MICROSATELLITE LETTERS Isolation and characterization of polymorphic microsatellite loci in the endangered San Diego fairy shrimp (Branchinecta sandiegonensis) Joelle M. Andrews • Andrew J. Bohonak • Marie A. Simovich Received: 11 November 2013 / Accepted: 25 November 2013 / Published online: 6 December 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract A set of ten polymorphic microsatellite loci were isolated and characterized for the endangered San Diego fairy shrimp (Branchinecta sandiegonensis). These loci were amplified on a set of 24 fairy shrimp collected from vernal pools located throughout San Diego County. The loci selected are highly variable across a wide sam- pling range (3–29 alleles per locus, 3–20 heterozygotes observed). After directly testing for Mendelian inheritance through family screens, eight markers did not show evi- dence of null alleles. This novel set of microsatellite markers will be useful in future genetic studies to assess intraspecific diversity, population connectivity and mating patterns. Keywords Vernal pools Á Fairy shrimp Á Anostraca Á Branchinecta Á Microsatellites Á Population genetics The San Diego fairy shrimp (Branchinecta sandiegonensis) inhabits a narrow, discontinuous range of vernal pool habitat on coastal chaparral-covered mesas and inland foothills of southern California, and is the most commonly found fairy shrimp species in this range (Eriksen and Belk 1999). Loss of vernal pool habitat in San Diego due to human disturbance has been extensive, with only small remnants of most vernal pool landscapes remaining (Bau- der and McMillan 1998). As a result, Branchinecta sand- iegonensis was listed as an endangered species (U.S. Fish and Wildlife Service [USFWS] 1997) among other sensi- tive species unique to these habitats. Here, we report the isolation and characterization of polymorphic B. sandie- gonensis microsatellite loci that will be useful in future population genetic studies. Genomic DNA from 10 adult B. sandiegonensis indi- viduals, each from a different population, was extracted using the DNeasy Blood and Tissue kit (Qiagen), and sent to Genetic Identification Services (GIS; Chatsworth CA, USA) to develop eight enriched microsatellite libraries with CA-, GA-, AAC-, CAG-, AAG-, ATG-, TACA-, and TAGA- repeats. Following digestion of genomic DNA with HindIII restriction enzyme, fragments 350–700 bp long were subjected to magnetic bead capture using biotinylated capture molecules. Enriched fragments were ligated into pUC19 plasmids and electroporated into E. coli. Colonies containing the insert were isolated and plasmid DNA was amplified via PCR. Resulting PCR products were sequenced using Amersham’s DYEnamic TM ET Termina- tor Cycle Sequencing Kit, followed by electrophoresis on an Applied Biosystems Model 377 DNA Sequencer in order to detect the presence of microsatellites. The eight enrichment libraries yielded a total of 98 cloned sequences containing microsatellites, and 66 of these sequences have suitable flanking regions. Primers were designed and tested by GIS against fifteen individuals and genomic library DNA. From these 66 sequences, 20 loci were found in preliminary screens to be polymorphic. All polymorphic loci underwent additional quality control screens to ensure that they were suitable for population genetic analysis. Additional primer sets for each locus were designed using J. M. Andrews (&) Á A. J. Bohonak Department of Biology, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-4614, USA e-mail: jswerdna23@aol.com A. J. Bohonak e-mail: abohonak@mail.sdsu.edu M. A. Simovich Department of Biology, University of San Diego, 5998 Alcala ´ Park, San Diego, CA 92110, USA e-mail: simo@sandiego.edu 123 Conservation Genet Resour (2014) 6:401–403 DOI 10.1007/s12686-013-0103-6