Chloroplast Barcodes Research Articles

Enriching barcoding markers in environmental samples utilizing a phylogenetic probe design: Insights from mock communities

AbstractHybridization capture is an emerging method making use of short oligonucleotide baits to enrich DNA libraries for genomic fragments of specific organisms thus enabling detection of their presence in environmental samples. Although it offers a primer‐independent alternative to metabarcoding, little empirical work has been dedicated to characterizing the underlying biases and coupled implications for biological interpretation. Moreover, few published bioinformatic pipelines are available for designing polynucleotide capture baits from a reference sequence collection. We designed RNA‐baits specifically targeting two chloroplast barcoding genes matK and rbcL to reveal the plant taxonomic diversity present in a given environmental sample. Our approach leverages the sensitivity of hybridization capture and the capacity of high‐throughput DNA sequencing instruments. It builds on a new and universal method based on ancestral sequence reconstruction, ultimately limiting the number of bait‐probes required and reducing experimental costs, while accessing high levels of taxonomic diversity. Our bait‐set selectively targets four main plant orders (Fagales, Pinales, Asterales, and Poales), representing ~18% of all described vascular plants. This is achieved through the use of only 4084 baits, each 80 nucleotides in length (80‐mer), capturing ~1.0–1.6 k nucleotide sequences from each taxon. Tests on mock communities revealed important factors influencing capture efficiency and relative abundance estimates, including GC‐content, the overall target length per taxa, and the bait density and mean number of mismatches to the bait sequence. Our results show that hybridization capture, like metabarcoding, requires caution when interpreting results quantitatively within (paleo)‐ecological studies. Biases detected in this work have the potential to be mitigated with bait designs that avoid extreme base compositional biases and balancing bait targets across taxa. However, we strongly recommend the use of mock communities and read simulations to quantify the accuracy of taxonomic representation when using new bait designs.

Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Assessment of genetic diversity and phylogenetic relationship of local coffee populations in southwestern Saudi Arabia using DNA barcoding.

The genetic diversity of local coffee populations is crucial to breed new varieties better adapted to the increasingly stressful environment due to climate change and evolving consumer preferences. Unfortunately, local coffee germplasm conservation and genetic assessment have not received much attention. Molecular tools offer substantial benefits in identifying and selecting new cultivars or clones suitable for sustainable commercial utilization. New annotation methods, such as chloroplast barcoding, are necessary to produce accurate and high-quality phylogenetic analyses. This study used DNA barcoding techniques to examine the genetic relationships among fifty-six accessions collected from the southwestern part of Saudi Arabia. PCR amplification and sequence characterization were used to investigate the effectiveness of four barcoding loci: atpB-rbcl, trnL-trnF, trnT-trnL, and trnL. The maximum nucleotide sites, nucleotide diversity, and an average number of nucleotide differences were recorded for atpB-rbcl, while trnT-trnL had the highest variable polymorphic sites, segregating sites, and haploid diversity. Among the four barcode loci, trnT-trnL recorded the highest singleton variable sites, while trnL recorded the highest parsimony information sites. Furthermore, the phylogenetic analysis clustered the Coffea arabica genotypes into four different groups, with three genotypes (KSA31, KSA38, and KSA46) found to be the most divergent genotypes standing alone in the cluster and remained apart during the analysis. The study demonstrates the presence of considerable diversity among coffee populations in Saudi Arabia. Furthermore, it also shows that DNA barcoding is an effective technique for identifying local coffee genotypes, with potential applications in coffee conservation and breeding efforts.

Molecular phylogeny and evolution of inflorescence types in Eperua.

The Amazonian hyperdominant genus Eperua (Fabaceae) currently holds 20 described species and has two strongly different inflorescence and flower types, with corresponding different pollination syndrome. The evolution of these vastly different inflorescence types within this genus was unknown and the main topic in this study. We constructed a molecular phylogeny, based on the full nuclear ribosomal DNA and partial plastome, using Bayesian inference and maximum likelihood methods, to test whether the genus is monophyletic, whether all species are monophyletic and if the shift from bat to bee pollination (or vice versa) occurred once in this genus. All but two species are well supported by the nuclear ribosomal phylogeny. The plastome phylogeny, however, shows a strong geographic signal suggesting strong local hybridization or chloroplast capture, rendering chloroplast barcodes meaningless in this genus. With our data, we cannot fully resolve the backbone of the tree to clarify sister genera relationships and confirm monophyly of the genus Eperua. Within the genus, the shift from bat to bee and bee to bat pollination has occurred several times but, with the bee to bat not always leading to a pendant inflorescence.

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.

A DNA barcode reference library for CITES listed Malagasy Dalbergia species.

On Madagascar, the illegal and unsustainable exploitation and illegal international trade of Dalbergia (rosewood) precious woods remain a serious conservation problem. Members of this genus are at high risk of extinction as a consequence of logging, mining, and slash and burn agriculture. Morphological identification of these Malagasy species is difficult in the absence of flowers and fruits, especially in the case of cut trees, sawn wood, and finished product. In this study, we use molecular barcoding to identify the Dalbergia species with the intent to contribute to the control of their illegal trade. Thirty-six Dalbergia samples representing 12 Malagasy species of which 11 have high commercial value, were collected to test the efficacy of a region of the plastid genome (rbcL) and a nuclear-transcribed ITS for barcoding. These widely used markers, as well as DNA barcoding gaps, "best match" and "best close match" approaches, and the neighbor-joining method were employed. All samples were amplified and sequenced using the two markers. Using a single locus, the "best match" and "best close match" approaches revealed that ITS has high discriminatory power within the tested Malagasy species. The combination of rbcL + ITS revealed 100% species discrimination. This study confirms that ITS alone and in combination with chloroplast barcode rbcL allow non-ambiguous identification for the 12 species studied. The results contribute to the development of DNA barcoding as a useful tool to identify Malagasy Dalbergia and suggest that the approach developed should be expanded to all 56 potentially exploited species in reference to international CITES requirements and the sustainable management of valuable resources.

Assessment of classification ability for rbcL and trnL barcode in some Stemonaceae samplescollected from northern Vietnam

Stemona tuberosa Lour. a precious medicinal plant that belongs to the Stemonaceae family, has high applicability to human life. Extracts from leaves or roots of this herb contain many bioactive compounds with great value for both medical application and economy. However, there is not enough data related to evaluation and classification for Stemona species in Vietnam. Therefore, in this study, the authors used the DNA barcoding method to evaluate at the molecular level for four samples of Stemona tuberosa Lour. in northern Vietnam compared with other reference sequences from other species in the Stemonaceae family from GenBank based on two chloroplast barcodes rbcL and trnL. The primary results of genetics distance analysis and phylogenetic trees showed the close relationship between the four studied samples with Stemona tuberosa Lour. (sequenced from GenBank). The trnL with 1100 bp in length also proves the better classification ability of Stemona than the rbcLregion with 600 bp in length. These outcomes would be the first promising step for further studies about Stemona and other herbs, thus contributing to both scientific research and practical applicability demands as classification and conservation.

DNA BARCODING LIBRARY DATABASE OF AQUILARIA MEMBER AND GYRINOPS MEMBER

Aquilaria and Gyrinops are the primary agarwood producer on international trade. For authentication and standardization purposes, it is essential to carry DNA barcoding studies of these genera. DNA barcoding studies on plants need a database of several regions on the plant genome that could act as a barcoding marker. These DNA barcoding markers could be divided into Chloroplasts barcoding and Nuclear barcoding. Several markers have been used for DNA barcoding study of agarwood producer species, including trnL-trnF, matK, rbcL, rpoC1, ycf1 (Chloroplast barcoding), and ITS (Nuclear barcoding). This review breakdown the availability of those DNA barcoding markers on the online genebank database for Aquilaria and Gyrinops. Aquilaria genus has 12 species members, while Gyrinops genus has six species members. The sequence of region trnL-trnF is the only barcoding marker covering all 12 species members of Aquilaria and six species members of Gyrinops. Both ITS and matK have covered nine species among 12 total species members of Aquilaria. The rbcL, rpoC1, and ycf1, respectively, have covered eight, five, and four species members of Aquilaria. Most of the barcoding markers have covered three species members of Gyrinops except for ITS (5 species) and rpoC1 (1 species). However, Gyrinops members have no ycf1 sequence on genebank database. Based on sequence availability on the genebank database, it could be concluded that the trnL-trnF region is the most promising DNA barcoding marker for the Aquilaria and Gyrinops members especially for the phylogenetic analysis purpose.

Chemotaxonomic Monitoring of Genetically Authenticated Amomi Fructus Using High-Performance Liquid Chromatography–Diode Array Detector with Chemometric Analysis

Amomi Fructus is widely used to treat digestive disorders, and Amomum villosum, A. villosum var. xanthioides, and A. longiligulare are permitted medicinally in national pharmacopeias. However, there are a variety of adulterants present in herbal markets owing to their morphological similarities to the genuine Amomum species. Forty-two Amomi Fructus samples from various origins were identified using internal transcribed spacer and chloroplast barcoding analyses, and then their chromatographic profiles were compared using chemometric analysis for chemotaxonomic monitoring. Among the Amomi Fructus samples, A. villosum, A. longiligulare, A. ghaticum, and A. microcarpum were confirmed as single Amomum species, whereas a mixture of either these Amomum species or with another Amomum species was observed in 15 samples. Chemotaxonomic monitoring results demonstrated that two medicinal Amomum samples, A. villosum and A. longiligulare, were not clearly distinguished from each other, but were apparently separated from other non-medicinal Amomum adulterants. A. ghaticum and A. microcarpum samples were also chemically different from other samples and formed their own species groups. Amomum species mixtures showed diverse variations of chemical correlations according to constituent Amomum species. Genetic authentication-based chemotaxonomic monitoring methods are helpful in classifying Amomi Fructus samples by their original species and to distinguish genuine Amomum species from the adulterants.

Identification of Suitable Barcodes for Specifically Detecting Adulterants of Turnip (Brassica rapa) and Radish (Raphanus sativus) in Maca (Lepidium meyenii)

Maca (Lepidium meyenii Walp.), an edible medicinal plant and popular dietary supplement, is native to South America in the Andes mountains of Peru. However, maca is frequently adulterated due to its high nutritional and medicinal value, which compromises its safety and effectiveness. In the present study, maca and its two adulterants, Brassica rapa (turnip) and Raphanus sativus (radish), were identified by one nuclear barcode (ITS) and four chloroplast barcodes (matK, psbA-trnH, rbcL, and trnL-trnF). All five DNA barcodes were perfectly identified in maca, turnip, and radish. Out of the five tested barcodes, trnL-trnF could be regarded as the ideal barcode for detecting turnip/radish adulteration in maca as it generated different sequence length among these three species, it was further validated by mixing turnip/radish into maca at various proportions, and trnL-trnF efficiently and sensitively detected adulteration even at a very low level (0.5% of adulteration).

DNA barcoding of Oryza: conventional, specific, and super barcodes

Key messageWe applied the phylogenomics to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.Rice (genus Oryza) is one of the most important crops in the world, supporting half of the world’s population. Breeding of high-yielding and quality cultivars relies on genetic resources from both cultivated and wild species, which are collected and maintained in seed banks. Unfortunately, numerous seeds are mislabeled due to taxonomic issues or misidentifications. Here, we applied the phylogenomics of 58 complete chloroplast genomes and two hypervariable nuclear genes to determine species identity in rice seeds. Twenty-one Oryza species were identified. Conspecific relationships were determined between O. glaberrima and O. barthii, O. glumipatula and O. longistaminata, O. grandiglumis and O. alta, O. meyeriana and O. granulata, O. minuta and O. malampuzhaensis, O. nivara and O. sativa subsp. indica, and O. sativa subsp. japonica and O. rufipogon.D and L genome types were not found and the H genome type was extinct. Importantly, we evaluated the performance of four conventional plant DNA barcodes (matK, rbcL, psbA-trnH, and ITS), six rice-specific chloroplast DNA barcodes (psaJ-rpl33, trnC-rpoB, rps16-trnQ, rpl22-rps19, trnK-matK, and ndhC-trnV), two rice-specific nuclear DNA barcodes (NP78 and R22), and a chloroplast genome super DNA barcode. The latter was the most reliable marker. The six rice-specific chloroplast barcodes revealed that 17% of the 53 seed accessions from rice seed banks or field collections were mislabeled. These results are expected to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

Meeting caribou in the alpine: Do moose compete with caribou for food?

The Atlantic-Gaspésie caribou (Rangifer tarandus caribou) is an endangered, isolated population that has been declining for decades in response to intensive logging. Timber harvesting has led to a significant increase in moose (Alces americanus) densities and has triggered numerical and functional predator responses. Moose are now frequently observed at higher altitudes in preferential caribou habitat. Despite extensive range overlap between these two species across Canada, few studies have precisely assessed the potential role of exploitative competition. We assessed the potential overlap between their diets during summer, a key period for energy and nutrient acquisition in cervids. We collected faeces from both species along an altitudinal gradient (200 m–1250 m asl) and used plant chloroplast barcoding sequence trnL to reconstruct diet at the species level. Plant species composition differed between caribou and moose samples, with cervid species explaining 40% of the variation in dissimilarity. We noted slight variations in dissimilarity between species between months and along an altitudinal gradient. Some species that are almost exclusively eaten by moose (speckled alder, wild redcurrant) or by caribou (common juniper, common horsetail) contributed the most to the dissimilarity between diets. The potential for food competition appears relatively low, even at increased moose densities, possibly as a result of past competition. This separation in food niche could also be explained by an imperfect segregation of species since the proportion of time spent in different strata of altitude is unequal. High moose densities thus appear to be more harmful to caribou due to shared predators, but a low level of competition for a few food items could contribute to the decline of this endangered population if some caribou are nutritionally stressed.

Combining stable isotope analysis with DNA metabarcoding improves inferences of trophic ecology.

Knowing what animals eat is fundamental to our ability to understand and manage biodiversity and ecosystems, but researchers often must rely on indirect methods to infer trophic position and food intake. Using an approach that combines evidence from stable isotope analysis and DNA metabarcoding, we assessed the diet and trophic position of Anthene usamba butterflies, for which there are no known direct observations of larval feeding. An earlier study that analyzed adults rather than caterpillars of A. usamba inferred that this butterfly was aphytophagous, but we found that the larval guts of A. usamba and two known herbivorous lycaenid species contain chloroplast 16S sequences. Moreover, chloroplast barcoding revealed high sequence similarity between chloroplasts found in A. usamba guts and the chloroplasts of the Vachellia drepanolobium trees on which the caterpillars live. Stable isotope analysis provided further evidence that A. usamba caterpillars feed on V. drepanolobium, and the possibilities of strict herbivory versus limited omnivory in this species are discussed. These results highlight the importance of combining multiple approaches and considering ontogeny when using stable isotopes to infer trophic ecology where direct observations are difficult or impossible.

Chemodiversity of Calophyllum inophyllum L. oil bioactive components related to their specific geographical distribution in the South Pacific region.

BackgroundDifferent parts of the tree Calophyllum inophyllum L. (nuts, leaves, roots, bark, fruits, nut oil and resin) are used as traditional medicines and cosmetics in most of the Pacific Islands. The oil efficiency as a natural cure and in traditional cosmetics has been largely described throughout the South Pacific, which led us to investigate C. inophyllum’s chemical and genetic diversity. A correlative study of the nut resin and leaf DNA from three distinct archipelagos in the South Pacific was carried out in order to identify diversity patterns in C. inophyllum across the South Pacific.MethodsCalophyllum inophyllum plants were sampled from French Polynesia, New Caledonia and Fiji. We extracted tamanu oil (nut oil) resin for chemo-diversity studies and sampled leaf tissues for genetic studies. We applied an analysis method designed for small quantities (at a microscale level), and used High Performance Liquid Chromatography (HPLC) to establish the chemo-diversity of tamanu oil resin. In-house standards were co-eluted for qualitative determination. Genetic diversity was assessed using chloroplast barcoding markers (the Acetyl-CoA carboxylase (accD) gene and the psaA-ycf3 intergenic spacer region).ResultsOur HPLC analysis revealed 11 previously known tamanu oil constituents, with variability among plant samples. We also isolated and characterized two new neoflavonoids from tamanu oil resin namely, tamanolide E1 and E2 which are diastereoisomers. Although genetic analysis revealed low genetic variation, our multivariate analysis (PCA) of the tamanu oil resin chemical profiles revealed differentiation among geographic regions.ConclusionWe showed here that chromatographic analysis using formalized in-house standards of oil resin compounds for co-elution studies against oil resin samples could identify patterns of variation among samples of C. inophyllum, and discriminate samples from different geographical origins.

Evaluation of cpDNA Barcodes in Selected Medicinal Plants of Mt. Arayat National Park, Pampanga, the Philippines

Objective: One common problem affecting herbal remedies is the incorrect identification of plants due to almost similar morphological characters. A promising technique to ensure correct identification of herbal medicine is the use of DNA barcoding where the gene markers either from Chloroplast (cpDNA) or Nucleus (nrDNA) are commonly used to authenticate plants. In this study, three chloroplast barcodes; matK, rbcL and trnL-F were evaluated to authenticate 20 selected medicinal plants of Mt. Arayat National Park (MANP), Pampanga, The Philippines. Methods: The genetic materials from the 20 selected medicinal plants were isolated, amplified, sequenced and aligned. All generated DNA barcodes were subjected to Kimura two-parameter model to calculate intergeneric and intrageneric variations while discriminatory power was determined using BLAST, neighbour-joining (NJ) and Wilcoxon two-sample test. Results: The results show 19 plant samples were successfully amplified. However, rbcL gave the highest sequencing success rate and identification rate up to species level over matK and trnL-F. Though, the three-cpDNA barcodes produced monophyletic clades revealing distinct association among species. The intergeneric divergences of rbcL, matK and trnL-F are significantly higher than their respective intrageneric divergences. This means the threecpDNA barcodes can effectively discriminate one species from another. Conclusion: The order of efficiency among the DNA barcodes is rbcL>matK>trnL-F. Nevertheless, the three-plastid barcodes could be used for practical and accurate authentication of medicinal plants while the baseline data in this study can be used for proper conservation of medicinal plants. Key words: BLAST, DNA barcoding, Medicinal plants, Monophyletic, Phylogeny.

A strategy for developing high-resolution DNA barcodes for species discrimination of wood specimens using the complete chloroplast genome of three Pterocarpus species

A method for extraction of wood DNA and a strategy for designing high-resolution barcodes for wood were developed. Ycf1b was the prioritized barcode to resolve the Pterocarpus wood species studied. DNA barcoding, an effective tool for wood species identification, mainly focuses on universal barcodes and often lacks high resolution to differentiate species, especially for closely related taxa within the same genus. Therefore, more highly informative DNA barcodes need to be identified. This study is the first to report a strategy for developing specific DNA barcodes of wood tissues. The complete chloroplast genomes of leaf samples of three Pterocarpus species, i.e., P. indicus, P. santalinus, and P. tinctorius, were sequenced, and thereafter, the most variable DNA regions were identified on the scale of the complete chloroplast genomes. Finally, wood DNA was extracted from 30 wood specimens of the three Pterocarpus species, and DNA recovery rates of the selected regions were tested for applicability to verification on the wood specimens studied. The seven regions with the most variation (rpl32-ccsA, rpl20-clpP, trnC-rpoB, ycf1b, accD-ycf4, ycf1a, and psbK-accD) were identified from the chloroplast genome by quantifying nucleotide diversity (Pi > 0.02), which was remarkably higher than that of the plant universal barcodes (rbcL, matK, and trnH-psbA) and the previously reported barcodes (ndhF-rpl32 and trnL-F) used for phylogenetic analysis in Pterocarpus. After comprehensive evaluation of species discrimination ability and applicability, the ycf1b region performed well in terms of the recovery success rate (76.7%) and species identification (100%) for wood specimens of the three Pterocarpus species, and was identified as the preferred high-resolution chloroplast barcode for selected Pterocarpus species. It will offer technical support for curbing illegal timber harvesting activities and for conserving endangered and valuable wood species.

Our love-hate relationship with DNA barcodes, the Y2K problem, and the search for next generation barcodes.

DNA barcodes are very useful for species identification especially when identification by traditional morphological characters is difficult. However, the short mitochondrial and chloroplast barcodes currently in use often fail to distinguish between closely related species, are prone to lateral transfer, and provide inadequate phylogenetic resolution, particularly at deeper nodes. The deficiencies of short barcode identifiers are similar to the deficiencies of the short year identifiers that caused the Y2K problem in computer science. The resolution of the Y2K problem was to increase the size of the year identifiers. The performance of conventional mitochondrial COI barcodes for phylogenetics was compared with the performance of complete mitochondrial genomes and nuclear ribosomal RNA repeats obtained by genome skimming for a set of caddisfly taxa (Insect Order Trichoptera). The analysis focused on Trichoptera Family Hydropsychidae, the net-spinning caddisflies, which demonstrates many of the frustrating limitations of current barcodes. To conduct phylogenetic comparisons, complete mitochondrial genomes (15 kb each) and nuclear ribosomal repeats (9 kb each) from six caddisfly species were sequenced, assembled, and are reported for the first time. These sequences were analyzed in comparison with eight previously published trichopteran mitochondrial genomes and two triochopteran rRNA repeats, plus outgroup sequences from sister clade Lepidoptera (butterflies and moths). COI trees were not well-resolved, had low bootstrap support, and differed in topology from prior phylogenetic analyses of the Trichoptera. Phylogenetic trees based on mitochondrial genomes or rRNA repeats were well-resolved with high bootstrap support and were largely congruent with each other. Because they are easily sequenced by genome skimming, provide robust phylogenetic resolution at various phylogenetic depths, can better distinguish between closely related species, and (in the case of mitochondrial genomes), are backwards compatible with existing mitochondrial barcodes, it is proposed that mitochondrial genomes and rRNA repeats be used as next generation DNA barcodes.

Molecular phylogeny and evolution of tropical vegetal genus Coccinia (Cucurbitaceae) using chloroplast barcode markers and its implications for speciation patterns

Evaluation of molecular phylogeny employing barcode markers for a population analysis has revealed the association between character evolution and diversification. The present research study endeavors to estimate the molecular phylogeny and evolution amongst 30 geographically distant land races of Coccinia grandis using chloroplast barcode markers. On the basis of sequences of ­C. grandis landraces and sister genera’s of Cucurbits; phylogeny and evolutionary distances were estimated using bootstrap method of UPGMA and ML with MEGA and DAMBE software. The rbcL appear to be evolving marginally amongst land races studied, while matK loci exhibited several parsimonically insignificant variations. The ycf5 locus resolved efficiently the phylogeny at both intraspecies and intergeneric levels. The values of Tajima’s relative rate test, Nei’s genetic distance and gamma distribution, within C. grandis accessions and among Cucurbits denoted plausibility of occurrences of random population expansion, slow ontogenic transitions and high consanguinity percentage among core barcode regions.The present study thus lays a platform for researchers working with vegetatively propagated plants to plan strategies for exchange of varieties, their maintenance, utilization and enhancement for further crop development and propagation.

Molecular Authentication of the Traditional Medicinal Plant "Lakshman Booti" (Smithia conferta Sm.) and Its Adulterants through DNA Barcoding.

Background:Smithia conferta Sm. is an annual herb widely used in Indian traditional medical practice and commonly known as “Lakshman booti” in Sanskrit. Morphological resemblance among the species of genus Smithia Aiton. leads to inaccurate identification and adulteration. This causes inconsistent therapeutic effects and also affects the quality of herbal medicine.Aim:This study aimed to generate potential barcode for authentication of S. conferta and its adulterants through DNA barcoding technique.Materials and Methods:Genomic DNA extracted from S. conferta and its adulterants was used as templates for polymerase chain reaction amplification of the barcoding regions. The amplicons were directed for sequencing, and species identification was conducted using BLASTn and unweighted pair-group method with arithmetic mean trees. In addition, the secondary structures of internal transcribed spacer (ITS) 2 region were predicted.Results:The nucleotide sequence of ITS provides species-specific single nucleotide polymorphisms and sequence divergence (22%) than psbA-trnH (10.9%) and rbcL (3.1%) sequences. The ITS barcode indicates that S. conferta and Smithia sensitiva are closely related compared to other species.Conclusion:ITS is the most applicable barcode for molecular authentication of S. conferta, and further chloroplast barcodes should be tested for phylogenetic analysis of genus Smithia.SUMMARY The present investigation is the first effort of utilization of DNA barcode for molecular authentication of S. conferta and its adulterants. Also, this study expanded the application of the ITS2 sequence data in the authentication. The ITS has been proved as a potential and reliable candidate barcode for the authentication of S. conferta. Abbreviations used: BLASTn: Basic Local Alignment Search Tool for Nucleotide; MEGA: Molecular Evolutionary Genetic Analysis; EMBL: European Molecular Biology Laboratory; psbA-trnH: Photosystem II protein D1- stuctural RNA: His tRNA gene; rbcL: Ribulose 1,5 bi-phosphate carboxylase/oxygenase large subunit gene.

Efficient Identification of the Forest Tree Species in Aceraceae Using DNA Barcodes.

Aceraceae is a large forest tree family that comprises many economically and ecologically important species. However, because interspecific and/or intraspecific morphological variations result from frequent interspecific hybridization and introgression, it is challenging for non-taxonomists to accurately recognize and identify the tree species in Aceraceae based on a traditional approach. DNA barcoding is a powerful tool that has been proposed to accurately distinguish between species. In this study, we assessed the effectiveness of three core standard markers (matK, rbcL and ITS) plus the chloroplast locus trnS-trnG as Aceraceae barcodes. A total of 231 sequences representing 85 species in this forest family were collected. Of these four barcode markers, the discrimination power was highest for the ITS (I) region (50%) and was progressively reduced in the other three chloroplast barcodes matK (M), trnS-trnG (T) and rbcL (R); the discrimination efficiency of the ITS marker was also greater than any two-locus combination of chloroplast barcodes. However, the combinations of ITS plus single or combined chloroplast barcodes could improve species resolution significantly; T+I (90.5% resolution) and R+M+T+I (90.5% resolution) differentiated the highest portion of species in Aceraceae. Our current results show that the nuclear ITS fragment represents a more promising DNA barcode marker than the maternally inherited chloroplast barcodes. The most efficient and economical method to identify tree species in Aceraceae among single or combined DNA barcodes is the combination of T+I (90.5% resolution).