Microsatellite primers designed from pyrosequences to compare two subspecies of surf clam, Spisula solidissima
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Microsatellite primers designed from pyrosequences to compare two subspecies of surf clam, Spisula solidissima Matthew P. Hare • Harmony Borchardt-Wier
Received: 9 June 2014 / Accepted: 9 July 2014 / Published online: 19 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract It was recently discovered that two cryptic subspecies of surf clam co-occur in near-shore waters of southern New England, Spisula solidissima solidissima and Spisula solidissima similis. The latter form was previously known only from south of Cape Hatteras. We developed and multiplexed nine fluorescent microsatellite markers that can be scored in both subspecies to compare genetic diversity, test for hybrids, or inform assignment tests. Keywords 454 pyrosequence Windowmasker Msatcommander Georgia Massachusetts Recently, genetic analyses demonstrated that a commercially fished surf clam population in Long Island Sound is not an ecotype of the oceanic surf clam, Spisula solidissima solidissima, as previously believed (Hare et al. 2010). Instead, it is a disjunct population of the ‘‘southern’’ surf clam, (Spisula solidissima similis), a genetically distinct subspecies with previously known populations found only south of Cape Hatteras. These subspecies are morphologically cryptic, so genetics will be an important tool for documenting the extent of S.s.similis in southern New England, testing for hybridization where the two taxa co-occur, and dating the northern populations to test whether their origin is recent enough to be associated with climate change. Genomic DNA of one specimen per subspecies was supplied to Evolutionary Genetics Core Facility at Cornell University for microsatellite enrichment, barcoded library preparation and 454 pyrosequencing (Roche GS-FLX Titanium) using methods described in Andres and M. P. Hare (&) H. Borchardt-Wier Department of Natural Resources, Cornell University, Ithaca, NY 14853, USA e-mail: [email protected]
Bogdanowicz (2011). A total of 56,763 barcoded 454 reads from S.s.similis were assembled into 25,139 contigs using SeqMan Pro (Lasergene v 8.1.1, DNASTAR, Inc.). For S.s.solidissima there were 117,044 reads assembled into 48,402 contigs. Msatcommander (Faircloth 2008) was used to identify contigs with at least 5 simple tandem repeats (STR) and design PCR primers. Four loci were selected for testing by scanning Msatcommander output for loci with relatively large expected PCR amplicon length, containing a moderate number of STR repeats, low to moderate contig read counts, and with primer sequences occurring in only 1–2 S.s.similis contigs (i.e., nonrepetitive; loci Ssim3823, Ssim1436, Ssim9323, Ssim10007). For the rest, local BLASTn was used to find microsatellite flanking sequences represented in contig fasta files from both subspecies. Windowmasker (Morgulis et al. 2006) was used to find and mask highly repetitive sequence in S.s.solidissima contigs before blasting them against S.s.similis contigs and saving hits with e-value \1e-10. Hits were considered for m
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