DNA Barcoding and Metabarcoding of Complex Matrices

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Technologies and Resources for Genetics".

Deadline for manuscript submissions: closed (10 February 2019) | Viewed by 53429

Special Issue Editors


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Guest Editor
Department of Bioscience and Biotechnology of University of Milano-Bicocca, I-20126 Milano, Italy
Interests: plant biodiversity; agrobiodiversity; molecular identification; food traceability
Special Issues, Collections and Topics in MDPI journals

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Guest Editor
ZooPlantLab, Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.za della Scienza 2, 20126-I Milano, Italy
Interests: integrative taxonomy; DNA barcoding; DNA metabarcoding; metazoans; diet analysis; food authenticity; high-throughput sequencing; ecosystem services
Special Issues, Collections and Topics in MDPI journals

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Guest Editor
Plant Evolution and DNA Metabarcoding Group, Natural History Museum, University of Oslo, 0315 Oslo, Norway
Interests: molecular identification of plants; plant DNA metabarcoding; plant phylogenomics; herbal authenticity; food and drug safety; Illegal plant trade

Special Issue Information

Dear Colleagues,

DNA barcoding is a molecular and bioinformatics tool that aims at identifying organisms’ species. The rationale of this approach is quite simple: Through the analysis of the variability in a single or in a few standard molecular marker(s), it is possible to discriminate biological entities. In the last fifteen years, this approach has largely been used to address taxonomic issues, as well as to achieve a reliable traceability system for raw materials, processed food items and other commercial products. Recently, due to the continuous advances in the field of high throughput sequencing, more and more applications have become possible in the field of molecular identification, especially with the development of DNA metabarcoding. This powerful approach enables simultaneous detection of multiple species in complex biological matrices. DNA metabarcoding can be adopted with high reliability and sensitivity, from environmental samples (e.g., soil, water and feces) to food products. Moreover, this revolutionizing identification strategy is also useful to analyze highly-processed materials containing degraded DNA, such as plant extracts, manufactured food, cosmetics or drugs plant-based products. Both DNA barcoding and metabarcoding demand suitable bioinformatics tools to achieve an efficient data processing and well-populated and verified public (or private) databases for the identification.

This Special Issue is devoted to the advances in DNA barcoding and metabarcoding to analyze complex matrices with the main goals of i) elucidating the technical and bioinformatical advances currently available for these tools; ii) defining the possible fields of application; and iii) the effectiveness of these approaches in the research and technology transfer strategy. The editors also invite submissions on advanced approaches, including DNA metagenomics, target-enrichment sequencing, and marker mining from genome skimming data.

We cordially invite researchers working actively in these fields to submit their original research manuscripts to this Special Issue.

Prof. Massimo Labra
Dr. Andrea Galimberti
Assoc. Prof. Hugo de Boer
Guest Editors

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Keywords

  • Botanicals
  • Diet and Microbiome characterization
  • DNA taxonomy
  • Environmental matrices
  • Fast identification approaches
  • Food Authentication and traceability
  • Food matrices
  • High Throughput Sequencing
  • Molecular markers

Published Papers (11 papers)

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Research

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17 pages, 2738 KiB  
Article
Single Fragment or Bulk Soil DNA Metabarcoding: Which is Better for Characterizing Biological Taxa Found in Surface Soils for Sample Separation?
by Laura M. Boggs, Melissa K. R. Scheible, Gustavo Machado and Kelly A. Meiklejohn
Genes 2019, 10(6), 431; https://doi.org/10.3390/genes10060431 - 6 Jun 2019
Cited by 5 | Viewed by 4691
Abstract
In forensic geology casework, sample size typically limits routine characterization of material using bulk approaches. To address this, DNA-based characterization of biological taxa has received attention, as the taxa present can be useful for sample-to-sample comparisons and source attribution. In our initial work, [...] Read more.
In forensic geology casework, sample size typically limits routine characterization of material using bulk approaches. To address this, DNA-based characterization of biological taxa has received attention, as the taxa present can be useful for sample-to-sample comparisons and source attribution. In our initial work, low biodiversity was captured when DNA barcodes were Sanger-sequenced from plant and insect fragments isolated from 10 forensic-type surface soils. Considering some forensic laboratories now have access to massively parallel sequencing platforms, we assessed whether biological taxa present in the same surface soils could be better characterized using DNA metabarcoding. To achieve this, plant and animal barcodes were amplified and sequenced on an Illumina® MiniSeq for three different DNA sample types (n = 50): individual fragments used in our initial study, and 250 and 100 mg of bulk soil (from the 10 sites used in the initial study). A total of 572 unique target barcode sequences passed quality filtering and were used in downstream statistical analyses: 54, 321, and 285 for individual fragments, 100 mg, and 250 mg bulk soil samples, respectively. Plant barcodes permitted some spatial separation of sample sites in non-metric multidimensional scaling plots; better separation was obtained for samples prepared from bulk soil. This study confirmed that bulk soil DNA metabarcoding is a better approach for characterizing biological taxa present in surface soils, which could supplement traditional geologic examinations. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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11 pages, 1459 KiB  
Article
Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature
by Wenjun Jiang, Li Ren, Mengyue Guo, Nitin Mantri, Sha Zhao and Xiaohui Pang
Genes 2019, 10(5), 397; https://doi.org/10.3390/genes10050397 - 22 May 2019
Cited by 10 | Viewed by 3559
Abstract
Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its [...] Read more.
Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5′-CGCTTTGCGACGCTCCCCTCCCTCCC-3′) on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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19 pages, 3483 KiB  
Article
Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures
by Peng Zhang, Chunsheng Liu, Xiasheng Zheng, Lan Wu, Zhixiang Liu, Baosheng Liao, Yuhua Shi, Xiwen Li, Jiang Xu and Shilin Chen
Genes 2019, 10(5), 343; https://doi.org/10.3390/genes10050343 - 7 May 2019
Cited by 8 | Viewed by 3788
Abstract
DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has [...] Read more.
DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has been used to address this authentication issue for the past few years, but conventional NGS technology is hampered in application due to its short sequencing reads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) via long-read sequencing, is employed for the identification of biological compositions in herbal compound formulas in adequate and well controlled studies. By directly sequencing the full-length amplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, the biological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the same time, clone-dependent Sanger sequencing was carried out as a parallel control. Further, another formula—Sanwei-Jili-San (SJS)—was analyzed with genes of ITS2 and CO1. All the ingredients in the samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenous species were also checked, some of which were considered as common contaminations in these products. Methodology analysis demonstrated that this method was sensitive, accurate and reliable. FLMB, a superior but feasible approach for the identification of biological complex mixture, was established and elucidated, which shows perfect interpretation for DNA barcoding that could lead its application in multi-species mixtures. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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20 pages, 1352 KiB  
Article
DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region
by Caroline Howard, Eleanor Hill, Marco Kreuzer, Purvi Mali, Eva Masiero, Adrian Slater and Tiziana Sgamma
Genes 2019, 10(4), 286; https://doi.org/10.3390/genes10040286 - 9 Apr 2019
Cited by 13 | Viewed by 4168
Abstract
There is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region [...] Read more.
There is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 103 ITS copies µL−1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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13 pages, 1147 KiB  
Article
Food Tracking Perspective: DNA Metabarcoding to Identify Plant Composition in Complex and Processed Food Products
by Antonia Bruno, Anna Sandionigi, Giulia Agostinetto, Lorenzo Bernabovi, Jessica Frigerio, Maurizio Casiraghi and Massimo Labra
Genes 2019, 10(3), 248; https://doi.org/10.3390/genes10030248 - 25 Mar 2019
Cited by 38 | Viewed by 6183
Abstract
One of the main goals of the quality control evaluation is to identify contaminants in raw material, or contamination after a food is processed and before it is placed on the market. During the treatment processes, contamination, both accidental and economically motivated, can [...] Read more.
One of the main goals of the quality control evaluation is to identify contaminants in raw material, or contamination after a food is processed and before it is placed on the market. During the treatment processes, contamination, both accidental and economically motivated, can generate incongruence between declared and real composition. In our study, we evaluated if DNA metabarcoding is a suitable tool for unveiling the composition of processed food, when it contains small trace amounts. We tested this method on different types of commercial plant products by using tnrL marker and we applied amplicon-based high-throughput sequencing techniques to identify plant components in different food products. Our results showed that DNA metabarcoding can be an effective approach for food traceability in different type of processed food. Indeed, the vast majority of our samples, we identified the species composition as the labels reported. Although some critical issues still exist, mostly deriving from the starting composition (i.e., variable complexity in taxa composition) of the sample itself and the different processing level (i.e., high or low DNA degradation), our data confirmed the potential of the DNA metabarcoding approach also in quantitative analyses for food composition quality control. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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11 pages, 911 KiB  
Article
Development of a DNA Barcoding-Like Approach to Detect Mustard Allergens in Wheat Flours
by Jessica Frigerio, Roberta Pellesi, Valerio Mezzasalma, Fabrizio De Mattia, Andrea Galimberti, Francesca Lambertini, Michele Suman, Sandro Zanardi, Andrea Leporati and Massimo Labra
Genes 2019, 10(3), 234; https://doi.org/10.3390/genes10030234 - 19 Mar 2019
Cited by 10 | Viewed by 4104
Abstract
The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour [...] Read more.
The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour (Triticum aestivum) can be contaminated by a wide range of species belonging to the Brassicaceae in the field or during grain harvests, storage, and processing. Among them, mustards (Brassica nigra, Brassica juncea and Sinapis alba) are well known allergenic species. Often, food quality laboratories adopt an ELISA approach to detect the presence of mustard species. However, this approach shows cross-reactivity with other non-allergenic species such as Brassica napus (rapeseed). In the last few years, DNA barcoding was proposed as a valid identification method, and it is now commonly used in the authentication of food products. This study aims to set up an easy and rapid DNA-based tool to detect mustard allergenic species. DNA barcoding (matK and ITS2) and chromosome markers (A6, B, C1 genome regions) were selected, and specific primers were validated on incurred reference food matrices. The developed test was proven to be able to distinguish mustard from rapeseed and wheat, overcoming cross-reactivity with Brassica napus. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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16 pages, 1623 KiB  
Article
Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products
by Denis O. Omelchenko, Anna S. Speranskaya, Andrey A. Ayginin, Kamil Khafizov, Anastasia A. Krinitsina, Anna V. Fedotova, Denis V. Pozdyshev, Viktoria Y. Shtratnikova, Evgenia V. Kupriyanova, German A. Shipulin and Maria D. Logacheva
Genes 2019, 10(2), 122; https://doi.org/10.3390/genes10020122 - 7 Feb 2019
Cited by 17 | Viewed by 4471
Abstract
Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from [...] Read more.
Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: (1) the DNA extraction method greatly influences amplification success; (2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and (3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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12 pages, 534 KiB  
Article
A Systematic Study on DNA Barcoding of Medicinally Important Genus Epimedium L. (Berberidaceae)
by Mengyue Guo, Yanqin Xu, Li Ren, Shunzhi He and Xiaohui Pang
Genes 2018, 9(12), 637; https://doi.org/10.3390/genes9120637 - 17 Dec 2018
Cited by 17 | Viewed by 4246
Abstract
Genus Epimedium consists of approximately 50 species in China, and more than half of them possess medicinal properties. The high similarity of species’ morphological characteristics complicates the identification accuracy, leading to potential risks in herbal efficacy and medical safety. In this study, we [...] Read more.
Genus Epimedium consists of approximately 50 species in China, and more than half of them possess medicinal properties. The high similarity of species’ morphological characteristics complicates the identification accuracy, leading to potential risks in herbal efficacy and medical safety. In this study, we tested the applicability of four single loci, namely, rbcL, psbA-trnH, internal transcribed spacer (ITS), and ITS2, and their combinations as DNA barcodes to identify 37 Epimedium species on the basis of the analyses, including the success rates of PCR amplifications and sequencing, specific genetic divergence, distance-based method, and character-based method. Among them, character-based method showed the best applicability for identifying Epimedium species. As for the DNA barcodes, psbA-trnH showed the best performance among the four single loci with nine species being correctly differentiated. Moreover, psbA-trnH + ITS and psbA-trnH + ITS + rbcL exhibited the highest identification ability among all the multilocus combinations, and 17 species, of which 12 are medicinally used, could be efficiently discriminated. The DNA barcode data set developed in our study contributes valuable information to Chinese resources of Epimedium. It provides a new means for discrimination of the species within this medicinally important genus, thus guaranteeing correct and safe usage of Herba Epimedii. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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15 pages, 1189 KiB  
Article
Trade in Zambian Edible Orchids—DNA Barcoding Reveals the Use of Unexpected Orchid Taxa for Chikanda
by Sarina Veldman, Seol-Jong Kim, Tinde R. Van Andel, Maria Bello Font, Ruth E. Bone, Benny Bytebier, David Chuba, Barbara Gravendeel, Florent Martos, Geophat Mpatwa, Grace Ngugi, Royd Vinya, Nicholas Wightman, Kazutoma Yokoya and Hugo J. De Boer
Genes 2018, 9(12), 595; https://doi.org/10.3390/genes9120595 - 30 Nov 2018
Cited by 6 | Viewed by 8119
Abstract
In Zambia, wild edible terrestrial orchids are used to produce a local delicacy called chikanda, which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which [...] Read more.
In Zambia, wild edible terrestrial orchids are used to produce a local delicacy called chikanda, which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which orchid species are traded on local markets, as orchid tubers are difficult to identify morphologically. In this study, the core land-plant DNA barcoding markers rbcL and matK were used in combination with nrITS to determine which species were sold in Zambian markets. Eighty-two interviews were conducted to determine harvesting areas, as well as possible sustainability concerns. By using nrITS DNA barcoding, a total of 16 orchid species in six different genera could be identified. Both rbcL and matK proved suitable to identify the tubers up to the genus or family level. Disa robusta, Platycoryne crocea and Satyrium buchananii were identified most frequently and three previously undocumented species were encountered on the market. Few orchid species are currently listed on the global International Union for the Conservation of Nature (IUCN) Red List. Local orchid populations and endemic species could be at risk of overharvesting due to the intensive and indiscriminate harvesting of chikanda orchids, and we therefore encourage increased conservation assessment of terrestrial African orchids. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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12 pages, 1072 KiB  
Article
Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis
by Nur Fadhila Fadzil, Alina Wagiran, Faezah Mohd Salleh, Shamsiah Abdullah and Nur Hazwani Mohd Izham
Genes 2018, 9(8), 408; https://doi.org/10.3390/genes9080408 - 12 Aug 2018
Cited by 16 | Viewed by 3898
Abstract
The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) [...] Read more.
The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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Review

Jump to: Research

16 pages, 1909 KiB  
Review
Tubulin-Based DNA Barcode: Principle and Applications to Complex Food Matrices
by Laura Morello, Luca Braglia, Floriana Gavazzi, Silvia Gianì and Diego Breviario
Genes 2019, 10(3), 229; https://doi.org/10.3390/genes10030229 - 18 Mar 2019
Cited by 8 | Viewed by 4576
Abstract
The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal [...] Read more.
The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods. Full article
(This article belongs to the Special Issue DNA Barcoding and Metabarcoding of Complex Matrices)
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