Efficiency of microsatellite isolation from orchids via next generation sequencing

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is costand time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16 plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.