Development and characterization of 24 microsatellite markers in a eublepharid gecko, Goniurosaurus kuroiwae
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MICROSATELLITE LETTERS
Development and characterization of 24 microsatellite markers in a eublepharid gecko, Goniurosaurus kuroiwae Takaki Kurita • Hiroaki Aoyama • Seikoh Saitoh Naoya Shinzato • Masanao Honda • Mamoru Toda
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Received: 6 October 2013 / Accepted: 14 October 2013 / Published online: 20 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The Ryukyu ground geckos, which comprise Goniurosaurus kuroiwae (sensu stricto) and four close relatives in the Ryukyu Archipelago, face conservation threats, and are listed as endangered in the Red List of both IUCN and Japan. We developed 24 microsatellite markers in G. kuroiwae and cross-amplified for another species, G. orientalis. All 24 and 22 loci were successfully amplified and genotyped in G. kuroiwae and G. orientalis, respectively. The number of alleles per locus and expected heterozygosity at polymorphic loci ranged from 1 to 11 and 0.050–0.891, respectively, in G. kuroiwae, and ranged from 1 to 15 and 0.050–0.932, respectively, in G. orientalis. Keywords Beads-enriched library Eublepharidae Next-generation sequencing The Ryukyu Archipelago
The Ryukyu ground geckos, Goniurosaurus kuroiwae (sensu stricto) and four close relatives, are ground dwelling
Electronic supplementary material The online version of this article (doi:10.1007/s12686-013-0074-7) contains supplementary material, which is available to authorized users. T. Kurita (&) Graduate School of Engineering and Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan e-mail: [email protected] H. Aoyama S. Saitoh N. Shinzato M. Toda Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan M. Honda Faculty of Biology, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
eublepharid geckos endemic to the Ryukyu Archipelago, Japan. All of these species face threats from habitat destruction, predation by introduced carnivorous mammals, and illegal hunting by pet traders. Management of speciesspecific conservation programs requires the knowledge of genetic diversity and population structure on each island in the Ryukyus, and to this end we developed 24 microsatellite loci for G. kuroiwae by use of a beads-enrichment technique and next-generation sequencing, and then crossamplified the microsatellites in its congener, G. orientalis. Total genomic DNA was extracted from muscle tissue of G. kuroiwae collected from Okinawa-jima Island. We conducted microsatellite enrichment using (AC)12, (AAC)9, and (AAAC)7 30 end biotinized oligonucleotide probes following Kurita et al.’s (2012) method. After recovery of DNA concentration by PCR, the PCR product of 300–800 bp was isolated using QIAquick Gel Extraction Kit (Qiagen). We conducted sub-cloning and Sangar sequencing for a part of the PCR product following Kurita et al.’s (2012) procedure, and prepared the rest of product through end repairing and adaptor ligation for the library of 454 GS Junior (Roche). The 454 GS Junior run yielded 67,652 reads, 12,408 o
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