The Internal Transcribed Spacer (ITS) Region and trnhH-psbA Are Suitable Candidate Loci for DNA Barcoding of Tropical Tree Species of India

Background

DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants.

Methodology and Principal Findings

Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species.

Conclusion

Although a conservative approach of a success rate of 60–70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.     

Evaluation of the DNA Barcodes in Dendrobium (Orchidaceae) from Mainland Asia

DNA barcoding has been proposed to be one of the most promising tools for accurate and rapid identification of taxa. However, few publications have evaluated the efficiency of DNA barcoding for the large genera of flowering plants. Dendrobium, one of the largest genera of flowering plants, contains many species that are important in horticulture, medicine and biodiversity conservation. Besides, Dendrobium is a notoriously difficult group to identify. DNA barcoding was expected to be a supplementary means for species identification, conservation and future studies in Dendrobium. We assessed the power of 11 candidate barcodes on the basis of 1,698 accessions of 184 Dendrobium species obtained primarily from mainland Asia. Our results indicated that five single barcodes, i.e., ITS, ITS2, matK, rbcL and trnH-psbA, can be easily amplified and sequenced with the currently established primers. Four barcodes, ITS, ITS2, ITS+matK, and ITS2+matK, have distinct barcoding gaps. ITS+matK was the optimal barcode based on all evaluation methods. Furthermore, the efficiency of ITS+matK was verified in four other large genera including Ficus, Lysimachia, Paphiopedilum, and Pedicularis in this study. Therefore, we tentatively recommend the combination of ITS+matK as a core DNA barcode for large flowering plant genera.     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1) ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642～645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。     

Evaluation of Four Commonly Used DNA Barcoding Loci for Chinese Medicinal Plants of the Family Schisandraceae

Many species of Schisandraceae are used in traditional Chinese medicine and are faced with contamination and substitution risks due to inaccurate identification. Here, we investigated the discriminatory power of four commonly used DNA barcoding loci (ITS, trnH-psbA, matK, and rbcL) and corresponding multi-locus combinations for 135 individuals from 33 species of Schisandraceae, using distance-, tree-, similarity-, and character-based methods, at both the family level and the genus level. Our results showed that the two spacer regions (ITS and trnH-psbA) possess higher species-resolving power than the two coding regions (matK and rbcL). The degree of species resolution increased with most of the multi-locus combinations. Furthermore, our results implied that the best DNA barcode for the species discrimination at the family level might not always be the most suitable one at the genus level. Here we propose the combination of ITS+trnH-psbA+matK+rbcL as the most ideal DNA barcode for discriminating the medicinal plants of Schisandra and Kadsura, and the combination of ITS+trnH-psbA as the most suitable barcode for Illicium species. In addition, the closely related species Schisandra rubriflora Rehder & E. H. Wilson and Schisandra grandiflora Hook.f. & Thomson, were paraphyletic with each other on phylogenetic trees, suggesting that they should not be distinct species. Furthermore, the samples of these two species from the southern Hengduan Mountains region formed a distinct cluster that was separated from the samples of other regions, implying the presence of cryptic diversity. The feasibility of DNA barcodes for identification of geographical authenticity was also verified here. The database and paradigm that we provide in this study could be used as reference for the authentication of traditional Chinese medicinal plants utilizing DNA barcoding.     

The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.     

Plant DNA barcodes can accurately estimate species richness in poorly known floras

Background

Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (∼70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology.

Methodology/Principal Findings

Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species.

Conclusions/Significance

We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways.     

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest.     

Study of molecular genetic diversity and evolutionary history of medicinally important endangered genus Chlorophytum using DNA barcodes

This study was aimed to authenticate and present phylogenetic relationship among 19 species of genus Chlorophytum using DNA barcoding. In all, 107 accessions were analyzed with eight plastid (matK, rbcL, trnH-psbA, rpoC1, ycf5, rpoB, atp and psbK-psbI) and six nuclear (ITS) markers. The matK and rbcL were found to be ideal markers for identification and discrimination of Chlorophytum species. Phylogenetic analysis based on matK and rbcL sequences resolved the species in two major clades. All markers, except matK and rbcL, showed ambiguous reads and paralogy in analysis. DGGE analysis showed the presence of pseudogenes and/or co-amplification in these markers, which caused poor sequence quality. Phylogeny and probable evolution of genus Chlorophytum was proposed on the basis of cytological, morphological and genetic information.     

Applying DNA barcodes for identification of plant species in the family Araliaceae

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.     

SNP Typing for Germplasm Identification of Amomum villosum Lour. Based on DNA Barcoding Markers

Amomum villosum Lour., produced from Yangchun, Guangdong Province, China, is a Daodi medicinal material of Amomi Fructus in traditional Chinese medicine. This herb germplasm should be accurately identified and collected to ensure its quality and safety in medication. In the present study, single nucleotide polymorphism typing method was evaluated on the basis of DNA barcoding markers to identify the germplasm of Amomi Fructus. Genomic DNA was extracted from the leaves of 29 landraces representing three Amomum species (A. villosum Lour., A. xanthioides Wall. ex Baker and A. longiligulare T. L. Wu) by using the CTAB method. Six barcoding markers (ITS, ITS2, LSU D1–D3, matK, rbcL and trnH-psbA) were PCR amplified and sequenced; SNP typing and phylogenetic analysis were performed to differentiate the landraces. Results showed that high-quality bidirectional sequences were acquired for five candidate regions (ITS, ITS2, LSU D1–D3, matK, and rbcL) except trnH-psbA. Three ribosomal regions, namely, ITS, ITS2, and LSU D1–D3, contained more SNP genotypes (STs) than the plastid genes rbcL and matK. In the 29 specimens, 19 STs were detected from the combination of four regions (ITS, LSU D1–D3, rbcL, and matK). Phylogenetic analysis results further revealed two clades. Minimum-spanning tree demonstrated the existence of two main groups: group I was consisting of 9 STs (ST1–8 and ST11) of A. villosum Lour., and group II was composed of 3 STs (ST16–18) of A. longiligulare T.L. Wu. Our results suggested that ITS and LSU D1–D3 should be incorporated with the core barcodes rbcL and matK. The four combined regions could be used as a multiregional DNA barcode to precisely differentiate the Amomi Fructus landraces in different producing areas.     

Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear‐cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH‐psbA and trnL‐F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra‐ and inter‐specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH‐psbA (100%), trnL‐F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH‐psbA and trnL‐F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.     

Two New Potential Barcodes to Discriminate Dalbergia Species

DNA barcoding enables precise identification of species from analysis of unique DNA sequence of a target gene. The present study was undertaken to develop barcodes for different species of the genus Dalbergia, an economically important timber plant and is widely distributed in the tropics. Ten Dalbergia species selected from the Western Ghats of India were evaluated using three regions in the plastid genome (matK, rbcL, trnH-psbA), a nuclear transcribed spacer (nrITS) and their combinations, in order to discriminate them at species level. Five criteria: (i) inter and intraspecific distances, (ii) Neighbor Joining (NJ) trees, (iii) Best Match (BM) and Best Close Match (BCM), (iv) character based rank test and (v) Wilcoxon signed rank test were used for species discrimination. Among the evaluated loci, rbcL had the highest success rate for amplification and sequencing (97.6%), followed by matK (97.0%), trnH-psbA (94.7%) and nrITS (80.5%). The inter and intraspecific distances, along with Wilcoxon signed rank test, indicated a higher divergence for nrITS. The BM and BCM approaches revealed the highest rate of correct species identification (100%) with matK, matK+rbcL and matK+trnH-psb loci. These three loci, along with nrITS, were further supported by character based identification method. Considering the overall performance of these loci and their ranking with different approaches, we suggest matK and matK+rbcL as the most suitable barcodes to unambiguously differentiate Dalbergia species. These findings will potentially be helpful in delineating the various species of Dalbergia genus, as well as other related genera.     

DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau

DNA barcoding, the identification of species using one or a few short standardized DNA sequences, is an important complement to traditional taxonomy. However, there are particular challenges for barcoding plants, especially for species with complex evolutionary histories. We herein evaluated the utility of five candidate sequences — rbcL, matK, trnH-psbA, trnL-F and the internal transcribed spacer (ITS) — for barcoding Rhodiola species, a group of high-altitude plants frequently used as adaptogens, hemostatics and tonics in traditional Tibetan medicine. Rhodiola was suggested to have diversified rapidly recently. The genus is thus a good model for testing DNA barcoding strategies for recently diversified medicinal plants. This study analyzed 189 accessions, representing 47 of the 55 recognized Rhodiola species in the Flora of China treatment. Based on intraspecific and interspecific divergence and degree of monophyly statistics, ITS was the best single-locus barcode, resolving 66% of the Rhodiola species. The core combination rbcL+matK resolved only 40.4% of them. Unsurprisingly, the combined use of all five loci provided the highest discrimination power, resolving 80.9% of the species. However, this is weaker than the discrimination power generally reported in barcoding studies of other plant taxa. The observed complications may be due to the recent diversification, incomplete lineage sorting and reticulate evolution of the genus. These processes are common features of numerous plant groups in the high-altitude regions of the Qinghai-Tibetan Plateau.     

DNA barcoding of Pedicularis L. (Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

Abstract One application of DNA barcoding is species identification based on sequences of a short and standardized DNA region. In plants, various DNA regions, alone or in combination, have been proposed and investigated, but consensus on a universal plant barcode remains elusive. In this study, we tested the utility of four candidate barcoding regions (rbcL, matK, trnH‐psbA, and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae). Amplification and sequencing was successful using single primer pairs for rbcL, trnH‐psbA, and ITS, whereas two primer pairs were required for matK. Patterns of sequence divergence commonly showed a “barcoding gap”, that is, a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species, respectively. Considering primer universality, ease of amplification and sequencing, and performance in discriminating species, we found the most effective single‐region barcode for Pedicularis to be ITS, and the most effective two‐region barcode to be rbcL + ITS. Both discriminated at least 78% of the 88 species and correctly identified at least 89% of the sequences in our sample, and were effective in placing unidentified samples in known species groups. Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis, a species‐rich cosmopolitan clade much in need of revision, as well as ecological studies in its center of diversity, the Hengduan Mountains region of China.     

DNA barcoding of Gaultheria L. in China (Ericaceae: Vaccinioideae)

Abstract Four DNA barcoding loci, chloroplast loci rbcL, matK, trnH‐psbA, and nuclear locus internal transcribed spacer (ITS), were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P‐distance, Wilcoxon signed rank test, and tree‐based analyses. This study included 186 individuals from 89 populations representing 30 species. For all individuals, single locus markers showed high levels of sequencing universality but were ineffective for species resolvability. Polymerase chain reaction amplification and sequencing were successful for all four loci. Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH‐psbA. A combination of matK and ITS was the most efficient DNA barcode among all studied regions, however, they do not represent an appropriate candidate barcode for Chinese Gaultheria, by which only 11 out of 30 species can be separated. Loci rbcL, matK, and trnH‐psbA, which were recently proposed as universal plant barcodes, have a very poor capacity for species separation for Chinese Gaultheria. DNA barcodes may be reliable tools to identify the evolutionary units of this group, so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

How Effective Are DNA Barcodes in the Identification of African Rainforest Trees?

Background

DNA barcoding of rain forest trees could potentially help biologists identify species and discover new ones. However, DNA barcodes cannot always distinguish between closely related species, and the size and completeness of barcode databases are key parameters for their successful application. We test the ability of rbcL, matK and trnH-psbA plastid DNA markers to identify rain forest trees at two sites in Atlantic central Africa under the assumption that a database is exhaustive in terms of species content, but not necessarily in terms of haplotype diversity within species.

Methodology/Principal Findings

We assess the accuracy of identification to species or genus using a genetic distance matrix between samples either based on a global multiple sequence alignment (GD) or on a basic local alignment search tool (BLAST). Where a local database is available (within a 50 ha plot), barcoding was generally reliable for genus identification (95–100% success), but less for species identification (71–88%). Using a single marker, best results for species identification were obtained with trnH-psbA. There was a significant decrease of barcoding success in species-rich clades. When the local database was used to identify the genus of trees from another region and did include all genera from the query individuals but not all species, genus identification success decreased to 84–90%. The GD method performed best but a global multiple sequence alignment is not applicable on trnH-psbA.

Conclusions/Significance

Barcoding is a useful tool to assign unidentified African rain forest trees to a genus, but identification to a species is less reliable, especially in species-rich clades, even using an exhaustive local database. Combining two markers improves the accuracy of species identification but it would only marginally improve genus identification. Finally, we highlight some limitations of the BLAST algorithm as currently implemented and suggest possible improvements for barcoding applications.     

Development of DNA barcodes for selected Acacia species by using rbcL and matK DNA markers

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.     

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.