Efficiency of microsatellite isolation from orchids via 454 sequencing

Background/Question/Methods

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 cost- and time- intensive. Recent advancement in next generation DNA sequencing techniques has expedited efficient isolation of microsatellites, and this technique has been successfully applied in various organisms including plants. 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 conventional methods. No published reports are available on isolation of orchid microsatellites by using high-throughput sequencing. For the first time in orchids, we report isolation of microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides) using 454 GS-FLX sequencing. For this, we combined the DNA of two species for enrichment and sequencing to reduce the cost. These two species represent two subfamilies within the Orchidaceae, but both are perennial orchids native to North America. Both are insect-pollinated and each is presumed to be outcrossing. Little or no information on population genetics of these species is available and microsatellites were not available for either species until now.

Results/Conclusions

From 1/16^th plate that was subjected to sequencing, 15,473 fragments containing at least one SSR were generated. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides; di-nucleotide SSRs contained at least eight repeats and tri- and tetra-nucleotides at least seven repeats. Among the three repeat types, the average length was higher for tri-nucleotides (102 bp) than for the di- (86 bp) or tetra-nucleotides (68bp). While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. Forty-four primer pairs were selected, based on our stringent criteria, for PCR tests. Of these, 20 showed clear bands of expected size in C. kentuckiense and 10 in P. ophioglossoides including four primer pairs that worked for both species. Previously reported attempts of orchid SSR development utilizing conventional methods typically yielded fewer than 100 SSRs per species which ultimately resulted in fewer than 10 useable loci in each species. Results demonstrate that the efficiency of microsatellite isolation is much higher with 454 GS-FLX sequencing technique in comparison to the conventional methods. Additionally, the combined DNA approach for enrichment and sequencing helped reduce the cost of microsatellite isolation for orchids and may possibly be applied to other organisms.