Article

DNA Barcodes: Methods and Protocols

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

DNA barcoding, a new method for the quick identification of any species based on extracting a DNA sequence from a tiny tissue sample of any organism, is now being applied to taxa across the tree of life. As a research tool for taxonomists, DNA barcoding assists in identification by expanding the ability to diagnose species by including all life history stages of an organism. As a biodiversity discovery tool, DNA barcoding helps to flag species that are potentially new to science. As a biological tool, DNA barcoding is being used to address fundamental ecological and evolutionary questions, such as how species in plant communities are assembled. The process of DNA barcoding entails two basic steps: (1) building the DNA barcode library of known species and (2) matching the barcode sequence of the unknown sample against the barcode library for identification. Although DNA barcoding as a methodology has been in use for less than a decade, it has grown exponentially in terms of the number of sequences generated as barcodes as well as its applications. This volume provides the latest information on generating, applying, and analyzing DNA barcodes across the Tree of Life from animals and fungi to protists, algae, and plants.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The Internal transcribed spacer regions (ITS) of nuclear ribosomal DNA and combination of chloroplast DNA barcode regions rbcL, matK are the most effective for amplification. cpDNA has been the most extensively used as the core DNA barcode for land plants, based on the availability of the sequencing efficiency and high level of taxonomic resolution [17]. ...
... ITS1 is located between the 18S and 5.8S RNA genes, while ITS2 is between the 5.8S and 28S RNA genes [18]. The rbcL plastid DNA region encodes a large subunit of ribulose bisphosphate carboxylase, the key enzyme carbon-fixation in the dark phase of photosynthesis [17]. The matK gene encodes the maturase K, an enzyme of intron splicing in chloroplasts, one of the most rapidly evolving plastid coding genes in angiosperms [19]. ...
Article
Full-text available
Biological diversity preservation is important concept in modern science and environmental policy of Uzbekistan, improving the status of global biodiversity. Flora of Uzbekistan accounts over 4380 species of vascular plants. 378 taxa representing approximately 9% of currently recognized species diversity of Uzbekistan are considered as a national endemics. The present study aimed at species identification and inventory of wild, rare and endangered plant species based on molecular genetic techniques. For effective species identification and high results reliability, the genotyping of three-four loci of the combinations nuclear and chloroplast DNA-barcodes was performed for all studied species. The genotyping of marker sequences ITS, rbcL, trnL-trnF, matK for Dracocephalum adylovii I.I. Malzev is conducted the first time in Uzbekistan. The reliable similarity in the BLAST database is determined at the genus taxonomic level. 78 consensus nucleotide sequences were obtained for Lamiaceae species. The results of molecular phylogenetic analysis based on marker sequence encoding the matK fragment are presented for representative of three subfamilies: Lamioideae, Nepetoideae, Scutellarioideae recorded in the flora of Uzbekistan. Currently, the solution of biological diversity preservation is global. The possibility of successful application of DNA-barcoding for ecological monitoring is demonstrated.
... Genomic DNA of each mosquito samples was extracted following the procedures of Kress and Erickson (2012) [20]. Before DNA extraction adult females were prevented from feeding on animal blood and then 60 samples of adult females were used for total DNA extraction. ...
... Genomic DNA of each mosquito samples was extracted following the procedures of Kress and Erickson (2012) [20]. Before DNA extraction adult females were prevented from feeding on animal blood and then 60 samples of adult females were used for total DNA extraction. ...
Article
Full-text available
Some species of mosquitoes such as Culex pipiens complex are survive at different habitat conditions. They are considered of medically important vectors for some diseases, which causes a huge financial and medical problems to humans. From this point of view, species identification is the first step in the control of Culex pipiens complex. Previously identification of Culex pipiens complex is mainly done on the basis of a wide variety of morphological and biological characteristics. This can be difficult because diagnostic morphological features are often varied so little between species. So that in the present work, Culex species identification is performed by using DNA sequencing assay based on the mtDNA cytochrome oxidase-I (COX-I) gene. 56 mosquitoes specimens were collected from different infected areas in Baljurashi province, Al Baha, Saudi Arabia and grown for several generations in lab. COX-I gene amplification was carried out using the universal primers LCO1490 and HCO2198. Only 35 mosquito specimens had good amplified COXI target gene sequenced. These DNA sequences were compared with available sequences using basic alignment search tool in NCBI-Nucleotide database. Most of the examined specimens have high matching result in gene-bank with LC102132. However specimens T6, 8, 9, 15, 21, 45 & 51 have high similarity with JQ958371 Culex pipiens strain cf-3 and also specimen T35 has high similarity with KJ500032 Culex pipiens isolate 2AF. In conclusion, Sequence alignment and phylogenetic analysis of mitochondrial COX-I gene can be used as molecular tool for identification of Culex species.
... The DNA-barcoding for fish species identification is a standard method developed by University of Guelph (ON, Canada) for seafood species identification and the method is currently used by the US Food and Drug Administration (FDA) (Handy et al., 2011). The method relies on the sequencing of the cytochrome c oxidase subunit I gene (COI) and the comparison of the sequence data with FDA standard barcodes or reference sequences housed in GenBank and/or the Barcode of Life Database (BOLD) (Kress and Erickson, 2012). In addition to DNA barcoding, PCR-based assays targeting the shrimp species-specific regions of COI and 16S ribosomal RNA genes have been published (Ortea et al., 2012;Sharma et al., 2020;Wilwet et al., 2021;Korzik et al., 2020;Pascoal et al., 2011). ...
Article
Full-text available
The Atlantic white shrimp (Litopenaeus setiferus) is of great economic importance to the United States and risk being substituted with imported species due to a shortage in domestic production. To improve the current methods used for the identification of the Atlantic white shrimp species, we designed and validated a robust multiplex PCR-lateral flow assay for the onsite identification of L. setiferus. The standardized assay was validated using a miniaturized, low-cost PCR instrument with 68 shrimp, prawn, and fish samples, spread over fourteen seafood species. L. setiferus was simultaneously amplified by the multiplex assay to give three visual bands, which distinguished it from other species having either one or two bands on the dipstick. The standardized assay showed 100% inclusivity for target L. setiferus samples, 100% exclusivity for non-target samples and can be completed in less than two hours. The assay standardized in this study can be used for onsite testing of L. setiferus samples at processing facilities, restaurants, and wholesalers' facilities.
... In any case, in light of the perceptions of our arrangement, it was shown that there were varieties in a few locales, at this point, were deficient in separating the examples. As per Kress and Erickson (2012), the CO1 quality can recognize the taxa up to species level on account of the exceptionally rationed varieties of the locale. These varieties of nucleotides can be utilized by a person that recognizes the species. ...
Article
Full-text available
Sutrisnawati, Ramadhan A, Trianto M. 2022. Molecular identification of Oncomelania hupensis lindoensis, snail intermediate hosts of Schistosoma japonicum from Central Sulawesi, Indonesia. Biodiversitas 23: 5989-5994. Schistosomiasis is a zoonotic parasitic disease with Oncomelania hupensis lindoensis, intermediate snail hosts of Schistosoma japonicum. The spread of O. hupensis lindoensis snail habitat was found in the three areas of the Napu, Bada, and Lindu Highlands with an infection rate above 1%. Oncomelania hupensis lindoensis is primarily cryptic species that are morphologically difficult to identify and distinguish from other species. Consequently, it can be confused with the naming of species. One of the molecular approaches that can be used to identify the Oncomelania spp. quickly and accurately is DNA barcoding using the COI mitochondrial gene. However, the research on identifying Oncomelania spp. in Indonesia is still very limited. Therefore, this study aimed to identify six Oncomelania spp. from Napu and Lindu, Central Sulawesi using COI mitochondrial gene as a molecular marker for DNA barcoding. The method used in this study was a Polymerase Chain Reaction (PCR) method with universal primers, LCO-F and HCO-R. The data obtained were then analyzed using GeneStudio, DNASTAR, BLAST, Identification Engine, Mesquite, MEGAX, and BEAST. The analysis was conducted to obtain similarity, genetic distance and reconstruct a phylogenetic tree. The result revealed that all six samples of Oncomelania spp. collected from Napu and Lindu were identified in one species, namely Oncomelania hupensis lindoensis. This research is very important to be carried out regularly periodically so that it can be used as a basis for Schistosomiasis control program data and related sectors to eradicate snails effectively, efficiently, and on target.
... The negative side of morphological characterization includes limited ability to distinguish the diversity and the presence of environmental factor effects (Wang et al. 2010). Identification based on molecular markers is more accurate and generates more relevant data (Kress and Erickson 2012). The molecular marker identification could also be used to differentiate the genotype of each evaluated accession (Li et al. 2018), and it can potentially be generated using any plant tissue (Kumar et al. 2016;Lee et al. 2016a). ...
Article
Full-text available
Lukman, Dinarti D, Siregar UJ, Turjaman M, Sudarsono. 2022. Characterization and identification of agarwood-producing plants (Aquilaria spp.) from North Aceh, Indonesia, based on morphological and molecular markers. Biodiversitas 23: 4861-4871. Agarwood is one of the non-timber export commodities important as raw materials for bioindustry. There are 26 agarwood-producing plant species in Indonesia belonging to three families (Thymelaeaceae, Euphorbiaceae, and Fabaceae). This study aims to identify the agarwood-producing plant species in North Aceh District, Aceh Province, Indonesia, based on their morphological and molecular characters. The evaluated morphological characteristics include the bark and leaf structures of endemic plants. Molecular characterization was carried out by sequencing of matK, ITS, and trnL-trnF genes. The collected data were analyzed using the appropriate software, such as R software, BlastX, Geneious Prime and MEGA X. The evaluated 66 accessions were clustered into two major groups based on their morphology. Group 1 consisted of 10.61% of the evaluated accessions is identified as Aquilaria beccariana. Group 2 was further classified into two subclusters (Group 2a and 3b). Group 2a consisted of 10,61% of the accessions as A. malaccensis and Group 2b consisted of 78,79% of the accessions as A. microcarpa. All samples are identified as A. malaccensis based on the matK DNA sequences. Meanwhile, Groups 1 are identified as A. beccariana and Group 2a and 2b as A. microcarpa based on the ITS sequences. Based on the trnL-trnF sequences, Groups 1 and 2a was identified as A. malaccensis and Group 2b as A. microcarpa.
... This technique comprehends two methodological steps. Initially, the DNA barcode library of known species is constructed, and then the barcode sequence of the unknown sample is matched against this library for identification purposes [42]. ...
Article
Full-text available
Fungi are amongst the most abundant and diverse organisms. Despite being widely known for their adverse role in food spoilage or as pathogens for humans, animals, or plants, they also present several beneficial effects. Fungi contribute to human well-being due to their role as decomposers, degrading decay matter into smaller molecules which can be easily used by other ecosystem members. These organisms can produce medicinal compounds or modulate protective immune responses in human intestine. Fungi intervene in diverse food processes or act as a food supply. Due to fungal diversity, the unequivocal identification of these organisms is crucial to increasing their practical applications and decreasing their adverse effects. The process of identification could be achieved through the integral sequencing of fungi genomes. However, this procedure would be time-consuming and rather cost-inefficient. Therefore, several molecular markers have been developed to overcome these limitations. The chronology of DNA-based molecular markers development can be divided into three main steps: (1) prior to the development of the PCR technique (RFLP); (2) after the development of the PCR technique (RAPD, AFLP, ISSR, VNTR, SNP, InDels, and DNA barcoding); (3) after the development of the massive parallel sequencing technique (Metabarcoding and WGS). Therefore, the present review covers an overview of the most recently developed molecular markers used for fungal detection and identification.
... Bat species were identified by external morphological characteristics as previously described [27,28]. For the lyssavirus positive case, the bat's species was further confirmed through the DNA barcoding [29][30][31]. ...
Article
Full-text available
Bat lyssaviruses were identified in Taiwan's bat population during 2016-2017. The lyssa-virus surveillance system was continuously conducted to understand the epidemiology. Through this system, the found dead bats were collected for lyssavirus detection by direct fluorescent anti-body test and reverse transcription polymerase chain reaction. Three bats were identified as positive during 2018-2021. A novel lyssavirus, designated as Taiwan bat lyssavirus 2, was detected in a Nyc-talus plancyi velutinus. This lyssavirus had less than 80% nucleotide identity in the nucleoprotein (N) gene with other lyssavirus species, forming a separate branch in the phylogenetic analysis. The other two cases were identified in Pipistrellus abramus (Japanese pipistrelles); they were identified to be similar to the former lyssavirus identified in 2016-2017, which was renominated as Taiwan bat lys-savirus 1 (TWBLV-1) in this study. Even though one of the TWBLV-1 isolates showed high genetic diversity in the N gene compared with other TWBLV-1 isolates, it may be a TWBLV-1 variant but not a new species based on its high amino acid identities in the nucleoprotein, same host species, and same geographic location as the other TWBLV-1.
... Samples were identified using morphological keys (Ali and Ripley, 1983;Roberts, 1991;Grzimek's Encycloclpaedia, 2002). Keel muscle tissues were taken and preserved separately in 70 % alcohol in plastic bottles (Kress and Erickson, 2012) and stored at room temperature at Centre for Bioresource Research (CBR) for molecular analysis. Voucher specimens were deposited at Museum of CBR. ...
Article
Full-text available
Authors' Contribution SF and FN conceived and designed the study; FN executed the experiment, analyzed the tissue samples, analyzed and interpreted the data. SF and FA critically reviewed and FN revised the manuscript for important intellectual contents and approved the final version. Corvidae is a species rich and morphologically diverse family of the order Passeriformes (Aves), generally well identified by barcodes globally. Species identification and phylogenetic analysis through DNA barcodes using mitochondrial COI gene (cytochrome c oxidase subunit I) was aimed for samples of birds collected from different regions of Pakistan. Mitochondrial DNA was successfully extracted from keel tissue and Folmer region of COI gene comprising ~650 bps was amplified using universal primers and PCR products were confirmed by 1% agarose gel electrophoresis. Sequencing was carried out by Sanger's method and BLAST analysis identified these samples as five species (Urocissa flavirostris, Dendrocitta vagabunda, Corvus splendens, Corvus corax and Corvus macrorhynchos) from three genera of family Corvidae. The nucleotide sequences were submitted to the Barcode of Life Data System (BOLD) and the eligible sequences were assigned the Barcode Index Numbers (BINs). The COI gene sequence data of 8 species of family Corvidae were retrieved from GenBank (NCBI) database and phylogenetic relationship was established in these 13 barcodes. K2-parameter distances were calculated on MEGA7 software with overall average distance of 0.141 calculated at 1000 bootstrap repetitions. Phylogenetic tree reconstructed by neighbour joining method discriminated all species into two distinct clades initially and then into subclades based on similarity and variations in their sequences. Species of the same genera were grouped into one clade as genus Corvus. DNA barcoding was proved to be an effective tool for species molecular identification and their phylogenetic analysis during this study which may help in identification and biogeographic studies of birds in future.
... DNA barcoding is new, efficient, quick, low-cost, and standard technique for the fast identification and evaluation of plant and animal species based on DNA sequence from a small fragment of the whole genome in a rapid, accurate [14][15][16][17][18]. DNA barcoding can help to detect species, quick identification of any species that are possibly novel to science and to report the essential ecological and evolutionary questions as a biological instrument [19][20][21][22][23][24][25]. DNA barcoding are frequently promoted for their facility to enhance the accessibility of scientific information and new knowledge to the public and non-specialists [26,27]. ...
Article
Full-text available
Background DNA barcoding have been considered as a tool to facilitate species identification based on its simplicity and high-level accuracy in compression to the complexity and subjective biases linked to morphological identification of taxa. MaturaseK gene ( MatK gene) of the chloroplast is very vital in the plant system which is involved in the group II intron splicing. The main objective of this study is to determine the relative utility of the “ MatK ” chloroplast gene for barcoding in 15 legume as a tool to facilitate species identification based on their simplicity and high-level accuracy linked to morphological identification of taxa. Methods and Results MatK gene sequences were submitted to GenBank and the accession numbers were obtained with sequence length ranging from 730 to 1545 nucleotides. These DNA sequences were aligned with database sequence using PROMALS server , Clustal Omega server and Bioedit program. Maximum likelihood and neighbor-joining algorithms were employed for constructing phylogeny. Overall, these results indicated that the phylogenetic tree analysis and the evolutionary distances of an individual dataset of each species were agreed with a phylogenetic tree of all each other consisting of two clades, the first clade comprising (Enterolobium contortisiliquum, Albizia lebbek), Acacia saligna , Leucaena leucocephala, Dichrostachys Cinerea, (Delonix regia, Parkinsonia aculeata), (Senna surattensis, Cassia fistula, Cassia javanica) and Schotia brachypetala were more closely to each other, respectively. The remaining four species of Erythrina humeana, (Sophora secundiflora, Dalbergia Sissoo, Tipuana Tipu) constituted the second clade. Conclusion Moreover, their sequences could be successfully utilized in single nucleotide polymorphism or as part of the sequence as DNA fragment analysis utilizing polymerase chain reaction in plant systematic. Therefore, MatK gene is considered promising a candidate for DNA barcoding in the plant family Fabaceae and provides a clear relationship between the families.
... The primary goal for DNA-based identification of taxa such as DNA barcoding is to be able to identify a taxon only by using a short DNA sequence (Hebert et al., 2003;Kress and Erickson, 2012;L€ ucking et al., 2020. This also has implications in the ecological study of fungi (Põlme et al., 2020). ...
Article
Full-text available
Intragenomic variation is the molecular variation within the genome among repetitive DNA. As a multigene family, nuclear ribosomal DNA (rDNA) has been widely used in fungal taxonomy for their ease in amplification and suitable variability to attain various levels of taxonomic resolution. At the intraspecific level, rDNA is believed to be under concerted evolution and the internal transcribed spacers (ITS) region is actually accepted as a universal barcoding marker for fungi. However, documentation of intragenomic variation of rDNA indicated that it can be problematic in species delimitation and identification. Fungal taxonomic studies have not generally taken into account the intragenomic variation of rDNA in a systematic manner. In this review, our objective is to address the definition, the origin and the mechanisms for maintenance of intragenomic variation, as well as its implication in the domain of fungal molecular taxonomy, particularly for species delimitation, identification and DNA barcoding. With advanced sequencing technologies (second and third generations), we also addressed how these technologies can be used to study the intragenomic variation of rDNA and also how the intragenomic variation will impact on DNA barcoding via high-throughput sequencing.
... DNA barcodes based on the mitochondrial cytochrome oxidase I (COI) gene were generated using standard procedures at the Biodiversity Institute of Ontario, University of Guelph and Colorado State University from a leg of a pinned specimen (Hebert et al. 2003Craft et al. 2010;Wilson 2012). Primers used for amplification and sequencing included LepF1, MLepF1, LepR1, MLepR2, C_LepFolF, and C_LepFolR (Hajibabaei et al. 2006, Hernández-Triana et al. 2014. ...
Article
Full-text available
Eight new species of Eucosmocydia Diakonoff are described and illustrated from the Afrotropical region: E. pappeana Brown and Razowski, new species (TL: Kenya); E. deinbolliana Brown and Razowski, new species (TL: Kenya); E. ugandensis Aarvik, new species (TL: Uganda); E. lecaniodiscana Brown and Razowski, new species (TL: Kenya); E. nigeriana Brown and Razowski, new species (TL: Nigeria); E. pan­coviana Brown and Razowski, new species (TL: Kenya); E. kirimiriana Brown and Razowski, new species (TL: Kenya); and E. macabensis Brown and Razowski, new species (TL: Mauritius). Three additional species are transferred to the genus: E. hymenosa (Razowski, 2013), new combination (TL: Nigeria); E. chlorobathra (Meyrick, 1911), new combination (TL: Seychelles); and E. trigonoptila (Meyrick, 1921), new combination (TL: Mozambique). We also transfer to the genus E. catamochla (Meyrick, 1932), new combination (TL: Indonesia), the first species recorded outside the Afrotropical region. We recognize two species groups in Eucosmocydia, and this contribution focuses on the oedipus Diakonoff, 1988 group (n = 13 species), the males of which are characterized by a unique flattened lobe from the base of the hindwing. Six species from Kenya were reared exclusively from native fruit of Sapindaceae; E. mixographa (Meyrick) was formerly reported from Fabaceae and Euphorbiaceae.
... DNA barcoding is being described as a new method employed for identifying any species, and it is now fairly being applied to taxa involving fungi and oomycetes (Kress et al., 2012). DNA barcode typically refers to a short, distinctive DNA sequence pattern of about 400-800 bp which are amplified, sequenced, and set apart for characterization by specific software to distinguish and identify species (Gao and Chang, 2013). ...
Chapter
Full-text available
This chapter focuses on doubling the farmer's income by practicing protected cultivation techniques specialized in vegetable cultivation
... Important developments of molecular methods have allowed fundamental advances in many fields of mycology (Morange et al., 1998). DNA barcoding (Kress and Erickson, 2012) has emerged as a tool to help differentiate and identify fungal collections, and may reveal morphologically and ecologically unpredictable systematic relationships (e.g., Gómez-Zapata et al., 2021). It has become the norm in taxonomy and phylogenetic studies, and is also being used in other subfields such as ethnomycology (Teke et al., 2018) and paleomycology (Bernicchia et al., 2006). ...
Article
Full-text available
Understanding and describing the diversity of living organisms is a great challenge. Fungi have for a long time been, and unfortunately still are, underestimated when it comes to taxonomic research. The foundations were laid by the first mycologists through field observations. These important fundamental works have been and remain vital reference works. Nevertheless, a non-negligible part of the studied funga escaped their attention. Thanks to modern developments in molecular techniques, the study of fungal diversity has been revolutionized in terms of tools and knowledge. Despite a number of disadvantages inherent to these techniques, traditional field-based inventory work has been increasingly superseded and neglected. This perspective aims to demonstrate the central importance of field-based research in fungal diversity studies, and encourages researchers not to be blinded by the sole use of molecular methods.
... Phylogenetic tree developed from phylogenetic hypothesis, normally based upon the substitution of homologus nucleotides (bases), usually vary among the different taxa. In the phylogenetic trees different clades (grouping of two or more taxa) formed are based upon the length of the branches that ultimately connects taxa or clades with their common ancestral origin (Kress & Erickson, 2012). For detailed analysis of the plant species through DNA Barcode technique, scientists put emphasis in Consortium for Barcode of Life (CBOL) the chloroplast genes, rbcL and matK (Chase et al., 2007) which serve as the core barcodes for plant species, along with supplement barcodes trnH-psbA and ITS which serve as intergenic sequence and ribosomal gene respectively (Chase et al., 2005;Kress et al., 2005). ...
Article
Full-text available
DNA Barcode Analysis was carried out as new taxonomic approach to reaffirm the identity and the phylogenetic relationship of five families of order Rosales by using three molecular markers such as rbcL, matK and trnH-psbA. This study will serve as contribution to the previous taxonomic position of the order Rosales on the basis of morphological characters as well as DNA sequence data. According to the DNA Barcode data cladogram of order Rosales indicated that family Moraceae formed an individual clade in all Neighbour Joining trees formed on the basis of rbcL, matK, trnH-psbA, rbcL+matK and rbcL+matK+trnH-psbA while in previous studies it was sister to Urticaceae and Cannabaceae. Another change was also observed in the placement of Family Rosaceae that did not form a separate clade as it did in previous studies but it was sister to Cannabaceae, Rhamnaceae and Urticaceae and embedded among these families. Our study does not support the previous taxonomic position of order Rosales based on morphological characters and some DNA sequences which are not standard barcodes now. The PCR amplification success rate was found highest 96% for rbcL followed by matK 64% while lowest for trnH-psbA 58%. The gene sequencing tree was successfully constructed when markers were used both singly and in combinations. The phylogenetic tree analysis demonstrated that, separate clusters forming species with more than 50% bootstrap value were the discriminate species, indicating high reliability of evolutionary relationship among the taxa. In the present study it was found that all three DNA Barcode markers when employed in combination exhibited maximum discriminatory power amongst all the studied species in the families of order Rosales.
... Five adult flesh fly samples were used for total DNA extraction. The genomic DNA extraction and purification was performed from one or two legs of these flies as per Kress and Erickson (2012) and also in accordance with our previous optimized protocol for DNA extraction from the whole mosquito and fly (Abdella et al., 2018). Each adult flesh fly was homogenized in 50 µL of insect lysis buffer (16.5 g GuSCN, 12 mL 0.5M EDTA pH 8.0, 6 mL 1 M tris-HCl pH 8.0, 1 mL triton X-100, 10 mL tween-20 and final volume made 200 mL by double distilled water). ...
Article
MITOCHONDRIAL COX-1 BARCODING FOR THE IDENTIFICATION AND PHYLOGENETIC ANALYSIS OF Sarcophaga (Liosarcophaga) dux RECORDED FROM JEDDAH CITY OF SAUDI ARABIA Abdullah Al Ghamdi1 , Tariq S. Alghamdi2 , Mohamed. N. Bakr 3,4, Khalid Al Ghamdi1 , Jazem Mahyoub1 , Touseef Amna2 * and Ehab M. Abdella2,5,* 1Biology Department, Faculty of Science, King Abdulaziz University, Jeddah (Saudi Arabia) 2Department of Biology, Faculty of Science, Albaha University, Albaha (Saudi Arabia) 3Department of Biological Science, Graduate School of Science, Hiroshima University, Hiroshima (Japan) 4Department of Pollution, National Institute of Oceanography and Fisheries, Cairo (Egypt) 5Zoology Department, Faculty of Science, Beni Suef University, Beni Suef (Egypt) *e-mail: touseefamna@gmail.com , ehabbdella@gmail.com (Received 1 November, 2021; accepted 31 December, 2021) ABSTRACT This study was aimed to identify and phylogenetically analyze the sarcophagid flies from Jeddah (Saudi Arabia) on the basis of mitochondrial cytochrome-C-oxidase subunit-1 (COX-1) gene. Five flesh fly samples were collected from different locations of Jeddah region. All the samples were first morphologically identified as Sarcophaga dux and confirmed on the basis of COX-1 gene amplification and sequencing techniques using universal primers LCO1490 and HCO2198. For phylogenetic analysis, 5 sequences were checked by COX-1 service on barcode of life data system (BOLD) database. To determine the phylogenetic relationships and genetic variability between 5 collected and BOLD samples, large Neighbor-joining tree comprising of the collected samples and all the available S. dux BOLD samples were constructed. The 32 most related BOLD sequences were used to construct maximum likelihood (ML) and Neighbor-joining trees. The phylogenetic analysis of COX-1 gene sequences revealed significant match with S. dux sequences from BOLD database: GQ254446 (Australia), MG780139 (India), AY879255 (China), JN869983 (Malaysia) and KC249664 (Egypt). This is the first report of the flesh fly species recorded in the Western regions of Saudi Arabia. Results indicated that the genetic diversity and phylogenetic relationship analysis of the collected S. dux samples have an important role in understanding the evolutionary processes of Sarcophaga cryptic species in Western regions of Saudi Arabia. Keywords: Genetic diversity, mitochondrial COX-1 gene, phylogenetic analysis, Sarcophaga dux
... Five adult flesh fly samples were used for total DNA extraction. The genomic DNA extraction and purification was performed from one or two legs of these flies as per Kress and Erickson (2012) and also in accordance with our previous optimized protocol for DNA extraction from the whole mosquito and fly (Abdella et al., 2018). Each adult flesh fly was homogenized in 50 µL of insect lysis buffer (16.5 g GuSCN, 12 mL 0.5M EDTA pH 8.0, 6 mL 1 M tris-HCl pH 8.0, 1 mL triton X-100, 10 mL tween-20 and final volume made 200 mL by double distilled water). ...
... For plants, DNA barcoding has truly become a universal tool for hypothesis testing by expanding the ability to identify a species at all stages of its life history (i.e., fruits, seeds, seedlings, mature individuals both fertile and sterile) from damaged or preserved specimens, as well as environmental samples with multiple species. Accordingly, DNA barcodes have been applied to address fundamental questions in ecology, evolution, and conservation biology, such as: how are species assembled in communities; what is the extent and specificity of multispecies interactions in well-studied and previously poorly known environments; and where are the most evolutionarily rich habitats for priority conservation and natural area protection in this age of habitat degradation [4,5]. With regard to the applied users of taxonomy, DNA barcodes also serve as a means to identify regulated species, invasive species, and endangered species. ...
Article
Full-text available
DNA barcoding has transformed the fields of ecology, evolution, and conservation by providing a rapid and effective tool for species identification. The growth of DNA barcodes as a resource for biologists has followed advances in computational and sequencing technology that have enabled high-throughput barcoding applications. The global DNA barcode database is expanding to represent the diversity of species on Earth thanks to efforts by international consortia and expanding biological collections. Today, DNA barcoding is instrumental in advancing our understanding of how species evolve, how they interact, and how we can slow down their extirpation and extinction. This review focuses on current applications of DNA barcode sequences to address fundamental lines of research, as well as new and expanding applications of which DNA barcoding will play a central role.
... Five adult flesh fly samples were used for total DNA extraction. The genomic DNA extraction and purification was performed from one or two legs of these flies as per Kress and Erickson (2012) and also in accordance with our previous optimized protocol for DNA extraction from the whole mosquito and fly (Abdella et al., 2018). Each adult flesh fly was homogenized in 50 µL of insect lysis buffer (16.5 g GuSCN, 12 mL 0.5M EDTA pH 8.0, 6 mL 1 M tris-HCl pH 8.0, 1 mL triton X-100, 10 mL tween-20 and final volume made 200 mL by double distilled water). ...
... Eluted genomic DNA was stored at −20 • C until the PCR analysis. Subsequently, 18S ribosomal partial sequences were amplified from purified genomic DNA using the Universal Folmer primers LCO 1490 (5 -GGT CAA CAA ATC ATA AAG ATA TTG G-3 ) as a forward primer and HCO2198 (5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 ) as a reverse primer [16,17]. A polymerase chain reaction (PCR) was carried out using a total volume of 25 µL master mix solutions (14 µL of ddH 2 O, 2.5 µL of Promega PCR buffer, 3 µL of Promega MgCl 2 solutions, 1 µL of Promega dNTP, 1 µL of each forward and reverse primers, 2 µL of DNA template and 0.5 µL of Promega Go Taq DNA polymerase). ...
Article
Full-text available
Fish parasites such as Caligus clemensi are a serious concern for cultured fish in many regions of the world, including Malaysia. This study was designed to elucidate the parasites’ prevalence and intensity coupled with the morphology and molecular identification of C. clemensi on cultured Lutjanus erythropterus in Jerejak Island, Penang, Peninsular Malaysia. The study was carried out on 200 fish specimens of cultured L. erythropterus obtained from the GST group aquaculture farm. Parasites were collected from the infested part of L. erythropterus fish, and their prevalence and intensity were determined. The parasites were identified morphologically using a field emission scanning electron microscope. Molecular studies were performed through PCR amplification and sequencing. MEGA 5 was used to construct a phylogenetic tree using the pairwise distance method. The results showed that only the C. clemensi parasite was found prevalent on L. erythropterus fish with a prevalence and mean intensity (S.D) of 198 (99%) and 36.4 ± 12.2, respectively. The prevalence varied significantly with respect to fish length (p < 0.05). The nucleotide BLAST sequence for 18S ribosomal RNA partial sequences showed 97% with 100% query similarity, E-value 0 with C. clemensi with the accession number DQ123833.1. Conclusively, C. clemensi remains a major parasite of L. erythropterus in the study area.
... It has been portrayed as a universal tool that can be linked to any kind of biological or biodiversity information (Galimberti et al., 2013). DNA barcoding systems utilize a short, standardized region (between 400 and 800 base pairs) to identify species (Kress & Erickson, 2012). The technique is based on the assumption that interspecies variation should exceed intraspecies variation: this difference (i.e. the barcode gap) of a standardized region is exploited for species level identification. ...
Article
Full-text available
The increased utilization of metrology resources and expanded application of its’ approaches in the development of internationally agreed upon measurements can lay the basis for regulatory harmonization, support reproducible research, and advance scientific understanding, especially of dietary supplements and herbal medicines. Yet, metrology is often underappreciated and underutilized in dealing with the many challenges presented by these chemically complex preparations. This article discusses the utility of applying rigorous analytical techniques and adopting metrological principles more widely in studying dietary supplement products and ingredients, particularly medicinal plants and other botanicals. An assessment of current and emerging dietary supplement characterization methods is provided, including targeted and non-targeted techniques, as well as data analysis and evaluation approaches, with a focus on chemometrics, toxicity, dosage form performance, and data management. Quality assessment, statistical methods, and optimized methods for data management are also discussed. Case studies provide examples of applying metrological principles in thorough analytical characterization of supplement composition to clarify their health effects. A new frontier for metrology in dietary supplement science is described, including opportunities to improve methods for analysis and data management, development of relevant standards and good practices, and communication of these developments to researchers and analysts, as well as to regulatory and policy decision makers in the public and private sectors. The promotion of closer interactions between analytical, clinical, and pharmaceutical scientists who are involved in research and product development with metrologists who develop standards and methodological guidelines is critical to advance research on dietary supplement characterization and health effects.
... The method used of DNA Barcoding can be used effectively to figure out the identity of the tissue that is being examined and to compare the overall gene structure of the invasive species to tell the differences between the native species on a genetic level [7]. The slight disparities can be used to indicate why one species is more effective in their environment and how this is able to outcompete the native species. ...
Article
Full-text available
Corbicula fluminea is an invasive species that has been observed to outcompete the native clams at the American River located near Sacramento in the Central Valley in California. We hypothesized that C. Fluminea has advantages exhibited physically including utilization of filter-feeding methods and relative spacing of its cirri as compared to the native American River clams. To investigate what makes the species so successful, we tested C. Fluminea versus the native clams in algal and E. coli environments to predict the relative advantage of a filter feeder. In addition, we used computer programs to digitally analyze the spacing between the actual cirri, which help bivalves capture food particles, of the two species. The findings pointed towards C. Fluminea's inherent advantage in both physical and genetic traits over the native clams species which allowed it to flourish and successfully invade the American River ecosystem. However, the species' genetic findings are found through DNA analysis.
... DNA barcoding is a powerful molecular tool for species identification that uses one or several conservative standard DNA sequences in organisms as a marker to identify species [12][13][14]. With the development of the latest cutting-edge technology, TCM research has entered the era of "Herbgenomics", which reflects the application of DNA barcoding technology in TCM research [15]. ...
Article
Full-text available
Based on the gradual transformation from wild growth to artificial cultivation, the accurate authentication of licorice seeds contributes to the first committed step of its quality control and is pivotal to ensure the clinical efficacy of licorice. However, it is still challenging to obtain genetically stable licorice germplasm resources due to the multi-source, multi-heterozygous, polyploid, and hybrid characteristics of licorice seeds. Here, a new method for determining the heterozygosity of licorice seed mixture, based on the various sites, and finding the composition characteristics of licorice seed is preliminarily designed and proposed. Namely, high-throughput full-length multiple DNA barcodes(HFMD), based on ITS multi-copy variation exist, the full-length amplicons of ITS2, psbA-trnH and ITS are directly sequenced by rDNA through the next-generation sequence(NGS) and single-molecule real-time (SMRT) technologies. By comparing the three sequencing methods, our results proved that SMRT sequencing successfully identified the complete gradients of complex mixed samples with the best performance. Meanwhile, HFMD is a brilliant and feasible method for evaluating the heterozygosity of licorice seeds. It shows a perfect interpretation of DNA barcoding and can be applied in multi-base multi-heterozygous and polyploid species.
... Taxon-specific primers targeting highly conserved regions of the genome are used to amplify sample DNA in a thermocycler [44]. The sample is then sent to a Next Generation Sequencer. ...
Chapter
Full-text available
State-of-the-art tools are revolutionizing protected area (PA) manager approaches to biodiversity monitoring. Effective strategies are available for test site establishment, data collection, archiving, analysis, and presentation. In PAs, use of new technologies will support a shift from primarily expert-based to automated monitoring procedures, allowing increasingly efficient data collection and facilitating adherence to conservation requirements. Selection and application of appropriate tools increasingly improve options for adaptive management. In this chapter, modern biodiversity monitoring techniques are introduced and discussed in relation to previous standard approaches for their applicability in diverse habitats and for different groups of organisms. A review of some of today's most exciting technologies is presented, including environmental DNA analysis for species identification; automated optical, olfactory, and auditory devices; remote sensing applications relaying site conditions in real-time; and uses of unmanned aerial systems technology for observation and mapping. An overview is given in the context of applicability of monitoring tools in different ecosystems, providing a theoretical basis from conceptualization to implementation of novel tools in a monitoring program. Practical examples from real-world PAs are provided.
... These failures are also detected in phylogenetic trees constructed by COI, implying they have a very close kinship (Figure 6). Different from the universal bar code consisting of a series of vertical bars that are printed on commercial products, DNA barcodes are genetic markers seeking to use sequence message of any biological sample, regardless of morphological identification of the sample, to address questions relating to taxonomy, ecology and evolution (33). A considerable amount of studies have evaluated the performance of a variety of barcode markers with respect to both their ease of PCR amplification and their capacity to delineate species (34). ...
Article
Full-text available
Background: Accurate taxonomic identification is the cornerstone for monitoring, conservation and management of ecological resources. China has the highest biodiversities and the richest species assemblages in the world, but is lacking in sufficient assessment to the abundant genetic variability. DNA barcoding is a proven tool employing sequence information for rapid and unambiguous species delineation. However, the ability of barcodes to distinguish species that are archaic and distinctive evolutionary lines remains largely untested.Methods: In order to investigate the resources of terrestrial animals in China, regions from mitochondrial COI and 16S are barcoded for 395 specimens belonging to 54 selected species, many of which are indigenous representatives in danger. High success rate of PCR amplification is achieved by using universal COI and 16S primers with many numts pseudogenes co-amplified from mammalian samples.Results: Application of barcodes to flag species is generally straightforward since no COI or 16S haplotypes are shared between closely related species. Barcoding gap, species resolution and phylogenetic relationships relying on our barcode libraries are further compared using distance and tree based approaches.Conclusion: Results show that the discriminatory power of the two barcode markers could differentiate on a case-by-case basis, and also suggest a careful consideration of the nuclear numts for barcoding studies as they might provide a new understanding for evolution.
... gov/genbank/). 7 The specific gene is designated as a "barcode" for different groups of organisms. 8 A DNA region that serves as a barcode must be sequenceable with universal primers that anneal to conservative regions around it and span multiple species while possessing small variation within a species. ...
Article
Full-text available
DNA structure has been leveraged in a variety of facets that allow scientists to perform a range of assays, including ones for identification of species, establishing evolutionary relationships between taxa, or even identifying individuals. Here, we present a DNA barcoding method as practical, hands-on approach that connects several experimental techniques in one sequence to teach the principles behind DNA isolation, purification, PCR, sequencing, and phylogeny analysis. Our set of exercises is designed for a teaching university laboratory setting. The three laboratory class assignments utilize DNA from a mushroom (can be purchased at a supermarket) and provide a pipeline to guide students through the process of identifying an unknown sample, like in many research laboratories. The third assignment can be used as a stand-alone exercise on phylogeny analysis and can be taught remotely. Students explore the theory behind the standard molecular techniques and apply it in a hands-on setting that involves experimental design, sample preparation, and use of hallmark molecular instruments.
... Earlier studies have been focused on elucidation of genetic distinction among mulberry species and varieties by There were reports on phylogenetic relationship between a little number of local indian genotypes using ISSR markers (Vijayan and Chatterjee, 2003), eleven species of M. alba L. by ISSR and RAPD technique (Prem et al., 2004), and Chinese wild and cultivated mulberry cultivars based on ISSR and SSR markers (Zhao et al., 2006 ). By employing RAPD markers, the work has been also performed to reveal genetic relationship among some mulberry genotypes ( Besides, ITS sequence has been used as a potential nuclear DNA marker to discriminate each other on the level of species in most plants (Walker and David, 2012). Recent study revealed that the nuclear ribosomal ITS sequence was more e cient than the chloroplastic psbA-trnH sequence in differentiating medicinal plant Boerhavia diffusa This study aimed to elucidate genetic distinction among four mulberry species such as M. mongolica Schneid., M. bombycis Koidz., M. alba Linn. ...
Preprint
Full-text available
Mulberry ( Morus spp.) is a cross-pollinating and highly hybridized plant of which productivity are greatly varied in different varieties. We analysed molecular distinction among four mulberry species and varieties cultivated in DPR Korea by using nuclear ribosomal internal transcribed spacer ( ITS ) sequences and inter simple sequence repeat (ISSR) markers. ITS sequences didn’t represent a remarkable interspecific distinction among four mulberry species used in our study, suggesting that it could not be employed to identify them. ISSR analysis using 16 random primers generated 158 different markers ranging from 100 to 4000 bp in size. The results showed the inter-specific genetic variation (55.34%) was slightly higher than intra-specific genetic variation(44.66%), with comparatively low average number of migrants per generation (Nm) among populations (0.3886). Using ISSR primers selected in this study, in the future, the suitable breeding strategy might be established in raising of elite mulberry varieties on the basis of interspecific hybridization.
... PCR amplification was carried out using two primer pairs, namely, BirdF1 and COIbirdR2 for COX I [7]; L15662 and H16065 for Cyt b [8]. PCR amplification was carried out in a total reaction volume of 25 μL using ×2 PCR Master Mix (Emerald, TaKara, Japan) primers and 50-100 ng DNA on 2720 thermal cycler (Applied Biosystems, Massachusetts, USA). ...
Article
Full-text available
Background and aim: India has large varieties (recognized, unrecognized) of native chickens (Desi) scattered throughout the country, managed under scavenging system different from commercial chicken breeds. However, they are less investigated for genetic diversity they harbor. The present study was planned to evaluate genetic diversity among two native chicken populations of North Gujarat (proposed Aravali breed) and South Gujarat (Ankleshwar breed). Aravali chicken, a distinct population with unique characters different from the registered chicken breeds of India is under process to be registered as a new chicken breed of Gujarat, India. Materials and methods: Two mitochondrial markers, namely, cytochrome oxidase c subunit I (COX I) and cytochrome b (Cyt b) genes were studied across 10 birds from each population. Methodology included sample collection (blood), DNA isolation (manual), polymerase chain reaction amplification of mitochondrial genes, Sanger sequencing, and purification followed by data analysis using various softwares. Results: Haplotype analysis of the COX I gene unveiled a total eight and three haplotypes from the Aravali and Ankleshwar populations, respectively, with haplotype diversity (Hd) of 92.70 % for the Aravali and 34.50% for the Ankleshwar breed. Haplotype analysis of the Cyt b gene revealed a total of four haplotypes from the Aravali population with 60% Hd and no polymorphism in Ankleshwar breed. The phylogenetic analysis uncovered Red Jungle Fowl and Gray Jungle Fowl as prime roots for both populations and all domestic chicken breeds. Conclusion: Study findings indicated high genetic variability in Aravali chicken populations with COX I mitochondrial marker being more informative for evaluating genetic diversity in chickens.
... Surrounding atmosphere plays a critical role in the development and growth of bamboo, hence, this barcoding technology can help the farmer in choosing the species that are the best for growth and development of bamboo in specific climatic conditions [137]. DNA standardized barcode is additionally utilized for building DNA barcode library which further utilized for contrasting and recognizing DNA barcode sequences recuperated from obscure DNA samples [138]. This technology is not only used for identification and classification of species but also has to protect endangered species, preserving natural resources, pharmaceutical, wildlife forensic, authentication of natural health products, illegitimate trading and even amusing activities [74,90,139,140]. ...
Article
Full-text available
Bamboo, a gramineous plant belonging to the family Poaceae , comprises of 1575 species from 116 genera across the globe. It has the ability to grow and evolve on degraded land and hence, can be utilized in the various applications as an alternative for plastic and wood. DNA barcoding, a long genomic sequence, identifies barcode region which shows species-specific nucleotide differences. This technology is considered as advanced molecular technique utilized for characterization and classification of the various species by applying distinctive molecular markers. Recent investigations revealed the potential application of various barcode regions such as matK , rbcL , rpoB , rpoC1 , psbA-trnH , and ITS2 , in identification of many bamboo species from different genus. In this review we comprehensively discussed the relevance of DNA barcoding as a tool in classification/identification of various bamboo species. We highlighted the methodology, how this advance technology overcomes the challenges associated with traditional methods along with prospects for future research.
... The DNA barcode is an approach that aims to perform a rapid identification of species of a taxonomic group, based on the amplification of a fragment of the sequence of the mitochondrial gene cytochrome c oxidase subunit I (COX1) and its comparison to those previously sequenced from morphologically identified specimens (Kress andErickson 2012, Hebert et al. 2003). As the efficacy of COX1 in the discrimination of fish species has previously been demonstrated (Thu et al. 2019, Ude et al. 2020, Ward et al. 2005, this can be very useful for the identification of Colombian ichthyofauna, considering the high diversity of fish in the country and the rate of ten species of freshwater fishes described per year (DoNascimiento et al. 2017). ...
Preprint
The Barcoding was proposed motivated by the mismatch between the low number of taxonomists that contrasts with the large number of species, the method requires the construction of reference collections of DNA sequences that represent existing biodiversity. Freshwater fishes are key indicators for understanding biogeography around the world. Colombia with 1610 species of freshwater fishes is the second richest country in the world in this group. However, genetic information of the species continues to be limited, the contribution to a reference library of DNA barcodes for Colombian freshwater fishes highlights the importance of biological collections and seeks to strengthen inventories and taxonomy of such collections in future studies. This dataset contributes to the knowledge on the DNA barcodes and occurrence records of 96 species of Freshwater fishes from Colombia. The species represented in this dataset correspond to an addition to BOLD public databases of 39 species. Forty-nine specimens were collected in Atrato bassin and 708 in Magdalena-Cauca bassin during the period of 2010 to 2020, two species (Loricariichthys brunneus and Poecilia sphenops) are considered exotic to the Atrato, Cauca and Magdalena basins and four species (Oncorhynchu mykiss, Oreochromis niloticus, Parachromis friedrichsthalii and Xiphophorus helleri) are exotic to Colombian hydrogeographic regions. All specimens are deposited in the CIUA collection at University of Antioquia and have their DNA barcodes made publicly available in the Barcode of Life Data System (BOLD) online database and the distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).
... This system uses short gene regions and standardized. [8][9][10]. DNA barcode also reliable for studying phylogenetic relationship among living things. ...
Article
Full-text available
Bamboos are versatile plants with many usages. Indonesia has a high diversity of Bamboos species and has been using them for a long time. As multipurpose plants, they are vulnerable to over exploration. Much of the bamboo land has been converted into housing or agricultural land. Development and conservation need to be balanced, to protect the genetic diversity, bamboo in particular. We studied the relationship among four genera of bamboo that mostly found in Indonesia using gene that presents in chloroplast DNA, ndhH . DNA from twenty-four samples from Bambusa, Dendrocalamus, Gigantochloa and Schizostachyum were extracted and amplified using specific ndhH primer. Sequencing results were analysed with BioEdit 7.2 software. Phylogenetic tree was build using raxmlGUI 2.0 software. The result showed that ndhH gene generated good sequence quality. It has small variation among samples. The result suggested that ndhH was able to differentiate some species within Schizostachyum , however; it was not a success for others.
... There are various steps involved in DNA barcoding that follows appropriate collection of samples from the plant tissues and passes through DNA extraction then sequencing, data editing and final barcode compare to barcode library. It finally leads to discovery of many species [11]. ...
Article
Wrasses are among the most interesting and numerically abundant fish families and are widely distributed in deep and coastal marine waters. A survey was undertaken to study the diversity of labrids along the southwest coast of India between December 2020 to January 2022. A total of 98 specimens belonging to the family Labridae were collected during the study period, including nine species classified under five genera. The species included Cheilinus chlorourus, Halichoeres timorensis, Halichoeres marginatus, Halichoeres zeylonicus, Labroides dimidiatus, Stethojulis trilineata, Stethojulis interrupta, Thalassoma lunare and Thalassoma jansenii. The species were identified through morphological studies and DNA barcoding using the mitochondrial cytochrome oxidase I (COI) gene. The analyses aimed to document the morphological and molecular variations of species from different localities. The resultant molecular data were compared with morphological identifications and additional sequence data from GenBank. The results concluded that generated mtDNA sequences of wrasses matched closely with the available sequences in the GenBank. The studied species were nested in clades with the nearest related species with low Pairwise distances. The study recommends stock assessment of the labrids of India, as they are heavily exploited for both as food and ornamental fish species.
Chapter
In China, the sea cucumber has a long history as a food. The nutrients of sea cucumbers abound in polysaccharides, lipids, proteins, and a variety of vitamins and minerals. In recent years, the sea cucumber industry has been growing rapidly, and sea cucumber products are becoming increasingly diversified, entailing the establishment of high-quality standards and detection systems. These standards and systems are crucial for increasing production, market supervision, combating counterfeit and shoddy products, and thus protecting consumers’ rights and interests. Rapid and precise identification of sea cucumber species, traceability of production techniques (covering origin, breeding procedure, etc.), and detection and quantification of practical components in sea cucumber products serve as the foundation for constructing standard quality-control systems. This can help to standardize the whole value chain of the sea cucumber business, from breeding to processing, deep processing, and sales. This chapter will discuss how to identify sea cucumber species, track their origins, detect the practical components, and establish the relevant product quality-control systems and standards.KeywordsSea cucumberNutritional valueSpecies identificationQuality management systemStandard
Article
Full-text available
Taro (Colocasia esculenta L. Schott, Araceae), an ancient root and tuber crop, is highly polygenic, polyphyletic, and polygeographic in nature, which leads to its rapid genetic erosion. To prevent the perceived loss of taro diversity, species discrimination and genetic conservation of promising taro genotypes need special attention. Reports on genetic discrimination of taro at its center of origin are still untapped. We performed DNA barcoding of twenty promising genotypes of taro indigenous to the northeastern hill region of India, deploying two chloroplast-plastid genes, matK and rbcL, and the ribosomal nuclear gene ITS2. The secondary structure of ITS2 was determined and molecular phylogeny was performed to assess genetic discrimination among the taro genotypes. The matK and rbcL genes were highly efficient (>90%) in amplification and sequencing. However, the ITS2 barcode region achieved significant discrimination among the tested taro genotypes. All the taro genotypes displayed most similar sequences at the conserved matK and rbcL loci. However, distinct sequence lengths were observed in the ITS2 barcode region, revealing accurate discriminations among the genotypes. Multiple barcode markers are unrelated to one another and change independently, providing different estimations of heritable traits and genetic lineages; thus, they are advantageous over a single locus in genetic discrimination studies. A dynamic programming algorithm that used base-pairing interactions within a single nucleic acid polymer or between two polymers transformed the secondary structures into the symbol code data to predict seven different minimum free energy secondary structures. Our analysis strengthens the potential of the ITS2 gene as a potent DNA barcode candidate in the prediction of a valuable secondary structure that would help in genetic discrimination between the genotypes while augmenting future breeding strategies in taro.
Chapter
Traditional Chinese medicine (TCM) researches become more and more dependent on the multi-omics data mining, to reveal the intricate and important patterns hidden in TCM preparations and their links with human diseases.
Chapter
Fungi is a group of eukaryotic and multicellular heterotrophs with a wide range of diversity of phenotypic characters. Therefore, the traditional practice of identifying fungi solely based on morphological characters is incomplete and outdated. With the advancement of molecular biology and bioinformatics, molecular data surge, i.e., DNA and proteins based on which fungal species can be identified. This chapter discusses DNA barcoding techniques using ITS, nuclear ribosomal subunits, protein-coding genes, secondary DNA markers, and DNA taxonomy to identify fungal species and their placement across different taxonomic levels. In addition to the list of curated molecular databases, this chapter provides a step-wise procedure for identifying extremophilic fungi.KeywordsDNA barcodingDNA taxonomyIdentification of extremophilic fungi
Article
Exploration on DNA-based investigations is frequently obscured by being constrained to work in suboptimal conditions that incorporate minimal sampling regimes, crucial tissue type, and amount. As nucleic acids are the source to explain different inquiries in DNA-involved investigations there is a need for a trendy strategy to affirm entire genome from complicated heterogeneous vegetation. In this verification, authors modified CTAB DNA extracting method to develop a fast and precise protocol for dehydrated biological material and extremely processed commercial samples. The tea plant, Camellia sinensis, has the importation of barely recognizable raw product when fermented and high adulteration risk commercially is the advantage noted to study the plant. To reaffirm the success of the present problem the DNA extracted from the protocol is subjected to molecular manipulations like sequencing of DNA with CBOL recommended plant barcoding markers like rbcL, matK, trnH-psbA, rpoC, rpoB, ycf1, ITS2 and created a standard reference library for south Indian tea clones to authenticate unknown taxa. This protocol was standardized with the initial screening of ten south Indian tea clones and five commercial samples. After successful trials, the modified CTAB manual was applied to 40 dehydrated tea cultivars and ten commercially available black dust and green tea. Authors have also verified the tea cultivars sequences extracted by this method are applicable to generate DNA barcodes. The tested method's success in extremely processed specimens proves the modified CTAB method as a promising alternative among all reported CTAB methods and costly isolation kits for incomplete specimens.
Article
Full-text available
The Barcode of Life initiative was originally motivated by the large number of species, taxonomic difficulties and the limited number of expert taxonomists. Colombia has 1,610 freshwater fish species and comprises the second largest diversity of this group in the world. As genetic information continues to be limited, we constructed a reference collection of DNA sequences of Colombian freshwater fishes deposited in the Ichthyology Collection of the University of Antioquia (CIUA), thus joining the multiple efforts that have been made in the country to contribute to the knowledge of genetic diversity in order to strengthen the inventories of biological collections and facilitate the solution of taxonomic issues in the future. This study contributes to the knowledge on the DNA barcodes and occurrence records of 96 species of Colombian freshwater fishes. Fifty-seven of the species represented in this dataset were already available in the Barcode Of Life Data System (BOLD System), while 39 correspond to new species to the BOLD System. Forty-nine specimens were collected in the Atrato River Basin and 708 in the Magdalena-Cauca asin during the period 2010-2020. Two species ( Loricariichthys brunneus (Hancock, 1828) and Poecilia sphenops Valenciennes, 1846) are considered exotic to the Atrato, Cauca and Magdalena Basins and four species ( Oncorhynchus mykiss (Walbaum, 1792), Oreochromis niloticus (Linnaeus, 1758), Parachromis friedrichsthalii (Heckel, 1840) and Xiphophorus helleri Heckel, 1848) are exotic to the Colombian hydrogeographic regions. All specimens are deposited in CIUA and have their DNA barcodes made publicly available in the BOLD online database. The geographical distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).
Chapter
Full-text available
The threats posed by deadly biological weapons, viz. insects and pests, are now easily combated by the use of latest technologies available in the field. Besides conventional methods, genetic manipulation of lethal genes to create sterile insect is one of the popular methods used in controlling insects and pests.
Chapter
Full-text available
Insects occupy the largest part of the phylum Arthropoda and manifest tremendous diversity; although some species of insects are beneficial, others are a problem to humans, as they reduce crop production, cause food losses and spread diseases. Therefore, it is important to maintain the pest population below the level of economic threshold to reduce the economic losses. Insect pests are developing resistance against insecticides; it became a challenge to improve understanding of the factors driving pest adaptation and evolution. With the surge of sequence information, researchers are accessing data to infer the biological questions and concentrate on genome sequencing to understand gene expression, gene regulation, quantification, genetic traits and gene disruption. Implementation of bioinformatics techniques are providing meta-omic tools for insect-microorganism interactions, synthesizing target-oriented insecticides and establishing pest’s evolutionary history. In this proposed chapter, we discuss the efficacy and utility of bioinformatics techniques in insect pest management. For instance: (1) analysis of insecticide resistance proteins using a computational tool (DIRProt), an ACE tool for insect resistance mutations, and using bioinformatics tools to detect gene arrangement accountable for adaptation; (2) OffTargetFinder software provides species-specific RNAi design to manage insect pests, sterile insect technique with RNAi and InsectBase platform for comparative genomic analysis on gene families, pathways and orthologs; (3) ConFind (conserved region finder) is for conserved sequence analysis and interpretation, and CryGetter automates the retrieval of Cry protein; and (4) using the gene disruption techniques, clustered regularly interspaced short palindromic repeats (CRISPR), and population suppression techniques.
Preprint
Full-text available
DNA barcodes have been considered as a tool to facilitate species identification based on their simplicity and high-level accuracy compression to the complexity and subjective biases linked to morphological identification of taxa. MaturaseK gene “ MatK” of the chloroplast is very crucial in the plant system which is involved in the group II intron splicing. The main objective of this current study is determining the relative utility of the “ MatK” chloroplast gene for barcoding in fifteen legume trees by both single region and multiregional approaches. The chloroplast “ MatK” gene sequences were submitted to GenBank and accession numbers (GenBank: LC602060 , LC602154 , LC602263 , LC603347 , LC603655 , LC603845 , LC603846 , LC603847 , LC604717 , LC604718 , LC605994 , LC604799 , LC605995 , LC606468 , LC606469 ) were obtained with sequence length ranging from 730 to 1545 nucleotides. These DNA sequences were aligned with database sequence using PROMALS server , Clustal Omega server and Bioedit program. Also, the maximum likelihood and neighbor-joining algorithms for phylogenetic reconstruction using the MEGA-X program were employed. Overall, these results indicated that the phylogenetic tree analysis and the evolutionary distances of an individual dataset of each species were agreed with a phylogenetic tree of all each other consisting of two clades, the first clade comprising (Enterolobium contortisiliquum, Albizia lebbek), Acacia saligna , Leucaena leucocephala, Dichrostachys Cinerea, (Delonix regia, Parkinsonia aculeata), (Senna surattensis, Cassia fistula, Cassia javanica) and Schotia brachypetala were more closely to each other, respectively. The remaining four species of Erythrina humeana, (Sophora secundiflora, Dalbergia Sissoo, Tipuana Tipu) constituted the second clade. Therefore, MatK gene is considered promising a candidate for DNA barcoding in plant family Fabaceae and providing a clear relationship between the families. Moreover, their sequences could be successfully utilized in single nucleotide polymorphism (SNP) or part of the sequence as DNA fragment analysis utilizing polymerase chain reaction (PCR) in plant systematic.
Article
Full-text available
Prior studies of butterfly diversification in the Neotropics have focused on Amazonia and the tropical Andes, while southern regions of the continent have received little attention. To address the gap in knowledge about the Lepidoptera of temperate South America, we analyzed over 3,000 specimens representing nearly 500 species from Argentina for a segment of the mitochondrial COI gene. Representing 42% of the country’s butterfly fauna, collections targeted species from the Atlantic and Andean forests, biodiversity hotspots that were previously connected but are now isolated. We assessed COI effectiveness for species discrimination and identification and how its performance was affected by geographic distances and taxon coverage. COI data also allowed to study patterns of genetic variation across Argentina, particularly between populations in the Atlantic and Andean forests. Our results show that COI discriminates species well, but that identification success is reduced on average by ~20% as spatial and taxonomic coverage rises. We also found that levels of genetic variation are associated with species’ spatial distribution type, a pattern which might reflect differences in their dispersal and colonization abilities. In particular, intraspecific distance between populations in the Atlantic and Andean forests was significantly higher in species with disjunct distributions than in those with a continuous range. All splits between lineages in these forests dated to the Pleistocene, but divergence dates varied considerably, suggesting that historical connections between the Atlantic and Andean forests have differentially affected their shared butterfly fauna. Our study supports the fact that large-scale assessments of mitochondrial DNA variation are a powerful tool for evolutionary studies.
Article
Full-text available
Metabarcoding has proven to be a powerful tool to assess ecological patterns and diversity from different habitats. Terrestrial invertebrate diversity is frequently based on bulk samples, which require comparatively high sampling effort. With environmental DNA (eDNA) metabarcoding, field sampling effort can be reduced while increasing the number of recovered organism groups. However, a proof of concept is missing for several invertebrate groups, hampering the development of best-practice advice for these groups. This study aims to provide recommendations on key aspects for the processing of soil samples, from sampling effort to choice of DNA extraction method and marker genes. This study uses eDNA metabarcoding as a tool for assessing invertebrate biodiversity in soil samples, specifically comparing two DNA extraction methods (with and without a lysis step) and two genes, 18S and COI markers. The results show that the choice of marker and DNA extraction method (including a lysis step) significantly affect species detection rates and concomitantly observed invertebrate community composition. Combining methods, by using larger amounts of starting material and including a lysis step resulted in an increase of invertebrate species numbers. Together, these methods improved the detection of species with known lower population densities and allowed the assessment of temporary mesofauna. Furthermore, the choice of marker significantly influenced the diversity levels found. The 18S marker allowed the detection of a higher number of annelid and nematode OTUs, while the COI marker was more suitable for detecting changes in arthropod community structure, especially at the species level. This study makes significant advances to the field of invertebrate biodiversity assessment, particularly using metabarcoding tools by addressing several methodological considerations that are key for accurate ecological appraisals.
Article
The correct scientific identification of the plant material is crucial to the safety and efficacy of herbal products. Trillium govanianum Wall. ex D. Don, a pharmaceutically prized medicinal plant species endemic to the Himalaya, has been recently subjected to large-scale extraction in the wild due to increasing market demand. Consequently, adulteration and/or substitution of the traded material of T. govanianum with closely-related plants such as Paris polyphylla Sm. has become a quality control problem for the herbal industry. Therefore the present study aimed to (i) develop a reference DNA barcode of T. govanianum and its potent adulterant P. polyphylla for correct identification, (ii) and check adulteration of market samples of T. govanianum. Reference DNA barcode library of T. govanianum and P. polyphylla was successfully established, using Internal transcribed spacer (ITS), Maturase K (matK), chloroplast intergenic spacer (trnH-psbA), and ribulose-bisphosphate carboxylase (rbcL) regions. The ITS, matK and trnH-psbA were found to be ideal reference barcodes for T. govanianum, while ITS and trnH-psbA were suitable for P. polyphylla. All the trade samples (i.e. dried rhizomes), investigated during the present study, got putatively identified with the respective reference barcodes; and thereby indicating the ITS and trnH-psbA regions as the potential DNA barcodes for the identification of the trade samples/adulterants. The ITS region showed the highest mean intra- and inter-specific distances, which proves its high efficiency in differentiating closely related species. Phylogenetic trees were also constructed following the neighbor-joining (NJ) method, based on ITS, rbcL, and trnH-PsbA regions of Trillium and Paris species, which distinguished Trillium species from those of Paris. The availability of a novel DNA barcode for this important medicinal plant species will be helpful for correct identification of its raw plant material, checking illegal trade, and to regulate its sustainable collection from the natural habitats.
Article
The genus Ocimum L. (Lamiaceae) is composed of aromatic plants known for their peculiar strong smell added to their close similarities in morphological attributes, increase attention and applications as ethnobotanicals of culinary and medicinal use. There are reports of difficulty in Ocimum taxonomy. Seedling morphology and molecular characterization of some Ocimum accessions collected from Southwest states of Nigeria were carried out to delimit members of the genus. Morphological measurements were carried out on the seedlings characters and recorded. Molecular characterization was carried out on their fresh leaf‐samples. Statistical analyses of quantitative seedling data were performed using SAS/PC1 version 9.01. Seedling morphology reveals phanerocotylar epigeal with foliaceous cotyledon type; reniform and flabellate cotyledon shape, obcordate and obtuse cotyledon apex, cordate and truncate cotyledon base. There is a significant mean difference in all the quantitative seedling characters at p<0.05. Blast query reveals the accessions as five species exhibiting 99 % homologous identities with previous submissions in the GenBank database.
Article
Here, we report for the first time a comprehensive survey of the DNA barcoding of commercially important groupers in Aceh waters, Indonesia using the cytochrome oxidase subunit I (COI) gene. The fish samples were collected from major fish landing sites and fish markets in nine districts in Aceh, that covered the western and eastern coastal ecosystems of Aceh. In total, the 144 COI sequences revealed 26 grouper species with most represented by multiple specimens. As expected, genetic distances increased from lower to higher taxonomic levels: 0 %–1.27 % within species, 3.79 %–19.96 % within genus and 13.84 %–24.15 % within family. Out of the total species, 23 (88.5 %), were categorised as Least Concern (LC) and three species (11.5 %) were Data Deficient (DD) under the IUCN Red List. Furthermore, four species were categorized under ‘decreasing population trend’ (Epinephelus bleekeri, E. coioides, Plectropomus leopardus, and Variola albimarginata). This study is a significant contribution to the DNA barcode library of groupers in this region and expansion of the Indonesian and the global barcode entries in general.
Chapter
Molecular markers are defined as any stable and inherited variation, which is quantifiable or detectable by a suitable method and can be subsequently used to detect the presence of a specific genotype or phenotype. Molecular markers are a potential aid to animal breeding, which helps in identifying the genetic makeup of an animal and thereby predicting its performance. The methods used to define molecular markers include Restriction Fragment Length Polymorphisms (RFLPs), Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Microsatellites, Minisatellites, Single Nucleotide Polymorphisms (SNPs) and many more. The most widely used application of marker systems in breeding is Marker-Assisted Selection (MAS). Utilization of marker-based information for genetic improvement of animals depends on the choice of an appropriate marker. In the future, molecular markers may serve as a potential tool for the evaluation and manipulation of existing germplasm in order to select and create desired traits in animals which, in turn, results in genetic improvement of animals.
Article
Full-text available
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
Article
Full-text available
Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.
Article
Full-text available
Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter-and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.
Article
Full-text available
The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon - UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.
Article
Full-text available
Species number, functional traits, and phylogenetic history all contribute to characterizing the biological diversity in plant communities. The phylogenetic component of diversity has been particularly difficult to quantify in species-rich tropical tree assemblages. The compilation of previously published (and often incomplete) data on evolutionary relationships of species into a composite phylogeny of the taxa in a forest, through such programs as Phylomatic, has proven useful in building community phylogenies although often of limited resolution. Recently, DNA barcodes have been used to construct a robust community phylogeny for nearly 300 tree species in a forest dynamics plot in Panama using a supermatrix method. In that study sequence data from three barcode loci were used to generate a well-resolved species-level phylogeny. Here we expand upon this earlier investigation and present results on the use of a phylogenetic constraint tree to generate a community phylogeny for a diverse, tropical forest dynamics plot in Puerto Rico. This enhanced method of phylogenetic reconstruction insures the congruence of the barcode phylogeny with broadly accepted hypotheses on the phylogeny of flowering plants (i.e., APG III) regardless of the number and taxonomic breadth of the taxa sampled. We also compare maximum parsimony versus maximum likelihood estimates of community phylogenetic relationships as well as evaluate the effectiveness of one- versus two- versus three-gene barcodes in resolving community evolutionary history. As first demonstrated in the Panamanian forest dynamics plot, the results for the Puerto Rican plot illustrate that highly resolved phylogenies derived from DNA barcode sequence data combined with a constraint tree based on APG III are particularly useful in comparative analysis of phylogenetic diversity and will enhance research on the interface between community ecology and evolution.
Article
Full-text available
DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.
Article
Full-text available
The assembly of DNA barcode libraries is particularly relevant within species-rich natural communities for which accurate species identifications will enable detailed ecological forensic studies. In addition, well-resolved molecular phylogenies derived from these DNA barcode sequences have the potential to improve investigations of the mechanisms underlying community assembly and functional trait evolution. To date, no studies have effectively applied DNA barcodes sensu strictu in this manner. In this report, we demonstrate that a three-locus DNA barcode when applied to 296 species of woody trees, shrubs, and palms found within the 50-ha Forest Dynamics Plot on Barro Colorado Island (BCI), Panama, resulted in >98% correct identifications. These DNA barcode sequences are also used to reconstruct a robust community phylogeny employing a supermatrix method for 281 of the 296 plant species in the plot. The three-locus barcode data were sufficient to reliably reconstruct evolutionary relationships among the plant taxa in the plot that are congruent with the broadly accepted phylogeny of flowering plants (APG II). Earlier work on the phylogenetic structure of the BCI forest dynamics plot employing less resolved phylogenies reveals significant differences in evolutionary and ecological inferences compared with our data and suggests that unresolved community phylogenies may have increased type I and type II errors. These results illustrate how highly resolved phylogenies based on DNA barcode sequence data will enhance research focused on the interface between community ecology and evolution.
Article
Full-text available
We propose in this paper to use three regions of plastid DNA as a standard protocol for barcoding all land plants. We review the other markers that have been proposed and discuss their advantages and disadvantages. The low levels of variation in plastid DNA make three regions necessary; there are no plastid regions, coding or non-coding, that evolve as rapidly as mitochondrial DNA generally does in animals. We outline two, three-region options, (1) rpoC1, rpoB and matK or (2) rpoC1, matK and psbA-trnH as viable markers for land plant barcoding.
Article
Full-text available
The selection of a DNA barcode in plants has been impeded in part due to the relatively low rates of nucleotide substitution observed at the most accessible plastid markers. However, the absence of consensus also reflects a lack of standards for comparing potential barcode markers. While many publications have suggested a host of plant DNA barcodes, the studies cannot be readily compared with each other through any quantitative or statistical parameter, partly because they put forward no single compelling rationale relevant to the adoption of a DNA barcode in plants. Here, we argue that the efficacy of any particular plant DNA barcode selection should reflect the anticipated performance of the resulting barcode database in assignment of a query sequence to species. While legitimate scientific disagreement exists over the criteria relevant to "database performance", the notion gives a unifying rationale for prioritizing selection criteria. Accordingly, we suggest a measure of barcode efficacy based on the rationale of database performance, "the probability of correct identification" (PCI). Moreover, the definition of PCI is left flexible enough to handle most of the scientific disagreement over how to best evaluate DNA barcodes. Finally, we consider how different types of barcodes might require different methods of analysis and database design and indicate how the analysis might affect the selection of the most broadly effective barcode for land plants.
Article
Full-text available
Short-sequence fragments ('DNA barcodes') used widely for plant identification and inventorying remain to be applied to complex biological problems. Host-herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcodes amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included-more than 10 per cent of the known Australian fauna-feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions.
Article
Full-text available
The Barcode of Life Data System (bold) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. bold is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of bold, discusses their functional capabilities, and concludes by examining computational resources and future prospects.
Article
Full-text available
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
Article
Full-text available
Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.
Article
Full-text available
Astraptes fulgerator, first described in 1775, is a common and widely distributed neotropical skipper butterfly (Lepidoptera: Hesperiidae). We combine 25 years of natural history observations in northwestern Costa Rica with morphological study and DNA barcoding of museum specimens to show that A. fulgerator is a complex of at least 10 species in this region. Largely sympatric, these taxa have mostly different caterpillar food plants, mostly distinctive caterpillars, and somewhat different ecosystem preferences but only subtly differing adults with no genitalic divergence. Our results add to the evidence that cryptic species are prevalent in tropical regions, a critical issue in efforts to document global species richness. They also illustrate the value of DNA barcoding, especially when coupled with traditional taxonomic tools, in disclosing hidden diversity.
Article
Full-text available
Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes. • angiosperm • internal transcribed spacer • Plummers Island • species identification • trnH-psbA
Article
Full-text available
Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.
Article
Full-text available
An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the 'Barcode of Life Initiative', to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1 = CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life.
Article
Full-text available
A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.
Article
Full-text available
DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a “DNA barcoding gap” is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes. • CITES • Kruger National Park • Mesoamerica
Article
The Barcode of Life Data System (BOLD) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of BOLD , discusses their functional capabilities, and concludes by examining computational resources and future prospects.
Article
DNA barcodes have been successfully applied to a limited number of animal groups with the application of the mitochondrial gene, cytochrome c oxidase subunit 1. Recently two DNA regions, the plastid trnH-psbA spacer and nuclear ribosomal ITS region, have been shown to have potential as an identification barcode for land plants, although with some significant drawbacks. The ideal barcode should be relatively short in length (∼700 bp), more variable between than within species, and easily amplifiable with universal primers. Building on current success, ongoing investigations are searching for the best barcode to apply to all land plants. Once established, a plant barcode may be effectively used in biodiversity inventories, conservation assessments, and applied forensic investigations. Advances in sequencing technology and the completion of the DNA barcode library have the potential to provide the public with increased access to information about the natural world.
Ten species in one: DNA barcoding reveals cryptic species in the neotropical ­skipper butterfly
  • Pdn Hebert
  • Eh Penton
  • Jm Burns
Foreword: how to conserve wild plants? Give the world the power to read them Plant conservation: a natural history approach
  • Dh Janzen
Commentary DNA barcoding: genes, genomics, and bioinformatics
  • Wj Kress
  • Dl Erickson
Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding
  • P Taberlet
  • E Coissac
  • F Pompanon
A two-locus global DNA barcode for land plants: the ­coding
  • Wj Kress
  • Dl Erickson
Foreword: how to conserve wild plants? Give the world the power to read them
  • D H Janzen
  • DH Janzen