Molecular Ecology Resources (2008) 8, 881–883 doi: 10.1111/j.1755-0998.2008.02097.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Isolation and characterization of polymorphic microsatellite and COI loci from the whelk Kelletia kelletii CROW WHITE* and ROBERT J. TOONEN† *Department of Ecology, Evolution & Marine Biology, University of California, Santa Barbara, CA 93106, USA, †Hawaii Institute of Marine Biology, University of Hawaii at Manoa, Kaneohe, HI 96744, USA Abstract There is considerable interest in the genetic structure of Kelletia kelletii because of its economic, ecological and scientific importance. To that end, we developed species-specific primers which amplify mitochondrial cytochrome c oxidase subunit I as well as 13 hyper- variable nuclear microsatellite loci. Using dye-labelled primers, the microsatellite loci can be co-amplified in two multiplex polymerase chain reactions and scored simultaneously on an automated sequencer. Keywords: COI, gastropoda, Kellet’s whelk, Kelletia kelletii, microsatellite, multiplex PCR Received 3 October 2007; revision accepted 3 December 2007 Kellet’s whelk, a subtidal gastropod in US and Mexico Pacific waters, is a rapidly increasing fishery species (Aseltine-Neilson et al. 2006), a significant predator in kelp forest ecosystems (Halpern et al. 2006), a possible indicator species linked to El Niño conditions (Zacherl et al. 2003), and the focus of microchemistry analyses estimating its larval dispersal patterns (Zacherl 2005). Given considerable interest in the genetic structure of Kelletia kelletii because of its economic, ecological and scientific importance, we report here development of highly polymorphic microsatellite markers as well as species-specific primers for the mito- chondrial cytochrome c oxidase subunit I (COI) locus for K. kelletii. We isolated microsatellite-containing sequences from dynabead-enriched DNA libraries following Glenn & Schable’s protocol (2005). Briefly, we extracted ~2 μg high molecular weight genomic DNA from ~25 mg foot tissue using the QIAGEN DNeasy kit, digested it using the restriction enzyme RsaI (New England Biolabs), and ligated double-stranded SuperSNX forward and reverse linkers. Fragments containing microsatellite sequences were hybrid- ized onto complementary, biotinylated oligonucleotides combined into two mixed probes: a low-temperature probe consisting of [(ATC) 8 G(AGG) 6 G(CCG) 5 (AAT) 10 (AAG) 8 (ACT) 8 (AAC) 8 G(ACG) 6 C(ACC) 6 C(AGC) 6 ] hybridized at 45 °C, and a high-temperature probe consisting of [(AAGC) 5 (AACC) 5 (AACG) 5 (AAGG) 5 (ATCC) 5 ] hybridized at 50 °C. Hybridized microsatellite-containing fragments were captured by Streptavidin M-280 Dynabeads (Dynal), isolated with a magnet, amplified using the SuperSNX forward primer and cloned using the One Shot TOP10 kit (Invitrogen). We isolated 96 colonies from each probe. We polymerase chain reaction (PCR)-amplified 187 colonies using M13 forward and reverse primers. These amplicons were sequenced in both directions using an Applied Biosystems (ABI) 3130xl automated sequencer with BigDye Terminator version 3.1 sequencing reagent, then edited and aligned using sequencher version 4.7 (Gene Codes). Of the 187 sequenced products, 26 from the low- and 13 from the high-temperature probe had no obvious micro- satellite repeats, and 38 (low) and 13 (high) lacked sufficient flanking sequences to develop primers. Of the 97 remaining sequences, primer 3 (Rozen & Skaletsky 2000) was used to design primers for 59 loci, 51 of which were ordered from Integrated DNA Technologies (IDT) and tested via agarose gel electrophoresis. Electrophoresis of dye-labelled primers (6-FAM from IDT; all others from ABI) for all 51 microsat- ellite loci was subsequently performed on an ABI PRISM 3100 and scored with genemapper version 4.0 (ABI). Using DNA extractions from eight K. kelletii individuals collected across the species’ range, 25 of the 51 loci amplified reliably and were polymorphic. Additional microsatellite markers were developed at the University of California, Davis in parallel with Correspondence: Crow White, Fax: 805-893-7612; E-mail: crowsfeather @gmail.com