Plant DNA Barcoding Research Articles

The low-copy nuclear gene Agt1 as a novel DNA barcoding marker for Bromeliaceae

BackgroundThe angiosperm family Bromeliaceae comprises over 3.500 species characterized by exceptionally high morphological and ecological diversity, but a very low genetic variation. In many genera, plants are vegetatively very similar which makes determination of non flowering bromeliads difficult. This is particularly problematic with living collections where plants are often cultivated over decades without flowering. DNA barcoding is therefore a very promising approach to provide reliable and convenient assistance in species determination. However, the observed low genetic variation of canonical barcoding markers in bromeliads causes problems.ResultIn this study the low-copy nuclear gene Agt1 is identified as a novel DNA barcoding marker suitable for molecular identification of closely related bromeliad species. Combining a comparatively slowly evolving exon sequence with an adjacent, genetically highly variable intron, correctly matching MegaBLAST based species identification rate was found to be approximately double the highest rate yet reported for bromeliads using other barcode markers.ConclusionIn the present work, we characterize Agt1 as a novel plant DNA barcoding marker to be used for barcoding of bromeliads, a plant group with low genetic variation. Moreover, we provide a comprehensive marker sequence dataset for further use in the bromeliad research community.

First Phylogeny of Bitterbush Family, Picramniaceae (Picramniales).

Picramniaceae is the only member of Picramniales which is sister to the clade (Sapindales (Huerteales (Malvales, Brassicales))) in the rosidsmalvids. Not much is known about most aspects of their ecology, geography, and morphology. The family is restricted to American tropics. Picramniaceae representatives are rich in secondary metabolites; some species are known to be important for pharmaceutical purposes. Traditionally, Picramniaceae was classified as a subfamily of Simaroubaceae, but from 1995 on, it has been segregated containing two genera, Picramnia and Alvaradoa, with the recent addition of a third genus, Nothotalisia, described in 2011. Only a few species of the family have been the subject of DNA-related research, and fewer than half of the species have been included in morphological phylogenetic analyses. It is clear that Picramniaceae remains a largely under-researched plant group. Here we present the first molecular phylogenetic tree of the group, based on both chloroplast and nuclear markers, widely adopted in the plant DNA barcoding. The main findings are: The family and its genera are monophyletic and Picramnia is sister to two other genera; some clades corroborate previous assumptions of relationships made on a morphological or geographical basis, while most parts of the molecular topology suggest high levels of homoplasy in the morphological evolution of Picramnia.

Determination of Place of Residence Using the Gene Information of Plants Carried by the Human Body.

Objective To identify the plant species using the DNA sequence of plant pollen from lung tissues of a unidentified body, infer the possible long-term places of residence of the deceased according to the distribution area of the pollen in the lung tissues, therefore narrow the scope of criminal investigation and provide clues for case solving. Methods Lung tissues were extracted from the deceased, total DNA was extracted by the mCTAB method. Gene fragments of the two plant DNA barcodes, matK and rbcL, were acquired using specific primers for amplification, then sequenced. The DNA sequences of target gene fragments were acquired through bioinformatics analysis. The sequences were combined with reference sequence data. Phylogenetic analysis was made to identify the species that the DNA sequences belonged to. The places where the deceased could have lived for a long time were inferred, according to the distribution information of plant species. Results Gene fragments of 32 plant species which belonged to 31 genera of 27 families were in the lung tissues of the deceased. Among them, plants of 9 genera that had certain indicative function were mainly endemic plants from Hainan, Guangdong, Guangxi and Yunnan. These results showed that the deceased may have stayed in these areas for a long time before death. After further investigation, the victim was confirmed to have come from a county in southern Guangxi, which was in accordance with the research results. Conclusion The method of using gene information of plants from lung tissues of human bodies to infer places of residence can assist inference of the places where the deceased could have lived for a long time. The present study may also provide new ideas for locating sources of the corpses in cases with unidentified victims.

Genomic analysis of chloroplast matK and rbcL gene from Flacourtia inermis Roxb for plant DNA barcoding

Genomic analysis of chloroplast matK and rbcL gene from Flacourtia inermis Roxb for plant DNA barcoding

Genomic analysis of chloroplast matK and rbcL gene from Flacourtia inermis Roxb for plant DNA barcoding

Identification of medicinally significant plant species is helpful for their utilization as medicine. Flacourtia inermis Roxb are important medicinal plant. Aim: To Genotypic characterization of Flacourtia inermis through DNA sequence of matK and rbcL gene. Flacourtia inermis Roxb DNA was isolated using NucleoSpin® Plant II Kit (Macherey-Nagel gene extraction) Kit. Isolated DNA was amplified by using PCR (matK and rbcL gene primers). Amplified matK and rbcL gene was sequenced by ABI 3500 DNA analyzer and the sequence quality was checked using sequence scanner software v1. The results exposed basic variability of the sequences. The matK and rcbL gene phylogenic tree was constructed using MEGA 6 software. Flacourtia inermis Roxb DNA was successfully isolated using NucleoSpin® Plant II Kit (Macherey-Nagel gene extraction) Kit and confirmed by agarose gel electrophoresis. Isolated matK and rbcL genomic DNA was successfully amplified by using PCR. From the matK sequence, BLASTn analysis result Flacourtia inermis species showed 99.20% relationship with Flacourtia jangomas and rbcL BLASTn analysis result of Flacourtia inermis species showed100% relationship with Flacourtia jangomas at NCBI database. From this study, the result concluded the matK and rcbL genomes can be used for DNA identification of Flacourtia inermis. The information obtained from the study would be helpful for further research involving genotypic identification of Flacourtia sp.

Species Identification of Oaks (Quercus L., Fagaceae) from Gene to Genome

Species identification of oaks (Quercus) is always a challenge because many species exhibit variable phenotypes that overlap with other species. Oaks are notorious for interspecific hybridization and introgression, and complex speciation patterns involving incomplete lineage sorting. Therefore, accurately identifying Quercus species barcodes has been unsuccessful. In this study, we used chloroplast genome sequence data to identify molecular markers for oak species identification. Using next generation sequencing methods, we sequenced 14 chloroplast genomes of Quercus species in this study and added 10 additional chloroplast genome sequences from GenBank to develop a DNA barcode for oaks. Chloroplast genome sequence divergence was low. We identified four mutation hotspots as candidate Quercus DNA barcodes; two intergenic regions (matK-trnK-rps16 and trnR-atpA) were located in the large single copy region, and two coding regions (ndhF and ycf1b) were located in the small single copy region. The standard plant DNA barcode (rbcL and matK) had lower variability than that of the newly identified markers. Our data provide complete chloroplast genome sequences that improve the phylogenetic resolution and species level discrimination of Quercus. This study demonstrates that the complete chloroplast genome can substantially increase species discriminatory power and resolve phylogenetic relationships in plants.

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30min at room temperature (20°C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

The Use of DNA Barcoding to Assess Phylogenetic β-Diversity in Mid-Subtropical Evergreen Broad-Leaved Forests of China

The application of quantifying phylogenetic information into measures of forest β-diversity is increasing for investigating the underlying drivers of community assembly along environmental gradients. In terms of assessing evolutionary inferences of community processes, a variety of plant DNA barcodes has been widely used in phylogenetic diversity measurements. However, relatively few studies have evaluated the effectiveness of DNA barcodes with using nuclear region in estimating phylogenetic β-diversity, particularly for communities in tropical or subtropical forests. In this study, we employed DNA barcodes combing with the nuclear region to construct the community phylogeny and examined the patterns of phylogenetic β-diversity of three mid-subtropical evergreen broad-leaved forests (EBLFs) in South China. Three phylogenetic construction methods were performed, including a Phylomatic-generated tree and two ML trees based on the combination of rbcL +matK +ITS with or without a constrained tree. Our results showed that the DNA barcodes including nuclear ITS constructed a highly resolved phylogenetic tree, but the application of a constrained tree had little influence on estimation of phylogenetic diversity metrics (mean pairwise distances and mean nearest taxon distances) based on branch lengths. Using both metrics and their standardized effect size metrics, we found that the patterns of phylogenetic β-diversity in mid-subtropical forests were non-random. There was a slight decline of phylogenetic β-diversity with increasing latitudes, but no trend was found along the altitude gradient. According to the analysis of variation partition, both environmental filtering and dispersion limitation could explain the variation of phylogenetic dissimilarity between communities in mid-subtropical EBLFs of China. Our results highlight the importance of neutrality and the niche conservatism in structuring the patterns of species diversity in subtropical woody communities.

The use of plant DNA barcoding coupled with HRM analysis to differentiate edible vegetables from poisonous plants for food safety

Poisonous Urobotrya siamensis Hiepko has caused deaths of consumers due to misidentification in several cases in past years. The poisonous plant shares the vernacular name in Thai “Phak Wan” with two vegetables, Melientha suavis Pierre and Sauropus androgynus (L.) Merr. The morphology of young leaves among these species is also similar. Therefore, consumer safety could be a major concern. We applied DNA barcoding coupled with high-resolution melting (Bar-HRM) analysis to differentiate the poisonous species from the edible species. Four regions of DNA barcodes, rbcL, matK, ITS2 and the psbA-trnH intergenic spacer, were analysed. Bar-HRM primer pairs were designed based on variable sites of the DNA sequence of the chloroplast matK gene. Distinct melting temperatures for U. siamensis, M. suavis and S. androgynus were revealed at 77.94 ± 0.06 °C, 77.47 ± 0.06 °C and 76.80 ± 0.00 °C, respectively, with confidence levels >99%. The method sensitively identified the poisonous U. siamensis species at levels as low as 0.01 ng of DNA template. Bar-HRM analysis was capable of identifying seedlings or young plants from local nurseries and proved to be an efficient method for detection of food processed by heat treatment, including 5 min of stir-frying and 15 min of boiling. This study is the first report showing that Bar-HRM analysis for safety concerns effectively differentiated poisonous plants from edible plants. Moreover, Bar-HRM analysis may be further applied to other vegetables for quality control, from crop production to food control processes.

DNA barcoding of a complex genus, Aesculus L. (Sapindaceae) reveals lack of species-level resolution

Aesculus L. is a small genus of horticulturally important trees and shrubs, comprising 13–19 species. Frequent hybridization among species, particularly in cultivation, has contributed to taxonomic confusion and difficulties in the identification of plants. In this study, we evaluated three widely employed plant DNA barcode loci, matK, and the entire ITS region (ITS1+5.8S+ITS2) as well as subunit ITS2 for 50 individuals representing 13 species of Aesculus, excluding only A. wangii (=A. assamica). In contrast to the plastid matK region, both the ITS and ITS2 loci displayed low levels of species discrimination, especially in our “first hit” BLASTn searches. We also presented the phylogeny of Aesculus based on matK and the entire ITS region, with additional matK and ITS sequences from GenBank. Our results show that Aesculus chinensis, A. flava, A. glabra, A. pavia, and A. sylvatica are probably not monophyletic. Furthermore, with the widest taxon coverage until now, the current study highlights the importance of sampling multiple individuals, not only for DNA barcoding, but also for phylogenetic studies.

A case study in forensic soil examination from China

Abstract Soil examination can provide useful forensic information about the spatial location and human activities of a suspect. Soil is widely used in criminal investigations. In a case that occurred in the countryside of Jilin Province, in the NE of China, soil was found adhering to clothing on a body. Examination of plant debris in the soil using plant DNA barcoding technique found it to be ginseng root, which indicated that the soil might have come from a ginseng plantation. In the first instance, it helped in finding the place where the body was initially buried. Then a comparison was made between the soil recovered from the body and from the ginseng plantation. Composite analysis of minerals, pollen types and elements provided additional information to assist in making comparisons. Trace amounts of soil located on the body played an important role in locating the burial site and was regarded as the most valuable evidence in convicting the suspect of murder even without the suspect's DNA being available.

Barcoding of estuarine macrophytes and phylogenetic diversity of estuaries along the South African coastline.

Plant DNA barcoding serves as an effective approach to building community phylogenies and increasing our understanding of the factors that determine plant community assemblages. The aims of the study were to (i) barcode macrophytes with high estuarine fidelity and (ii) to determine the phylogenetic diversity (PD) of selected South African estuaries for conservation prioritisation. Three DNA barcoding gene regions (rbcLa, matK, and trnH-psbA) were assessed, and community phylogenies were constructed for 270 estuaries. Generally, the matK barcode had the greatest discrimination success rate of 67.4% (parsimony informative sites = 418). Closely related species formed clades that also represent estuarine habitat types. Estuaries with high phylogenetic diversity along the southeast coast were associated with a combination of mangrove and salt marsh habitats. Species richness was strongly and significantly correlated with PD (r = 0.93; p < 0.000). Based on mean pairwise distance (MPD), more temperate estuaries (56) showed significant phylogenetic clustering compared to subtropical estuaries (24) (p < 0.05). Similarly, based on mean nearest taxon distance (MNTD), significant phylogenetic clustering was highest in temperate estuaries (50) compared to subtropical estuaries (12) (p < 0.05). This suggests that the coexistence of plant species in estuaries is structured by both biotic and abiotic interactions.

Plant DNA Barcodes

Abstract—The search for universal plant DNA barcodes has proved to be a big challenge. A single locus (or combination of several loci) that can be used for species identification has yet to be revealed, though the (rbcL + matK+ ITS) combination recommended in 2009 as the standard makes it possible to assign a species to the corresponding genus. The variability of some markers differs in different taxonomic groups and usually makes it possible to select the DNA barcode (sometimes even a mini-barcode) for a specific group, especially for applied tasks. Next-generation sequencing (NGS) methods make it possible to obtain a large number of extended DNA barcodes (sequences of complete chloroplast genomes and ribosomal genes), which allows the researcher to overcome the limitations of standard DNA barcodes. It is important that NGS technologies significantly enhance the possibility of the use of herbarium specimens. The search for plant DNA barcodes is ongoing.

Current State and Prospects of DNA Barcoding and DNA Fingerprinting in the Analysis of the Quality of Plant Raw Materials and Plant-Derived Drugs

Abstract—This paper reviews the use of molecular genetic methods (DNA barcoding and DNA fingerprinting) for the authentication of plant raw materials and herbal medicinal products. ITS2 is shown to be the most effective DNA barcode for plants. This marker allows researchers to identify even highly degraded DNA, which is especially important in the case of herbal preparations and biologically active additives. The main problem of traditional DNA barcoding via Sanger sequencing is that it is impossible to read a barcode without preliminary cloning of an amplificate when a plant mix includes a large amount of various adulterants and excipients of plant origin, since species-specific chromatograms are superimposed on each other. Thus, NGS sequencing of ITS2 amplicons is a preferable method for the DNA barcoding of plant materials. This method can be used to analyze highly degraded DNA in multicomponent plant mixes and can be used to identify all their ingredients. The review also covers such DNA fingerprinting techniques as RFLP, RFLP-PCR, RAPD, RAPD-SCAR, AFLP-PCR, and ISSR.

Plant DNA-barcode library and community phylogeny for a semi-arid East African savanna.

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA-barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL-F, trnH-psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra- and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack "barcode gaps", defined as disparities between the maximum intra- and minimum interspecific genetic distances. We found barcode gaps for 72%-89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode-gap presence or absence for all of the markers in combination and for matK, trnL-F, and trnH-psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.

Comparison of Methods To Examine Diet of Feral Horses from Noninvasively Collected Fecal Samples

Feral horses (Equus ferus caballus) have become abundant on public lands in the American West, particularly over the past 10 yr. In areas where they are overabundant, there is risk of habitat degradation. Most previous studies on diet and habitat use of feral horses were conducted more than 20 yr ago; rangelands have changed considerably in that time, so it is useful to revisit horse diets. We conducted a study to examine the diet of feral horses using noninvasive methods and subjectively compare diet analysis techniques. We collected feral horse fecal samples from a sagebrush/pinyon-juniper ecosystem in Colorado in May, August, and October 2014. We analyzed 30 fecal samples from each collection session by both microhistology and plant DNA barcoding. Both microhistology and plant DNA barcoding results indicated horse diet consisted primarily of graminoids (78.5% and 68.8%, respectively, both of which are in greater proportion than availability based on ecological site descriptions); however, the two methods differed in species composition of grasses. Similar to other studies, microhistological analyses underestimated the proportion of forbs in the diet compared with plant DNA barcoding analyses, which showed a surprisingly high contribution of forbs to the diet compared with previous studies. Our results suggest plant DNA barcoding analyses have great potential, although both methods have inherent biases.

Potential Spread of Cheatgrass and Other Invasive Species by Feral Horses in Western Colorado

The invasive grass cheatgrass (Bromus tectorum L.) presents major challenges for land management and habitat conservation in the western United States. Feral horses (Equus ferus caballus) have become overabundant in some areas of the West and can impact fragile semiarid ecosystems. Amid ongoing efforts to control cheatgrass in the Great Basin, we conducted a study to determine if feral horses contribute to the spread of cheatgrass through distribution via their feces. We collected feral horse fecal samples from Little Book Cliffs Herd Management Area in western Colorado in 2014. Fecal samples were dried, and 20 from each of 3 collection sessions were cultivated to examine germination success. Six species germinated from 18 samples (30%; mostly one plant per sample where germination occurred), including cheatgrass from 8% of samples. In a separate study we examined the diet of this same horse population using fecal plant DNA barcoding. Plant species that germinated were rare in the diet and germinated from fewer samples than expected relative to their detection in the diet. Our results suggest that feral horses could be contributing to cheatgrass propagation. Native ungulates and domestic cattle also have this potential. Although management of all large ungulates is necessary to mitigate cheatgrass spread, control of feral horse numbers is particularly necessary.

Sensitive detection of Piper nigrum L. adulterants by a novel screening approach based on qPCR

The spice made from the fruits of Piper nigrum L. (Piperaceae) has high economic value since the beginnings of international trade. Because its price has been increasing, adulterations with papaya seeds, cayenne pepper and maize flour were reported. These have been screened by methodologies dedicated to the detection of single adulterants lacking sensitivity and specificity. Herein we propose a specific, highly-sensitive, high-throughput and affordable qPCR-based methodology for the detection of P. nigrum contaminants (Carica papaya, Zea mays and Capsicum annuum) using plant DNA barcodes trnL and psbA-trnH. The method enables the specific detection of contaminants in a short time with low limits of detection (LOD6 values of 1, 2 and 10 Haploid Genome Equivalents). A market survey (29 samples) revealed 41% of samples contaminated, though about ¾ at very low levels indicating accidental contamination. The proposed tool will contribute to the improvement of quality of this much traded spice.

EMBL2checklists: A Python package to facilitate the user-friendly submission of plant and fungal DNA barcoding sequences to ENA

BackgroundThe submission of DNA sequences to public sequence databases is an essential, but insufficiently automated step in the process of generating and disseminating novel DNA sequence data. Despite the centrality of database submissions to biological research, the range of available software tools that facilitate the preparation of sequence data for database submissions is low, especially for sequences generated via plant and fungal DNA barcoding. Current submission procedures can be complex and prohibitively time expensive for any but a small number of input sequences. A user-friendly software tool is needed that streamlines the file preparation for database submissions of DNA sequences that are commonly generated in plant and fungal DNA barcoding.MethodsA Python package was developed that converts DNA sequences from the common EMBL and GenBank flat file formats to submission-ready, tab-delimited spreadsheets (so-called ‘checklists’) for a subsequent upload to the annotated sequence section of the European Nucleotide Archive (ENA). The software tool, titled ‘EMBL2checklists’, automatically converts DNA sequences, their annotation features, and associated metadata into the idiosyncratic format of marker-specific ENA checklists and, thus, generates files that can be uploaded via the interactive Webin submission system of ENA.ResultsEMBL2checklists provides a simple, platform-independent tool that automates the conversion of common DNA barcoding sequences into easily editable spreadsheets that require no further processing but their upload to ENA via the interactive Webin submission system. The software is equipped with an intuitive graphical as well as an efficient command-line interface for its operation. The utility of the software is illustrated by its application in four recent investigations, including plant phylogenetic and fungal metagenomic studies.DiscussionEMBL2checklists bridges the gap between common software suites for DNA sequence assembly and annotation and the interactive data submission process of ENA. It represents an easy-to-use solution for plant and fungal biologists without bioinformatics expertise to generate submission-ready checklists from common DNA sequence data. It allows the post-processing of checklists as well as work-sharing during the submission process and solves a critical bottleneck in the effort to increase participation in public data sharing.

Rapid detection of several endangered agarwood-producing Aquilaria species and their potential adulterants using plant DNA barcodes coupled with high-resolution melting (Bar-HRM) analysis

Abstract Aquilaria is an endangered agarwood-producing genus that is currently protected by international laws. The agarwood trade is strictly monitored to prevent illegal harvesting, which has caused high demand for this natural product. Other plant sources of similar appearance or fragrance as agarwood are used as adulterant species in counterfeit products. To promote species identification via the DNA barcoding technique, the existing DNA barcoding database in our laboratory was enriched with seven plant barcoding sequences from a commercially important Aquilaria species (Aquilaria beccariana) and seven adulterant species (Cocos nucifera, Dalbergia latifolia, Pinus contorta var. latifolia, Santalum album, Strychnos ignatii, Thuja sp. and Terminalia catappa). DNA barcoding with high-resolution melting analysis (Bar-HRM) showed that the mini-barcode internal transcribed spacer 1 (ITS1) was an effective gene locus that allows for a rapid and species-specific detection of Aquilaria and their adulterants, while four other mini-barcodes (rbcL, trnL intron, ITS2 and 5.8s) functioned as a support and a crosscheck for the barcoding results. The accuracy of the Bar-HRM technique in species origin identification was further assessed with seven agarwood blind specimens. The Bar-HRM technique is a potential tool for validating agarwood-species origin and detecting products with adulterant species.