Ethnobotany genomics - use of DNA barcoding to explore cryptic diversity in economically important plants

The ethnobotany genomics concept is founded on the idea of 'assemblage' of biodiversity knowledge. This includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). Ethnobotany genomics is defined as exploring the variation in genomic sequences from many species, and here we present some of our recent work that demonstrates the potential benefits of this approach for ethnobotanical research with economic implications. DNA barcoding was used to identify Acacia and nutmeg taxa that are economically important to society-at-large. Furthermore we identified considerable variation that is recognized by several indigenous cultures. The impacts of ethnobotany genomics will extend well beyond biodiversity science. Explorations of the genomic properties across the expanse of life are now possible using DNA barcoding to assemble sequence information for a standard portion of the genome from large assemblages of species. Perhaps the most important contribution is major barcode projects will leave an important legacy; a comprehensive repository of highquality DNA extracts that will facilitate future genomic investigations.

We present here the first use of DNA barcoding in a new approach to ethnobotany we coined "ethnobotany genomics". This new approach is founded on the concept of 'assemblage' of biodiversity knowledge, which includes a coming together of different ways of knowing and valorizing species variation in a novel approach seeking to add value to both traditional knowledge (TK) and scientific knowledge (SK). We employed contemporary genomic technology, DNA barcoding, as an important tool for identifying cryptic species, which were already recognized ethnotaxa using the TK classification systems of local cultures in the Velliangiri Hills of India. This research is based on several case studies in our lab, which define an approach to that is poised to evolve quickly with the advent of new ideas and technology. Our results show that DNA barcoding validated several new cryptic plant species to science that were previously recognized by TK classifications of the Irulas and Malasars, and were lumped using SK classification. The contribution of the local aboriginal knowledge concerning plant diversity and utility in India is considerable; our study presents new ethnomedicine to science. Ethnobotany genomics can also be used to determine the distribution of rare species and their ecological requirements, including traditional ecological knowledge so that conservation strategies can be implemented. This is aligned with the Convention on Biological Diversity that was signed by over 150 nations, and thus the world's complex array of human-natural-technological relationships has effectively been re-organized.

Contents: Introduction Inventing Life: Intellectual Property and the New Biology Alison McLennan and Matthew Rimmer PART I: A HISTORY OF BIODISCOVERY 1. Of Plants, Pills, and Patents: Circulating Knowledge Eva Hemmungs Wirten PART II: MEDICINE, BIOTECHNOLOGY, AND GENOMICS 2. Bilski v. Kappos and Biotechnology Patents: Back to the Future? Yann Joly and Francis Hemmings 3. The Current State of Patent Eligibility of Medical and Biotechnology Inventions in the United States Joshua D. Sarnoff 4. Patent Law, the Emerging Biotechnologies and the Role of Language in Subject Matter Expansionism Graham Dutfield PART III: BIOBANKS, BIOINFORMATICS AND BIOBRICKS 5. Standards for Biobank Access and Intellectual Property Dianne Nicol and Richard Gold 6. The 1000 Genomes Project Donna M. Gitter 7. Building with Biobricks: Constructing a Commons for Synthetic Biology Research Alison McLennan PART IV: GENETICS, STEM CELLS, AND NANOTECHNOLOGY 8. Regulating Gene Regulation: Patenting Small RNAs Adam Bostanci, Jane Calvert and Pierre-Benoit Joly 9. Stem Cell Patents: Looking for Serenity Amina Agovic 10. Cosmo, Cosmolino: Patent Law and Nanotechnology Alison McLennan and Matthew Rimmer PART V: BIODIVERSITY, FOOD SECURITY, AND CLIMATE CHANGE 11. Patenting The Kakadu Plum and the Marjarla Tree: Biodiscovery, Intellectual Property and Indigenous Knowledge Sarah Holcombe and Terri Janke 12. Climate-Ready Crops: Intellectual Property, Agriculture, and Climate Change Matthew Rimmer 13. The Doomsday Vault: Seed Banks, Food Security, and Climate Change Matthew Rimmer Bibliography Index

DNA barcoding is a sequencing-based method that can be used for the identification of fish species in a regulatory setting. The objective of this study was to compare modified versions of three DNA extraction kits (i.e., Qiagen DNeasy Blood and Tissue Kit, Sigma-Aldrich Extract-N-Amp Kit; and Life Technologies MagMax-96 DNA Multi-Sample Kit) and two polymerase chain reaction (PCR) setup methods (manual vs. automated) for use in DNA barcoding, with a focus on minimizing time, costs, and labor. DNA was extracted from 83 fish products using each of the three kits and the results were compared based on sequencing success and sequencing quality parameters. A subset of 14 fish products was also tested in triplicate to compare PCR setup methods. Initially, reduced sequencing success was observed with the MagMax Kit (88 %) compared to the other two kits (95–96 %); however, after PCR and sequencing were repeated for DNA samples that initially failed, all three methods showed very high sequencing success (98–99 %). Overall, the modified Extract-N-Amp Kit offered the greatest reduction in time and costs, while the DNeasy Blood and Tissue Kit produced sequences with the highest quality and highest initial success rates. Automation of the PCR setup process resulted in slightly greater success (100 %) compared to manual PCR setup (98 %), and reduced the potential for human error that may result from manual pipetting. The results of this study demonstrate the advantages of incorporating rapid and/or automated methods into the DNA barcoding workflow, especially with regard to high-throughput operations.

This study purposed to use information and communication technologies for documenting indigenous farming knowledge for improved preservation, accessibility and use in Kilifi County, Kenya. The objectives that guided this study were to assess the awareness and perception of the study community regarding use of ICTs in preservation and management of indigenous farming knowledge, to explore available ICTs tools that can capture and document indigenous farming knowledge, to advance the important role a library repository could play in preservation, management, storage and dissemination of indigenous farming knowledge and to identify barriers and concerns related to IFK preservation, accessibility and use. The outcome of this research is a knowledge asset of captured indigenous farming experiences, processes, and insights to contribute to a pool of indigenous farming knowledge for learning and scaling up preservation and public utilization. This study was conducted in all the seven Sub Counties in Kilifi County namely Malindi, Magarini, Kilifi North, Kilifi South, Ganze, Kaloleni and Rabai where a sample size of ninety eight respondents that was derived using Krejcie and Morgan formula n=X2NP (1-P)/e2 (N-1) +X2P (1-P) that is used when a population is more than ten thousand (10,000) were targeted. The research instruments that were used included questionnaires, interview schedule, personal observation, storytelling and focus group discussions which were recorded using information and communication technology resources such as video recording to come up with a knowledge asset of indigenous farming knowledge experiences for uploading into the County of Kilifi Public Library repository was realized. Cronbach’s alpha was used to test the validity of the instruments. Secondary data was collected from County of Kilifi Demographic Reports, Kilifi County Development plan, County Government of Kilifi Agricultural Sector Development Programme and reputable databases. Data analysis involved the use of inferential statistics using Statistical Package for Social Science (SPSS) and tables of means and standard deviation which were used to present the data. The findings of the study are that indigenous farming knowledge is very valuable and has assisted the community in food security and needs to be passed down to the younger generation. Farmers’ awareness and perception of the role of ICT in preservation of IFK is very good and agreed that if IFK is not documented, it may disappear as they died. The farmers were aware of ICTs tools to capture, document and disseminate indigenous farming experiences for improved preservation and accessibility in Public Libraries in Kilifi County including mobile phones, radios, television, computers, internet, memory cards, social media technologies, iPads and flash discs can be used. The Sub County Agricultural Officers and Librarians have the qualification and experience required to collaborate with the Kaya Elders (Farmers) to document and preserve the IFK for posterity. The public library repository can be a knowledge asset in the preservation, management and dissemination of documented indigenous farming experiences and provide free access to indigenous knowledge information resources, providing places for access to researchers of indigenous knowledge, training users on accessing indigenous knowledge resources and allowing farmers to observe indigenous knowledge practices by offering demonstration site in the library compound. However, there were barriers and concerns including climate change, use of certified seeds instead of indigenous seeds, its accessibility, government introduction of early maturing seeds due to prolonged drought, people’s perception of it being primitive knowledge, its none documentation and inaccessibility, poor preservation, knowledge gaps left by dying indigenous knowledge owners that were noted that can be surmounted by documenting indigenous knowledge practices, creating awareness on indigenous knowledge resources, mentorship (the old passing knowledge to the young), creating platforms to allow access to indigenous knowledge as well as creating indigenous knowledge databases, mainstreaming it into our formal education, building awareness on indigenous farming knowledge, community based indigenous knowledge maintenance, creating national indigenous knowledge inventories and securing intellectual property of indigenous farming knowledge. The study recommended indigenous farming knowledge be incorporated to scientific farming knowledge by embedding it in ICTs tools such as mobile phones, and social media technologies that will enhance its accessibility and mainstreaming with scientific knowledge, educating people on value of indigenous farming knowledge by public libraries’ embracing their role of creating awareness through creation of more platforms including indigenous farming knowledge databases and revamping public libraries to become viable indigenous farming knowledge assets by empowering the public libraries in acquiring, preserving, managing and disseminating IFK in the form of books, audio visual resources, technical skills, human skills, demonstration gardens and adult education learners and double their effort in creating the necessary awareness for them to achieve their intended purpose.

DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species.

BackgroundThe past several years have seen a flurry of papers seeking to clarify the utility and limits of DNA barcoding, particularly in areas such as species discovery and paralogy due to nuclear pseudogenes. Heteroplasmy, the coexistence of multiple mitochondrial haplotypes in a single organism, has been cited as a potentially serious problem for DNA barcoding but its effect on identification accuracy has not been tested. In addition, few studies of barcoding have tested a large group of closely-related species with a well-established morphological taxonomy. In this study we examine both of these issues, by densely sampling the Hawaiian Hylaeus bee radiation.ResultsIndividuals from 21 of the 49 a priori morphologically-defined species exhibited coding sequence heteroplasmy at levels of 1-6% or more. All homoplasmic species were successfully identified by COI using standard methods of analysis, but only 71% of heteroplasmic species. The success rate in identifying heteroplasmic species was increased to 86% by treating polymorphisms as character states rather than ambiguities. Nuclear pseudogenes (numts) were also present in four species, and were distinguishable from heteroplasmic sequences by patterns of nucleotide and amino acid change.ConclusionsHeteroplasmy significantly decreased the reliability of species identification. In addition, the practical issue of dealing with large numbers of polymorphisms- and resulting increased time and labor required - makes the development of DNA barcode databases considerably more complex than has previously been suggested. The impact of heteroplasmy on the utility of DNA barcoding as a bulk specimen identification tool will depend upon its frequency across populations, which remains unknown. However, DNA barcoding is still likely to remain an important identification tool for those species that are difficult or impossible to identify through morphology, as is the case for the ecologically important solitary bee fauna.

Preface

There are more than 200,000 marine species worldwide. These include many important economic species, such as large yellow croaker, ribbonfish, tuna, and salmon, but also many potentially toxic species, such as blue-green algae, diatoms, cnidarians, ctenophores, Nassarius spp., and pufferfish. However, some edible and toxic species may look similar, and the correct identification of marine species is thus a major issue. The failure of traditional classification methods in certain species has promoted the use of DNA barcoding, which uses short, standard DNA fragments to assist with species identification. In this review, we summarize recent advances in DNA barcoding of toxic marine species such as jellyfish and pufferfish, using genes including cytochrome oxidase I gene (COI), cytochrome b gene (cytb), 16S rDNA, internal transcribed spacer (ITS), and Ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL). We also discuss the application of this technique for improving the identification of marine species. The use of DNA barcoding can benefit the studies of biological diversity, biogeography, food safety, and the detection of both invasive and new species. However, the technique has limitations, particularly for the analysis of complex objects and the selection of standard DNA barcodes. The development of high-throughput methods may offer solutions to some of these issues.

Classical way of practicing taxonomy is in endangered race in the era of genomics. In recent years taxonomy became fashionable that is owing to the revolutionary approaches in taxonomy called DNA barcoding. It is a novel approach that has generated optimism in enhancing biodiversity assessments. In DNA barcoding, complete data can be retrieved from a single specimen regardless of life stage or morphological characters. The core idea behind the DNA barcoding is the fact of minor variation in highly conserved region of DNA during the evolution within the species. Sequences have successfully been utilized for DNA barcoding which include cytoplasmic mitochondrial DNA (cox1), chloroplast DNA (trnL-F, matK, ndhF, atpB and rbcL) and nuclear DNA (ITS and housekeeping genes). Now it has been used for diverse applications such as biodiversity assessment, life history and ecological studies, forensic analysis and many more. In this chapter, we discuss the significance and utility of DNA barcoding in various fields.

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.

DNA barcoding is a new term introduced in to scientific literatures by Hebert and coworkers almost a decade ago. The concept of barcoding alone is well-known to the public: a series of black bars printed on many commercial products (Universal Product Code), which are used to distinguish different products. Advances made in molecular biology and molecular techniques late 20th century e.g. sequencing technologies, has inspired scientists to apply barcoding concept to all domains of life by using the unique nature of DNA for each single species, in order to generate a comprehensive library of living organisms on the planet earth. Such an ambitious initiative would result in a global DNA barcode database which will be valuable for biological scientists, medical, governmental and legal agencies as a mean of identification. The first initiative for DNA barcoding was funded in Canada and later on several DNA barcoding campaigns came in to the scene. The International Barcode of Life consortium (http://ibol.org/) was established in 2004. It is an international initiative devoted to develop DNA barcoding as a global standard for the identification of biological species. The Consortium for the Barcode of Life (CBOL), an international consortium coordinated from Smithsonian Institute in Washington (USA), was established to promote DNA barcoding, coordinate efforts and generally oversee the standardization process. DNA barcoding is a technique for discriminating species through analysis of sequence data, i. e. short sequences of genetic material in the genome that are unique to that organism are used to identify species mainly through PCR amplification by using primers for the broadest- possible target taxonomic group. The usefulness of DNA barcodes for proper discrimination of species was first demonstrated in animals. A 648 nucleotide base pair length region from the mitochondrial cytochrome c oxidase 1 (CoI) gene was used to identify different animal species; such that, this short sequence has emerged as the standard barcode region for higher animals. The important criteria for barcode loci are effective species-level identification – achieved when interspecific variation exceeds intraspecific –, universality, good sequence quality and coverage. Several global DNA barcoding campaigns have been established to target specific taxonomic groups such as such as plants, fungi, protists, bacteria and different entities of the kingdom animal including fishes, brides, insects, nematodes, mammals etc. In most cases in animals, CoI provides adequate resolution. However, in plants, fungi the substitution rates of this gene are much slower, and scientists are actively searching for barcode genes. For example the nuclear ribosomal Internal Transcribed Spacer (ITS) region has been proposed as universal DNA barcode marker for Fungi by the Fungal Barcoding Consortium published. However, in certain groups of fungi the ITS region fail to discrimination species; such that, secondary barcode loci will be needed for the proper delineation of species in question. One of the most important issues in DNA barcoding is standardization. A DNA barcode is not the same as a DNA sequence. For a barcode, the sequence should stem from a voucher specimen with the voucher being accessible in public collections and the trace files on which the sequences are based should be publically available. The quality and uniformity of data in databases is very crucial for the success of DNA barcodes as a universal molecular identification key. To achieve this goal, a set of guidelines and protocols should be set from collecting species to storing molecular data. The final goal of DNA barcoding project is to create a barcode reference library, where sequence data must be integrated with well characterized taxonomic units. Reference sequences are the core component of the DNA

Scientists widely agree that species extinction has heavily accelerated in the last decades. A grave problem for the conservation of diversity is the still very fragmentary knowledge of the ecology of most species. Attempts of sustainable management and conservation must integrate local communities and their traditional knowledge. Management decisions need to include the high importance of natural resources in providing building materials, food and medicines for rural as well as urbanized communities. The traditional use of plant resources, particularly of non-timber products like medicinal plants, has deep roots not only in indigenous communities, but is practiced in a wide section of society. The use of medicinal herbs is often an economically inevitable alternative to expensive western medicine. The base knowledge of this traditional use is passed from one generation to the next. Especially the medical use represents a highly dynamic, always evolving process, where new knowledge is constantly being obtained, and linked to traditional practices. An increased emphasis is being placed en possible economic benefits especially of the medicinal use of plant products instead of pure timber harvesting-an approach particularly appealing to countries with difficult economic conditions. Examples from Eastern Africa and South America are being used to show the effects of integrated approaches to conserve biological and cultural diversity.

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples. We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample. Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples. The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.

Abstract. Partovi R, Iranbakhsh A, Sheidai M, Ebadi M. 2020. The use of DNA barcoding to avoid adulteration in olive plant leaf products. Asian J For 5: 42-47. The leaves of olive plant species Olea europeae, and O. europeae var. cuspidata have been used for medicinal perfused in Iran. The first species leaves have been used to control the blood pressure, while the leaves of wild olive have been used for abortion by locals. Our preliminary inspection of the medicinal plant market revealed that the leaves of these two olive species are sold mistakenly to the consumers and their health might be at risk. Therefore, we permed this investigation to produce DNA barcodes for correct identification of these two olive species and also identify the potential adulteration in our local market. We used Internal transcribed spacer (ITS), as well as plastid genome trnL-F intergenic spacer and ribosomal protein L16 (rpL16) sequences. These sequences after alignment and curation produced DNA barcodes that can differentiate the two olive species from each other. The phylogenetic trees constructed also separated the samples of these olive species and confirmed the potential use of these short DNA sequences for olive barcoding. The present study revealed that some of the local shops mistakenly sell the wild olive leaves instead of the cultivated olive leaf to be used for blood pressure. This mistake endangers the health of pregnant women consumers if they carry a child. We suggest using a combination of nuclear ITS and plastid intergenic spacer (trnL-F and rpL16) regions for DNA barcoding of olive plants to avoid leaf product adulteration.