Ribosomal Internal Transcribed Spacer Research Articles

DNA barcoding of East Asian Amentotaxus (Taxaceae): Potential new species and implications for conservation

AbstractDNA barcoding is a useful tool for species identification using standardized genomic DNA fragments. The genus Amentotaxus, consisting of five or six species, is confined to South China, Northeast India, Laos, and Vietnam. All species have been assessed as globally or nationally threatened. However, there is uncertainty about the number of species involved, especially in the border areas of southern China, northern Vietnam, and Laos. We selected five DNA barcodes (rbcL, matK, trnH‐psbA, trnL‐F, and internal transcribed spacer (ITS)) to evaluate their discrimination ability in this genus, and to investigate the current taxonomy of Amentotaxus. Our results indicate that all the selected barcoding regions showed a high level of universality for PCR and sequencing. When six species are recognized, the nuclear ribosomal DNA region ITS and the chloroplast DNA region trnL‐F used on their own provided the highest identification success (60%). Two barcode combinations that included either ITS or trnL‐F had the same species discrimination ability. Combinations using additional barcodes did not improve the species identification success. When only five species are recognized, with A. hatuyenensis T. H. Nguyen treated as a synonym of A. yunnanensis H. L. Li, the discrimination rate rises to 100%. Our results also indicate that recent collections from Yunnan province, China, Lao Cai province, Vietnam, and Laos may represent a potential new species. The findings from this study will be very useful for the formulation of appropriate conservation strategies for threatened Amentotaxus species in national and trans‐boundary regions.

Molecular systematics of southern African monkey orange Strychnos L. (Loganiaceae)

Strychnos is the largest genus of the Loganiaceae with about 200 species distributed across Africa (including Madagascar), the Americas, Australia and Asia. Recent molecular phylogenetic effort at elucidating relationships globally provided a useful overview for the genus, based on the internal transcribed spacer (ITS) data. However, an understanding of evolutionary and ecological patterns at regional levels is better served by fine scale phylogenetic analysis to resolve species complexes for conservation and allied reasons. In this study, we use plastid (trnL-trnF, trnS-trnG) and nuclear ribosomal ITS sequence data to infer phylogenetic patterns among members of southern Africa Strychnos. We also evaluate sectional validity of the current classification for African members with ITS sequence data. Our findings support the monophyly of Strychnos, although several of the sections are not monophyletic, thus raising the need for sectional reappraisal of the genus. S. xantha is sister to our expanded representation of southern African taxa, while S. aculeata is sister to all African taxa. The uncertain relationships among S. innocua, S. madagascariensis and S. gerrardii were partly resolved in the phylogenetic analysis of combined datasets. S. innocua is sister to the other two species in a well-supported clade. S. gerrardii and S. madagascariensis are also sister taxa that are not yet reciprocally monophyletic, but possess other features to distinguish them. The trnS-trnG marker revealed a relatively large indel event of potential taxonomic value in section Densiflorae. There is also a clear ecological signal in both the plastid and nuclear datasets, as both consistently placed forest species at the base of the phylogeny, and savanna species in more derived positions. We addressed the adaptive significance of such signals as they relate to fruit and growth forms, and submit proposals towards a comprehensive sectional revision of African Strychnos.

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows

Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

Analysis of the relationship between ribosomal DNA ITS sequences and active components in Rhodiola plants.

Rhodiola plants are a valuable resource in traditional Chinese medicine. The objective of this study was to evaluate the correlation between ribosomal DNA internal transcribed spacer (ITS) sequences and the three active components in Rhodiola plants. For this, we determined ITS sequence polymorphisms and the concentrations of active components salidroside, tyrosol, and gallic acid in different Rhodiola species from the Tibetan Plateau. In a total of 23 Rhodiola samples, 16 different haplotypes were defined based on their ITS sequences. Analysis of the active components in these same samples revealed that salidroside was not detected in species with haplotypes H4, H5, or H10, tyrosol was not detected with haplotypes H3, H5, H7, H10, H14, or H15, and gallic acid was detected in with all haplotypes except H14 and H15. In addition, the concentrations of salidroside, tyrosol and gallic acid varied between samples with different haplotypes as well as those with the same haplotype, implying that no significant correlation exists between haplotype and salidroside, tyrosol or gallic acid concentrations. However, a statistically significant positive correlation was observed for among these three active components.

Phylogenetic relationship in Fritillaria spp. of Iran inferred from ribosomal ITS and chloroplast trnL-trnF sequence data

The genus Fritillaria embraces up to 165 taxa in the family Liliaceae, most of which are of high medicinal and ornamental value and importance. In this study, 44 specimens of the genus representing 9 species were collected from their natural habitats located in 10 provinces of Iran. Phylogenetic analysis was performed based on DNA sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal cistron and the trnL-trnF regions. The phylogeny was constructed using the neighbor joining inference method. Results indicate that the examined samples were evidently diverged into 2 distinct clades. Members of the subgenera Fritillaria and Rhinopetalum formed one clade while the other clade contained the subgenera Theresia and Petilium. There can be seen a high degree of similarity between the only yellow-colored crown imperial specimen and the red-colored specimens. The endemic species of Fritillaria straussii, Fritillaria zagrica and Fritillaria kotschyana which their status within the subgenera known in the genus Fritillaria has been remained undefined, fell into the subgenus Fritillaria. The clades also had relatively reasonable distribution patterns based on the genetic structure, geographical conditions and climate specifications. This study revealed the feasibility of the ITS and trnL-trnF DNA sequence for phylogeny of the genus Fritillaria. This is the first phylogenetic analysis of Fritillaria spp. in Iran.

First Report of Laurel Oak as a Host for the Pecan Root-Knot Nematode, Meloidogyne partityla, in Florida

Roots of laurel oak (Quercus laurifolia Michx.), member of the family Fagaceae, were found to be heavily galled by the pecan root-knot nematode, Meloidogyne partityla, in two separate home gardens between 2010 and 2012, in Alachua Co., FL. Distinct round galls were observed on secondary and tertiary roots. Internally, root-knot nematode females were clearly visible when the roots were thinly sliced and egg masses were seen protruding from the root surfaces. The nematode species identification was performed using morphology of the male stylet, selected characters of the second-stage juveniles (J2), female perineal patterns, and esterase (EST) and malate dehydrogenase (Mdh) isozyme phenotypes. Morphology of perineal patterns of females, body, stylet, and tail length of the J2 and males all matched those of the original description of M. partityla (2). A swollen deeply grooved rectum was observed in the J2. The male stylet had a blunt tip with a prominent thickening at the junction between the cone and shaft. The stylet knobs of males and females were bipartite, each incised by a deep medium longitudinal groove (2). The isozyme phenotypes (EST = Mp3; Mdh = N1a) were consistent with those previously reported for M. partityla from Florida (1). Mitochondrial DNA (mtDNA) (3) and ribosomal internal transcriber spacer (ITS) DNA (4) of females were amplified to further confirm the nematode species identification. The mtDNA amplification using the C2F3/1108 primer set (3) and the ITS amplification using a recently available M. partityla specific primer set (4) produced fragments of approximately 530 bp and 550 bp, respectively. These were consistent with those already reported for this nematode species. This first report of a plant host for the pecan root-knot nematode outside of the family Juglandaceae indicates that the nematode may have migrated from Quercus species to pecan trees during the period when orchards were being established in Florida. To our knowledge, this is the first report of the pecan root-knot nematode infecting laurel oak.

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.

Taxonomic review of the umbelliferous taxa Heracleum moellendorffii complex in Korea based on molecular phylogenies of nuclear ribosomal ITS sequences

Taxonomy of umbelliferous taxa Heracleum moellendorffii complex has been unclear in their species delimitation in the far-eastern Asian regions. In both Korea and China Heracleum moellendorffii was adopted for their description while H. sphondylium was chosen to describe Japanese Heracleum. From Genbank accessions, taxa collected from Kamtchatka and Promorskiy, Russia were defined as H. maximum, endemic taxon to North America. In this study, we reviewed the taxonomy of Heracleum moellendorffii complex in Korea and neighboring countries on the basis of molecular phylogenies derived from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) regions. From three Korean accessions of Heracleum investigated in this study, two types of ITS sequences were obtained; two accessions were related to Chinese H. moellendorffii var. moellendorffii and North American H. maximum without forming a clade while the other one was identical to accession from H. maximum from Primorskiy, Russia. In the other hand, Japanese H. moellendorffii (=H. sphondylium ssp. sphondylium var. nipponicum in the flora of Japan) was closely related to H. maximum accessions from Korea and Russia, not nested within the clade comprising several subspecies of H. sphondylium. In order to delimit species boundaries among putatively closely related Heracleum species in fareastern Asian countries, more samples and much more rapidly evolved DNA regions must be investigated with interpretation of morphological and anatomical features.

The evolution and utility of ribosomal ITS sequences in Bambusinae and related species: divergence, pseudogenes, and implications for phylogeny

Ribosomal internal transcribed spacer (ITS) sequences are commonly used for phylogenetic reconstruction because they are highly reiterated as components of rDNA repeats, and hence are often subject to rapid homogenization through concerted evolution. Concerted evolution leads to intragenomic uniformity of repeats even between loci on nonhomologous chromosomes. However, a number of studies have shown that the ITS polymorphism within individuals is quite common. The molecular systematics of Bambusinae and related species were recently assessed by different teams using independently generated ITS sequences, and the results disagreed in some remarkable features. Here we compared the ITS sequences of the members of Bambusa s. l., the genera Dendrocalamus, Dinochloa, Gigantochloa, Guadua, Melocalamus, Monocladus, Oxytenanthera, Thyrsostachys, Pleioblastus, Pseudosasa and Schizostachyum.We have reanalysed the ITS sequences used by different research teams to reveal the underlying patterns of their different results. After excluding the sequences suspected to represent paralogous loci, a phylogenetic analysis of the subtribe Bambusinae species were performed using maximum parsimony and maximum-likelihood methods. The implications of the findings are discussed. The risk of incorporating ITS paralogues in plant evolutionary studies that can distort the phylogenetic signal should caution molecular systematists.

Out of the Qinghai–Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae)

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippophaë rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.

Investigating the ecology and evolution of cryptic marine nematode species through quantitative real‐time PCR of the ribosomal ITS region

The presence of morphologically similar but genetically distinct species has impacted biogeographical and ecological paradigms. In marine sediments, free-living nematodes form one of the most abundant and diverse faunal groups. Inferring the importance of nematode diversity for ecosystem functioning requires species-level identification, which is hampered by the lack of easily observable diagnostic characters and the presence of cryptic species. New techniques are urgently needed to adequately study the ecology and evolution of cryptic species. The aim of the present study was to evaluate the potential of a quantitative real-time PCR (qPCR) assay using the internal transcribed spacer (ITS) region of the ribosomal DNA to detect and quantify cryptic species of the R.(P.) marina complex. All primer pairs proved to be highly specific, and each primer pair was able to detect a single juvenile in a pool of 100 nematodes. C(t) values were significantly different between developmental stages for all species except for PmIII. Despite differences between developmental stages, a strong correlation was observed between the amount of extracted DNA and the number of nematodes present. Relative and absolute quantification estimates were comparable and resulted in strong positive correlations between the qPCR estimate and the actual number of nematodes present in the samples. The qPCR assay developed here provides the ability to quickly identify and quantify cryptic nematode species and will facilitate their study in laboratory and field settings.

Molecular Phylogenetics ofExophialaSpecies Isolated from Korea

BackgroundRecently, identification of fungi have been supplemented by molecular tools, such as ribosomal internal transcribed spacer (ITS) sequence analysis. According to these tools, morphological Exophiala species was newly introduced or redefined.ObjectiveThis study was designed to investigate the phylogenetics based on ribosomal ITS sequence analysis from clinical Exophiala species isolated in Korea.MethodsThe strains of Exophiala species were 4 clinical isolates of phaeohyphomycosis agents kept in the department of dermatology, Dongguk University Medical Center(DUMC), Gyeongju, Korea. The DNAs of total 5 strains of Exophiala species were extracted by bead-beating method. Polymerase chain reaction of ITS region using the primer pairs ITS1-ITS4, was done and phylogenetic tree contributed from sequences of ITS region from 5 Korean isolates including E. dermatitidis CBS 109154 and comparative related strains deposited in GenBank.ResultsThe strains of Exophiala species were 3 strains of E. dermatitidis, 1 strain of E. jeanselmei and 1 strain of Exophiala new species. Among the 3 subtypes (type A, B, C) of E. jeanselmei, E. jeanselmei DUMC 9901 belonged to type B. Of the 2 main types of E. dermatitidis (type A, B) and 3 subtypes of E. dermatitidis type A (A0, A1 and A2), two strains (E. dermatitidis CBS 709.95, E. dermatitidis CBS 109154) belonged to A0 subtypes, 1 strain (E. dermatitidis DUMC 9902) A1 subtype, respectively.ConclusionPhylogenetic analysis of ITS region sequence provided useful information not only for new species identification but for the subtyping and origin of Exophiala species.

Phylogeny and taxonomy of the Pyrenaria complex (Theaceae) based on nuclear ribosomal ITS sequences

The generic limits in the Pyrenaria complex are discussed based on morphological and molecular evidence. The phylogeny of the Pyrenaria complex was reconstructed using parsimony and Bayesian analyses of sequence data from the nuclear internal transcribed spacer (ITS) region. The Pyrenaria complex is shown to be monophyletic and is sister to the Camellia–Polyspora clade. None of the previously recognized genera (including Pyrenaria, Tutcheria, Parapyrenaria and Sinopyrenaria) is monophyletic, and the species of these genera are scattered and nested within the Pyrenaria complex, suggesting that the four genera should be combined as Pyrenaria sensu lato. This study indicates a close relationship among the genera within the Pyrenaria complex, Camellia and Polyspora, and reveals that Camellia has a closer relationship with Polyspora than with the Pyrenaria complex.

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.

Molecular Systematic Study of Chrysosplenium Series Pilosa (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.

First Report of Xylella fastidiosa in Peach in New Mexico.

Xylella fastidiosa is a gram-negative bacterium that causes disease in a wide variety of plants such as grapes, citrus trees, oleanders, and elm and coffee trees. This bacterium is xylem limited and causes disease symptoms such as leaf scorch, stunting of plant growth, branch dieback, and fruit loss. The presence of X. fastidiosa was previously reported in New Mexico where it was found to be infecting chitalpa plants and grapevines (3). In the summer of 2010, peach (Prunus persica (L.) Batsch) trees from two locations in northern New Mexico exhibited leaf deformity and stunting, dark green venation, slight mottling, and branch dieback. Preliminary viral diagnostic screening was performed by Agdia (Elkhart, IN) on one symptomatic tree and it was negative for all viruses tested. Three trees from two different orchards tested positive for X. fastidiosa by ELISA and PCR analysis using X. fastidiosa-specific primer sets HL (1) and RST (2). Bacterial colonies were also cultured from these samples onto periwinkle wilt media. Eight colonies obtained from these three plants tested PCR positive using the X. fastidiosa-specific primers. The 16S ribosomal and 16S-23S rRNA internal transcribed spacer (ITS) region (557 nucleotides) (GenBank Accession No. HQ292776) along with the gyrase region (400 nucleotides) (GenBank Accession No. HQ292777) was amplified from the peach total DNA samples and the bacterial colonies. Sequencing analysis of these regions indicate that the X. fastidiosa found in peach is 100% similar to other X. fastidiosa multiplex isolates including isolates from peach, pecan, sycamore, and plum trees and 99% similar to the X. fastidiosa isolates previously found in New Mexico. Further analysis of the 16S ribosomal and 16S-23S rRNA ITS sequences with maximum likelihood phylogenetic analysis using Paup also groups the peach isolates into the X. fastidiosa multiplex subspecies. The gyrase sequence could not be used to differentiate the peach isolates into a subspecies grouping because of the lack of variability within the sequence. This X. fastidiosa multiplex subspecies could possibly be a threat to the New Mexico pecan industry since pecan infecting X. fastidiosa isolates belong to the same bacterial subspecies. It is not known if X. fastidiosa subspecies multiplex isolates from peach are capable of infecting pecans but they are closely genetically related. It is interesting to note that the isolates from peach are different than previously described X. fastidiosa isolates in New Mexico that were infecting chitalpa and grapes (3). X. fastidiosa has previously been described in peach; the disease is called "phony peach". The peach trees exhibited stunting and shortened internodes as reported for "phony peach". They also exhibited slight mottling and branch dieback that may be due to the environment in New Mexico or perhaps they are also exhibiting mineral deficiency symptoms in association with the X. fastidiosa disease. To our knowledge, this is the first report of X. fastidiosa in peach in New Mexico.

Candida northwykensis sp. nov., A Novel Yeast Isolated from the Gut of the Click Beetle Melanotus villosus

Two yeast morphotypes, BET 4(T) and BET 7, were isolated from the gut of click beetle Melanotus villosus. Click beetles were collected from the decaying timber within the woodlands of North Wyke Research, South West England, UK (latitude, 50°46'29″N; longitude, 3°55'23″W). Morphotype BET 7 was identified as Debaryomyces hansenii, and the other morphotype, BET 4(T), was found to differ from Priceomyces castillae and Priceomyces haplophilus, its closest phylogenetic neighbours, by 5.0% with respect to the nucleotide sequence of the D1/D2 domain of the large-subunit (LSU) rRNA gene, and by 8.0% with respect to the ribosomal internal-transcribed spacer (ITS) region. BET 4(T) also differ from P. castillae and P. haplophilus in a number of different phenotypic characteristics. Thus, based on the unique nucleotide sequences of its D1/D2 domain and ITS region, its physiological characteristics and an inability to sporulate, strain BET 4(T) is assigned the status of a new species of Candida, for which the name Candida northwykensis sp. nov., is proposed. The type strain is BET 4(T) (NCYC 3525(T) = CBS 11370(T)).

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

Abstract DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character‐based method, tree‐based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor‐joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character‐based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character‐based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.

Phylogeny of Korean Hornbeam ( Carpinus turczaninovii ) based on nuclear ribosomal ITS sequence

The genus Carpinus belonging to Coryloideae, Betulacea e, has significant economic and ornamental importance. This study was undertaken with the aim to understand the genetic diversity among eighteen isolates of Carpinus turczaninovii collected from different geographical regions of Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences, to compare the infraspecificphylogenetic relationships among C. turczaninovii in Korea, and some known Carpinus plants. The size variation of sequenced rRNA ITS regions was not seen, with 215, 162, 222 bp of ITS1 region, 5.8S rRNA gene, ITS2 region, respectively. However, some certain nucleotide variations resulted in genetic diversity. In the genus Carpinus, C. turczaninovii closely genetic with Castanopsis kawakam ii, Carpinus orientalis, Carpinus monbeigiana , and Calyptranthes polynenra formed one monophyletic clade, while Carpinus betulus and Carpinus laxiflora , respectively formed one monophyletic clade. In the present ITS sequence analysis, Carpinus fangiana, Carpinus coreana, Carpinus japonica , and Carpinus caroliniana were considered as Out Group, compared to C. turczaninovii group. The highest intraspecific variation was found between C. caroliniana and C. japonica. The least intraspecific variation was obtained between C. turczaninovii and C. coreana . The results will help further understanding Carpinus infraspecies population and their phylogenetic analysis. Key words: Carpinus, Hornbeam, Carpinus turczaninovii, molecular identification, nuclear ribosomal RNA, internal transcribed spacer, infraspecific-phylogenetic relationship.

Genetic variation in the ribosomal internal transcribed spacers of Aphanomyces astaci Schikora from Finland

Ribosomal Internal Transcribed Spacers (ITS) have been the most popular source of DNA data for phylogenetics and PCR detection. In this study, we investigated intraspecific and intragenomic DNA polymorphism of ribosomal ITS regions of the crayfish plague causing pathogen Aphanomyces astaci Schikora, groups Pacifastacus I and Astacus. A total of 36 ITS regions of A. astaci were sequenced from 18 new isolates and 31 polymorphic sites were analysed and compared. We also compared data produced in this study to previously published A. astaci sequences in the NCBI GenBank. We observed a 3.2% polymorphism level in 23 cloned sequences. Even though polymorphism was observed in ITS regions, Finnish A. astaci isolates could not be grouped (into either PsI or As) based on these differences.