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Identifying plant, fungal, and animal ingredients in a specific mixture remains challenging during the limitation of PCR amplification and low specificity of traditional methods. Genomic DNA was extracted from mock and pharmaceutical samples. Four type of DNA barcodes were generated from shotgun sequencing dataset with the help of a local bioinformatic pipeline. Taxa of each barcode was assigned by blast to TCM-BOL, BOLD, and GenBank. Traditional methods including microscopy, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were carried out according to Chinese pharmacopoeia. On average, 6.8 Gb shotgun reads were sequenced from genomic DNA of each sample. Then, 97, 11, 10, 14, and one operational taxonomic unit (OTU) were generated for ITS2, psbA-trnH, rbcL, matK, and COI, respectively. All the labeled ingredients including eight plant, one fungal, and one animal species were successfully detected in both the mock and pharmaceutical samples, in which Chebulae Fructus, Poria, and Fritilariae Thunbergia Bulbus were identified via mapping reads to organelle genomes. In addition, four unlabeled plant species were detected from pharmaceutical samples, while 30 genera of fungi, such as Schwanniomyces, Diaporthe, Fusarium were detected from mock and pharmaceutical samples. Furthermore, the microscopic, TLC, and HPLC analysis were all in accordance with the standards stipulated by Chinese Pharmacopoeia. This study indicated that shotgun metabarcoding could simultaneously identified plant, fungal, and animal ingredients in herbal products, which has the ability to serve as a valuable complement to traditional methods.

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.

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.

BackgroundInternal 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 findingsIn 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.ConclusionsIdentification 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.

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 Fisher's 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.

BackgroundThe 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 findingsHere, 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.ConclusionsThe 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.