Development and characterization of twenty-three microsatellite markers for the freshwater minnow Santa Ana speckled dac
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TECHNICAL NOTE
Development and characterization of twenty-three microsatellite markers for the freshwater minnow Santa Ana speckled dace (Rhinichthys osculus spp., Cyprinidae) using paired-end Illumina shotgun sequencing Schyler O. Nunziata • Stacey L. Lance • Kenneth L. Jones • Stacey A. Nerkowski Anthony E. Metcalf
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Received: 6 August 2012 / Accepted: 13 August 2012 / Published online: 31 August 2012 Ó Springer Science+Business Media B.V. 2012
Abstract We isolated and characterized a total of 23 microsatellite loci from the Santa Ana speckled dace, Rhinichthys osculus spp., a freshwater minnow restricted to southern California. Loci were screened in 24 individuals from five watersheds. The number of alleles per locus ranged from 7 to 25, observed heterozygosity ranged from 0.409 to 0.875, and the probability of identity values ranged from 0.005 to 0.081. These new loci will provide tools for examining taxonomic status, population structure, and management strategies. Keywords Rhinichthys osculus Illumina Microsatellite PAL_Finder PCR primers SSR STR
A member of the Cyprinidae, the species Rhinichthys osculus is among the most widely distributed fish species in the western United States (Moyle 2002). Within Southern California R. osculus spp. is recognized as the subspecies Santa Ana speckled dace. The Santa Ana speckled dace was once widely distributed throughout the Santa Ana, San Gabriel, and Los Angeles river systems, but presently inhabits small isolated streams and creeks in the Santa Ana
S. O. Nunziata S. L. Lance Savannah River Ecology Laboratory, University of Georgia, Aiken, SC 29802, USA K. L. Jones Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, CO 80045, USA S. A. Nerkowski A. E. Metcalf (&) Department of Biology, California State University, San Bernardino, San Bernardino, CA 92407, USA e-mail: [email protected]
and San Gabriel watersheds. The extent range occupied by the dace is restricted due to habitat fragmentation attributed to human activity (Swift et al. 1993) and to the aftermath of the effects of fire and flood (SAWPA 2004). The Santa Ana speckled dace was listed as a species of special concern in 1995 by the California Department of Fish and Game and as a sensitive species by the United States Forest Service in 1998. However, listing as a federally endangered species was denied due to a lack of peer reviewed description, including genetic analyses (U.S. FWS 1996). Total DNA was extracted from one individual of R. osculus, following the protocol in the—Eppendorf Phase Lock Gel Manual (Mouse Tail Genomic DNA Isolation Protocol) for use in isolation of microsatellite loci. An Illumina pairedend shotgun library was prepared by shearing 1 lg of DNA using a Covaris S220 and following the standard protocol of the Illumina TruSeq DNA Library Kit and using a multiplex identifier adaptor index. Illumina sequencing was conducted on the HiSeq with 100 bp paired-end reads. Five million of the resulting reads were analyzed with
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