Chloroplast DNA Barcodes Research Articles

APPLICATION OF THE DNA BARCODING METHOD FOR IDENTIFICATION AND TAXONOMIC ANALYSIS OF THE LAMIACEAE FAMILY

The Lamiaceae family is one of the largest and most important among the aromatic plants, including many species with pronounced pharmacological properties. Plants of this family, such as mint, rosemary and thyme, are widely used in traditional and modern medicine due to their therapeutic and biochemical properties. The importance of correct species identification is emphasized by the need to preserve the biodiversity and sustainable use of the resources of this family. In this work, plants of Lamiaceae family growing in the territory of the Karaganda region were studied by DNA barcoding. The studies were conducted on the basis of nucleotide sequences of rbcL and trnH-psbA chloroplast DNA barcodes using the methods of paired genetic distances, BLASTn and phylogenetic tree based on the analysis of maximum parsimony. The high efficiency of the rbcL locus was demonstrated for the identification of higher taxonomic groups, such as subfamilies and tribes, while the intergenic spacer trnH-psbAhasproved to be effectiveat the level of speciesandgenus level.

Development of specific barcodes for identification of Aloe species based on chloroplast DNA barcoding

DNA barcoding is a widely used taxonomic tool for correctly identifying plant species. The chloroplast matK and rbcL are some of the most extensively studied variable coding genes of angiosperms and have been recommended as a universal barcode for terrestrial plants. Genus Aloe contains around 500 species, and most Aloe species have extensive traditional medicinal usage worldwide, viz., A. arborescens, A. ferox, A. maculate, and A. vera. This study evaluated the two chloroplast DNA barcodes (matK and rbcL) to develop a theoretical base for species identification and germplasm conservation of Aloe species. The Maximum Parsimony analysis was conducted to study the evolutionary relatedness of Aloe sequences using matK and rbcL sequences obtained from the NCBI nucleotide database. The results revealed that 49 Aloe accessions were segregated into four major clades using matK phylogenetic analysis. Likewise, the 29 Aloe accessions were allocated into two clades based on the rbcL phylogeny. Additionally, specific barcodes have been developed for the following species: A. vera, A. boylei, A. ciliaris, A. karasbergensis, and A. sinkatana based on SNP (Single Nucleotide Polymorphism) site analysis of barcode sequences and the corresponding DNA QR (quick response) code of each sequence was generated. This study provides a unique method for species identification of the genus Aloe and indicates the possibility of correctly identifying, discriminating, and documenting the Aloe species.

Insights into the phylogenetic relationships and species boundaries of the Myricaria squamosa complex (Tamaricaceae) based on the complete chloroplast genome

Myricaria plants are widely distributed in Eurasia and are helpful for windbreak and embankment protection. Current molecular evidence has led to controversy regarding species boundaries within the Myricaria genus and interspecific phylogenetic relationships between three specific species—M. bracteata, M. paniculata and M. squamosa—which have remained unresolved. This study treated these three unresolved taxa as a species complex, named the M. squamosa complex. The genome skimming approach was used to determine 35 complete plastome sequences and nuclear ribosomal DNA sequences for the said complex and other closely related species, followed by de novo assembly. Comparative analyses were conducted across Myricaria to identify the genome size, gene content, repeat type and number, SSR (simple sequence repeat) abundance, and codon usage bias of chloroplast genomes. Tree-based species delimitation results indicated that M. bracteata, M. paniculata and M. squamosa could not be distinguished and formed two monophyletic lineages (P1 and P2) that were clustered together. Compared to plastome-based species delimitation, the standard nuclear DNA barcode had the lowest species resolution, and the standard chloroplast DNA barcode and group-specific barcodes delimitated a maximum of four out of the five species. Plastid phylogenomics analyses indicated that the monophyletic M. squamosa complex is comprised of two evolutionarily significant units: one in the western Tarim Basin and the other in the eastern Qinghai-Tibet Plateau. This finding contradicts previous species discrimination and promotes the urgent need for taxonomic revision of the threatened genus Myricaria. Dense sampling and plastid genomes will be essential in this effort. The super-barcodes and specific barcode candidates outlined in this study will aid in further studies of evolutionary history.

The large Gunnera's (G. tinctoria and G. manicata) in Europe in relation to EU regulation 1143/2014.

Incorrect labelling of plants in the horticultural trade and misidentification is widespread. For the inspection services of the EU member states, correct identification of G. tinctoria has become important since the species was added to the List of Union concern in accordance with EU regulation 1143/2014 in August 2017. In the horticultural trade Gunnera plants are generally of modest dimensions and rarely flowering, so that the major distinguishing morphological characters for the identification of the two large species, G. tinctoria and G. manicata, are missing. As G. tinctoria is included in the EU regulation, its trade is prohibited, although the closely related species, G. manicata is not included on the list. Given that it is often difficult to distinguish between these two large herbaceous species using morphological attributes we used standard chloroplast DNA barcode markers, supplemented at a later stage by ITS markers. Plant material of putative G. tinctoria or G. manicata was obtained from the native and introduced range, both from "wild" sources, botanical gardens, and the horticultural trade. In western Europe plants circulating in the horticultural trade turned out to be predominantly G. tinctoria, with only one plant in cultivation identified as true G. manicata and the G. manicata found in botanical gardens was a hybrid recently described as G. x cryptica.

Genotypic and Phenotypic Diversity of Endemic Golden Camellias Collected from the North of Vietnam.

Golden Camellias have recently been used as a food, cosmetic, and traditional medicine in China and Vietnam. Forty-two species have natural distribution in Vietnam, of which thirty-two species were considered endemic species of this country. The morphology of leaves and flowers of these species were similar; therefore, their taxonomic identification usually needed experts and the authentication has often been confused among species. Our study aims to describe the genetic diversity and the relationship of six species Camellia phanii, Camellia tamdaoensis, Camellia tienii, Camellia flava, Camellia petelotii and Camellia euphlebia by using three chloroplast DNA-barcodes: matK, rbcL and trnH-psbA. We also clarified the significant differences in anatomical characteristics of midvein and blade of their leaves, which suggested the possibility to use these criteria in taxonomy. In addition, preliminary chemical profiles of the methanolic extracts of leaves from six Golden Camellias such as total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC) and chlorogenic acids content (TCGAs) also showed the diversity among them. Interestingly, the discrimination on the catechins profile among six species followed the same tendency with the genetic distance on the phylogeny tree suggesting that catechins (i. e., discriminative catechins) can be biomarkers for the chemotaxonomy of these six Golden Camellias.

Assessment of plant species diversity (Lamiaceae Lindle.) in Uzbekistan based on DNA barcoding

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.

The Efficiency of DNA Barcoding in the Identification of Afromontane Forest Tree Species

The identification of flowering plants using DNA barcoding proposed in last decades has slowly gained ground in Africa, where it has been successfully used to elucidate the systematics and ecology of several plant groups, and to understand their evolutionary history. Existing inferences on the effectiveness of DNA barcoding to identify African trees are mostly based on lowland forests, whereas adjacent montane forests significantly differ from the latter floristically and structurally. Here, we tested the efficiency of chloroplast DNA barcodes (rbcLa, matK, and trnH-psbA) to identify Afromontane Forest tree species in a 20.28 ha permanent plot in Ngel Nyaki, Taraba state, Nigeria. We collected, identified, and vouchered 274 individuals with diameter at breast height ≥ 1 cm belonging to 101 morphospecies, 92 genera, and 48 families. rbcLa and matK used alone or in combination performed better than in lowland forests, with the best species discrimination obtained with the two-locus combination of matK + rbcLa. The intragenic spacer trnH-psbA was too variable to align and could not be tested using the genetic distance method employed. Classic DNA barcode can be a powerful tool to identify Afromontane tree species, mainly due to the non-prevalence in these communities of species—rich genera (low species-to-genus ratio) that constitute the biggest challenge of DNA barcoding of flowering plants.

Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae).

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panaxvietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

In silico Identification and Assessment of Potential Chloroplast DNA Barcodes for Discriminating Brassicaceous Species using Machine Learning Algorithms

Complete chloroplast genome sequences of 89 Brassicaceous species (42 genera) were used for identification and assessment of potential DNA barcodes at genus and species levels. Sliding windows analysis was performed on the aligned file to identify hyper-variable regions based on nucleotide diversity(π). Out of 23 identified hyper-variable regions, 3 coding regions i.e. ycf1, ndhF and ndhA and 3 combinations of coding and non-coding regions i.e. ‘ndhH-rps15, rps15, rps15-ycf1, ycf1’; ‘ccsA/ycf5, ccsA/ ycf5-ndhD, ndhD’ and ‘ndhE-ndhG, ndhG, ndhG-ndhI, ndhI’ were selected for sequence enrichment and assessed using six supervised machine learning algorithms i.e. J48, Jrip, SMO, Naive Bayes, Random Forest and KNN using WEKA along with distance based method using ‘nearneighbour’ function in SPIDER. It was observed that ycf1 was the most efficient region for discriminating Brassicaceous species with average identification rate of 76% and maximum identification rate of 86% at species level. The other three regions i.e. ndhA, ‘ndhH-rps15,rps15,rps15-ycf1,ycf1’ and ‘ccsA/ycf5,ccsA/ycf5-ndhD,ndhD’ were found to be more efficient than well established markers i.e. matK and rbcL and hence can be used as potential DNA barcodes for family Brassicaceae. Supervised machine learning algorithms i.e. SMO, Random Forest and KNN along with distance based method SPIDER(NN) were shown to be more efficient and stable as compared to Jrip, J48 and Naive Bayes.

De Novo Assembly and Species-Specific Marker Development as a Useful Tool for the Identification of Scutellaria L. Species.

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S. indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S. indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

Phylogenomic relationships and species identification of the olive genus Olea (Oleaceae)

AbstractThe olive genus Olea includes c. 30–40 taxa in three subgenera (Olea, Tetrapilus, and Paniculatae) within the family Oleaceae. Historically, the Olea genus was classified into four groups that were overall well supported by reconstructed phylogenies, despite incomplete sampling of subgenus Tetrapilus and poor resolution within clades. These analyses also showed that the genus was not monophyletic. Reliable identification of Olea species is important for both their conservation and utilization of this economically important genus. In this study, we used phylogenomic data from genome skimming to resolve relationships within Olea and to identify molecular markers for species identification. We assembled the complete plastomes, and nrDNA of 26 individuals representing 13 species using next‐generation sequencing and added 18 publicly available accessions of Olea. We also developed nuclear SNPs using the genome skimming data to infer the phylogenetic relationships of Olea. Large‐scale phylogenomic analyses of 138 samples of tribe Oleeae supported the polyphyly of Olea, with Olea caudatilimba and Olea subgenus Tetrapilus not sharing their most recent common ancestor with the main Olea clade (subgenus Paniculatae and subgenus Olea). The interspecific phylogenetic resolution was poor owing to a possible rapid radiation. By comparing with the plastome data, we identified the markers ycf1b and psbE‐petL as the best Olea‐specific chloroplast DNA barcodes. Compared with universal barcodes, specific DNA barcodes and super‐barcode exhibited higher discriminatory power. Our results demonstrated the power of phylogenomics to improve phylogenetic relationships of intricate groups and provided new insights into barcodes that allow for accurate identification of Olea species.

Research Progress in Plant Molecular Systematics of Lauraceae.

Simple SummaryLauraceae, as an angiosperm group with high ecological and economic value, has been widely studied. With the development of science and technology, the research of Lauraceae has changed from morphology to molecular systematics. Our paper reviewed the molecular phylogeny of Lauraceae in recent years. From the aspects of gene fragments, chloroplast genome and DNA barcodes, we mainly discussed the establishment of Cinnamomeae in the ‘Core Lauraceae’ and the tribal controversial genera (Actinodaphne, Sassafras, Caryodaphnopsis, Neocinnamomum and Cassytha). We think that the whole genome and inflorescence characteristics are the breakthrough to solve the tribal problem of Lauraceae. Using reliable molecular and morphological evidence to reconstruct the phylogenetic relationship of Lauraceae will provide an important theoretical basis for the rational utilization of Lauraceae resources, the development of potential resources and the protection of rare plants.Lauraceae is a large family of woody plants with high ecological and economic value. The tribal and generic division and phylogenetic relationship of Lauraceae have long been controversial. Based on morphological and molecular evidence, phylogenetic relationships within the Cinnamomeae, Laureae and Perseeae tribes, also called ‘the Core Lauraceae’, have arisen particular attention. In this review, we comprehensively collated the literatures on the phylogeny of Lauraceae published in recent years and summarized progress made in molecular systematic researches employing gene fragments, chloroplast genomes and DNA barcodings analyses. We clarified the phylogenetic relationships and main controversies of ‘the Core Lauraceae’, the systemic position of fuzzy genera (Neocinnamomum, Caryodaphnopsis and Cassytha) and the development of chloroplast genome and DNA barcodes. We further suggested and proposed the whole genome analysis and different inflorescence types would be possible to provide more information for further research on phylogenetic relationships and taxonomy of Lauraceae.

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.

Genetic Diversity among Selected Medicago sativa Cultivars Using Inter-Retrotransposon-Amplified Polymorphism, Chloroplast DNA Barcodes and Morpho-Agronomic Trait Analyses.

Alfalfa (Medicago sativa L.) is a major forage crop of family Fabaceae and is frequently cultivated in Egypt. The present study is concerned with the genetic discrimination of fifteen alfalfa cultivars from three different countries (Egypt, Australia, and USA) using two molecular approaches: inter-retrotransposon-amplified polymorphism (IRAP) markers and two chloroplast DNA barcodes matK and the trnH in addition to the analysis of fifteen morpho-agronomic traits. The genetic relatedness, based on analysis of IRAP marker polymorphism and produced using eleven primers by clustering via principal component analysis (PCA) and multivariate heatmap biostatistical methods differentiated the two Egyptian cultivars EGY1-Ismailia1 and EGY2-Nubaria1 from the three Australian and seven American cultivars, with some distinction of the cv. USA6-SW9720 and cv. AUS4-SuperFast. The results were also supported by the sequence analysis of the matK and the trnH genes on the genetic relatedness between eight cultivars. Moreover, it might be suggested that breeding lines from M. sativa cultivars may provide novel insights and a better understanding of the domestication of M. sativa genetic diversity. The classification of the eight cultivars, as revealed by morpho-agronomic traits, confirmed the close genetic relationship between the two Egyptian cultivars and indicated some resemblance between them and the AUS2-Siri Nafa, whereas the two American cultivars, USA1-Super supreme and USA4-Cuf101, were clearly isolated from a cluster of other three cultivars USA7-SW9628, USA8-Magna901, and USA9-Perfect. The results are useful sources of genetic information for future breeding programs in crop development and open new possibilities of producing M. sativa lines harboring high forage quality, productivity, and resistance to biotic and abiotic stresses.

Seed Total Protein Profiling in Discrimination of Closely Related Pines: Evidence from the Pinus mugo Complex.

The Pinus mugo complex includes several dozen closely related European mountain pines. The discrimination of specific taxa within this complex is still extremely challenging, although numerous methodologies have been used to solve this problem, including morphological and anatomical analyses, cytological studies, allozyme variability, and DNA barcoding, etc. In this study, we used the seed total protein (STP) patterns to search for taxonomically interesting differences among three closely-related pine taxa from the Pinus mugo complex and five more distant species from the Pinaceae family. It was postulated that STP profiling can serve as the backup methodology for modern taxonomic research, in which more sophisticated analyses, i.e., based on the DNA barcoding approach, have been found to be useless. A quantitative analysis of the STP profiles revealed characteristic electrophoretic patterns for all the analyzed taxa from Pinaceae. STP profiling enabled the discrimination of closely-related pine taxa, even of those previously indistinguishable by chloroplast DNA barcodes. The results obtained in this study indicate that STP profiling can be very useful for solving complex taxonomic puzzles.

Exploring Parthenium weed biotypes by chloroplast DNA barcode analysis

Abstract Background: Parthenium weed (Parthenium hysterophorus L.) is an invasive weed that has invaded vast regions of Pakistan in a relatively very short period of a decade or two, threatening the crop fields of the agrarian fed country. Parthenium hysterophorus L. is native of central South America and Gulf of Mexico, has now turned out to be a weed of global significance due to its alarming invasions and profuse spread in approximately all parts of the world. Its invasion is probably due to the contamination of its seeds in the imported grains from other countries of the world. Objective: During comprehensive sampling from Pakistan and Australia, it was observed that parthenium weed accessions exhibited several distinct morphological features present at different geographical regions. Therefore this study focuses on the use of plastid DNA barcodes (psbA-trnH) to evaluate the extent of variations in nucleotide sequences between the parthenium weed sampled accessions. Methods: The variability or genetic diversity was evaluated through sequencing of the amplified products and data was subjected to phylogenetic analysis in Molecular Evolutionary Genetic Analysis (MEGA; version 6.06) software. Results: In Maximum Likelihood tree, mainly two clades with three subdivisions are evident which showed increased heterogenity. The results of sequence based markers showed 12 haplotypes of P. hysterophorus populations (having two parsimony informative sites) with 10 indels and a few SNPs (single nucleotide polymorphisms). Conclusions: The results advocate that there have been multiple introductions of parthenium weed into Pakistan.

An Economical Approach to Distinguish Genetically Needles of Limber from Whitebark Pine

Whitebark pine is difficult to distinguish from limber pine when seed cones are not present. This is often the case because of young stand age, growth at environmental extremes, or harvesting by vertebrate species. Developing an economical genetic identification tool that distinguishes non-cone-bearing limber from whitebark pine, therefore, could aid many kinds of research on these species. Phylogenetic studies involving limber and whitebark pine suggest that chloroplast DNA sequences differ between these species. We therefore wanted to identify chloroplast loci that could differentiate limber from whitebark pine trees by taking an economical approach involving restriction-site analysis. We generated chloroplast DNA barcode sequences sampled from limber and whitebark pine trees that we identified using attached seed cones. We searched for nucleotide differences associated with restriction endonuclease recognition sites. Our analyses revealed that matK and the psbA-trnH spacer each readily amplified and harbored multiple DNA-sequence differences between limber and whitebark pine. The matK coding sequence of whitebark pine has a BsmAI restriction site not found in limber pine. The psbA-trnH spacer of limber pine has two PsiI restriction sites, neither of which is found in whitebark pine. DNA-sequence and restriction-site analysis of the psbA-trnH spacer from 111 trees showed complete congruence between visually and genetically identified limber (n = 68) and whitebark (n = 43) pine trees. We conclude that restriction site analysis of the chloroplast psbA-trnH spacer and matK involves both minimal technical expertize and research funds. These findings should be of value to foresters interested in species identification and distribution modeling, as well as the analysis of fossil pine pollen, given that gymnosperms transmit chloroplast DNA paternally.

Applicability of chloroplast DNA barcodes for wood identification between Santalum album and its adulterants

Abstract The East Indian sandalwood, Santalum album L., is known for its fragrant heartwood and extractive oils. The increasing demand for the valuable sandalwood products has led to illegal and excessive logging, and there are other Santalum species in the commercial market as substitutes (adulterants). Improved tools for the identification of Santalum species are needed to control this situation. In this study, four chloroplast DNA (cpDNA) regions were screened (matK, psbA-trnH, trnK and trnL) on 35 vouchered specimens covering five Santalum species, i.e. Santalum acuminatum, S. album, Santalum lanceolatum, Santalum murrayanum and Santalum spicatum. The goal of this study was to test the species discrimination ability by means of both the TaxonDNA and neighbor-joining (NJ) methods. The results indicate that the combination of psbA-trnH+trnK gave the best performance for discrimination (100%) of the studied Santalum species concerning the discrimination ability and recovery rate. Meanwhile, six unvouchered wood specimens were retrieved and accurately identified at the species level based on the recommended DNA barcodes. The DNA barcode method is now ready for application in the monitoring of the trade of this valuable resource, and provides an effective approach for wood species identification and product authentication.

Molecular Identification of Three Aquilaria (Thymelaeaceae) Species through DNA Barcoding.

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medicine, perfumes and incense. The species endemic to China, Aquilaria yunnanensis, is often misidentified as the two valuable species, Aquilaria sinensis and Aquilaria crassna. In present study, three DNA barcodes (internal transcribed spacer (ITS), maturase K gene (matK) and trnL-trnF) were used to evaluate whether these genes can be used to discriminate the three species, and evaluate the phylogenetic relationship between the three Aquilaria species. For accurate identification of the three Aquilaria species, a total of 26 nucleotide variations were detected when comparing the three DNA barcodes. We found that A. sinensis is closely related to A. crassna based on combination of nuclear and chloroplast DNA barcodes, and is closely related to A. yunnanensis based on chloroplast DNA barcodes. Taken together, we suggest that the combination of ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.

Effectiveness of the DNA barcoding approach for closely related conifers discrimination: A case study of the Pinus mugo complex

DNA barcoding is a standard and efficient method, frequently used for identification, discrimination and discovery of new species. Although this approach is very useful for classifying the world's biodiversity, little is known about its usefulness in barcoding at lower taxonomic level and its discrimination rate for closely related species, like conifers. In this study, we compared the genetic variation of eight chloroplast DNA barcode regions (matK, rbcL, trnH-psbA, trnL-trnF, rpl20-rps18, trnV, ycf1, ycf2) in 17 conifers - three closely related pines from Pinus mugo complex and 14 more distant conifers representing two genera and four sections of the Pinaceae family. The discrimination rate for a single and for multiple DNA barcode regions analyzed in this study was estimated using the Tree-Building and PWG-Distance methods. The usefulness of the DNA barcoding approach for analyzing and resolving taxonomic inconsistency among closely related and more phylogenetically distant conifers was evaluated and discussed.