Molecular Ecology Resources (2008) 8, 1088–1090 doi: 10.1111/j.1755-0998.2008.02186.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Isolation and characterization of 11 polymerase chain reaction primers for microsatellite loci for the Chilean marine isopod Excirolana hirsuticauda P. A. HAYE and S. MARCHANT Departamento de Biología Marina, Facultad de Ciencias del Mar, Universidad Católica del Norte and Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Larrondo 1281, Casilla 117, Coquimbo, Chile Abstract The Chilean isopod Excirolana hirsuticauda is a marine benthic brooder with wide distri- butional range and low potential for long-distance dispersal. Eleven microsatellite markers were developed for E. hirsuticauda using enriched libraries. Characterization of those loci in 35 individuals from Playa Blanca beach showed high allelic diversity with a mean of 10.9 alleles per locus. The average expected and observed heterozygosities were 0.65 and 0.41. These microsatellite loci are the first published for any Excirolana species and should be useful to study the genetic structure of E. hirsuticauda. Keywords: Chile, connectivity, genetic diversity, Isopoda Received 15 December 2007; revision accepted 29 January 2008 Excirolana hirsuticauda is a marine benthic isopod endemic to the Chilean coasts and inhabits from Hornitos (22°29′S, 70°5′W) to Cucao (42°43′S, 74°14′W) (~2156 km). They live in the intertidal and subtidal sediment and like all isopods, they brood their eggs and release juveniles that can crawl. Thus, E. hirsuticauda does not have a larval dispersive stage. Also, given their small body size, these organisms have limited long-distance dispersal through swimming and crawling. Given the above, little gene flow between beaches along the coast of Chile is expected. Molecular markers such as microsatellites, that are codominant and highly polymorphic, should be useful to analyse the micro and macrogeographical genetic structure of this species. Herein, we report the isolation and characterization of 11 microsatellite loci for E. hirsuticauda. Restricted DNA fragments from 17 individuals of E. hirsuticauda collected from intertidal in Puñihuil (41°55′S, 74°00′ W) were enriched for four common microsatellite motifs (ATG, CAG, CAGA and TAGA) (Zane et al. 2002) by Genetic Identification Services (www.genetic-id-services.com). Fragments of 350–700 bp were ligated in to the BamHI site of the pUC19 plasmid to create microsatellite-enriched libraries. Ligation products were introduced into Escherichia coli strain DH5α (ElectroMAX, Invitrogen) by electroporation. After the transformation and recovery incubation in SOC broth (Invitrogen), 20% of glycerol was added to the final volume. Plating of 5 μL of this product produced between 100 and 300 recombinant colonies (more than 10 000 re- combinant cells/mL). Bluogal/IPTG/ampicillin–Luria-Bertani (BIA–LB) agar plates were used to isolate colonies for sequencing. For this, white colonies from spread stock plates were transferred in to a new BIA–LB agar plate and then incubated overnight at 37 °C in a 96-well block using 2× LB broth. Plasmid DNA was purified using Millipore Multiscreen MAFB NOB plates. A two-step 10-μL polymer- ase chain reaction (PCR) was performed to sequence the clones using M13 primers. The first step was to prepare a PCR mix with 1× PCR buffer, 3 mm MgCl 2 , 0.83 mm of dNTP mix, 1.5 μm of each primer, 0.0042 mg/mL of RNase and 4 μL of a picked colony, which was set to 100 °C for 10 min to lyse the bacteria and then at 37 °C to soak. The second step was to add 5 μL of a hot-start solution (1× PCR buffer and 0.375 U/μL of BIOTaq DNA polymerase) to the pre- pared mix and place it in the thermocycler for 21 cycles (94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s) with a final extension step at 72 °C for 2 min. DNA sequencing was performed in an Applied Biosystems PRISM 377 DNA Correspondence: P. A. Haye, Departamento de Biología Marina, Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Casilla 117, Coquimbo, Chile. Fax: +56-51-209821; E-mail: phaye@ucn.cl