DNA Barcoding Technology Research Articles

Authentication of Small Berry Fruit in Fruit Products by DNA Barcoding Method

Small berry fruit products are gaining an expanded market due to their high nutrition value. However, the authenticity of products is challenged by adulteration and mislabeling. To establish an accurate and robust method for identifying both known and unknown fruit species in small berry fruit products, DNA barcoding technology based on Sanger sequencing was adopted. To overcome the influence of processing conditions on DNA recovery, mini-barcodes of rbcL and ITS and a medium-barcode of psbA-trnH were applied. To identify ingredients in products containing mixed species, plasmid cloning was applied to separate mixed barcodes. The method established in this paper could detect 1% to 10% target species in mixed fruit juice.

Aire-dependent thymic FoxP3+ regulatory T cells in Type 1 Diabetes

Abstract Aire expression within the specialized mTECs has been found to be important for enforcing negative selection of T cells, however, mounting evidence has demonstrated the importance of Aire in Treg differentiation and development. Autoimmune diseases like T1D arise from the failure to restrain immune responses against self. Utilizing a novel mouse model, where the Aire locus drives the selected expression of the high affinity DT receptor, revealed a significant reduction in the number of FoxP3+ Tregs within the thymus following treatment. Thus, we hypothesize that alterations in the thymic self-antigen expression and presentation via ablation of Aire-expressing mTECs, will affect the repertoire and function of Aire-dependent FoxP3+ Tregs. New high-throughput platforms and single-cell DNA barcoding technology will allow us to assess several thousand individual Tregs that have undergone Aire-dependent selection in the thymus. The identification of these unique clones and gene signatures will be further interrogated retrogenically, allowing for qualitative assessment of their function within the model of T1D. The antigen specificities of the Aire-dependent TCRs will be assessed via screening against peptide libraries using MHCII molecule strongly associated with NOD mouse T1D. We anticipate identification of novel sequences that are recognized by Tregs, which may lead to discovery of new self-peptides that contribute to T1D. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent FoxP3+ Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.

Identification of Plastrum Testudinis used in traditional medicine with DNA mini-barcodes

The carapace of the tortoise Chinemys reevesii is an ingredient of “Guijia”, a traditional Chinese medicine. However, C. reevesii is difficult to raise in aquaculture and is rare in the wild. Counterfeit tablets are made from carapaces of other species. In addition to C. reevesii, other species including Mauremys sinensis, Indotestudo elongate and Trachemys scripta have been used in Plastrum Testudinis as well. After processing, these carapaces are difficult to identify on the basis of morphological characteristics, which impedes law enforcement. Our study used DNA barcoding technology to identify C. reevesii and its substitutes. We extracted concentrated genomic DNA for PCR amplification. Based on the analysis of 61 full-length COI sequences, we designed four pairs of mini-barcode primers: Tu-A, Tu-B, Tu-C and Tu-D. The Tu-B primers sequenced genomic DNA with a success rate of 76.47%, and the Tu-D primers sequenced genomic DNA with a success rate of 88.24%. The identification efficiency of these two mini-barcodes was 70.59% and 64.71%, and the overall identification efficiency was approximately 76.47%. Similarly, a set of mini barcode systems was generated, which may provide an effective and low-cost method for the identification of authentic tortoise shells.

DNA barcoding as a regulatory tool for seafood authentication in Canada

Since 2013, DNA barcoding has been in use by the Canadian Food Inspection Agency (CFIA) to speciate finfish fillets sold and consumed in Canada. We retrospectively assessed both the usefulness of DNA barcoding technology as a regulatory tool and the usefulness of the publicly available Barcode of Life Database (BOLD) in supporting the application of this technology. Speciation results for 354 finfish products collected from across Canada between 2013 and 2016 as part of the routine inspections and sampling plans of the CFIA were analyzed to determine the success rate of positive identification, and the level of mislabelling. The overall success rate was 93.2%. The rate of mislabelling was 14.8%, which is in the low end of mislabelling rates reported in the literature. Among the mislabelled samples, there was a case where an endangered species (IUCN Red List) was sold under another name. DNA barcoding as applied by the CFIA identified mislabelled seafood products in Canada. We also conducted a gap analysis comparing public DNA barcode sequences in BOLD against the seafood species listed in the CFIA “Fish List”. As of March 26th, 2018, 67 species names out of 829 unique species names on the CFIA Fish List were not associated with any records in BOLD. To best support CFIA's regulatory application of DNA barcoding, BOLD could be improved by the addition of taxonomically authenticated sequences for the 67 species lacking barcoding data, and by maintaining a collection of sequences exclusively for use in regulatory testing.

Investigation on Species Authenticity for Herbal Products of Celastrus Orbiculatus and Tripterygum Wilfordii from Markets Using ITS2 Barcoding.

Herbal material is both a medicine and a commodity. Accurate identification of herbal materials is necessary to ensure the safety and effectiveness of medication. With this work, we initiated an identification method to investigate the species authenticity for herbal products of Celastrus orbiculatus and Tripterygum wilfordii utilizing DNA barcoding technology. An ITS2 (internal transcribed spacer two) barcode database including 59 sequences was successfully established to estimate the reliability of species-level identification for Celastrus and Tripterygium. Our findings showed that ITS2 can effectively and clearly distinguish C. orbiculatus, T. wilfordii and its congeners. Then, we investigated the proportions and varieties of adulterant species in the herbal markets. The data from ITS2 region indicated that 13 (62%) of the 21 samples labeled as “Nan-she-teng” and eight (31%) of the 26 samples labeled as “Lei-gong-teng” were authentic; the remaining were adulterants. Of the 47 herbal products, approximately 55% of the product identity were not in accordance with the label. In summary, we support the efficacy of the ITS2 barcode for the traceability of C. orbiculatus and T. wilfordii, and the present study provides one method and reference for the identification of the herbal materials and adulterants in the medicinal markets.

Identification of bear bile powder and its adulterants by using DNA barcoding technology

To identify the precious bile powder and its adulterants by DNA barcoding, and establish its standard experimental process to ensure the safe and effective utilization. Total twelve sequences from samples of bear bile powder which come from Ursus thibetanus for DNA extraction, PCR(polymerase chain reaction) and sequence, then using CodonCode Aligner V 7.0.1 shear primer region to obtain COI sequence. The COI sequences of U. arctos and their adulterants were obtained from GenBank. MEGA7.0 software was applied for analyzing mutation, calculating intraspecific and interspecific K2P(Kimura 2-Parameter) genetic distance and constructing the Neighbor-joining tree(NJ). The results showed that the maximum K2P genetic distance of bear bile powder of U. thibetanus and U. arctos are far less than minimum K2P genetic distance within its adulterants species, and the results of NJ tree demonstrated that each species could be distinguished from the counterfeits obviously. DNA barcoding is a safe, convenient and reliable technique for species identification, and it is important to establish the standard sequence of COI sequences for animal medicines.

DNA barcoding technology to identity spawn from the Jiangmen marine environment in summer

PDF HTML阅读 XML下载 导出引用 引用提醒 基于DNA条形码技术对江门沿岸海域夏季鱼卵的鉴定 DOI: 作者: 作者单位: 1. 中国水产科学研究院南海水产研究所, 农业农村部南海渔业资源开发利用重点实验室, 广东 广州 510300;2. 上海海洋大学 水产与生命学院, 上海 201306 作者简介: 张楠(1987-),男,助理研究员,从事水产种质资源研究.E-mail:zhangnhs@163.com 通讯作者: 中图分类号: S917 基金项目: 科技部科技基础性工作专项（2013FY110700）；中国水产科学研究院中央级公益性科研院所基本科研业务费专项（2013A11）；农业部重大财政专项（NFZX2013）；国家科技基础条件平台建设运行项目（2017DKA30470）. DNA barcoding technology to identity spawn from the Jiangmen marine environment in summer Author: Affiliation: 1. Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs;South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China;2. College of Aqua-life Science and Technology, Shanghai Ocean University, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:以江门沿岸海域夏季采集到的鱼卵研究对象，利用DNA条形码技术分析鉴定鱼卵种类。本研究获得鱼卵个体有效线粒体COI序列信息217个，测序成功率为68.5%。经BOLD数据库比对分析，成功鉴定鱼卵5目14科19属20种（未知种2种）；种内遗传距离为0~0.006，平均遗传距离为0.002，种间遗传距离为0.149~0.325，平均遗传距离为0.255，种间遗传距离为种内遗传的128倍；鱼卵样品以鲈形目数量最多，占51.8%；鲱形目次之，占24.8%；其中鱼卵优势种为黄斑鲾（Megalaspis cordyla）、粗鳞鮻（Harpadon nehereus）、亚洲鱚（）。本次调查川山群岛东部海域获得鱼卵数量、鱼卵种类数均最多，是江门沿岸海域日本鳀、龙头鱼的主要产卵场；黄茅海、镇海湾西部水域均未获得鱼卵，可能与水域环境变化及陆源污染导致产卵环境破坏、亲体量减少有关。该研究结果揭示了江门沿岸海域夏季鱼卵分布格局，为其制定有效的产卵场保护措施提供依据，同时表明DNA条形码技术能有效地对鱼卵进行种类鉴定，可被广泛地应用于我国沿海海域鱼卵鉴定工作。 Abstract:The main purpose of this study was to analyze and identify the species of fish eggs from the Jiangmen marine environment in summer, based on DNA barcoding technology. In the present study, we acquired a total of 217 effective mitochondrial COI sequences from fish eggs, and the success rate of sequencing was up to 68.5%. After the alignment of the BOLD database, we found that besides 2 species, a total of 5 orders, 14 families, 19 genera, and 20 species were identified. Statistical analyses showed that the interspecific genetic distances were 0~0.006, and mean interspecific genetic distance was 0.002, whereas the intraspecific genetic distances were 0.149~0.325, and mean intraspecific genetic distance was 0.255, which was 128-fold higher than the former. Among the fish eggs, the most abundant order was Perciformes (51.8%), followed by Clupeiformes (24.8%); the most abundant species was Megalaspis cordyla, , and . In this investigation, the most abundant fish eggs were found in eastern Chuanshan Archipelago, both in numbers and species. The Chuanshan Archipelago is also known as the main spawning site of . However, nearly no fish eggs were found in the Huangmao Bay area or western Zhenhai Bay, possibly owing to environmental disruption and a decrease of effective parent number, triggered by environmental variation and contamination from land-sourced pollutants. In general, we revealed the distribution pattern of fish eggs from the Jiangmen marine environment in summer, which provided a theoretical basis for developing effective protective measures for spawning sites. Meanwhile, the results also showed that DNA barcoding technology could be effective and broadly applied to identify the species of fish eggs in the marine habitats of our country. 参考文献 相似文献 引证文献

DNA Barcoding and Identification of Medicinal Plants in the Kingdom of Bahrain

Authentication of medicinally important plants is essential for increasingly demands of herbal remedies worldwide. DNA barcoding technology is currently gaining importance as a reliable tool for plant species identification, although one barcode gene is not enough in the exceptions. Short sequence diversity of standardized specific coding gene regions of rbcLa and matK of plastid genome together with noncoding ribosomal internal transcribed spacer 2 (ITS2) marker is used as barcode to compare and differentiate plant species. The success of obtaining sequences of the 29 analyzed plants distributed in 21 families using three different barcode genes rbcLa, matK and ITS2 were 97%, 79% and 75% respectively. Multiple sequence alignment confirmed the medicinal plants at species level by 89.28%, 86.32% and 60.86% obtained through rbcLa, ITS2 and matK barcodes sequences respectively. The genetic distance between sequence pairs (GD) and percentage identity (PI) is compared to analyze the plant identity at species level. The phylogenic trees constructed to show the relatedness and distance of the analyzed plants in the history of evolution by the analysis of richness of clades. The construction of DNA barcode library of desert medicinal plants is an introductory research arena in Kingdom of Bahrain in helping the routine identification of plants, and developing guidelines for detection of adulterants in herbal medicines as well as protection of biodiversity.

Integrative description of two new species of Malagasy chirping giant pill-millipedes, genus Sphaeromimus (Diplopoda: Sphaerotheriida: Arthrosphaeridae)

The species-rich giant pill-millipedes (Sphaerotheriida) often represent a microendemic component of Madagascar’s mega-invertebrate fauna. Of the chirping genus Sphaeromimus de Saussure & Zehntner, 1902, ten species have been described. Here, we describe two new species of Sphaeromimus integratively, combining light microscopy, scanning electron microscopy, DNA barcoding and micro-CT technology for the first time in a taxonomic description of a giant pill-millipede. S. kalambatritra sp. nov. and S. midongy sp. nov. are the first giant pill-millipedes collected and described from the mountainous rainforests of Kalambatritra and Midongy. Both species show island gigantism compared to their congeners. Our analysis of the mitochondrial COI gene shows that the two species are related to one another with a moderate genetic distance (9.4%), while they are more closely related to an undetermined specimen from the forest of Vevembe (6.3% and 8.4%). They stand in a basal position with S. ivohibe Wesener, 2014 and S. musicus (de Saussure & Zehntner, 1897). The four aforementioned species share a high number of stridulation ribs on the male harp. Our micro-CT analysis provides a look into the head of S. kalambatritra sp. nov. and shows that non-destructive CT methods are a useful tool for studying the inner morphology of giant pill-millipedes.

Profile of Daniel H. Janzen

When Daniel Janzen gave his acceptance speech for the 1997 Kyoto Prize, he disappointed many people who had come to hear a preeminent biologist discuss iconic studies, from ant–acacia mutualism to seed predation (1, 2). “They expected a talk about science, but I’d switched to conservation,” explains Janzen, DiMaura Professor of Conservation Biology at the University of Pennsylvania. Janzen, who was elected to the National Academy of Sciences in 1992, and his wife, tropical ecologist Winnie Hallwachs, have spent 35 years reframing the idea of national parks into that of collaboration between forests and societies. “The biology is easy, but we have to study society,” explains Janzen. “For me, it’s all human ecology.” Together with thousands of partners, they have developed and expanded Area de Conservacion Guanacaste (ACG) in Costa Rica into 168,000 ha of land and sea that, Janzen notes, protect 2.6% of the world’s biodiversity, and are now using DNA barcoding technology to create a library of species (3). Daniel Janzen and Winnie Hallwachs. Image courtesy of Maureen A. Donnelly (Florida International University, Miami, FL). Growing up in Minnesota in the 1940s, Janzen quickly became comfortable in nature. “I shot my first pheasant off the back steps, but I could get the bus off the front steps to the Minneapolis public library,” he recalls. “I had the best of both worlds.” The son of a wildlife management administrator, Janzen’s fascination with the tropics began at the age of 14 on a butterfly-collecting trip to Mexico. In 1962, with a BS degree from the University of Minnesota and having entered the entomology PhD program at the University of California, Berkeley, Janzen returned to Mexico, looking for a thesis project. “In those days, rule number one for ecology graduate students was that you did all your own research,” …

Development of environmental DNA (eDNA) methods for detecting high-risk freshwater fishes in live trade in Canada

Preventing the arrival, establishment, and spread of aquatic invasive species is an important step in protecting our aquatic environments. The use of detection tools, like DNA barcoding technologies, high-throughput sequencing and environmental DNA (eDNA) monitoring, is becoming increasingly important in preventing the introduction of potential invasive species. The combination of eDNA with realtime PCR (qPCR) provide the opportunity to have a rapid and specific detection. In this study, we developed a DNA sequence library that has sufficient depth and species coverage such that high-risk species can be confidently discriminated from legitimately imported and native species. A total of 12 species-specific qPCR assays were developed for the detection of 13 potential invasive species (pAIS) in bulk water samples. Detection of these species was also compared with a HTS approach. We have demonstrated the high sensitivity of qPCR assays using eDNA at very low densities, suggesting we could detect a low number of individuals mixed with non-target species in a simulated live shipment. For the detection of a targeted list of species, qPCR is advantageous. The mini-barcodes developed in this project offered a good sensitivity of detection, and HTS is a discovery tool that can be desirable when unlisted or numerous species need to be identified.

Identification and distribution of fish eggs and larvae in the water-level fluctuation zone of Three Gorges Reservoir

PDF HTML阅读 XML下载 导出引用 引用提醒 三峡消落区鱼卵、仔稚鱼种类的鉴定及分布 DOI: 作者: 作者单位: 1. 中国水产科学研究院 长江水产研究所, 农业部淡水生物多样性保护重点实验室, 湖北 武汉 430223;2. 中国水产科学研究院 长江水产研究所, 农业部长江中上游渔业资源环境重点野外科学观测试验站, 湖北 武汉 430223;3. 重庆市水产科学研究所, 重庆 401121 作者简介: 阮瑞(1985-),男,博士,从事鱼类分子遗传学研究.E-mail:ruan.rui@163.com 通讯作者: 中图分类号: S93 基金项目: 农业部长江流域渔政管理办公室"三峡水库消落对消落区鱼类产卵孵化的影响调查与损失评估"项目. Identification and distribution of fish eggs and larvae in the water-level fluctuation zone of Three Gorges Reservoir Author: Affiliation: 1. Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China;Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Science, Wuhan 430223, China;2. Fishery Resources and Environmental Science Experimental Station of The Upper-Middle Reaches of Yangtze River, Ministry of Agriculture of China;Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China;3. Chongqing Fishery Science Institute, Chongqing 401121, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:三峡库区水位从每年3月的175 m逐步降低至6月的145 m，水位下降时间与库区部分鱼类产卵同步，对鱼类早期资源造成了严重的影响。本研究以距离大坝150 km至330 km范围的消落区为研究区域，通过普查方法收集暴露于水面或残留于浅滩的鱼卵和仔稚鱼，利用DNA条形码技术鉴定其种类，初步分析其鱼类组成和分布。通过对收集的样本进行mtCO I序列比对分析，可将其分为14种序列类型，其中10种类型鉴定到种的水平。鲤（）占测序样本比例最大，分别为50.0%、24.6%，且分布范围最广。在研究区域内，干流上游样品数量和种类均比下游多，另外，在支流磨刀溪采集了733粒鱼卵和108尾仔稚鱼、鉴定到9种鱼类，是本次研究中样品和种类最多的区域，表明磨刀溪受三峡库区消落影响最严重，间接暗示了该支流鱼类资源丰富，应给予重点保护。本研究旨在为全面评估三峡库区消落对鱼类早期资源的影响奠定基础。 Abstract:Three Gorges Dam is a vital project in the development and harnessing of the Yangtze River. Water-level fluctuation zone of Three Gorges Reservoir, which refers to the area along the Three Gorges Reservoir banks bounded by minimum (145 m) and maximum (175 m) water levels of the Three Gorges Reservoir, has provoked a series of ecological problems, which included soil erosion, reduction in ecosystem types, decrease in biodiversity and so on. However, effect of water level fluctuating on fish of early life history stages in the water-level fluctuation zone was not yet reported. In this study, the research area was between 150 km and 330 km from Three Gorges Dam, where fish eggs and larvae, exposed to water or remained in shallows, were collected. Based on distribution of sampling places, the research area was divided into 8 regions, which were mainstream (down), Meixi River, mainstream (middle), Shilu River, Modaoxi River, Tangxi River, mainstream (up), and Pengxi River. According to sampling time and place, and morphological characters of fish eggs and larvae, 132 samples were preserved, including 1735 fish eggs and 150 larvae. Twenty-four samples were collected in mainstream. But only one sample in respective mainstream (middle) and mainstream (down) included respective one and nine larvae, in which no fish egg was found. In Modaoxi River, a tributary of Three Gorges Reservoir, there were the most samples in this study, and 59 samples were collected including 733 fish eggs and 108 larvae. Due to insufficient morphological diagnostic characters in kraurotic fish eggs and larvae, it is easy to misidentify in the genus or species level. So, this study was employed DNA barcoding technology to identify the samples. DNA barcoding technology could be used as a rapid tool to distinguish morphologically similar species, and survey many uncertain species and species composition. Previous researches have shown that only molecular identification could guarantee identification of fish eggs and larvae to the species level. Through mtCO I sequence alignment, and based on mtCO I sequences similarity values (species:>99%, genus:92%-99%, family:85%-92%), the samples were distinguished to 14 types, of which 10 types were identified to species:Cyprinuscarpio, Carassiusauratus, Hemiculterleucisculus, Rhinogobius nagoyae, Rhinogobius giurinus, Opsariichthysbidens, Acheilognathusmacro-pterus, Pseudorasboraparva, Hypophthalmichthys molitrix, and Rhinogobius, Rhodeus, and Rhodeusocellatus and . If fish DNA barcode database of Yangtze River or Three Gorges Reservoir could be completely and accurately constructed, and new molecular markers could be developed for fishes that can be not distinguished by mtCO I barcode, it would help to further identify fish eggs and larvae in the water-level fluctuation zone and investigate fish resources of the Yangtze River. had the largest proportion in the samples, 50.0% and 24.6% respectively, and widely distributed. The number and species of samples in upstream of mainstream was more than downstream in the research area. It may be not only related to its topography, and also its fish resources. In Modaoxi River, the number and species of samples was the most in this research area, which contained nine fish species. The result showed that it was more affected from the water level fluctuating, at the same time, it also indicated that fish resource were rich in Modaoxi River, which should be got more attention in the future fish resources investigation and protection in Three Gorges Reservoir. This study employed DNA barcoding technology to identify fish eggs and larvae collected in partial water-level fluctuation zone, which would contribute to comprehensively assessing the effect of water level fluctuating on fish resources in water-level fluctuation zone of Three Gorges Reservoir. 参考文献 相似文献 引证文献

Modelling Therapy Resistance for the Identification of Treatment-Refractory Cell Population(s) in Human Glioblastoma

Despite aggressive multimodal therapy, human glioblastoma (hGBM), a highly malignant grade IV astrocytic tumour, remains incurable and inevitably relapses. Recent data has implicated intratumoral heterogeneity as the driver of therapy resistance and tumour relapse in hGBM. Thus models that capture the evolving hGBM biology in response to chemoradiotherapy will allow for the identification of cellular pathways that govern GBM therapy failure. In this study, we have developed a novel model to profile the clonal evolution of treatment naïve brain tumour initiating cell (BTIC) enriched hGBMs through chemoradiotherapy using: stem cell assays, BTIC marker expression and transcriptome analysis, immunohistochemistry, and cellular DNA barcoding technology. We report that treatment of hGBM BTICs leads to increased self-renewal capacity and higher transcript expression of stem cell genes Bmi1 and Sox2. Based on global transcriptome analysis of the in vitro treated hGBM, we also identify a hyper-aggressive form of glioma. Using our therapy-adapted hGBM-mouse xenograft model, we discover that despite tumour regression and increased mouse survival post-therapy, tumour relapse remains inevitable. The treatment-refractory cells again have increased self-renewal capacity and higher expression of Bmi1 and Sox2. Furthermore, by combining cellular DNA barcoding technology, which barcodes hGBM at single cell resolution, with our novel in vitro and in vivo therapy models, we are able to determine whether a pre-existing or a therapy driven subpopulation(s) seeds hGBM tumour relapse. Profiling the dynamic nature of heterogeneous hGBM subpopulations through disease progression and treatment may lead to the identification of novel therapeutic targets for the treatment of recurrent hGBM.

Evaluation of DNA barcode candidates for the discrimination of Artemisia L.

Because of the very similar morphologies and wide diversity of Artemisia L. varieties, they are difficult to identify, and there have been many arguments about the systematic classification Artemisia L., especially concerning the division of species. DNA barcode technology is used to rapidly identify species based on standard short DNA sequences. To evaluate seven candidate DNA barcodes (ITS, ITS2, psbA-trnH, rbcL, matK, rpoB, and rpoC1) regarding their ability to identify closely related species of the Artemisia genus in Xinjiang. The corresponding PCR amplification efficiency, detectable genetic divergence, identification efficiency and phylogenetic tree were assessed. We found that the internal transcribed spacer (ITS) region exhibited the highest interspecific divergence, which was significantly higher than the observed intraspecific variation and showed the highest identification efficiency, followed by ITS2, psbA-trnH and, finally, rpoB. matK, rbcL, and rpoC1 performed poorly in this evaluation. ITS, ITS2, and psbA-trnH were able to perfectly identify the tested species Artemisia annua, A. absinthium, A. rupestris, A. tonurnefortiana, A. austriaca, A. dracunculus, A. vulgaris, and A. macrocephala. Therefore, we propose the ITS, ITS2, and psbA-trnH regions as promising DNA barcodes for the closely related species of Artemisia L. in Xinjiang.

Barcoding the food chain: from Sanger to high-throughput sequencing

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace "environmental DNA", and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

Identification of Fritillariae bulbus from adulterants using ITS2 regions

Distinguishing the five types of Fritillariae bulbus (Fritillariae cirrhosae bulbus, Fritillariae ussuriensis bulbus, Fritillariae pallidiflorae bulbus, Fritillariae thunbergii bulbus and Fritillariae hupehensis bulbus) is difficult because of their similar morphological, physical, and chemical characteristics. These species, which are included in the Chinese Pharmacopoeia, exhibit different therapeutic effects. Therefore, we used internal transcribed spacer 2 (ITS2) regions as DNA barcode to distinguish the five types of Fritillariae bulbus from each other and their adulterants. A total of 151 samples belonging to five types of Fritillariae bulbus (a total of 11 species of Fritillaria) and two adulterant species were collected. The phylogeny was constructed using the neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) methods. Genomic DNAs were successfully extracted from all bulbus samples and obtaining high-quality ITS2 sequences via polymerase chain reaction optimization. The length of the ITS2 sequence of 12 Fritillaria species ranged from 235bp to 241bp, and the average GC contents were over 68%. Adulterants Bolbostemma paniculatum and Fritillaria maximowiczii could be distinguished from all Fritillariae bulbus species by using the distance and tree methods. The ML tree showed better topology than the NJ and MP trees. Members of each type of Fritillariae bulbus formed one clade, which was strongly supported with bootstrap values, except for Fritillariae thunbergii bulbus and Fritillariae hupehensis bulbus in the ML tree. Therefore, DNA barcoding technology based on ITS2 regions can be used to distinguish the five types of Fritillariae bulbus from each other and their adulterants except for Fritillariae thunbergii bulbus and Fritillariae hupehensis bulbus.

Sources of varieties and quality of circular Fructus Ligustri Lucidi

This study aimed to trace sources and quantitatively analyze the specnuezhenide content of circular Fructus Ligustri Lucidi for clinical use. Different specifications of Fructus Ligustri Lucidi were identified using DNA barcoding technology and the specnuezhenide content was analyzed by High Performance Liquid Chromatography (HPLC). The ITS sequence of circular Fructus Ligustri Lucidi was identical to that of standard privet, which was determined through botanical identification. ITS sequence similarity between circular Fructus Ligustri Lucidi and Fructus Ligustri Lucidi which was registered in NCBI ranged from 99.5% to 100%. The sequences of circular and other Fructus Ligustri Lucidi were clustered in a Neighbor-Joining tree with bootstrap value of 95, and these sequences could be distinguished from adulterants. Conforming to pharmacopoeia standard, the average specnuezhenide content of circular Fructus Ligustri Lucidi was higher than that of chicken waist Fructus Ligustri Lucidi. Circular Fructus Ligustri Lucidi derived from Ligustrum lucidum Ait. and the specnuezhenide content was higher in circular Fructus Ligustri Lucidi than that in chicken waist Fructus Ligustri Lucidi.

DNA Barcoding Identification of Kadsurae Caulis and Spatholobi Caulis Based on Internal Transcribed Spacer 2 Region and Secondary Structure Prediction.

Background:Kadsurae Caulis and Spatholobi Caulis have very similar Chinese names. Their commodities were hard to distinguish because their stems were very alike after dried and processed. These two herbal drugs were often mixed in clinical use.Objective:Authenticity assurance is crucial for quality control of herbal drugs. Therefore, it is essential to establish a method for identifying the two herbs.Materials and Methods:In this paper, we used the DNA barcoding technology, based on the internal transcribed spacer 2 (ITS2) regions, to differentiate Kadsurae Caulis and Spatholobi Caulis.Results:The ITS2 of these two herbs were very different. They were successfully differentiated using the DNA barcoding technique.Conclusions:DNA barcoding was a promising and reliable tool for the identification of medicinal plants. It can be a powerful complementary method for traditional authentication.SUMMARY The internal transcribed spacer 2 (ITS2) regions between Kadsurae Caulis and Spatholobi Caulis varied considerably, totally 139 variable sitesSample 1 was not Kadsurae Caulis as it labeled, but it should be Spatholobi Caulis in fact based on ITS2 regionThe secondary structure can also separate Kadsurae Caulis and Spatholobi Caulis effectivelyDNA barcoding provided an accurate and strong prove to identify these two herbs. Abbreviations used: CTAB: hexadecyltrimethylammonium bromide, DNA: deoxyribonucleic acid, ITS2:internal transcribed spacer 2, PCR: polymerase chain reaction

Differential Genomic Approach to Explore Phylogenetic Link

Different approaches are in progress to study phylogenetic relatedness and divergence among organisms. Genomic study is the most effective method for resolving phylogenetic relationships. Multiple genes are preferred over single gene sequence analysis and whole genome sequences are nowadays is the best nucleotide sequence-based approach to identify and classify the taxonomically complex group of organisms to deal with several phylogenetic issues. Various bioinformatics tools are providing valuable information to study significant evolutionary relationships such as next generation sequencing, proteomics etc. DNA barcoding technology is also recently used skill to study phylogeny and identify the morphologically ambiguous species by amplifying a small fragment of Cytochrome c Oxidase Subunit I (COI). We recently reported multiple gene phylogenies of Indian termites resulting in incongruent results. However, whole genome analysis will provide a significant outcome to resolve the diverse observations studied in these species.

Application of metabarcoding technology in studies of fungal diversity

Fungal diversity is threatened by climate change, land-use change, and environmental pollution, and requires urgent conservation action. Construction of the fungal species database is an important prerequisite for the study and conservation of fungal diversity. Recently developed DNA barcoding and metabarcoding technologies can provide accurate, rapid, and highly efficient identification on a large scale, and to a large extent compensate for the defects of traditional identification methods. In this paper, we review the application of metabarcoding in fungal species diversity assessment, the study of mechanisms underlying fungal diversity, and the reconstruction of fungal palaeoecology. We emphasize that the application of metabarcoding technology in fungal diversity studies is still in the primary phase, and greater efforts are needed in the construction of reliable reference databases, the optimization of experimental procedures, and updates of bioinformatics tools. Hence, we suggest enhancing cooperation among fungal taxonomists, ecologists, and computer technicians. They should work together to address problems in fungal diversity studies via metabarcoding, which would provide more sound scientific evidence for fungal diversity conservation on a large scale.