Linchun Shi

Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.

CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences

BackgroundThe complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need.ResultsWe have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR) are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities.ConclusionsCPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible fromhttp://www.herbalgenomics.org/cpgavas.

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA.

Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.

A renaissance in herbal medicine identification: from morphology to DNA

Numerous adverse reactions have arisen following the use of inaccurately identified medicinal plant ingredients, resulting in conditions such as aristolochic acid nephropathy and herb-induced poisoning. This problem has prompted increased global concern over the safety of herbal medicines. DNA barcoding, a technique aiming at detecting species-specific differences in a short region of DNA, provides a powerful new tool for addressing this problem. A preliminary system for DNA barcoding herbal materials has been established based on a two-locus combination of ITS2+psbA-trnH barcodes. There are 78,847 sequences belonging to 23,262 species in the system, which include more than 95% of crude herbal drugs in pharmacopeia, such as those of China, Japan, Korea, India, USA, and Europe. The system has been widely used in traditional herbal medicine enterprises. This review summarizes recent key advances in the DNA barcoding of medicinal plant ingredients (herbal materia medica) as a contribution towards safe and efficacious herbal medicines.

Utility of the trnH-psbA intergenic spacer region and its combinations as plant DNA barcodes: a meta-analysis.

Background The trnH–psbA intergenic spacer region has been used in many DNA barcoding studies. However, a comprehensive evaluation with rigorous sequence preprocessing and statistical testing on the utility of trnH–psbA and its combinations as DNA barcodes is lacking. Methodology/Principal Findings Sequences were searched from GenBank for a meta-analysis on the usefulness of trnH–psbA and its combinations as DNA barcodes. After preprocessing, we constructed full and matching data sets that contained 17 983 trnH–psbA sequences and 2190 sets of trnH–psbA, matK, rbcL, and ITS2 sequences from the same sample, repectively. These datasets were used to analyze the ability of trnH–psbA and its combinations to discriminate species by the BLAST and BLAST+P methods. The Fishers exact test was used to evaluate the significance of performance differences. For the full data set, the identification success rates of trnH–psbA exceeded 70% in 18 families and 12 genera, respectively. For the matching data set, the identification rates of trnH–psbA were significantly higher than those of the other loci in two families and four genera. Similarly, the identification rates of trnH–psbA+ITS2 were significantly higher than those of matK+rbcL in 18 families and 21 genera. Conclusion/Significane This study provides valuable information on the higher utility of trnH–psbA and its combinations. We found that trnH–psbA+ITS2 combination performs better or equally well compared with other combinations in most taxonomic groups investigated. This information will guide the optimal usage of trnH–psbA and its combinations for species identification.

Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae

Abstract  In 2009, the Consortium for the Barcode of Life (CBOL) recommended the combination of rbcL and matK as the plant barcode based on assessments of recoverability, sequencing quality, and levels of species discrimination. Subsequently, based on a study of more than 6600 samples belonging to 193 families from seven phyla, the internal transcribed spacer (ITS) 2 locus was proposed as a universal barcode sequence for all major plant taxa used in traditional herbal medicine. Neither of these two studies was based on a detailed analysis of a particular family. Here, Zingiberaceae plants, including many closely related species, were used to compare the genetic divergence and species identification efficiency of ITS2, rbcL, matK, psbK–psbI, trnH–psbA, and rpoB. The results indicate that ITS2 has the highest interspecific divergence and significant differences between inter‐ and intraspecific divergence, whereas matK and rbcL have much lower divergence values. Among 260 species belonging to 30 genera in Zingiberaceae, the discrimination ability of the ITS2 locus was 99.5% at the genus level and 73.1% at the species level. Thus, we propose that ITS2 is the preferred DNA barcode sequence for identifying Zingiberaceae plants.

DNA Barcode Goes Two-Dimensions: DNA QR Code Web Server

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, “DNA barcode” actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

Transcriptome analysis of the Ophiocordyceps sinensis fruiting body reveals putative genes involved in fruiting body development and cordycepin biosynthesis.

Ophiocordyceps sinensis is a highly valuable and popular medicinal fungus used as a tonic and roborant for thousands of years in traditional Asian medicine. However, unsustainable harvesting practices have endangered this species and very little is known about its developmental programming, its biochemistry and genetics. To begin to address this, the transcriptome of the medicinal O. sinensis fruiting body was analyzed by high-throughput. In this O. sinensis 454-EST dataset, four mating type genes and 121 genes that may be involved in fruiting body development, especially in signal transduction and transcription regulation, were discovered. Moreover, a model was developed for the synthesis of the primary medicinal compound, cordycepin, and the putative biosynthetic enzymes identified. This transcriptome dataset provides a significant new resource for gene discovery in O. sinensis and dissection of its valuable biosynthetic and developmental pathways.

Stability and Accuracy Assessment of Identification of Traditional Chinese Materia Medica Using DNA Barcoding: A Case Study on Flos Lonicerae Japonicae

DNA barcoding is a novel molecular identification method that aids in identifying traditional Chinese materia medica using traditional identification techniques. However, further study is needed to assess the stability and accuracy of DNA barcoding. Flos Lonicerae Japonicae, a typical medicinal flower, is widely used in China, Korea, and other Southeast Asian countries. However, Flos Lonicerae Japonicae and its closely related species have been misused and traded at varying for a wide range of prices. Therefore, Flos Lonicerae Japonicae must be accurately identified. In this study, the ITS2 and psbA-trnH regions were amplified by polymerase chain reaction (PCR). Sequence assembly was performed using CodonCode Aligner V 3.5.4. The intra- versus inter-specific variations were assessed using six metrics and “barcoding gaps.” Species identification was conducted using BLAST1 and neighbor-joining (NJ) trees. Results reveal that ITS2 and psbA-trnH exhibited an average intraspecific divergence of 0.001 and 0, respectively, as well as an average inter-specific divergence of 0.0331 and 0.0161. The identification efficiency of ITS2 and psbA-trnH evaluated using BLAST1 was 100%. Flos Lonicerae Japonicae was formed into one clade through the NJ trees. Therefore, Flos Lonicerae Japonicae can be stably and accurately identified through the ITS2 and psbA-trnH regions, respectively.

Identification of Lonicera japonica and its related species using the DNA barcoding method.

To choose a suitable DNA marker to authenticate the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae, seven candidate DNA bar codes (i.e., RBCL, MATK, PSBA-TRNH, ITS2, ITS, TRNL intron, and TRNL-F intergenic spacer) were tested on forty-four samples of LONICERA JAPONICA and its closely related species using the DNA barcoding method. We found that all seven candidate bar codes yielded 100 % PCR amplification efficiency and that the sequencing efficiency of the five other candidate bar codes was 100%, with the exception of ITS and ITS2. The highest interspecific divergence was provided by the PSBA-TRNH intergenic spacer, followed by the TRNL-F intergenic spacer based on six parameters and Wilcoxon signed rank tests. Through the inspection of the histograms of the barcoding gap, the distribution of the PSBA-TRNH intergenic spacer was well separated; and only this candidate DNA bar code possessed the highest species identification efficiency at 100 % by BLAST1 method. In conclusion, using the PSBA-TRNH intergenic spacer as a DNA bar code is suitable for the identification of the botanical origins of Flos Lonicerae Japonicae and Flos Lonicerae. This study may provide an important example for the authentication of the botanical origin of medicinal herbs listed in the Chinese Pharmacopoeia.