Evaluating The Feasibility Of Using Candidate DNA Barcodes In Discriminating Dicotyledon

With high levels of evolution, adaptation and diversities, the dicotyledon plants are widely distributed and are of great importantance of medicinal and biological values. The identification of different species is difficult especially based solely on morphological characteristics; additionally, the limitations in traditional taxonomy prevent these techniques from meeting the complicated demands of species recognition. DNA barcoding is a novel technology that uses a standard DNA sequence to facilitate simple and accurate species identification.DNA barcoding is a rapidly developing frontier technology, it has become a hotspot and has gained worldwide attentions in the scientific academica. The studies about how to select, evaluate and verify the appropriated DNA barcode candidate has been reported increasingly.Up until now, a consensus has not been reached regarding which DNA sequences can be used as the best plant barcodes. In 2009, the DNA regions rbcL, matK have been recommended by Plant working group of CBOL (the Consortium for the Barcode of Life) as DNA barcodes for plants, yet the successful identification rate of this barcoding combination is only 73% at specific level, the work is "far from perfect". In 2010, Chen et al. compared the practicality of using the suggested barcode sequences against a large number of medicinal plants and recommended ITS2 as a DNA barcode. But the study to evaluate these regions for species identification in the large plant taxon which includes a large number of closely related species is rarely reported. Thus, works of this area keep on performing.We tested the feasibility of using four proposed DNA regions(matK, rbcL, psbA-trnH and nrDNA ITS2, ITS) for discriminating plant species within Fabaceae (Choripetalae) and Asteraceae (Sympetalae), the two large families of dicotyledon which include a large number of closely related species.Our results demonstrate that ITS2 was useful in regards to its universality, sequence variation, "barcoding gap" and identification capability of the Fabaceae and Asteraceae family. In Fabaceae, twenty-four species found in the Chinese Pharmacopoeia, along with another sixty-six species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1,507 sequences derived from 1,126 species belonging to 196 genera. The study of "identification of Astragalus Plants in China using ITS2" further verified the power of ITS2.41 of 47 Astragalus close related species have been identified by ITS2. Besides, ITS2 secondary structure is of significance for the systematic and taxonomic study. Therefore, ITS2 may be a useful barcode for Astragalus species, with great application value. In Asteraceae, ITS2 performed best of other markers. We also explored the species discriminating power of ITS2 on a large-scale within Asteraceae using 3,490 sequences representing 2,315 species belonging to 494 different genera and found that it correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively (BLAST 1 method).matK is a useful barcode for Fabaceae species. Of 1,355 plant samples collected from 1,079 species belonging to 409 diverse genera, matK precisely identified approximately 80% and 96% of them at the species and genus levels, respectively. However, at present, the use of matK as a universal barcode remains problematic due to the absence of universal primers. The psbA-trnH plastid region is as a powerful marker to the authentication of Fabaceae medicinal plants. We correctly differentiate 22 species belonging to 19 genera listed in the Chinese Pharmacopoeia only using the psbA-trnH plastid region. Moreover, the psbA-trnH regions could also distinguish between 97 medicinal plant species of Fabaceae commonly used including their adulterants, and seven adulterants of outgroup species. Yet, there were some poor quality psbA-trnH sequences mainly due to the presence of long mononucleotide repeats. In addition, the psbA-trnH sequences in public database is not much, large-scale verification can not be done.Altogether, ITS2 region has already been recommended as a core marker for taxonomic classification and barcoding in dicotyledon based on its universal and high species identification rate within Fabaceae and Asteraceae. Furthermore, we propose that matK and psbA-trnH be used in combination with ITS2 as standard barcodes. This approach will significantly broaden the application of DNA barcoding to resolve classification problems at the genera and species levels of dicotyledon and provide public with a powerful tool for practical use.