Polymorphic microsatellite DNA markers for the marine gastropod Littorina subrotundata

Coastal habitats of the northern temperate zone are home to over 19 species of Littorina (Reid 1996). Due to its widespread abundance and phenotypic diversity, members of this genus have been the model system of choice for numerous ecological and evolutionary studies (Reid et al . 1996 and references therein). Although mitochondrial DNA has been useful in the study of population structure in Littorina , it is clear from recent data (Kyle & Boulding 1998) that faster-evolving markers are needed for fine-scale metapopulation analysis. To this end, we have isolated 12 polymorphic microsatellite loci for Littorina subrotundata (Carpenter 1864), a direct-developing species from the North Pacific. We report preliminary data evaluating their potential utility as high-resolution genetic markers for this model taxon. The general methods for the isolation of simple sequence loci outlined in Rassmann et al . (1991) were followed, with the following alterations. Total genomic DNA from the visceral mass of L. subrotundata was isolated via organic extraction with methylene chloride/ isoamyl alcohol and subsequent ethanol precipitation (Claxton et al . 1997). DNA from five individuals was combined, digested with the restriction enzymes Hae III and Rsa I and size selected using 1% agarose gel electrophoresis. DNA fragments of 300–1000 bp in length were ligated into the Eco RV site of the pZErO plasmid vector (Invitrogen) which was then transformed into Top F ′ Escherichia coli cells (Invitrogen) via electroporation using standard methods ( E. coli Pulser, Bio-Rad). Approximately 32 000 colonies were screened with [ γ 33 P]-ATP-labelled AAT 10 , AAAT 6 (hybridization T = 52 ° C), AAG 8 , AAC 9 and GATA 7 (hybridization T = 55 ° C) oligonucleotide probes according to the methods of Shaw (1997; P. W. Shaw, personal communication). DNA sequences of inserts from clones positive in two successive hybridizations were obtained using the manufacturer’s suggested cycle sequencing protocol for the ABI model 377 Version 2.1.1. using M13 sequencing primers and labelled ddNTPs. Of the 50 sequences obtained, 48 contained unique microsatellites composed of at least one trinucleotide repeat array. PCR (polymerase chain reaction) primers for 10 of these loci were designed using Primer V0.5 (Lincoln & Daly 1991) and Gene Runner V3.0 (Hastings software) programs and are reported in Table 1. Two dinucleotide loci, VAM-F1 and Table 2 Cross-species amplifications data for Cebus apella and Lagothrix lagotricha microsatellites. Neotropical primates. Successful amplification is indicated by the observed genotype (in bp) of the single sample per species that was analysed