TECHNICAL NOTE Development and characterization of 20 polymorphic microsatellite markers for the Texas hornshell, Popenaias popeii (Bivalvia: Unionidae), through next-generation sequencing Kentaro Inoue • Brian K. Lang • David J. Berg Received: 21 August 2012 / Accepted: 23 August 2012 / Published online: 6 September 2012 Ó Springer Science+Business Media B.V. 2012 Abstract We identified 28 microsatellite loci from Pop- enaias popeii, a freshwater mussel that has experienced population declines throughout its range. Twenty loci were polymorphic, with 4–10 alleles, observed heterozygosity values of 0.375–1.00, and 16 % of alleles shared by more than one population. These loci should be useful for describing population genetic diversity, which will facili- tate ongoing conservation efforts for P. popeii. Keywords 454 shotgun pyrosequencing  De novo sequencing  Freshwater mussels  Microsatellite primers Freshwater mussels (Unionoidea) are among the most endangered groups of animals in North America (Lydeard et al. 2004). Popenaias popeii (Lea 1857), the Texas horn- shell, is endemic to the Rio Grande drainages in the south- west US and northern Mexico, along with coastal drainages in northeastern Mexico (Howells et al. 1996; Strenth 2004). However, current US populations of P. popeii are limited to three rivers: lower Rio Grande and Devils River in Texas and a single 14 km reach of the Black River in New Mexico (Lang 2001); the status of Mexican populations is unknown (Strenth 2004; Karatayev et al. 2012). The New Mexico population is isolated from Texas populations due to large dams and unsuitable water quality (Howells et al. 1996; Lang 2001; Strenth 2004). Threats to this species are primarily anthropogenic activities including modification of physical conditions in streams, and reduction in water quality due to groundwater extraction and increased salinity levels result- ing from irrigation water returns. Population reduction and current threats have made this species a candidate for listing under the US Endangered Species Act (US Federal Register 2005). Although life history and population demography for this species have been studied (Smith et al. 2003; Levine et al. 2012), genetic assessment of extant populations is critical to inform conservation and management strategies. Development of highly variable microsatellite markers will allow such assessment of genetic structure within and among populations. High throughput, next-generation sequencing (also known as 454 shotgun pyrosequencing) allows creation of massive genomic sequences that provide opportunities to develop microsatellite markers (Guichoux et al. 2011). We developed microsatellite markers for P. popeii using this de novo sequencing approach. We extracted genomic DNA from one individual using a DNeasy Kit (Qiagen, Inc.). The Plant–Microbe Genomics Facility at Ohio State University performed shotgun pyrosequencing using a Roche 454 FLX Titanium Genome Sequencer, which produced a total of 161,714 reads averaging 394 bp in length corresponding to *63.8 Mbp generated from 1/4th of a picotiterplate. Putative microsatellite loci were identified from the dataset using MSATCOMMANDER v1.0.8 (Faircloth 2008) to design primers on flanking regions of microsatellite loci using PRIMER3 (Rozen and Skaletshky 2000) as its primer design engine and adding M13R or CAG tail sequence at the 5 0 -end of either forward or reverse primers. K. Inoue (&) Department of Zoology, Miami University, Oxford, OH 45056, USA e-mail: inouek@muohio.edu B. K. Lang New Mexico Department of Game and Fish, Santa Fe, NM 87507, USA D. J. Berg Department of Zoology, Miami University, Hamilton, OH 45011, USA 123 Conservation Genet Resour (2013) 5:195–198 DOI 10.1007/s12686-012-9766-7