TECHNICAL NOTE Isolation and characterization of 21 polymorphic microsatellite loci in the Japanese dace (Tribolodon hakonensis) Noriyuki Koizumi • Thomas W. Quinn • Myeongsoo Park • Jennifer Fike • Kazuya Nishida • Takeshi Takemura • Keiji Watabe • Atsushi Mori Received: 14 January 2011 / Accepted: 10 February 2011 / Published online: 19 February 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Twenty one polymorphic microsatellite loci for the Japanese dace (Tribolodon hakonensis) were isolated and characterized. The number of observed alleles per locus in 32 individuals ranged from 3 to 30. The observed and expected heterozygosities ranged from 0.125 to 0.969 and from 0.175 to 0.973, respectively. All loci conformed to Hardy–Weinberg equilibrium, no linkage disequilibrium was observed between pairs of loci and no loci showed evidence of null alleles. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species. Keywords Cyprinidae Á Microsatellite primers Á Genetic diversity Á Conservation Á Rural ecosystem The Japanese dace (Tribolodon hakonensis) is distributed throughout Japan with the exception of the Ryukyu Islands. Individuals inhabit streams from the stream mouth to upstream regions and are also found in adjoining agricultural canals. In spring and summer they spawn in shallow waters with gentle flow (Kawanabe and Mizuno 1989). This species is a commercial and game fish in some areas. Populations have been adversely impacted by river improvement and land consolidation projects (Moriyama et al. 2008) leading to the designation of the Japanese dace as a species of conservation concern. Although Hanzawa et al. (1987) identified twelve mitochondrial DNA haplotypes among three locations on Honshu Island, this information is insuf- ficient for investigating the intraspecific genetic diversity and detailed spatial structure of the populations within rural areas, prefectures, or regions. Therefore, we isolated and characterized 21 polymorphic microsatellite loci that could be useful for conservation genetic studies, following Koiz- umi et al. (2007a, b; 2009). Genomic DNA from a fin clip of an adult collected from the Nishikinu River, Tochigi Prefecture, was extracted using a standard phenol/chloroform procedure (Asahida et al. 1996). A partially enriched microsatellite genomic library was constructed using the protocol of Glenn and Schable (2005) with some modifications. Briefly, DNA was digested with the restriction enzyme RsaI (New England Biolabs) and was then ligated to double-stranded Super- SNX linkers. Linker-ligated DNA was denatured and hybridized to two biotinylated microsatellite probes [(CA) 12 and (CT) 12 ], and the resultant hybridized com- plexes were captured on streptavidin-coated beads (Dynal) treated with a blocking step (St. John and Quinn 2008). After washing unhybridized DNA away, the remaining DNA was eluted and amplified using the Polymerase Chain Reaction (PCR) following the procedure of Glenn and Schable (2005). PCR products were cloned using a TOPO- TA Cloning Kit (Invitrogen). Cloned inserts from 192 colonies were isolated and amplified using M13 primers (Glenn and Schable 2005), N. Koizumi (&) Á K. Nishida Á T. Takemura Á K. Watabe Á A. Mori National Institute for Rural Engineering, Tsukuba, Ibaraki 305-8609, Japan e-mail: koizumin@affrc.go.jp T. W. Quinn Department of Biological Sciences, University of Denver, Denver, CO 80208, USA M. Park Rural Research Institute, Ansan, Gyeonggi 426-908, Korea J. Fike U.S. Geological Survey, Fort Collins Science Center, Fort Collins, CO 80526-8118, USA 123 Conservation Genet Resour (2011) 3:565–567 DOI 10.1007/s12686-011-9405-8