Thomas W. Therriault Received: 20 July 2010 / Accepted: 21 July 2010 / Published online: 11 August 2010 Ó Her Majesty the Queen in Right of Canada 2010 Abstract 454 pyrosequencing reads were used to isolate microsatellites in the global marine ascidian invader, Didemnum vexillum. This method allows simple and cost- effective isolation of new markers from organisms without existing genomic information and, to our knowledge, has not been used before to develop a polymorphic microsat- ellite marker set. Loci had between two and eleven alleles and overall mean observed and expected heterozygosities of 0.57 and 0.62, respectively. These markers will greatly facilitate research required to develop control and mitiga- tion strategies for D. vexillum. Keywords Didemnidae Á Ascidiacea Á SSRs Á Next generation sequencing Á Tunicate invasions Á Microsatellite isolation Globally, invasive species are one of the greatest threats to native biodiversity; in the marine environment, invasive ascidians are causing increased ecological and economic concern (e.g. Lambert 2007). Recently, the colonial tuni- cate Didemnum vexillum (Didemnidae:Ascidiacea) has received much attention due to its global invasion history (Therriault and Herborg 2007). On Georges Bank, D. vexillum has formed extensive mats that smother native benthic communities (Valentine et al. 2007). Microsatellite markers can be used to characterize spatial and temporal patterns of dispersal by invasive species (e.g. Dupont et al. 2009; Darling and Folino-Rorem 2009), helping to eluci- date dispersal vectors and pathways and identify potential management options. Next-generation sequencing should enable genome-scale research even on non-model organisms (Hudson 2008). However, short read lengths, cost, and intensive bioinfor- matics requirements are obstacles that may hinder the uptake of these technologies. Here we use 454 pyrose- quencing for the isolation of microsatellites in D. vexillum, thus displaying how next-generation sequencing can meet a routine need of molecular ecology and conservation genet- ics laboratories. 454 technology generates the longest read- length of the next-generation sequencing methods, enabling the use of reads from a low genome coverage sequence survey for microsatellite isolation. To our knowledge, this method has not been used before to develop a polymorphic microsatellite marker set (but see Castoe et al. 2010). Didemnum vexillum samples were collected into 70–95% ethanol. DNA was extracted using a DNeasy Tissue Kit (QIAGEN). 454 sequencing using a Roche GS-FLX sequencer was done at the McGill University and Genome Quebec Innovation Centre. One plus one-eighth standard runs were done, generating over 1.3 million reads and over 400 million bp of data and yielding many more candidate loci than needed testing to arrive at a final set of twelve polymorphic ones (see below). Average read length was over 300 bp. The extent of genome coverage achieved is uncertain because genome size in D. vexillum is unknown; however, based on the 160 MB Ciona intestinalis genome (Simmen et al. 1998), we estimate about 2.69 coverage. Approximately 23% of the 454 reads were assembled into 25,506 contigs of 500 nucleotides or longer (mean 779 bp). We used a custom Perl script (available upon request) to search contigs and raw 454 reads for C. L. Abbott (&) Á A. Tabata Á T. W. Therriault Pacific Biological Station, Fisheries & Oceans Canada, 3190 Hammond Bay Rd, Nanaimo, BC V9T 6N7, Canada e-mail: cathryn.abbott@dfo-mpo.gc.ca D. Ebert Department of Botany, University of British Columbia, 3529-6270 University Blvd, Vancouver, BC V6T 1Z4, Canada 123 Conservation Genet Resour (2011) 3:79–81 DOI 10.1007/s12686-010-9294-2