Plant Dna Barcode Sequence Screening And Identification

DNA barcoding is a new technique that uses a short DNA sequence from a standardized and agreed-upon position in the genome as a molecular diagnostic for species-level identification, it is an effective complement for traditional taxonomy based on morphology. At present, the research and application of DNA barcoding in plants is in exploration stage, so the most important task is to select candidate genes or DNA regions and identify a suitable DNA barcode. In this study, we compared different candidate DNA barcodes from gymnosperms to evaluate their universality. In addition, we also aimed at two specific families (Rosaceae and Euphorbiaceae) to test the effectiveness of different candidate barcodes for identifying plants, which will accelerate the determination of universal DNA barcodes for plants and help to build up a comprehensive DNA barcode library. Based on the results of experimental research, the main results are as follows:1. In order to determine an effective DNA barcode for gymnosperms, seven DNA regions(psbA-trnH, rbcL, matK, rpoB, rpoCl, ITS and ITS2) were evaluated on four criteria including the success rates of PCR amplification, the extent of specific genetic divergence, the DNA barcoding gap and the ability for species discrimination. Our results suggest that ITS2 is most appropriate for barcoding gymnosperms among the seven loci tested. To further evaluate the ability of ITS2 for identifying gymnosperms, we also tested it in 888 gymnosperm samples collected from 502 species of 80 diverse genera in 12 families. ITS2 correctly identified 73% and 98% of them at the species and genus levels, respectively.2. We tested the applicability of four DNA regions (rbcL, matK, rpoCl and ITS2) as the barcodes for identifying species within Rosaceae. Based on assessments of the success rates of PCR amplification, the extent of specific genetic divergence, the DNA barcoding gap and the ability for species discrimination, our results suggest that ITS2 is the best of the four loci tested for barcoding Rosaceae. We further evaluated the effectiveness of ITS2 for identifying a wide range of species within Rosaceae. Of the 1410 plant samples collected from 893 species in 96 diverse genera, ITS2 successfully identified 78% and 100% of them at the species and genus levels, respectively. Moreover, we also evaluated the species identification ability of the ITS2 region in 12 genera that are rich in closely related species using the BLAST1 method. Although problems were encountered in the genera (Amelanchier, Alchemilla and Rosa), using ITS2 for species identification resulted in an 86%-97% success rate for five genera (Acaena, Polylepis, Pyrus, Malus, and Fragaria) and an 69%-76% success rate for four other genera (Crataegus, Cliffortia, Rubus and Prunus). For three genera(Cliffortia, Prunus and Rubus) with more than 100 species per genus in our samples, ITS2 also performed well with a>70% successful identification rate.3. We compared the specific genetic divergence, the DNA barcoding gap and the ability for species discrimination of four DNA regions (rbcL, matK, ITS and ITS2) of species within Euphorbiaceae, the present results affirmed that ITS/ITS2 is a potential barcode for Euphorbiaceae species. This study also provided a large-scale test to evaluate the effectiveness of ITS/ITS2 for differentiating species within Euphorbiaceae. For the 1,183 plant samples collected from 871 species in 66 diverse genera, ITS/ITS2 successfully identified>90% and 100% of them at the species and genus levels, respectively. Furthermore, we evaluated the two loci in 7 genera(Andrachne, Mallotus, Euphorbia, Croton, Phyllanthus, Macaranga and Glochidion) within Euphorbiaceae. For the samples collected, using ITS/ITS2 for species identification resulted in an 68-100% success rate for the genera. For two genera(Euphorbia and Croton) with more than 240 species in our sample, ITS/ITS2 also performed well with 88-99% successful identification rates.The research systematically analyzed and compared the effectiveness of different candidate DNA barcodes for identifying plants using gymnosperms, Rosaceae and Euphorbiaceae. The ITS2 locus is a powerful barcode among all three plant taxa tested, so our results strongly suggest that the ITS2 region should have a great potential to be used as one of the universal barcodes for plants. Our work contributes valuable information for identifying species in other plant taxonomic groups and promotes the determination of the standard DNA barcode for plants.