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Published on: June 23, 2012
Microsatellite marker development by multiplex ion torrent PGM sequencing: a case study of the endangered Odorrana
Takeshi Igawa1, Masafumi Nozawa2, Mai Nagaoka2
1From the Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan (Igawa, Komaki, and Sumida); the Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka, Japan (Nozawa); the Department of Genetics, The Graduate University of Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan (Nozawa); the Department of Health Sciences, Hiroshima Prefectural University, Hiroshima, Japan (Nagaoka and Fujii); and the Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan (Oumi). Takeshi Igawa is now at the Division of Development Science, Department of Developmental Science, Graduate School of International Development and Cooperation, Hiroshima University, Higashi-Hiroshima 739-8529, Japan. tigawa@hiroshima-u.ac.jp.
Abstract:
The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used next-generation sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the three-related species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies.
