Molecular Ecology Notes (2004) 4, 632 – 634 doi: 10.1111/j.1471-8286.2004.00742.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Development of Microsatellite Loci in Eulachon (Thaleichthys pacificus) K. H. KAUKINEN, K. J. SUPERNAULT and K. M. MILLER Department of Fisheries and Oceans Canada, Science Branch, Pacific Biological Station, Nanaimo, B.C., Canada V9R 5K6 Abstract The decline of eulachon spawning runs over the past decade has enhanced the need for management units that reflect the genetic structure of the species. We report the development of 14 novel, highly polymorphic di- and tetranucleotide loci for use in population studies. In a survey of over 200 individuals from two rivers from the Pacific NW, observed hetero- zygosities of the loci ranged from 0.54 to 0.95 and an average of 23 alleles per locus were resolved. All 14 microsatellite markers were in Hardy–Weinberg equilibrium. Keywords: library enrichment, Microsatellites, Osmeridae Received 18 March 2004; revision received 22 April 2004; accepted 16 June 2004 Eulachon, Thaleichthys pacificus, are small anadromous fish that occur from the southern Bering Sea to the Columbia River (Hay & McCarter 2000). These fish played an impor- tant role in the early economy of the First Nations people both as a food source and as a barter item. In recent years, there has been a sharp decline in eulachon spawning runs. As few as 30 runs remain, 15 of which are located within British Columbia (BC) (Hay & McCarter 2000). Effective management units for the conservation of eulachon popu- lations require information on the demographic structure of eulachons. Genetic studies have revealed only weak genetic structure in eulachons, indicative of a low level of imprinting in a species that spends limited time in freshwater residence. A mtDNA-based study by McLean et al . (1999) revealed a weak isolation by distance (IBD) mode of struc- ture among eulachon sampled from the Columbia River to Cook Inlet in central Alaska, with a degree of differenti- ation among sites more typical of marine than anadromous species. A follow-up study based on five minimally poly- morphic microsatellite loci revealed a greater degree of genetic variation among some sites, but the demographic units of structure were obscure and the patterns of differentiation did not support an IBD model (McLean & Taylor 2001). Together, these studies support the hypo- thesis that relative to other anadromous species, eulachons display low fidelity to natal spawning sites and contain relatively low levels of genetic variation, hence supporting the classification of eulachons into a single ESU. The present study was undertaken to develop microsatellite markers of eulachon, to determine whether eulachon are depauperate of genetic diversity and to access whether more highly poly- morphic microsatellite loci exist that could potentially resolve a higher level of demographic resolution among sites. A magnetic bead-based enrichment protocol similar to that described in McPherson et al . (2001) was used to iso- late tetranucleotide repeat loci from eulachon, with the following modifications: (1) 600–2000 bp fragment size selection following restriction digest and after the SNX- PCR prior to TA-cloning; (2) fewer SNX linker ligation cycles (five as opposed to 18); (3) additional PCR step after the linker ligation; (4) hybridization of the bead to the oligo at 48 ° C. Genomic eulachon DNA was prepared from liver tissue of a single adult individual. Initially, the library was enriched using GACA and GATA probes during the hybridization step. Of the 192 GACA enriched clones sequenced, 94 contained SSRs and primers to 16 potential loci were designed. The GATA enrichment was less successful, with only seven of the 48 clones sequenced containing GATA repeats; primers were designed to four of these. A second screening was conducted using AAC, CCTT, CCAT, GCAT, GGAT, CGAT, GACT, GATT, and TCAA oligonucleotide probes. Enrichment ranged from 0% (no SSRs isolated using CGAT, GACT, GATT and TCAA probes) to 50% (GGAT). Of the 144 enriched clones sequenced in the second screening, 40 contained micro- satellites, and primers for 11 potential loci were designed (Table 1). The level of enrichment for microsatellites aver- aged over all probes was 36% and was in stark contrast to the enrichment of other species of fish, invertebrates, and marine mammals, which averaged 61 – 88% (data not shown). Correspondence: K. M. Miller. E-mail: Millerk@dfo-mpo.gc.ca