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.     

Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.Cholangiocellular carcinoma (CCC)¹ is a malignant neoplasm that arises from the cholangiocytes, the epithelial cells lining the bile ducts. The tumors, consisting of a significant amount of fibrous stroma, are classified as intrahepatic, extrahepatic, or hilar according to their anatomic location. Most common are the Klatskin tumors, originating from the confluence of the right and left hepatic ducts (1). Compared with other types of cancer, CCC is a relatively rare disease, accounting for about 3% of all gastrointestinal malignancies (2). However, its incidence is increasing, and as a result of poor patient outcomes it has overtaken hepatocellular carcinoma as the main cause of death from a primary hepatobiliary tumor (3). Reasons for the high mortality rate (5-year survival rate of about 5%) (4) are the difficult diagnosis and limited treatment options. At present, extensive surgical resection of the extrahepatic bile ducts and parts of the liver or liver transplantation remain the only potentially curative treatment options, although most patients are considered inoperable at the time of diagnosis (5).In general, the diagnosis of CCC is made based on histomorphological evaluation of core biopsies or cytological specimens. However, distinction between CCC and benign diseases such as reactive bile ductules or bile duct adenomas can be challenging when based on conventional histology alone. Additionally, it may be difficult to distinguish CCC from metastatic adenocarcinoma in the liver, especially when it originates from the pancreas like pancreatic ductal adenocarcinoma. Therefore, specific immunohistochemical tissue markers for CCC would be highly beneficial for further validation of the diagnosis. In routine immunohistochemical diagnosis of CCC, so far, the detection of p53 (a product of a tumor suppressor gene) has proven useful, although its application is limited because of low sensitivity (6). The cytokeratins Ck7, Ck8, Ck18, and Ck19 have been reported to have sensitivities of between 80% and 97% for CCC cells, but at low specificities and a similar expression as in non-tumorous cholangiocytes (7). In addition, the tumor marker carcinoembryonic antigen, which is a commonly applied serum marker, has been used for immunohistochemical staining of CCC tissue. Although this was reported to be positive in 100% of the tested CCC sections, it also was immunoreactive in 60% of hepatocellular carcinomas (8). Recently, it has been shown that the polycomb group protein EZH2 may be useful for differential diagnosis of cholangiolocellular carcinoma (a subtype of CCC), bile duct adenomas, and ductular reaction. This, however, applies only to this certain type of CCC (9). Establishing reliable immunohistochemical tumor markers specific for CCC therefore remains a challenge.Several proteomic studies using different sample types and various techniques have been performed in order to identify CCC-specific proteins. The analysis of CCC cell lines, for example, has led to the identification of potential diagnostic and prognostic biomarker candidates (10–12). In addition, cell lines have been used to discover proteins predictive of the response to chemotherapy (13). Because results from cell culture experiments do not always reflect the actual conditions in the tumor, the use of patient samples can be advantageous. The most appropriate source of tumor-specific signals is tumor tissue, which in the past has been analyzed via two-dimensional electrophoresis (14) and mass-spectrometry-based proteomic approaches such as histology-directed MALDI-TOF-MS (15), Surface-enhanced laser desorption/ionization (SELDI) TOF-MS (16), or LC-MS/MS (17). So far, however, none of the potential biomarkers have been successfully implemented into clinical routine.Recently, we demonstrated that the application of two complementary techniques, two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass-spectrometry-based label-free LC-MS/MS, is an auspicious tactic for the discovery of novel biomarker candidates in hepatocellular carcinoma tissue (18). Here, we applied this well-established workflow as the initial step for the discovery of tissue markers that improve the differential diagnosis of intrahepatic CCC from benign bile duct diseases. In these experiments, CCC tumor tissue was compared with non-tumorous liver tissue (n = 8). Because this does not allow discrimination among different cell types such as hepatocytes, cholangiocytes, and tumor cells, an immunohistochemical determination of the cell-type-specific expression was subsequently performed for the most promising biomarker candidates. Stress-induced phosphoprotein 1, the protein showing the greatest specificity and sensitivity for CCC tumor cells, was verified as a suitable biomarker candidate for CCC in a larger patient cohort (n = 60).     

A Novel Method of Automatic Plant Species Identification Using Sparse Representation of Leaf Tooth Features

Automatic species identification has many advantages over traditional species identification. Currently, most plant automatic identification methods focus on the features of leaf shape, venation and texture, which are promising for the identification of some plant species. However, leaf tooth, a feature commonly used in traditional species identification, is ignored. In this paper, a novel automatic species identification method using sparse representation of leaf tooth features is proposed. In this method, image corners are detected first, and the abnormal image corner is removed by the PauTa criteria. Next, the top and bottom leaf tooth edges are discriminated to effectively correspond to the extracted image corners; then, four leaf tooth features (Leaf-num, Leaf-rate, Leaf-sharpness and Leaf-obliqueness) are extracted and concatenated into a feature vector. Finally, a sparse representation-based classifier is used to identify a plant species sample. Tests on a real-world leaf image dataset show that our proposed method is feasible for species identification.     

DNA条形码：物种分类和鉴定技术

当前,一项称为“生命的条形码”计划正在欧美等国展开,其目的是实现对地球上现存的约1000万物种进行快速和准确的鉴定。DNA条形码是一种利用短的DNA序列对物种进行鉴定的技术。对DNA条形码的概念和原理进行了介绍,举例说明了其在物种分类、遗传多样性及物种鉴定研究中广泛的利用价值,阐述了当前该领域的研究现状,对未来的发展方向进行了展望。     

Novel Sample Preparation for Mass Spectral Analysis of Complex Biological Samples

The ability to combine a selective capture strategy with on chip MALDI-TOF analysis allows for rapid, sensitive analysis of a variety of different analytes. In this overview a series of applications of capture enhanced laser desorption ionization time of flight (CELDI-TOF) mass spectrometry are described. The key feature of the assay is an off-chip capture step that utilizes high affinity bacterial binding proteins to capture a selected ligand. This allows large volumes of sample to be used and provides for a concentration step prior to transfer to a gold chip for traditional mass spectral analysis. The approach can also be adapted to utilize specific antibody as the basis of the capture step. The direct and indirect CELDI-TOF assays are rapid, reproducible and can be a valuable proteomic tool for analysis of low abundance molecules present in complex mixtures like blood plasma.     

Forensic DNA Analysis of Pacific Salmonid Samples for Species and Stock Identification

Identification of salmonid tissue samples to species or population of origin has been conducted for over 20 forensic cases in British Columbia. Species identification is based on published sequence variation in exon and intron regions of coding genes. Identification of source populations or regions is carried out using microsatellite and major histocompatibility complex allele frequency data collected from populations throughout the species range and with standard genetic stock identification (GSI) methods. Regional contributions to mixture samples are estimated using maximum likelihood mixture analysis and classification of individual genotypes is carried out with Bayesian methods. DNA has been obtained successfully from salmon scale samples, fresh, frozen and canned tissue samples and bloodstains in clothing. Results from DNA analyses have been instrumental in a number of convictions. A major benefit has been cost savings resulting from the number of guilty pleas entered after disclosure to the defendant of results from genetic testing. In two cases, GSI analysis resulted in exoneration of suspects under investigation for possible illegal sales of Fraser River sockeye salmon by substantiating their claim that the fish originated from the Skeena River watershed. DNA analysis has generally corroborated the species and stock identification carried out by fishery officers, but has revealed that species identification of samples from sources such as restaurants and fish plants can be erroneous. Forensic DNA analysis has facilitated the conviction of those who purchase fish not caught under the authority of licence, thus bringing those who buy fish illegally as well as those involved in illegal harvest and sales within the scope of law enforcement.     

12种园林植物耐荫性鉴定指标的筛选

在对遮荫条件下12种园林植物的生长增量、叶片厚度、比叶面积、叶片含水量、叶绿素a＋b含量、叶绿素a,b、类胡萝卜素含量、糖含量、过氧化物酶活性、日平均净光合速率、光补偿点、表观量子效率等生理生化指标测定基础上,对各项指标进行了主成分分析和逐步回归分析,结果表明生长增量、比叶面积、叶绿素a,b、过氧化物酶活性、目平均净光合速率等5个指标对植物的耐荫性有显著影响。采用所建立的最优回归方程对供试植物进行耐荫性预测及排序的结果与栽培试验观察结果相一致,表明这5个指标可作为快捷的植物耐荫性鉴定指标。     

Next-Generation Sequencing for Rodent Barcoding: Species Identification from Fresh,Degraded and Environmental Samples

Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.     

Identification of Novel Genetic Alterations in Samples of Malignant Glioma Patients

Glioblastoma is the most frequent and malignant human brain tumor. High level of genomic instability detected in glioma cells implies that numerous genetic alterations accumulate during glioma pathogenesis. We investigated alterations in AP-PCR DNA profiles of 30 glioma patients, and detected specific changes in 11 genes not previously associated with this disease: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3, and INPP5A. Further correlations revealed that 8 genes might play important role in pathogenesis of glial tumors, while changes in GP2, KCNG2 and KIR3DL3 should be considered as passenger mutations, consequence of high level of genomic instability. Identified genes have a significant role in signal transduction or cell adhesion, which are important processes for cancer development and progression. According to our results, LHFPL3 might be characteristic of primary glioblastoma, SGCG, HTR4, ITGB1, CPS1, PROS1 and INPP5A were detected predominantly in anaplastic astrocytoma, suggesting their role in progression of secondary glioblastoma, while alterations of PDE4D seem to have important role in development of both glioblastoma subtypes. Some of the identified genes showed significant association with p53, p16, and EGFR, but there was no significant correlation between loss of PTEN and any of identified genes. In conclusion our study revealed genetic alterations that were not previously associated with glioma pathogenesis and could be potentially used as molecular markers of different glioblastoma subtypes.     

Identification and Characterization of Potential Therapeutic Candidates in Emerging Human Pathogen Mycobacterium abscessus: A Novel Hierarchical In Silico Approach

Mycobacterium abscessus, a non-tuberculous rapidly growing mycobacterium, is recognized as an emerging human pathogen causing a variety of infections ranging from skin and soft tissue infections to severe pulmonary infections. Lack of an optimal treatment regimen and emergence of multi-drug resistance in clinical isolates necessitate the development of better/new drugs against this pathogen. The present study aims at identification and qualitative characterization of promising drug targets in M. abscessus using a novel hierarchical in silico approach, encompassing three phases of analyses. In phase I, five sets of proteins were mined through chokepoint, plasmid, pathway, virulence factors, and resistance genes and protein network analysis. These were filtered in phase II, in order to find out promising drug target candidates through subtractive channel of analysis. The analysis resulted in 40 therapeutic candidates which are likely to be essential for the survival of the pathogen and non-homologous to host, human anti-targets, and gut flora. Many of the identified targets were found to be involved in different metabolisms (viz., amino acid, energy, carbohydrate, fatty acid, and nucleotide), xenobiotics degradation, and bacterial pathogenicity. Finally, in phase III, the candidate targets were qualitatively characterized through cellular localization, broad spectrum, interactome, functionality, and druggability analysis. The study explained their subcellular location identifying drug/vaccine targets, possibility of being broad spectrum target candidate, functional association with metabolically interacting proteins, cellular function (if hypothetical), and finally, druggable property. Outcome of the present study could facilitate the identification of novel antibacterial agents for better treatment of M. abscesses infections.     

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57p^E17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.     

PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.     

Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.