High-Resolution Melting Analysis for the Discovery of Novel Single-Nucleotide Polymorphisms in Rainbow and Cutthroat Trout for Species Identification MOLLY T. MCGLAUFLIN* AND MATT J. SMITH International Program for Salmon Ecological Genetics, School of Aquatic and Fishery Sciences, University of Washington, 1122 Northeast Boat Street, Box 355020, Seattle, Washington 98195, USA JONATHAN T. WANG Applied Biosystems, 850 Lincoln Centre, Foster City, California 94404, USA SEWALL F. YOUNG Washington Department of Fish and Wildlife, 1111 Washington Street SE, Olympia, Washington 98501, USA NENG CHEN Department of Pathology, Stanford University, 3373 Hillview Avenue, Stanford, California 94305, USA YONG C. LEE Applied Biosystems, 850 Lincoln Centre, Foster City, California 94404, USA CARITA PASCAL AND LISA W. SEEB International Program for Salmon Ecological Genetics, School of Aquatic and Fishery Sciences, University of Washington, 1122 Northeast Boat Street, Box 355020, Seattle, Washington 98195, USA JUNKO STEVENS Applied Biosystems, 850 Lincoln Centre, Foster City, California 94404, USA JAMES E. SEEB* International Program for Salmon Ecological Genetics, School of Aquatic and Fishery Sciences, University of Washington, 1122 Northeast Boat Street, Box 355020, Seattle, Washington 98195, USA Abstract.—We describe the use of high-resolution melting (HRM), a recent enhancement to traditional DNA melting analyses for the characterization of polymerase chain reaction products, in the identification of 11 novel single-nucleotide polymorphisms (SNPs) for distinguishing between rainbow trout Oncorhynchus mykiss and cutthroat trout O. clarkii. Single-nucleotide polymorphisms provide an important tool for species identification in studies of hybridization and introgression between these two species, but until now the time-consuming and costly nature of DNA sequencing has limited identification of new markers. The ability of HRM to accurately discern nucleotide changes in a DNA sequence make it a cost- and time-effective alternative to traditional sequencing for the detection of novel SNPs. Hybridization, both natural and human-influenced, is a problem that plagues the conservation and manage- ment of trout species across their range. Hybrid individuals can be difficult to identify, are often misclassified, and complicate the dynamics and genetic composition of populations. Cutthroat trout Oncorhyn- chus clarkii is a polytypic species, consisting of 14 subspecies and a variety of life history forms widely distributed across the western United States (Behnke and Tomelleri 2002). Rainbow trout O. mykiss,a closely related species, also contains numerous sub- species and often overlaps with the range of native cutthroat trout (Behnke and Tomelleri 2002). Natural hybridization is known to occur between sympatric populations of rainbow and cutthroat trout in some areas where subspecies of the two have evolved in sympatry, in some cases reaching hybridization levels of 85% (Campton and Utter 1985; Kozfkay et al. 2007). This is particularly the case for coastal cutthroat trout O. c. clarkii) and steelhead (anadromous rain- bow trout O. mykiss), as well as some populations of * Corresponding author: jseeb@u.washington.edu Received June 13, 2009; accepted October 21, 2009 Published online March 4, 2010 676 Transactions of the American Fisheries Society 139:676–684, 2010 Ó Copyright by the American Fisheries Society 2010 DOI: 10.1577/T09-103.1 [Note]