Molecular Ecology Notes (2002) 2, 101–103 © 2002 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTE Characterization of 14 tetranucleotide microsatellite loci derived from Pacific herring J. B. OLSEN, C. J. LEWIS, E. J. KRETSCHMER, S. L. WILSON and J. E. SEEB Alaska Department of Fish and Game, Gene Conservation Laboratory, 333 Raspberry Road, Anchorage, Alaska 99518 –1599, USA Abstract Spawning aggregations of Pacific herring (Clupea pallasi) often exhibit significant interan- nual variation in allele frequencies of neutral gene markers. We isolated 14 tetranucleotide microsatellites to examine hypothetical processes that may produce this unique genetic sig- nal. We developed and tested primer pairs for each locus and then estimated locus varia- bility in samples (n = 60) from two populations. The number of alleles per locus ranged from five to 49. The expected heterozygosity across loci and populations ranged from 0.20 to 0.96. These microsatellites will be useful for estimating genetic variation in herring on a fine geographical scale. Keywords: microsatellites, Pacific herring, population genetics Received 19 August 2001; revision accepted 13 September 2001 Pacific herring (Clupea pallasi ) in the Gulf of Alaska and Bering Sea exhibit distinct life history and morphological traits as well as large and temporally stable variation at biochemical and molecular genetic markers (Grant & Utter 1984; Rowell et al. 1991; Seeb et al. 1999). In contrast, herring from spatially distinct spawning aggregations within these two sea basins often exhibit significant interannual genetic variation that is as great or greater than the genetic variation among herring from geographically proximate spawning locations (Seeb et al. 1999). This important finding is consistent with similar observations of temporal instability made in Atlantic herring ( Clupea harengus L., McQuinn 1997) and other pelagic marine fishes including California sardine (Sardinops sagax caeruleus) and northern anchovy (Engraulis mordax) and has been termed ‘chaotic patchiness’ (Hedgecock 1994). In order to evaluate the hypothetical processes producing this unique genetic signature in herring, we plan to examine patterns of genetic variation among age cohorts within and between spatially distinct but geographically proximate spawn- ing aggregates. O’Connell et al. (1998) describe five polymorphic microsatellites that provide sufficient statistical power to detect genetic structure among some neighbouring herring populations. Unfortunately, these five dinucleotide loci exhibit complicated shadow banding (‘stutter’) when amplified by polymerase chain reaction (PCR) and are difficult to genotype. We have isolated 14 polymorphic tetranucleotide microsatellites in herring that exhibit little or no stutter. Here we report the development of primers and estimates of variability for these novel microsatellites. Sequences for 19 novel DNA fragments ( ∼ 350 –550 bp) containing TAGA tetranucleotide microsatellites were identified by Genetic Identification Services (GIS, Chats- worth, CA) using an enrichment protocol similar to Edwards et al. (1996). Genomic DNA from a single Pacific herring was partially restricted with a cocktail of seven blunt-end cutting enzymes (RsaI, HaeIII, BsrB1, PvuII, StuI, ScaI, EcoRV). Fragments in the size range of 300 –750 bp were adapted and subjected to magnetic bead capture (CPG Inc., Lincoln Park, NJ), using (TAGA) 8 biotinylated cap- ture molecules (Integrated DNA Technology, Coralville, IA). Captured molecules were amplified and restricted with HindIII to remove the adapters. The resulting frag- ments were ligated into the HindIII site of pUC19. Recom- binant molecules were electroporated into Escherichia coli DH5alpha. Recombinant clones were selected at random for sequencing. Sequencing was performed in a MJ Research PTC-200 thermocycler using ABI Prism Taq DyeDeoxy™ terminator chemistry. The sequences were visualized on an ABI 373 DNA sequencer. Primer pairs for 19 sequences were designed using the program primer3 (Rozen & Skaletsky 1996). Unlabelled Correspondence: Jeffrey B. Olsen. Fax: 907-267-2442; E-mail: jeff_olsen@fishgame.state.ak.us