Two new highly polymorphic microsatellite loci and inadvertent minisatellite loci for Lymnaea auricularia

© The Malacological Society of London 2004 Microsatellite loci are useful tools for resolving population genetic histories. Because of their high mutation rates, they often reveal fine-scale variation between recently diverged populations that other markers (e.g. allozymes) may fail to detect. We report here on the development of species-specific microsatellite primers for Lymnaea (Radix) auricularia (Linnaeus, 1758) (Pulmonata, Lymnaeidae) which we undertook in order to recover the history of its arrival in Lake Baikal. Lymnaea auricularia is a palaearctic generalist not recorded from Baikal until around 1960, and only recently found in large numbers. Populations in Baikal are morphologically divergent from potential shallow water source populations (M. Stift, E. Michel, T.Ya. Sitnikova, K.Yu. Mamonova & D.Yu. Sherbakov, unpublished observations). This raises questions of population isolation, selection, and invasion dynamics that can be addressed with microsatellites. We also tested cross-species amplification of existing primers that were developed for the closely related Lymnaea truncatula. L. auricularia, Lymnaea truncatula and L. peregra form a well-supported clade in an 18S rDNA-based phylogeny. Genomic DNA was isolated from parasite-free foot tissue of L. auricularia using a modified CTAB method. For the isolation of the microsatellite loci we used a modified FIASCO method (Fast Isolation by AFLP of Sequences Containing repeats). DNA was digested with restriction enzymes MseI and TaqI, followed by adaptor ligation. Fragments were amplified by a polymerase chain reaction (AFLP-PCR). Biotin-labelled di-, triand tetra-nucleotide repeat sequences were hybridized to the PCR product and the hybridization complex was lifted out with streptavidin-coated magnetic spheres (Promega) . After washing, the bound DNA was eluted from the magnetic spheres and re-amplified. The DNA fragments were cloned using the plasmid pGEM– TEasy vector (Promega) and transformed into Escherichia coli JM109 High Efficiency Competent Cells (Promega). We screened the colonies for the presence of a repeat-insert using PCR with universal M13 primers and the biotinylated microsatellite sequence. Using alkaline lysis, we recovered plasmids from 72 insert-containing colonies and sequenced them using the Amersham sequencing kit. Sequencing reaction products were run on a Li-Cor 4200 automatic sequencer and analysed with E-Seq software (Li-Cor V.1.1) yielding 16 unique repeatcontaining sequences of both microsatellites (2-, 3-, 4-nucleotide repeats) and minisatellites (> 20-nucleotide repeats). We used the Primer3 program 9 to select primer pairs for these sequences. A total of nine sequences had suitable flanking regions for forward and reverse primer development. We tested these and the L. truncatula primer sets in polymerase chain reactions (PCR) with a total volume of 10 l containing approximately 10 ng DNA, 0.1 g BSA, 1 l PCR buffer (HT Biotechnology), 100 mM Tris–HCl, pH 9.0, 15 mM MgCl2, 500 mM KCl, 1% Triton X-100, 0.1 % (w/v) stabilizer), 10 M primer, 0.25 mM of each dNTP and 1U Taq (HT Biotechnology Ltd). Each reaction was exposed to the following temperature regime: an initial denaturation of 2 min at 94°C, followed by 35 cycles of amplification at 94°C for 30 s, 30 s at the optimal annealing temperature (Table 1), 45 s at 72°C, and an additional 10 min at 72°C. The PCR-products of all loci were visualized on agarose gels (1%) stained with ethidiumbromide. The minisatellite loci were immediately scored by hand. All three were monomorphic. For the microsatellite loci that amplified (two presented for the first time here, one of L. truncatula), PCR was repeated with IR-700 labelled primers. The PCR products were visualized on 6.5% polyacrylamide gels and run on a LiCor 4200 automatic sequencer. Three primer pairs produced interpretable and repeatable amplification products. We tested variability on samples from 11 populations (sample sizes ranging from five to ten) of L. auricularia from Lake Baikal and its RESEARCH NOTES