Design of Expressed-Sequence Tag-Linked Microsatellites for Non-model Aquacultured Species

Microsatellites physically linked to expressed-sequence tags (EST-SSRs) are an important resource for linkage mapping and comparative genomics in aquacultured species, and data-mining in publicly-available EST databases is a common strategy for EST-SSR discovery.  At present, however, many aquacultured species lack the species-specific genomic resources needed for efficient characterization of EST-SSRs.  This paper describes a phylogenetically-informed, comparative-genomics approach to EST-SSR discovery.  The approach employs existing genomic resources to efficiently design PCR primers by taking into account the degree to which flanking regions are conserved between taxa that are related phylogenetically at various levels to the species of interest. The utility of the approach is demonstrated using the red drum (Sciaenops ocellatus), an aquacultured species of economic importance in the United States and elsewhere.  The amplification success rate for primers designed for EST-SSRs was nearly 100% when using EST libraries from species in the same family and sub-order as red drum.  The primers developed also amplified EST-SSRs in a wide range of perciform fishes, suggesting a potential use in comparative genomics.  The study demonstrates that EST-SSRs can be efficiently developed for a species when limited species-specific sequence data are available by exploiting genomic resources from well-studied species, even those at extended taxonomic distances.  A software tool designed for the automation of this process is presented and the implications of this approach for linkage and comparative mapping in aquacultured species are discussed.