Characterization of polymorphic microsatellite markers for the tideland snail Batillaria flectosiphonata
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Characterization of polymorphic microsatellite markers for the tideland snail Batillaria flectosiphonata Hajime Itoh • Satomi Kamimura • Kimiko Hirose Shigeaki Kojima
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Received: 5 February 2015 / Accepted: 16 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Batillaria flectosiphonata is distributed in the Ryukyu Islands, south-western Japan, and shows genetic differentiation among island groups. This species is vulnerable to environmental fluctuations because the connectivity among the groups is low. Therefore, the conservation of each group is necessary to maintain their genetic diversity. Using next-generation sequencing, 10 microsatellite markers were developed for B. flectosiphonata. In Miyara Bay and the Sakashita River estuary, the numbers of alleles per locus were 1–9 and 1–8, and the expected heterozygosities were 0–0.859 and 0–0.655, respectively. Keywords Batillaria flectosiphonata Microsatellite Next-generation sequencing Batillaria snails are a major group in the tidal areas of Japan. Batillaria flectosiphonata is distributed in the Ryukyu Islands and according to a mitochondrial analysis, displays different genetic structures among island groups (Kojima et al. 2003). Therefore, each local population is presumed to be an evolutionarily significant unit and vulnerable to environmental fluctuations, such as coastal reclamation and future climate change, because the connectivity among the groups is low. However, most of the Electronic supplementary material The online version of this article (doi:10.1007/s12686-015-0452-4) contains supplementary material, which is available to authorized users. H. Itoh (&) K. Hirose S. Kojima Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8564, Japan e-mail: [email protected] S. Kamimura Graduate School of Urban Engineering, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi, Osaka 558-8585, Japan
habitats of Batillaria snails on Okinawajima Island are destined for development or have already been reclaimed (Okinawa Prefecture 2005), and B. flectosiphonata is designated a near-threatened species in some locations. Polymorphic loci are more useful for monitoring genetic diversity for species management and conservation than a single mitochondrial gene. Therefore, we developed 10 microsatellite markers for B. flectosiphonata using nextgeneration sequencing. Total DNA was extracted from a single individual of B. flectosiphonata collected from the Konase River estuary on Amami-Oshima Island. Its DNA (500 ng) was sheared by nebulization to a mean fragment size of approximately 400 bp and then converted into a sequence-ready template library with the Roche 454 Rapid Library protocol (Roche). Emulsion PCR was performed with the Lib-L emPCR kit (Roche). The Roche 454 GS Junior sequencer (Roche) was used for pyrosequencing, which generated 27,015 reads. In total, 1199 unique sequences exceeding 80 bp in length and containing microsatellite motifs consisting
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