Molecular Ecology Resources (2008) 8, 172– 174 doi: 10.1111/j.1471-8286.2007.01915.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Microsatellite markers for leatherside chubs Lepidomeda aliciae and Lepidomeda copei K. E. MOCK,* L. S. BJERREGAARD,* M. C. BELK,† C. ROWE * and J. B . JOHNSON†‡ *Department of Wildland Resources, Utah State University, Logan, UT 84322, USA, †Department of Biology and ‡Monte L. Bean Life Science Museum, Brigham Young University, Provo, UT 84602, USA Abstract We report the isolation and characterization of eight microsatellite markers in a rare desert cyprinid fish, the leatherside chub. This taxon has recently been divided into two species (Lepidomeda aliciae and Lepidomeda copei) based on genetic, ecological and morphological data, and we explore the utility of these microsatellite loci in both species. All eight loci show promise as highly polymorphic markers in L. aliciae, but only three of the markers appear to be useful in L. copei. Keywords: cyprinid, leatherside chub, Lepidomeda aliciae, Lepidomeda copei, microsatellite, primers, Utah Received 19 May 2007; revision accepted 19 June 2007 Leatherside chubs (previously Gila copei) are rare cyprinid fishes endemic to portions of western North America that have experienced dramatic population declines during the past century and have been extirpated from much of their historical range (Wilson & Belk 2001; Johnson et al . 2004). These species were originally characterized as a single taxon but have recently been shown to consist of two distinct species, Lepidomeda aliciae and Lepidomeda copei , corresponding to historical geographical subdivision (Johnson & Jordan 2000; Dowling et al . 2002; Johnson et al . 2004). The effective management of these taxa requires an understanding of gene flow and genetic diversity at the population and drainage levels, necessitating the devel- opment of polymorphic nuclear molecular markers. Micro- satellites would be particularly useful for the southern clade ( L. aliciae ) since it consists of several populations in multiple drainages, separated by barriers of varying age and porosity. Hence, we sought to develop a set of microsatellite markers for use in L. aliciae that might also be useful in L. copei . Fin clips collected from three L. aliciae individuals in each of three drainages (San Pitch, Provo and Sevier watersheds, Utah) and from three L. copei individuals from the Bear River, Wyoming were used for microsatellite development. DNA was extracted from these tissues following a salting-out protocol (Sunnucks & Hales 1996), and assessed using a 0.7% agarose gel stained with ethidium bromide. DNA from these individuals was pooled, and a microsatellite library was developed at the Savannah River Ecology Laboratory (SREL) following the protocol of Glenn & Schable (2005). From SREL, we obtained 96 forward and reverse sequences containing microsatellite tandem repeats. These sequences were ranked manually according to the motif, length and quality of repeats. Priming regions on candidate sequences were selected using primer 3 (Rozen & Skaletsky 2000) and primerselect software ( lasergene 5.1, DNASTAR). We screened these candidate primers and loci using polymerase chain reaction (PCR), optimizing reagent concentrations and reaction conditions. Based on this screening, we identified eight microsatellite loci that provided robust amplification, a high level of polymorphism, and which contained repeat motifs which were easily binned and scored (Table 1). Optimized PCR conditions for all loci included a reaction volume of 10 μ L containing 0.2 m m of each dNTP, 1.5 m m MgCl 2 , 1× PCR buffer (New England BioLabs), approximately 15 ng DNA template, and 0.2 U Taq DNA polymerase. PCR conditions for all loci consisted of an initial denaturation at 95 ° C for 2 min, followed by 30 cycles of 94 ° C for 30 s, 57 ° C for 30 s, and 72 ° C for 45 s. These cycles were followed by a final extension at 72 ° C for 7 min and a 4 ° C hold. For locus 27–5, we used an engineered 5 ′ primer tag (5 ′- CAGTCG- GGCGTCATCA) attached to the forward primer (Schable et al . 2002). The tag portion of the primer served as a bind- ing template for an additional primer (Boutin-Ganache et al . 2001). PCR conditions for this locus were identical to Correspondence: K. E. Mock, Fax: (435)797 3796; E-mail: karen.mock@usu.edu