Submitted to: European Weed Research Society Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: January 10, 2010
Publication Date: July 12, 2010
Citation: Jeanneau, M., Bon, M., Martin, J. 2010. Meaningful application of the new 454 large scale pyrosequencing technology (Roche GS-FLX 454) to the identification of microsatellites for small-scale research projects. 15th European Weed Research Society Symposium Proceedings, Kaspovar, Hungary 12-15 July 2010.
Technical abstract: Microsatellites or simple sequence repeats are DNA sequences that consist of tandem repeats of 1-6 nucleotides. Because of high levels of polymorphism, ease to use and co-dominance, they are generally seen as the most pertinent markers to study at a fine scale level the genetic structure and demographic history of invasive weed populations and their natural enemies. However, their development using the classical microsatellite-library by enrichment remain typically time consuming and labor intensive, impeding their generalization at the level of small-scale research projects. We considered in the present study the extent to which the new generation sequencing such 454 Life Sciences/Roche GS-FLX pyrosequencing based-technology, could lead to a rapid, more efficient and less costly way to identify microsatellites for a small-scale research project than the classical microsatellite-library by enrichment. This system relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in an oil-aqueous mixture emulsion. The DNA fixed to these beads is then amplified by PCR and each DNA-bound bead is placed into a fiber optic chip for pyrosequencing into the GS FLX System. DNA was sourced from Ceutorhynchus assimilis (Coleoptera: Curculionidae), one natural enemy of the invasive weed, Lepidium draba sp draba, (Brassicaceae). From the resulting 12,000 sequences data after one run only, we were able to identify 333 microsatellite markers that have sufficient flanking sequence available for primer design. In contrast, only 16 potential microsatellite markers were discovered following sequencing of 93 clones of a previous classical microsatellite-enriched library. This strategy is substantially more rapid, and economical than classical methods and can be easily applicable to small scale research project.