TECHNICAL NOTE The development of 24 polymorphic microsatellite loci for the endangered barred galaxias, Galaxias fuscus, through next generation DNA sequencing Adam D. Miller • Anthony van Rooyen • Renae M. Ayres • Tarmo A. Raadik • Peter Fairbrother • Andrew R. Weeks Received: 3 January 2012 / Accepted: 8 February 2012 / Published online: 28 February 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Barred galaxias, Galaxias fuscus, is an endan- gered freshwater fish confined to headwaters of mountain streams in the Goulburn River system in central Victoria, Australia. Next generation DNA sequencing was used to develop a suite of microsatellite markers that can be used for future population genetic assessments of G. fuscus.A total of 24 polymorphic loci were identified and 12 char- acterized using 30 individuals from a single sample locale. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.58; mean expected heterozygosity = 0.37) with no evidence of individual loci deviating significantly from Hardy–Wein- berg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indi- cated no evidence of null alleles across loci. The 12 markers characterized in the present study provide a valuable resource for future population genetic assessments and management of G. fuscus in Australia. Keywords Microsatellite development Á Next generation DNA sequencing Á Galaxias fuscus Á Barred galaxias Á Endangered freshwater fish Barred galaxias, Galaxias fuscus Mack, 1936 (Galaxiidae), is a small, scaleless, non-migratory freshwater fish endemic to headwaters of mountain streams in the south-eastern portion of the Goulburn River system in central Victoria, Australia. The species is currently listed as endangered under the Commonwealth Environment Protection and Biodiversity Conservation Act 1999, and a National Recovery Plan has been developed to facilitate the recov- ery and survival of this highly vulnerable species. As part of this plan a comprehensive population genetic analysis of G. fuscus across its current distribution was recommended to determine patterns of population genetic structure, gene flow, and genetic diversity (Raadik et al. 2010). Here we report the development and characterization of novel microsatellite markers that will facilitate future population genetic assessments of the species. The 454 next generation sequencing platform was used to identify microsatellite markers for G. fuscus. Approxi- mately 10 lg of genomic DNA was extracted from muscle tissue from a single G. fuscus specimen using a QIAGEN DNA Easy kit (Qiagen). DNA was subsequently processed by the Australian Genome Research Facility (AGRF) where it was nebulized, ligated with 454 sequencing primers and tagged with a unique oligo sequence allowing sequences to be separated from pooled species DNA sequences using post-run bioinformatic tools. The DNA sample was analyzed using high throughput DNA A. D. Miller (&) Á A. van Rooyen Á A. R. Weeks Cesar, 293 Royal Parade, Parkville, VIC 3052, Australia e-mail: amiller@cesaraustralia.com A. D. Miller Department of Zoology, The University of Melbourne, Parkville, VIC 3010, Australia A. D. Miller School of Life and Environmental Sciences, Deakin University, Warrnambool, VIC 3280, Australia R. M. Ayres Á T. A. Raadik Á P. Fairbrother Department of Sustainability and Environment, Arthur Rylah Institute for Environmental Research, Heidelberg, VIC 3084, Australia A. R. Weeks Department of Genetics, The University of Melbourne, Parkville, VIC 3010, Australia 123 Conservation Genet Resour (2012) 4:613–616 DOI 10.1007/s12686-012-9605-x