Evaluation of pyrosequencing for large-scale identification of plant species (grasses as a model)

Grasses (Poaceae) are among the most important and widely cultivated plants. Identification of grass species using morphological characters is a difficult task for nonspecialists, and it is not always an accurate method. DNA-based molecular markers have been widely used as an alternative towards more accurate identification of plant species. The molecular method that can be effective for large-scale identification of grass species, however, must satisfy requirements for specific amplification of DNA and reveal enough variability to distinguish species. The goal of this study was to evaluate the ability of pyrosequencing to detect SNP polymorphisms for an optimal discrimination of grass species. PCR amplifications and the sequencing of 19 fragments of chloroplast genes were performed using a pooled DNA template of 32 British native grass species to be identified and chloroplast DNA-specific universal primers. Based on amplicon size and sequence variance, the 6 most variable loci (psbE&F, rbcL. 1, ndhF. 1, ndhF. 2, ndhF. 3, and clpP&rps12) were selected to be targeted for pyrosequencing of the species analyzed. The pyrosequenced regions contained sufficient polymorphisms to allow the complete diagnosis of all species analyzed, except for Elymus caninus and Elytrigia repens, which had identical sequences unique from the remaining species. When targeted loci were pyrosequenced in representative species of the 2 economically important grass genera Festuca L. (8 species) and Poa L. (11 species), all species in each genus could be separated. Should the SNP markers developed here proved to be species-specific, they can provide a valuable tool for reliable diagnosis of grasses and applications (e.g., forensic botany) moving forward.