Comparison of the ITS Sequences of 5 Common <i>Potentilla</i> Species in Jilin Province of China

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.

The phylogenetic relationships of anastomosis groups (AG) of Rhizoctonia associated with Ceratobasidium and Thanatephorus teleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidium and Thanatephorus supported at least 12 monophyletic groupings within Ceratobasidium and Thanatephorus. There was strong support for separation of Ceratobasidium and Thanatephorus, however, six sequences representing different AG of Ceratobasidium grouped with certain sequences within the Thanatephorus clade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus (21 groups) and Ceratobasidium (10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidium may be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.

The phylogenetic relationships of anastomosis groups (AG) of Rhizoctonia associated with Ceratobasidium and Thanatephorus teleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidium and Thanatephorus supported at least 12 monophyletic groupings within Ceratobasidium and Thanatephorus. There was strong support for separation of Ceratobasidium and Thanatephorus, however, six sequences representing different AG of Ceratobasidium grouped with certain sequences within the Thanatephorus clade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus (21 groups) and Ceratobasidium (10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidium may be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.

BackgroundSpecies in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information.MethodsWe sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry.ResultsWe found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades.ConclusionsTwo species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

Data processing can mask biology: towards better reporting of fungal barcoding data?

Mating tests, internal transcribed spacer (ITS) sequence analysis, intergenic spacer 1-restriction fragment length polymorphism (IGS1-RFLP), IGS1 sequence analysis, and IGS2-RFLP analysis were carried out on isolates of 17 morphologically different Pleurotus mushrooms collected on Ferula sinkiangensis. The isolates were divided, based on mating tests and ITS sequence analysis, into two groups identical to P. eryngii var. ferulae and P. nebrodensis, respectively. Single spores from these two groups were incompatible, but those from P. eryngii var. ferulae and P. eryngii were compatible and combined to produce 56.25% dikaryon mycelia with clamp connections. The ITS of P. eryngii var. ferulae and P. nebrodensis (GenBank accession no. AY311408) were both 638 bp in size but differed by 3% in sequence. P. eryngii var. ferulae and P. eryngii (GenBank accession no. AY368658) were identical in ITS size and sequence. P. nebrodensis was the dominant population of Pleurotus mushroom growing on F. sinkiangensis. It exhibited genetic diversity. The two species could also be distinguished by IGSI-RFLP, similar to identification by mating tests and ITS sequence analysis. Difference in IGS1-RFLP existed between P. eryngii var. ferulae and P. nebrodensis. The sequence difference reached 2.28%. Both IGS1 size and IGS1-RFLP were similar among the different samples of P. nebrodensis. The 17 isolates were separated into five types based on IGS2 size and IGS2-RFLP, with both interspecies and extraspecies differences. P. nebrodensis exhibited polymorphism and was divided into four types. These results agreed with macroscopic differences. IGS2 might be the effective domain of genetically polymorphic ribosomal DNA in P. nebrodensis mushrooms found in Xinjiang, China.

Identification of dermatophytes using the traditional method is sometimes problematic because of atypical microscopic or macroscopic morphology. The aim of this study was to evaluate the feasibility of using sequencing of the ribosomal internal transcribed spacer (ITS)1 and ITS2 regions for identification of 17 dermatophyte species. The ITS regions of 188 strains (62 reference strains and 126 clinical isolates) were amplified by PCR and sequenced. Species identification was made by sequence comparison with an in-house database comprising ITS sequences of type or neotype strains or by blast searches for homologous sequences in public databases. Strains producing discrepant results between conventional methods and ITS sequence analysis were analysed further by sequencing the D1-D2 domain of the large-subunit rRNA gene for species clarification. The identification rates by ITS1 and ITS2 sequencing were higher than 97 %. Based on reference sequences of type or neotype strains, it was noted that most strains of Trichophyton mentagrophytes were misidentifications of Trichophyton interdigitale. In addition, barcode sequences were present in species of the Microsporum canis complex and Trichophyton rubrum complex. These barcode sequences are useful for species delineation when the results of ITS sequencing are ambiguous. In conclusion, ITS sequencing provides a very accurate and useful method for the identification of dermatophytes.

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.

In order to resolve the relationships among species from Angelica s.s. and its allied genera in East Asia, ITS (Internal Transcribed Spacer) sequences of 44 taxa were used to analyze their sequence divergence and to construct three phylogenetic trees in this paper. Three taxa were used as the outgroup. Some conclusions could be achieved as follows. (1) Angelica s.s., Czernaevia, and Coelopleurum were closely related, but the relationship between Ostericum and them was ulterior. It was also supported by analyses on fruit anatomy and chemical constitutes. Ostericum was proposed as a relatively independent genus. (2) Results from ITS sequences supported the view that Angelica s.s. was monophyletic and could be divided into several sections. (3) Results from ITS sequences and chemical constitutes indicated that the relationship between Peucedanum and Angelica s.s. was very close. (4) Results from ITS sequences as well as from conformation and chemical constitutes showed that the divergence of A. sinensis from other taxa of Angelica s.s. was great. The taxonomic position of A. sinensis should be reconsidered. (5) Both analyses of ITS sequences and chemical constitutes revealed that Ligusticum was not a natural group.

Thirty isolates of Apiosporina morbosa, which were isolated from wild chokecherry (Prunus virginiana), an ornamental cultivar of chokecherry with purple foliage (P. virginiana ‘Shubert Select‘), and domestic plum (Prunus domestica), were compared for morphological and molecular characteristics, using culture techniques and data from internal transcribed spacers (ITS) I and II, restriction fragment length polymorphisms (RFLP), and sequence-related amplified polymorphism (SRAP) analyses. Strains of A. morbosa grew as olive-green colonies that changed in about 2 weeks to a dark-brown colour. Cultures produced single- or two-celled and cylindrical conidia with tapering ends. Conidia had a mean size of 4.51 μm × 10.97 μm. The overall colony and conidial morphology in cultures of the A. morbosa isolates was generally similar to that of an isolate of Cladosporium herbarum. Pseudothecia were globose and produced clavate asci with unequally two-celled and club-shaped ascospores having mean sizes of 6.68–7.37 μm × 16.42–16.78 μm. RFLP markers produced by endonucleases DdeI, Bst98I, and VspI in the ITS region differentiated the pathogen isolates from other gall-associated fungi, but no difference was observed among the A. morbosa isolates, using these markers. The sequences of the ITS region had >99% similarity among 30 isolates of A. morbosa, and a total of eight unique genotypes were found, based on ITS sequence alignment. The most common genotype was manifested in 21 of 30 A. morbosa isolates, based on the ITS sequences. Three A. morbosa isolates had an identical sequence to that of an isolate of C. herbarum. A comparison with an isolate of Cladosporium cladosporioides showed that A. morbosa differed from C. cladosporioides by 3%, based on ITS sequences. SRAP analysis detected 18 unique genotypes and a high genetic diversity (H = 0.256) among 25 isolates of A. morbosa. The pathogen population isolated from wild chokecherry produced more unique genotypes and higher genetic diversity (H = 0.283) than the population from ‘Shubert Select’ (H = 0.165). However, an exact test indicated no differentiation between these two populations (P = 0.334) with a gene flow rate of 4.28. Most of the isolates from ‘Shubert Select’ were clustered in a subgroup in a phylogenetic dendrogram. Phylogenetic analysis of ITS sequences of the A. morbosa isolates in our study and related taxa extracted from GenBank revealed that A. morbosa had the closest genetic distance with Cladosporium spp.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.

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.

The dried plant material of medically important plant Actinidia eriantha especially when it remains in the form of powder often look morphologically similar to its related species. The lack of efficient methods to distinguish the authentic material from other similar species leads to chances of adulteration. The molecular authentication of herbal plant materials such as the internal transcribed spacer (ITS) sequences is considered as more reliable method compared to morphological traits. In this study, we aim to evaluate the potential of identification for roots of A. eriantha and its related species by ITS sequences. The lengths of ITS regions ranged from 624 to 636 bp with GC content ranging from 50.96% to 59.55%. A total of 194 variation sites and 46 haplotypes were formed in 185 samples. Among them, the roots of A. eriantha possessed specific sites at 85bp (C), 205bp (T), 493bp (C), 542bp (G), 574bp (C), 582bp (T) and 610bp (G), while A. hemsleyana, A. callosa, A. valvata and A. polygama have their own specific sites. The inter-specific genetic distance among 8 Actinidia species in the range 2.28% to 11.00%. The phylogenetic tree constructed with ITS, ITS1 and ITS2 region showed that the ITS sequences have higher potential for identification in 8 Actinidia species. However, as to A. eriantha, A. hemsleyana and A. valvata, these three barcodes have the same identification ability. The ITS regions indicated that different samples from same species can be grouped together, except for A. arguta and A. melanandrah. In conclusion, the ITS sequences can be used as an efficient DNA barcode for the identification of A. eriantha and its related species.

The dried plant material of medically important plant Actinidia eriantha especially when it remains in the form of powder often look morphologically similar to its related species. The lack of efficient methods to distinguish the authentic material from other similar species leads to chances of adulteration. The molecular authentication of herbal plant materials such as the internal transcribed spacer (ITS) sequences is considered as more reliable method compared to morphological traits. In this study, we aim to evaluate the potential of identification for roots of A. eriantha and its related species by ITS sequences. The lengths of ITS regions ranged from 624 to 636 bp with GC content ranging from 50.96% to 59.55%. A total of 194 variation sites and 46 haplotypes were formed in 185 samples. Among them, the roots of A. eriantha possessed specific sites at 85bp (C), 205bp (T), 493bp (C), 542bp (G), 574bp (C), 582bp (T) and 610bp (G), while A. hemsleyana, A. callosa, A. valvata and A. polygama have their own specific sites. The inter-specific genetic distance among 8 Actinidia species in the range 2.28% to 11.00%. The phylogenetic tree constructed with ITS, ITS1 and ITS2 region showed that the ITS sequences have higher potential for identification in 8 Actinidia species. However, as to A. eriantha, A. hemsleyana and A. valvata, these three barcodes have the same identification ability. The ITS regions indicated that different samples from same species can be grouped together, except for A. arguta and A. melanandrah. In conclusion, the ITS sequences can be used as an efficient DNA barcode for the identification of A. eriantha and its related species.

ABSTRACTSequence variation of ribosomal DNA internal transcribed spacers (ITS) among populations, species, and genera of the diatom genus Stephanodiscus was investigated. ITS 1 and ITS 2, including the 5.8S gene, were sequenced from geographically distant and nearby populations of S. niagarae Ehrenberg. In addition, repeats from S. hantzschii Grunow and Cyclotella meneghiniana Kützing were sequenced to determine the taxonomic range over which the ITS region could be used for diatom systematics. The morphologically distinct S. yellowstonensis Theriot & Stoermer, thought to have evolved from S. niagarae in Yellowstone Lake between 12,000 and 8000 years ago, also was sequenced to assess its relationship to nearby S. niagarae populations. The organization and relative sizes of ITS 1 and ITS 2 in Stephanodiscus species were similar to those reported for other eukaryotes. In general, ITS 2 was slightly larger and more variable than ITS 1. Cladistic analysis of ITS sequences did not resolve relationships of nearby S. niagarae and S. yellowstonensis populations. However, central North American S. niagarae populations were in a clade supported by two nucleotide changes. For Cyclotella, much of the ITS region was not alignable with that for Stephanodiscus species; therefore, generic‐level comparison within the Thalassiosiraceae may not be possible. The variation (95–96% similarity) between S. hantzschii and other Stephanodiscus species suggests that interspecific relationships could be assessed with ITS sequences. Although S. yellowstonensis is morphologically distinct from S. niagarae, no autapomorphic nucleotide sites were identified. Two S. niagarae populations (Heart and Lewis Lakes), however, did possess autapomorphic ITS sites.