nrDNA Internal Transcribed Spacer Sequences Research Articles

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods

Ethnopharmacological relevanceThe roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. Aim of this studyThis study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. Materials and methodsThe sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. ResultsThe plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. ConclusionThe nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

Species-specific molecular signature of Commiphora species of Saudi Arabia inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.

The deciduous habit and tendency to produce flowers prior to developing leaves, and a predominantly dioecious system of breeding in the genus Commiphora leads to difficulties in its taxonomic identification at species level. The characteristics of easy amplification by universal primer, shorter length and higher discrimination power at the species level makes the internal transcribed spacer (ITS) sequence of nuclear ribosomal DNA (nrDNA) to a smart gene for generating species-specific phylogenetic inferences in most of the plants groups. The present study deals the ITS sequence of nrDNA based molecular genotyping of seven species of the genus Commiphora of Saudi Arabia. The molecular phylogenetic analysis of ITS sequences of nrDNA of Commiphora species distributed in Saudi Arabia reveals the the occurrence of C. madagascariens in Saudi Arabia.

Primula jiugongshanensis sp. nov. (Primulaceae) from China, based on morphological and molecular evidence

Primula jiugongshanensis J. W. Shao, a new species from Mt Jiugongshan in Hubei Province, China, is described and illustrated. This species is a biennial glabrous herb with pinnately compound leaves, and affiliated to P. sect. Ranunculoides C. M. Hu. It is distinguished from other species of the section by outer simple leaves ovate, flowers distylous, corolla lobe apex conspicuously emarginate, and lack of bulblets. Molecular phylogenetic analysis of nrDNA internal transcribed spacer sequences (ITS) was conducted and the results also supported the recognition of P. jiugongshanensis as a new species. The species is known from only three separated unprotected populations and is categorized as ‘Endangered’ (EN).

Molecular phylogenetic analyses of internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA indicate monophyly of the genus <i>Phytolacca</i> L. (Phytolaccaceae)

Relationships within the family Phytolaccaceae sensu lato were examined based on internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA). The study revealed Phytolacca L. as taxonomically the most difficult genus in the family with completely unknown phylogeny. Molecular evidence was used from nrDNA ITS sequences of about 90% of the species for maximum parsimony analyses, and the molecular phylogenetic analyses defined a monophyletic Phytolacca. This first molecular phylogenetic study of Phytolacca concludes that the relationships among the species within the genus do not show harmony with the generic classification based on morphology. These results set the stage for a more detailed phylogenetic analysis of Phytolacca.Bangladesh J. Plant Taxon. 22(1): 1­–8, 2015 (June)

Molecular phylogeny of Asian Meconopsis based on nuclear ribosomal and chloroplast DNA sequence data.

The taxonomy and phylogeny of Asian Meconopsis (Himalayan blue poppy) remain largely unresolved. We used the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and the chloroplast DNA (cpDNA) trnL-F region for phylogenetic reconstruction of Meconopsis and its close relatives Papaver, Roemeria, and Stylomecon. We identified five main clades, which were well-supported in the gene trees reconstructed with the nrDNA ITS and cpDNA trnL-F sequences. We found that 41 species of Asian Meconopsis did not constitute a monophyletic clade, but formed two solid clades (I and V) separated in the phylogenetic tree by three clades (II, III and IV) of Papaver and its allies. Clade V includes only four Asian Meconopsis species, with the remaining 90 percent of Asian species included in clade I. In this core Asian Meconopsis clade, five subclades (Ia–Ie) were recognized in the nrDNA ITS tree. Three species (Meconopsis discigera, M. pinnatifolia, and M. torquata) of subgenus Discogyne were imbedded in subclade Ia, indicating that the present definition of subgenera in Meconopsis should be rejected. These subclades are inconsistent with any series or sections of the present classifications, suggesting that classifications of the genus should be completely revised. Finally, proposals for further revision of the genus Meconopsis were put forward based on molecular, morphological, and biogeographical evidences.

Phylogeny of the genus Potentilla (Rosaceae) in Iran based on nrDNA ITS and cpDNA trnL-F sequences with a focus on leaf and style characters’ evolution

The present survey reports maximum parsimony (MP) and Bayesian phylogenetic analyses of the genus Potentilla L. in Iran using the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnL-F intergenic region of chloroplast DNA. A total of 25 taxa from 9 sections, 2 intersectional hybrids, and 4 related genera of Iranian taxa plus 40 previously sequenced data were analyzed for both trnL-F and nrDNA ITS regions. Individual and combined analysis of ITS and trnL/F data revealed monophyly of the genus Potentilla. The Potentilla core group was composed of monophyletic subclades of the sections Persicae (Th.Wolf) Juz., Potentilla, Micranthae Sojak., and Speciosae (Th.Wolf) Juz.; unresolved branches of the sections Rivales Poeverl., Aureae (Rydb.) Juz., Terminales, (Döll) Gren. & Godr., and Pensylvanicae Poeverl.; and the American genera Ivesia and Horkelia. The evolutionary trends of some diagnostic morphological traits were evaluated. Anther structure, and style shape and position evolved independently while multiple gains and parallel evolutions were inferred for leaf division.

Historical biogeography of the Angelica group (Apiaceae tribe Selineae) inferred from analyses of nrDNA and cpDNA sequences

Abstract Biogeographical patterns and diversification processes of Asia‐centered angiosperm groups have been significantly affected by the multistage uplift of the Himalayas–Tibetan Plateau since the Late Tertiary. The divergence time of the largely East Asian Angelica group (Apiaceae, subfamily Apioideae, tribe Selineae) was initially analyzed using Beast and nrDNA internal transcribed spacer sequence data from 96 representatives of tribe Selineae and relatives. Further analyses of the biogeographical history of the Angelica group were carried out using Beast, S‐Diva, Rasp, and Lagrange on datasets containing all or some of the following loci: nrDNA internal and external transcribed spacers; cpDNA rps16 intron; and cpDNA rps16‐trnK, rpl32‐trnL, and trnL‐trnT intergenic spacers. The results suggested that the Angelica group was originally present in the East Palearctic during the global cooling of the late Middle Miocene (13.6 Mya) and that the Angelica s.s. clade originated in the same region at 10.2 Mya. Subsequent diversifications of the Angelica s.s. clade intensified in the East Palearctic during the middle Late Miocene (10.0–7.0 Mya) and in the eastern Himalayan Zone during the late Pliocene and Pleistocene (<4.0 Mya). These diversifications likely corresponded with plateau uplift‐driven climatic changes. Considering elevational reconstructions, the differential responses to altitude appear to be the primary factor explaining the recent radiation of the group in the eastern Himalayas. The North American species of the Angelica group were retrieved as polyphyletic and their migrations involved six independent dispersals to North America at least since the middle Late Miocene, including four times from northeast Asia and twice from Europe.

Reappraisal of Primula ranunculoides (Primulaceae), an endangered species endemic to China, based on morphological, molecular genetic and reproductive characters

The taxonomic status of Primula ranunculoides and other members of section Ranunuculoides is reappraised here based on data from morphological, reproductive and molecular characters. Multivariate analysis of morphological characters indicates that P. ranunculoides is a coherent species that can be distinguished from its sectional congeners P. cicutariifolia and P. merrilliana by the characters of simple kidney-shaped outer leaves and the unique clonal reproductive ability by which apices of the scape differentiate into bulblets at the late phases of flowering. Recognition of P. ranunculoides at the specific level is also supported by palynological characters, breeding system, cross pollination results and molecular phylogenetic analysis of nrDNA internal transcribed spacer sequences. A taxonomic revision of section Ranunuculoides is presented and a possible mechanism of speciation discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 338–349.

Genetic diversity of Kalopanax pictus populations in Korea based on the nrDNA ITS sequence

Kalopanax pictus is a long-lived deciduous pe- rennial tree in the family Araliaceae mainly distributed in the East Asia. In Korea, this species is of ecological and medical importance. Because typical populations of this species are small and distributed in patches, K. pictus has been considered as a narrow habitat species. To understand the genetic di- versity and population structure of this species, the sequence variation of the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region was analyzed among 18 different K. pictus populations in the present investigation. The nrDNA ITS sequences of Korean populations inves- tigated in this study showed identical of 616 bp in length, and no any nucleotide variation was found in the entire nrDNA ITS region sequence. This result suggested that the K. pictus populations in Korea might belong to the same isolate, and no mutation was found in the nrDNA ITS region. Compared with other known ITS sequence sources from K. pictus populations, only four variable nucleotide sites were found within the entire ITS region. Very narrow genetic diversity appearing in the population level of K. pictus makes us hypothesize that their relatively isolated habitats. The long-lived traits might be one main reason. However, another probability was that the nr- DNA ITS region might be noneffective in classifying po- pulations of K. pictus. Thus, to further understand the phy- logenetic relationship of K. pictus populations, more samplings should be performed based on more DNA sequences.

Molecular differentiation in Indian Citrus L. (Rutaceae) inferred from nrDNA ITS sequence analysis

Molecular differentiation in 24 accessions representing 19 taxa of Indian Citrus has been examined through sequence analysis of Internal Transcribed Spacer (ITS) region of nrDNA. Sequence length in the 24 accessions of Citrus taxa ranged from 512 to 665 bp (ITS1 & ITS2 partial and 5.8S complete sequence). The ITS sequences were very rich in G+C content ranging from 61.40 to 66.60% with an average of 64.2%. Genetic distance within Citrus group ranged from 0 to 13.4% with an average of 4.6%, showing moderate rate of nucleotide divergence. The phylogeny was inferred using the Maximum parsimony (MP) and Neighbor-Joining (NJ) methods. Both MP and NJ trees separated all the 24 accessions of Citrus into six distinct clusters. The disposition of all the accessions of Citrus in separate clusters in ITS-derived dendrograms was partly in accordance with the morpho-taxonomic affinities of the target taxa. This study supports the concept of Citrus medica (citron), C. reticulata (mandarin), and C. maxima (pummelo) as the basic species of the genus. However, ITS marker could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle’s Citrus classification system. The present study also supports the distinctiveness of C. indica (Indian wild orange), C. latipes (Khasi papeda) and C. hystrix (Melanesian papeda) as true species, besides elucidating the probable hybrid origin and relationships among the cultivated species/biotypes, such as Citrus ×aurantiifolia (sour lime) C. ×limon (lemon), C. ×taitensis (Indian rough lemon), C. limettioides (sweet lime), C. ×aurantium (including sour and sweet oranges and grapefruit), and other indigenous varieties of Indian origin: C. megaloxycarpa (sour pummelo), C. karna (karna orange), C. pseudolimon (Hill lemon), ‘Memang athur’, ‘Pummelo-lemon’ and ‘Kathairi nimbu’.

Genetic diversity among Salvia miltiorrhiza Bunge and related species inferred from nrDNA ITS sequences

To investigate the genetic diversity and phylogenetic relationships of Salvia miltiorrhiza and related species, we analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 7 accessions of Salvia miltiorrhiza and another 23 samples from other taxa within the genus Salvia by maximum parsimony and Bayesian inference analyses. There were 257 variation sites amounting to 40.8% of the total base pairs. All of the data revealed abundant genetic diversity in the genus Salvia. The results showed that the tested materials could be grouped into 3 clusters, and the 3 different occasions in Salvia were clustered into 1 clade, which suggests that the species from different occasions form independent lineages. We can easily distinguish S. miltiorrhiza from a variety of Salvia species in this manner. Moreover, according to cluster analysis, S. bowleyana, S. yunnanensis, and S. cavaleriei var. simplelefolia are good or potential germplasm resources for S. miltiorrhiza.

Genetic diversity and phylogenetic analysis of genus Paeonia based on nuclear ribosomal DNA ITS sequence

The genus Paeonia belongs to the family Paeoniaceae having significant medicinal and ornamental importance. The present investigation was undertaken with an aim to understand phylogenetic relationships of three Paeonia species (P. lactiflora, P. obovata, and P. suffruticosa) that are widely distributed in China, Korea, and Japan, using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence and to compare the phylogeny results with investigations reported earlier using existed sequences of the same species. The size variation obtained among sequenced nrDNA ITS region was narrow and ranged from 722 to 726 bp. The highest interspecific genetic distance (GD) was found between P. lactiflora and P. suffruticosa or P. obovata. The phylogram obtained using our nrDNA ITS sequences showed non-congruence with previous hypothesis of the phylogeny between section Paeonia and section Moutan of genus Paeonia. This result was supported by the phylogenetic relations showed in the phylogram constructed with existed sequences in NCBI. The present study suggested that P. obovata belonging to section Paeonia was phylogenetically closer to P. suffruticosa representing section Moutan of genus Paeonia than P. lactiflora belonging to section Paeonia. The main reason of the paraphyly of section Paeonia is thought to be nucleotide additivity directly caused by origin hybridization. This study provides more sequence sources of genus Paeonia, and will help for further studies in intraspecies population, and their phylogentic analysis and molecular evolution.

Phylogenetic relationships in Ranunculus species (Ranunculaceae) based on nrDNA ITS and cpDNA trnL-F sequences

The genus Ranunculus L., with a worldwide distribution, is the largest member of the Ranunculaceae. Here, nuclear ribosomal internal transcribed spacer (ITS) sequence data and chloroplast trnLF sequence data were used to analyze phylogenetic relationships among members of the annual and perennial (Group Praemorsa, Group Rhizomatosa, Group Grumosa and Group non-Grumosa) species of Ranunculus in Iran. In the strict consensus tree of nrDNA ITS sequence analyses, seven sub-clades were described, based on morphological, karyological, palynological, and ecological features. Within each clade, there were species belonging to more than one Group, and species of a single Group may fall into different clades, revealing that a classification based on underground system characters does not show natural interspecific relationships and must be revised. This is also apparent from trnL-F sequence analyses. Based on our results, the Praemorsa and Rhizomatosa Groups can be merged. The ITS sequence data show interspecific relationships more clearly than trnL-F sequence data. © 2011 Progress in Biological Sciences, Vol. 1, No.1, 41-47.

Assessment of genetic diversity among Indian populations of Cuscuta reflexa based on ITS sequences of nrDNA

Internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified from 30 populations of Cuscuta reflexa Roxb. collected from nine different geographical locations of India. Analysis of nucleotide sequences reflects the polymorphism among the populations and differentiated them regionally. Maximum Likelihood Analysis (MLA) conducted on the ITS datasets, revealed that clade showed 100% bootstrap support (together) and subclade showed grouping according to their geographic locations in all the sampled populations except the samples collected from West Bengal and Assam. Thus, such results showed the distribution of C. reflexa in different geographical regions which might have evolved under reproductive isolation while the genetic exchange could be the reason in case of C. reflexa’s populations distributed in the West Bengal and Assam.

The internal transcribed spacer rDNA specific markers for identification of Zanthoxylum piperitum

Genus Zanthoxylum which has significant medical importance belongs to the family Rutaceae. This investigation was aimed to identify total internal transcribed spacer (ITS) regions among the nuclear ribosomal DNA (nrDNA) to distinguish Zanthoxylum piperitum from Zanthoxylum sichinifolium . The nrDNA ITS sequence markers were developed by using universal ITS5/ITS4 primer pairs. Speciesspecific primers amplified the total ITS region sequence named ITS1-YL1 and ITS1-YL2. These were amplified efficiently when paired with universal primer ITS4 in Z. piperitum, but not in Z. schinifolium . ITS1-YL1/ITS4 or ITS1-YL2/ITS4 preferential amplification was shown to be particularly useful for detection and distribution of Z. piperitum from other plant species, especially Z. schinifolium . These primers are useful to study the structure of Rutaceae family. Such identifications will be helpful for phylogenetic analysis in intraspecies population of the genus Zanthoxylum. Key words: Zanthoxylum piperitum , rDNA Int-sp markers, phylogenetic relationship, ribosomal DNA, internal transcribed spacer.

Identification ofLonicera japonicaby PCR-RFLP and Allele-Specific Diagnostic PCR Based on Sequences of Internal Transcribed Spacer Regions

The flower buds of Lonicera japonica Thunb. are widely used in Chinese medicine for their anti-inflammatory prosperities. Some closely related species of L. japonica which have similar morphology but weaker biological activity are also used medicinally. The discrimination of L. japonica from its adulterants is currently limited to methods of morphology and chemical fingerprinting. The analysis of sequence variation in the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) has recently become an effective method for identification of medicinal herbs. In the present study, we found that nrDNA ITS sequences differ significantly between L. japonica and the 4 tested adulterant species. We identified a distinctive site which can be digested by restriction endonuclease ECO52 I and designed a pair of allele-specific primers for diagnostic PCR on the nrDNA ITS2 region. These two techniques provide accurate, effective, and rapid approaches to distinguish L. japonica from closely related species.

Phylogeny of Ptychostomum (Bryaceae, Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

Abstract The phylogeny of Ptychostomum was first undertaken based on analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal (nr) DNA and by combining data from nrDNA ITS and chloroplast DNA rps4 sequences. Maximum parsimony, maximum likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii (Schwägr.) J. R. Spence (≡Bryum funkii Schwägr.) is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well‐supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Müll. Hal. (≡B. inclinatum (Brid.) Turton ≡Bryum archangelicum Bruch & Schimp.), Bryum lonchocaulon Müll. Hal., Bryum pallescens Schleich. ex Schwägr., and Bryum pallens Sw. to Ptychostomum.

DNA taxonomy in morphologically plastic taxa: Algorithmic species delimitation in the Boodlea complex (Chlorophyta: Cladophorales)

DNA-based taxonomy provides a convenient and reliable tool for species delimitation, especially in organisms in which morphological discrimination is difficult or impossible, such as many algal taxa. A group with a long history of confusing species circumscriptions is the morphologically plastic Boodlea complex, comprising the marine green algal genera Boodlea, Cladophoropsis, Phyllodictyon and Struveopsis. In this study, we elucidate species boundaries in the Boodlea complex by analysing nrDNA internal transcribed spacer sequences from 175 specimens collected from a wide geographical range. Algorithmic methods of sequence-based species delineation were applied, including statistical parsimony network analysis, and a maximum likelihood approach that uses a mixed Yule-coalescent model and detects species boundaries based on differences in branching rates at the level of species and populations. Sequence analyses resulted in the recognition of 13 phylogenetic species, although we failed to detect sharp species boundaries, possibly as a result of incomplete reproductive isolation. We found considerable conflict between traditional and phylogenetic species definitions. Identical morphological forms were distributed in different clades (cryptic diversity), and at the same time most of the phylogenetic species contained a mixture of different morphologies (indicating intraspecific morphological variation). Sampling outside the morphological range of the Boodlea complex revealed that the enigmatic, sponge-associated Cladophoropsis ( Spongocladia) vaucheriiformis, also falls within the Boodlea complex. Given the observed evolutionary complexity and nomenclatural problems associated with establishing a Linnaean taxonomy for this group, we propose to discard provisionally the misleading morphospecies and genus names, and refer to clade numbers within a single genus, Boodlea.

Genetic relationship between Echinochloa crus‐galli and Echinochloa oryzicola accessions inferred from internal transcribed spacer and chloroplast DNA sequences

Barnyardgrass, hexaploid Echinochloa crus‐galli, is considered to arise from the hybridization between tetraploid Echinochloa oryzicola and an unknown diploid species. The genetic relationship between E. crus‐galli and E. oryzicola was examined to investigate the position of E. oryzicola in the evolutionary process of E. crus‐galli, based on the nuclear DNA internal transcribed spacer (ITS) and the chloroplast cpDNA trnT‐L, trnL intron, and trnL‐F regions. New World E. crus‐galli was clearly separated from Eurasian E. crus‐galli and showed a close relationship to the American taxa, Echinochloa crus‐pavonis and Echinochloa walteri, in both the ITS and chloroplast DNA. The nrDNA ITS sequences indicated no differentiation between the Eurasian E. crus‐galli and E. oryzicola, in contrast to their clear divergence in the cpDNA sequence. The present results suggest that E. oryzicola is the male donor of E. crus‐galli.

Phylogenetic study of Korean Geranium(Geraniaceae) based on nrDNA ITS sequences

Phylogenetic analyses were conducted to evaluate evolution and relationship of 16 taxa of Korean Geranium including 3 outgroups using ITS (internal transcribed spacer) squences of nuclear ribosomal DNA. Phylogenetic studies used most parsimony and neighbor-joining methods including bootstrapping and jackknifing analysis. As the result, Korean Geranium forms monophyletic group. In the parsimony tree G. koraiense var. hallasanense situated as the most basal clade and Erianthum group forms one clade by high bootstrap ans jackknife values (100% of bootstrap and jackknife values). G.dahuricum as one of the Krameri group is closely related with Palustre group by very weak relationship (37% of bootstrap and 44% of jackknife values) and the node collapse in the strict tree. G. Knuthii whick was one of wilfordii group is closely related with Koreanum group . G. sibiricum, one of Sibiricum group, is the most closest relationshipwith G. soboliferum and these species are sister to G. krameri. G. tripartitum and G. wilfordii which are wilfordii group are linked to G, nepalense, G, thunbergii f. pallidum and G. thunbergii. This result suggested that the phylogenetic analysis of ITS sequences should be useful to address phylogenetic questions on the genus Korean Geranium.