A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0–0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.     

Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences

An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL–F, and psbA–trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL + matK, psbA–trnH + ITS1, and trnL–F + ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

Evaluation of chloroplast and nuclear DNA barcodes for species identification in Terminalia L.

The genus Terminalia L. belongs to the Combretaceae family, which includes several medicinal and threatened species with high trade value. Species of Terminalia in India belong to four sections and species identification within the sections is considered to be complex due to the lack of sufficient taxonomical characters and the existence of morphotypes. Therefore, we tested the effectiveness of three chloroplast DNA barcodes (rbcL, matK, and trnH-psbA) and a nuclear DNA barcode (ITS2) for the discrimination of Terminalia species. A reference DNA barcode library consisting of 120 DNA barcodes from ten species of Terminalia was created. Intra-specific divergence was not observed among the accessions for any marker. Inter-specific divergence was highest in trnH-psbA (10.6%), followed by ITS2, matK and rbcL markers. The success of species differentiation by DNA barcodes was 100% with trnH-psbA, 80% with matK and ITS2, and 10% with rbcL. In the phylogenetic trees, the rbcL marker did not differentiate the species in any section. Two species from the section Catappa were not differentiated by matK and ITS2 markers. Only trnH-psbA resolved all the species and ranked the best among four markers for species identification. However, regarding species relationship studies, ITS2 was found to be better than other markers because it formed a separate clade for each section.     

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。     

DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

Background

Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.

Methodology/Principal Findings

In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.

Conclusions

Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.     

DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.     

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.     

DNA Barcode ITS Effectively Distinguishes the Medicinal Plant Boerhavia diffusa from Its Adulterants

Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant in India and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, a DNA barcoding technique has been applied to identify and distinguish B. diffusa from its closely-related species. The phylogenetic analysis was carried out for the four species of Boerhavia using barcode candidates including nuclear ribosomal DNA regions ITS, ITS1, ITS2 and the chloroplast plastid gene psbA-trnH. Sequence alignment revealed 26% polymorphic sites in ITS, 30% in ITS1, 16% in ITS2 and 6% in psbA-trnH, respectively. Additionally, a phylogenetic tree was constructed for 15 species using ITS sequences which clearly distinguished B. diffusa from the other species. The ITS1 demonstrates a higher transition/transversion ratio, percentage of variation and pairwise distance which differentiate B. diffusa from other species of Boerhavia. Our study revealed that ITS and ITS1 could be used as potential candidate regions for identifying B. diffusa and for authenticating its herbal products.     

DNA barcoding Chinese medicinal Bupleurum

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.     

Design of molecular markers for identification of species of the genus Chironomus (Diptera,Chironomidae)

Accurate identification and differentiation of species of the genus Chironomus based on their morphological features is a difficult problem. Unambiguous species identification by means of molecular markers is possible at any stage of the life cycle. Polymerase chain reaction (PCR) with species-specific primers was used to develop molecular markers (amplicons) for identification of Chironomus piger, Ch. dorsalis, and Ch. pseudothummi. Nucleotide sequences of the internal transcribed spacer region (ITS) of the locus coding for ribosomal RNA were used to design species-specific primers for these target species. Each of the species-specific primer pairs yielded species-specific amplicons (molecular markers) only with the DNA of target species: Ch. piger, Ch. dorsalis, and Ch. pseudothummi. Test PCRs with the DNA of eighteen Chironomus species confirmed the specificity of the primers obtained. The molecular markers produced in PCR with the designed species-specific primers permit reliable identification of Ch. piger, Ch. dorsalis, and Ch. pseudothummi and their differentiation from other species of the genus Chironomus.     

Genetic Diversity of Schistosoma haematobium Eggs Isolated from Human Urine in Sudan

The genetic diversity of Schistosoma haematobium remains largely unstudied in comparison to that of Schistosoma mansoni. To characterize the extent of genetic diversity in S. haematobium among its definitive host (humans), we collected S. haematobium eggs from the urine of 73 infected schoolchildren at 5 primary schools in White Nile State, Sudan, and then performed a randomly amplified polymorphic DNA marker ITS2 by PCR-RFLP analysis. Among 73 S. haematobium egg-positive cases, 13 were selected based on the presence of the S. haematobium satellite markers A4 and B2 in their genomic DNA, and used for RFLP analysis. The 13 samples were subjected to an RFLP analysis of the S. haematobium ITS2 region; however, there was no variation in size among the fragments. Compared to the ITS2 sequences obtained for S. haematobium from Kenya, the nucleotide sequences of the ITS2 regions of S. haematobium from 4 areas in Sudan were consistent with those from Kenya (> 99%). In this study, we demonstrate for the first time that most of the S. haematobium population in Sudan consists of a pan-African S. haematobium genotype; however, we also report the discovery of Kenyan strain inflow into White Nile, Sudan.     

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.     

“DNA signaturing” database construction for Tetradesmus species identification and phylogenetic relationships of Scenedesmus-like green microalgae (Scenedesmaceae,Chlorophyta)

Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST.     

Use of DNA sequences for Rickettsia identification in Ixodes ricinus ticks: the first detection of Rickettsia monacensis in Poland

The cosmopolitan tick Ixodes ricinus inhabiting Europe, including Poland, is a vector for many pathogens, such as various Rickettsia species, which spread to new territories. They are present mainly in the Mediterranean countries, but have also been found in Central Europe at increasing frequency. In the present study, the gltA gene, encoding citrate synthase, and an internal transcribed spacer (ITS) were employed to detect the DNA and identify the species of tick-borne pathogens of the Rickettsia genus. The presence of bacterial DNA was detected in 9.5% of the examined I. ricinus individuals. Based on the nucleotide sequences of the analysed genomic fragments, most pathogens were identified as Rickettsia helvetica, while Rickettsia monacensis was revealed in one case. We have described for the first time, to our knowledge, the occurrence of this species in Poland. Both markers employed in the experiments were successful in species identification of R. helvetica. The newly described species R. monacensis may be identified by the protein-coding gene, but the ITS nucleotide sequences proved insufficient.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.