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

Abstract

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.

Caps DNA Barcoding for Field Laboratory Identification of Grass Species (British Grasses as a Model)

“…The molecular method that can be effective for large scale identification of grass species, however, must be fast, reliable, and cost-effective high throughput method that reveals enough variability to distinguish species and at the same time generates highly conserved markers within species and implies specific amplification of DNA (Haider 2015). CAPS was proved useful for identification of several groups of plant species such as those of Aegilops L. and T. aestivum L. (Haider & Nabulsi 2008), Orchidaceae (Haider et al 2010), and Vicia subgenus (Fabaceae, Haider et al 2012).…”

“…Sanger sequencing (Haider 2016) and the more complicated pyrosequencing (Haider 2015) have been used recently for identification of grass species for which we present here the alternative CAPS technique that is much cheaper, faster, less complex, and utilises the technologies of ubiquitous PCR, digestion with endonucleases and agarose gel electrophoresis. When choosing among these three techniques, factors to consider include sensitivity, specificity, reproducibility, the limit of detection, turnaround time, ease of data interpretation, and cost (including instrument, reagent, etc.)…”

“…Amersham Biosciences, UK) and the concentration of each DNA template was set at 10 ng/μL.PCRs were performed in 20 µl reaction mixes comprising of 20 ng template DNA (2 µl @ 10 ng/µl), 15m M primer, and other reagents described byHaider (2015). We first used 16/19 universal primer pairs that were developed byHaider (2015) for targeting 16 regions of 13 genes of the chloroplast genomes of grasses to test for amplification on template DNAs from 117 British native grasses. These were: rps.4, psbE&psbF, rpoA.2, rbcL.…”