When Technology Meets Conservation: Increased Microsatellite Marker Production Using 454 Genome Sequencing on the Endangered Okaloosa Darter (Etheostoma okaloosae)

Abstract

High-throughput sequencing affords a cost and time effective means of obtaining large numbers of genetic markers for conservation studies. Here, we present thousands of novel microsatellite loci developed for the Okaloosa darter, Etheostoma okaloosae, an endangered percid fish. We sequenced more than 29 million bp using 454 whole genome shotgun sequencing and employed free user-friendly bioinformatics tools to screen for microsatellite loci and design appropriate primers. We tested 39 primer sets for polymorphism and ran population-level analyses on a population of Okaloosa darters. Of these, 30 markers were variable with an observed and expected heterozygosity of 0.382 and 0.430, respectively, and allele numbers ranging from 2 to 13. Comparisons against the zebra fish reference genome, Danio rerio, revealed that these loci represent an adequate chromosomal coverage of the darter genome, although total genomic coverage was only 2.4-3.3%. We also tested these loci on the brown darter, E. edwini, and identified loci that will be useful for hybridization studies between these taxa.