Variation In Internal Transcribed Spacer Research Articles

A LIKELY INTERSECTIONAL HYBRID IN VACCINIUM (ERICACEAE) ON SAN BRUNO MOUNTAIN, SAN MATEO COUNTY, CALIFORNIA

A population of Vaccinium from San Bruno Mountain in San Mateo County, California has been confused with V. cespitosum Michx. (V. sect. Myrtillus), a species documented from the mountain, since its initial collection in 1961. These plants resemble V. cespitosum and other species in V. sect. Myrtillus in several characters, but differ most notably in their evergreen habit and well developed calyx lobes. The latter characters are shared by V. ovatum Pursh (V. sect. Pyxothamnus), the only other species of Vaccinium known from San Bruno Mountain, suggesting that the population is a hybrid between V. cespitosum and V. ovatum. We used data from gross morphology, leaf anatomy, and DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and plastid matK and ndhF regions to test the hybrid status of this population. Hybrid status is supported by: 1) a plastid sequence profile identical to that of V. cespitosum from San Bruno Mountain and different from the profiles of all other Vaccinium samples in the study, 2) an ITS profile that differs from V. cespitosum from the mountain by only two polymorphic sites, and 3) the possession of gross morphological and anatomical characters that are either shared with one putative parent or the other, or are intermediate between them. Although ITS variation was non-additive and closely matched one of the putative parents, uniparental inheritance has been observed in other hybrids. Two novel gross morphological characters were also observed in the hybrid. The study documents a case of likely intersectional hybridization within Vaccinium, only rarely observed under natural conditions. Vaccinium cespitosum may have been outcompeted by the hybrid to the point of its extirpation from San Bruno Mountain. The population is here newly described as the nothospecies Vaccinium ×brunoense P.W.Fritsch, occurring in an area of only ca. 200 m2 with the number of individuals remaining uncertain because of clonal growth.

Molecular Phylogeography and Evolutionary History of the Endemic Species Corydalis hendersonii (Papaveraceae) on the Tibetan Plateau Inferred From Chloroplast DNA and ITS Sequence Variation

In response to past climatic changes, the species with different habits or adaptive traits likely have experienced very different evolutionary histories, especially for species that restricted to high mountain areas. In order to trace how Quaternary climatic oscillations affected range distributions and intraspecific divergence of such alpine plants on the Tibetan Plateau, here, we investigated maternally inherited chloroplast DNA (cpDNA) markers and biparentally inherited nuclear ribosomal internal transcribed spacer (ITS) DNA variations and aimed to explore the phylogeographic history of the endemic alpine species Corydalis hendersonii Hemsl. (Papaveraceae). We sequenced four cpDNA fragments (trnS-trnG, trnT-trnL, atpH-atpI, and psbE-petL) and also the nuclear (ITS) region in 368 individuals from 30 populations across the species' range. The network and phylogenetic analysis based on cpDNA variations identified 15 chlorotypes that cluster into three distinct clades. However, our nuclear DNA results demonstrated that there were four genetic/geographical groups within C. hendersonii. Some common and highly divergent cpDNA and ITS haplotypes were distributed in the populations of central and northeastern Tibetan Plateau, and the highest nucleotide diversity and genetic differentiation were detected in the central region. Demographic tests further indicated that the populations of southwestern and western Tibet may have experienced recent range expansion, which most likely occurred during the last glacial maximum (LGM) and continued its expansion after the beginning of the Holocene. These two different groups of this species may have derived from potential refugia that existed in the central and/or northeastern regions of Tibet during recent interglacial periods. In addition, our AMOVA analyses detected high genetic differentiation along with the whole sampling range. Also, distinct phylogeographic structures were detected among populations of C. hendersonii based on both of cpDNA and ITS variation. These findings shed new light on the importance of climatic oscillations during Quaternary and complex local topography as causes of intraspecific diversification and demographic changes within cold-tolerant herbs in the Tibetan Plateau biodiversity hotspot.

Variation in the Internal Transcribed Spacer Region of Phakopsora pachyrhizi and Implications for Molecular Diagnostic Assays.

Phakopsora pachyrhizi, the causal agent of soybean rust (SBR), is a global threat to soybean production. Since the discovery of SBR in the continental United States, quantitative polymerase chain reaction assays based on the internal transcribed spacer (ITS) ribosomal DNA locus were established for its rapid detection. However, insufficient data were initially available to test assays against factors that could give rise to misidentification. This study aimed to reevaluate current assays for (i) the potential for false-positive detection caused by nontarget Phakopsora species and (ii) the potential for false-negative detection caused by intraspecific variation within the ITS locus of P. pachyrhizi. A large amount of intraspecific and intragenomic variation in ITS was detected, including the presence of polymorphic ITS copies within single leaf samples and within single rust sori. The diagnostic assays were not affected by polymorphisms in the ITS region; however, current assays are at risk of false positives when screened against other species of Phakopsora. This study raises caveats to the use of multicopy genes (e.g., ITS) in single-gene detection assays and discusses the pitfalls of inferences concerning the aerobiological pathways of disease spread made in the absence of an evaluation of intragenomic ITS heterogeneity.

Disease-free monoculture farming by fungus-growing termites

Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis.

Genetic divergence of Platycerus hongwonpyoi (Coleoptera: Lucanidae) in South Korea

AbstractWe examined the genetic divergence of Platycerus hongwonpyoi Imura & Choe, 1989 in South Korea using the nuclear wingless (Wg) gene, internal transcribed spacer (ITS) region and mitochondrial cytochrome oxidase subunit I (COI) gene. We found no variation in Wg or ITS. Based on COI, P. hongwonpyoi was split into four well defined and one weakly supported clades, which were inferred to have diverged 2.11–1.33 Ma. The Platycerus hongwonpyoi population size seems to have decreased during the past several tens of thousands of years. The divergence times of major clades of P. hongwonpyoi were comparable with those involved in the speciation of certain Japanese species. Frequent overlapping of different clades at the same sites suggests the occurrence of secondary gene flow following differentiation in South Korea. In conclusion, the genus Platycerus underwent strikingly different divergence patterns in South Korea compared with Japan according to the disparate topographies of these two geographical areas.

Phylogenetic relationship and genetic differentiation of Populus caspica and Populus alba using cpDNA and ITS noncoding sequences

Populus caspica Bornm. (section Leuce and subsection Albida), one of the most endangered endemic tree species in the Hyrcanian Forest in Iran, has numerous morphological characteristics that are closely similar to Populus alba; to clarify their taxonomic relatedness and genetic differentiation and thus inform conservation strategies, we used the noncoding regions of chloroplast DNA (cpDNA; trnL-F and trnH-psbA) and the internal transcribed spacer (ITS). Leaf samples were collected from six populations across northern Iran. cpDNA and ITS fragments were amplified by universal primers using the PCR technique and directed sequencing. The results showed that P. caspica is genetically differentiated from P. alba, and two ITS variants were detected within some P. caspica individuals. Conflicts between topologies from ITS and plastid genomes were observed. High differentiation of P. caspica from the other Populus species shown in this study confirmed the diverging taxonomic status of this endangered species. We recommend in situ conservation measures (e.g., protected areas) for at least several populations of this species, especially in the plain regions of the Hyrcanian forest.

The genetic variation of Angiostrongylus cantonensis in the People\u2019s Republic of China

BackgroundThe People’s Republic of China (P.R. China) is the presumptive home range of the rat lungworm Angiostrongylus cantonensis, a major aetiological agent of human eosinophilic meningitis. We present a study of the genetic variation of A. cantonensis in P.R. China. Our aim was to deepen the current knowledge pertaining to its origin and global spread from a molecular perspective.MethodsAdult A. cantonensis were collected in the frame of a national survey and identified based on morphological criteria. Polymerase chain reaction (PCR) was employed to amplify the target DNA sequences (cytochrome c oxidase subunit I (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) and internal transcribed spacer (ITS)). The PCR product of cox1 was directly submitted to sequencing, while clone sequencing was used for nad1 and ITS. The identity of the samples was verified by comparing the sequences to those of accepted A. cantonensis specimens. The specific composition of substitutions in each gene was analysed, and the genotypes were compared based on the complete cox1, nad1 and ITS genes.ResultsWe characterised the complete mitochondrial genes cox1 and nad1 of 130 specimens and obtained 357 nuclear sequences containing two complete ITS (ITS1 and ITS2) and 5.8S rRNA of the same samples. All specimens were genetically confirmed as A. cantonensis. Two major groups (i.e. I and II) were identified according to the phylogeny of cox1 sequences. Group I could be further categorised into six distinct clades. Almost half of the specimens (47.7%) belong to the clade Ia and 22.3% to the group II. The former was widely distributed across the study region. A variable number of repeat units in three microsatellites was observed, resulting in considerable length variation in ITS. Intragenomic variation of ITS sequences was found in a large proportion of the samples. Genotyping showed a striking difference between mitochondrial DNA and ITS.ConclusionsOur results demonstrate that A. cantonensis is the only rat lungworm species in P.R. China and shows high genetic diversity. Results of diversity and genotyping of A. cantonensis can be impacted by the sequencing strategy and biomarker. Although ITS may be a valuable marker for interspecific identification, it is not suitable for studying the intraspecific variation of A. cantonensis due to its high intragenomic variation and current challenges for direct sequencing.

Intra- and inter-isolate variation of ribosomal and protein-coding genes in Pleurotus: implications for molecular identification and phylogeny on fungal groups

BackgroundThe internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and elongation factor 1-alpha (EF1α) are often used in fungal taxonomy and phylogenetic analysis. As we know, an ideal molecular marker used in molecular identification and phylogenetic studies is homogeneous within species, and interspecific variation exceeds intraspecific variation. However, during our process of performing ITS, RPB2, and EF1α sequencing on the Pleurotus spp., we found that intra-isolate sequence polymorphism might be present in these genes because direct sequencing of PCR products failed in some isolates. Therefore, we detected intra- and inter-isolate variation of the three genes in Pleurotus by polymerase chain reaction amplification and cloning in this study.ResultsResults showed that intra-isolate variation of ITS was not uncommon but the polymorphic level in each isolate was relatively low in Pleurotus; intra-isolate variations of EF1α and RPB2 sequences were present in an unexpectedly high amount. The polymorphism level differed significantly between ITS, RPB2, and EF1α in the same individual, and the intra-isolate heterogeneity level of each gene varied between isolates within the same species. Intra-isolate and intraspecific variation of ITS in the tested isolates was less than interspecific variation, and intra-isolate and intraspecific variation of RPB2 was probably equal with interspecific divergence. Meanwhile, intra-isolate and intraspecific variation of EF1α could exceed interspecific divergence. These findings suggested that RPB2 and EF1α are not desirable barcoding candidates for Pleurotus. We also discussed the reason why rDNA and protein-coding genes showed variants within a single isolate in Pleurotus, but must be addressed in further research.ConclusionsOur study demonstrated that intra-isolate variation of ribosomal and protein-coding genes are likely widespread in fungi. This has implications for studies on fungal evolution, taxonomy, phylogenetics, and population genetics. More extensive sampling of these genes and other candidates will be required to ensure reliability as phylogenetic markers and DNA barcodes.

The Phylogeny and Biogeography of Phyla nodiflora (Verbenaceae) Reveals Native and Invasive Lineages throughout the World

Phyla nodiflora is an herbaceous perennial and an enigmatic species. It is indigenous to the Americas but is considered a natural component of the flora in many areas and a weed in others. Our aim was to circumscribe the native range of P. nodiflora, to explore dispersal mechanisms and routes and to test the hypothesis that P. nodiflora is native outside of the Americas. Determining whether distributions are natural or human-induced has implications for decisions regarding weed control or conservation. We undertook phylogenetic analyses using sequence variation in nuclear DNA marker ITS (Internal Transcribed Spacer) for a global sample of 160 populations of P. nodiflora sourced from Asia, Australia, central America, the Mediterranean, southern North America, South America and Africa. Analyses included maximum likelihood, maximum parsimony, a Bayesian estimation of phylogeny and a parsimony network analysis which provided a genealogical reconstruction of ribotypes. We evaluated phylogenies against extensive historical and biogeographical data. Based on the sequences, 64 ribotypes were identified worldwide within P. nodiflora and considerable geographic structure was evident with five clades: one unsupported and the remaining weakly supported (bootstrap support ranging from 52% to 71%). Populations from central and southern North America formed the core area in the indigenous range and we have detected at least three native lineages outside of this range. Within Australia P. nodiflora is represented by at least one native lineage and several post-European introductions. Phyla nodiflora is one of the few species in the family Verbenaceae to have a pan-tropical native distribution, probably resulting from natural dispersal from America to Africa then to Australasia. However, it has also undergone human-mediated dispersal, which has obscured the native-origin of some ribotypes. These introductions present a risk of diluting the pan-tropical structure evident in this species and therefore they have important conservation implications.

Definition of Eight Mulberry Species in the Genus Morus by Internal Transcribed Spacer-Based Phylogeny.

Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus.

Genetic structure of Pseudo-nitzschia pungens (Bacillariophyceae) populations: Implications of a global diversification of the diatom

Pseudo-nitzschia pungens is a planktonic marine diatom known to be widespread in tropical and temperate coastal waters. We examined the population genetic structure of tropical Southeast Asian populations of P. pungens and compared it with those of northern and southern temperate populations. The secondary structures of the nuclear encoded internal transcribed spacer (ITS) region of 164 strains of P. pungens were modeled and analyzed. The tree revealed three ITS entities: clade I (comprised of P. pungens var. pungens) was distributed mainly in northern temperate waters; clade II (comprised of both P. pungens var. pungens and var. cingulata) was mainly from the NE Pacific; and clade III (comprised of both P. pungens var. pungens and var. aveirensis) was restricted to tropical and warm-temperate waters. Hybrids of both P. pungens var. pungens and var. cingulata co-occurred in clades I and II. Sixty haplotypes were revealed from the sequences of 164 strains. Haplotype diversity inferred from the median-joining network was in accordance with phylogenetic analysis, further supporting the grouping of the P. pungens haplogroups. Our results revealed limited gene flow between P. pungens from tropical and temperate waters, and significant population structure, as estimated by an analysis of molecular variance (AMOVA), with 75% of the total ITS variation found among populations (ФST=0.75). This study suggests that distinct environmental clines, such as ocean thermohaline circulation, have a potential for fragmenting and dispersing global populations of P. pungens. Formation of the Isthmus of Panama, in particular, is speculated to play a role in this allopatric differentiation in P. pungens populations worldwide.

Molecular diversity, phylogeography and genetic relationships of the Silene paradoxa group of section Siphonomorpha (Caryophyllaceae)

We investigate the genetic diversity and demographic patterns of the Silene paradoxa group from section Siphonomorpha, which includes the three species S. paradoxa, S. fruticosa and S. aristidis. Eight additional taxa, belonging to the same section, were also included in this study for comparative purposes: S. nutans, S. viridiflora and six species of the italica complex. The nuclear internal transcribed spacer (ITS) and the two chloroplast spacers trnH–psbA and trnS–trnG show that the paradoxa group constitutes a separate genetic lineage within section Siphonomorpha and includes three well-differentiated species, characterized by different levels of genetic diversity and demographic histories. All three species showed specific haplotypes and ITS variants, intraspecific variation and contrasting demographic histories. S. paradoxa probably followed a demographic expansion scenario, whereas S. fruticosa and S. aristidis most likely went through spatial expansions in the past. Chasmophyte species and species from open rocky habitats are found in both the paradoxa group and the italica complex, suggesting that the switch between the chasmophytic and open habitats occurred at least twice in the Siphonomorpha section.

ITS sequence variation and concerted evolution in the natural hybrid species Malus toringoides

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides, and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ∼ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ∼ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.

Molecular markers from three organellar genomes unravel complex taxonomic relationships within the coralline algal genus Chiharaea (Corallinales, Rhodophyta)

The use of molecular markers in taxonomic studies has become a standard practice in biology. However, consensus on which markers to use at the species level is lacking because evolutionary lineages show differences in divergence rates between organellar genomes. Ideally, researchers use multiple lines of evidence when first describing a species, such as the incorporation of several molecular markers from varied genomes (mitochondrion, plastid and nucleus). This study examined species boundaries in the red algal genus Chiharaea. We used five molecular markers, with at least one marker from each genome, coupled with thorough morphological analyses. We recognized three species in Chiharaea (C.americana, C. rhododactyla sp. nov., C. silvae) and two forms (C. americana f. americana and C. americana f. bodegensis (H.W. Johansen) stat. nov.). For C. americana f. americana and C. americana f. bodegensis differentiation based on morphological data was reflected in the plastid-encoded large subunit of RuBisCO (rbcL), but was not concordant with either the mitochondrial cytochrome c oxidase subunit 1 (COI-5P) or nuclear internal transcribed spacer (ITS) sequence data. We suggest that this discordance is indicative of ongoing hybridization and introgression between populations of C. americana f. americana and C. americana f. bodegensis. In addition, we used a PCR assay with ITS specific primers to amplify multiple ITS variants for collections assignable to C. americana indicating that there is genetic variability within ITS copies most likely due to introgression, crossing over and/or the retention of ancestral variants.

Multiple Divergent ITS1 Copies Were Identified in Single Tomato Genome Using DGGE Analysis

The intra-genomic variation in the internal transcribed spacer (ITS) region has led to misleading conclusions in the evolutionary analysis of plants; understanding this variation is critical for correct evolutionary analysis based on ITS sequences. To reveal the ITS variation in tomato, entire copies of ITS1 sequences within tomato species were separated using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. ITS1 copies varied significantly in sequence composition, but not in sequence length within the same tomato cultivar. DNA sequence similarity of the ITS1 copies was 77–100 %. Moreover, AT and GC contents in ITS1 copies from each tomato cultivar were significantly different, ranging from 50.4 to 64.3 % for GC and from 35.7 to 49.6 % for AT. However, the length variation of ITS1 was insignificant, ranging from 279 to 282 bp. Multiple copies of divergent ITS1 present in the tomato genome indicate that some copies may be paralogues. In conclusion, DGGE technique is a reliable and novel approach to reveal the entire ITS copy variation and the possible evolutionary relationship of tomato.

Geographical variation and systematics of the tetraploid marsh orchid Dactylorhiza majalis subsp. sphagnicola (Orchidaceae) and closely related taxa

Dactylorhiza majalis subsp. sphagnicola is an allotetraploid marsh orchid derived from parents closely similar to present-day D. incarnata and the western European form of D. maculata subsp. maculata, suggesting that it has a postglacial origin. It extends from northwestern continental Europe into areas formerly covered by the Weichselian ice sheet in mid-Scandinavia. Here, we studied the variation at both the plastid and nuclear marker systems to describe the geographical variation in subsp. sphagnicola and its evolutionary history. We investigated whether subsp. sphagnicola is affected by secondary hybridization and gene flow from its parental lineages or from other allotetraploid marsh orchids, and we also compared subsp. sphagnicola with other allotetraploids of similar origins. We analysed 492 plants from 50 populations. Thirty-seven populations were collected as potential Dactylorhiza majalis subsp. sphagnicola, five as subsp. sesquipedalis (D. elata), one as D. elata subsp. brennensis, one as subsp. calcifugiens, one as subsp. occidentalis and the remaining five as populations with some affinity to subsp. lapponica (including D. traunsteineri). All populations were analysed for plastid haplotypes and nuclear internal transcribed spacer (ITS) allele frequencies, and a subset of 43 populations was analysed for five nuclear microsatellite loci. Dactylorhiza majalis subsp. sphagnicola was dominated by a single plastid haplotype that was also dominant in western European D. maculata subsp. maculata, and most of the alternative haplotypes differed by only one mutation from the dominant one. There was more variation in nuclear microsatellites and ITS, and the variation was geographically structured in these markers. Subspecies occidentalis and calcifugiens shared haplotypes with subsp. sphagnicola, whereas subsp. sesquipedalis and brennensis had other haplotypes. Dactylorhiza majalis subsp. sphagnicola may have a postglacial origin within its present continental distribution. It has incorporated genetic material from D. maculata subsp. maculata by secondary hybridization and introgression, and some northern populations have assimilated strongly divergent haplotypes from the northeastern form of D. maculata subsp. maculata. Subspecies sphagnicola has also evolved morphologically divergent local populations in the north that do not differ from the typical populations in genetic markers. It may form mixed populations with other allotetraploid subspecies of D. majalis and, at least at one site, it has become integrated with subsp. lapponica, demonstrating that independently derived allotetraploids may contribute to a common gene pool. Subspecies calcifugiens seems to be derived from subsp. sphagnicola, and further studies based on a larger sample may confirm that it is better recognized as a variety. The so-called D. elata subsp. brennensis is of hybrid origin and combines markers from subsp. sesquipedalis with markers from the D. majalis core complex, possibly subsp. majalis. The new combination Dactylorhiza majalis subsp. sesquipedalis (Willd.) H.A.Pedersen & Hedren comb. nov. is provided. (C) 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168, 174193. (Less)

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

Phylogenetic Analysis of Subgenus Vigna Species Using Nuclear Ribosomal RNA ITS: Evidence of Hybridization among Vigna unguiculata Subspecies

Molecular phylogeny among species belonging to subgenus Vigna (genus Vigna) was inferred based on internal transcribed spacer (ITS) sequences of 18S-5.8S-26S ribosomal RNA gene unit. Analysis showed a total of 356 polymorphic sites of which approximately 80% were parsimony informative. Phylogenetic reconstruction by neighbor joining and maximum parsimony methods placed the 57 Vigna accessions (belonging to 15 species) into 5 major clades. Five species viz. Vigna heterophylla, Vigna pubigera, Vigna parkeri, Vigna laurentii, and Vigna gracilis whose position in the subgenus was previously not known were placed in the section Vigna. A single accession (Vigna unguiculata ssp. tenuis, NI 1637) harbored 2 intragenomic ITS variants, indicative of 2 different types of ribosomal DNA (rDNA) repeat units. ITS variant type-I was close to ITS from V. unguiculata ssp. pubescens, whereas type-II was close to V. unguiculata ssp. tenuis. Transcript analysis clearly demonstrates that in accession NI 1637, rDNA repeat units with only type-II ITS variants are transcriptionally active. Evidence from sequence analysis (of 5.8S, ITS1, and ITS2) and secondary structure analysis (of ITS1 and ITS2) indicates that the type-I ITS variant probably does not belong to the pseudogenic rDNA repeat units. The results from phylogenetic and transcript analysis suggest that the rDNA units with the type-I ITS may have introgressed as a result of hybridization (between ssp. tenuis and ssp. pubescens); however, it has been epigenetically silenced. The results also demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. unguiculata.

Nuclear rDNA evidence for major lineages of helenioid Heliantheae (Compositae)

Abstract Results of phylogenetic analyses of nuclear 18S–26S rDNA internal transcribed spacer (ITS) region sequences for representatives of most genera of helenioid Heliantheae and various members of Heliantheae s.s. (sensu stricto) and Eupatorieae help to clarify major lineages and relationships in the clade corresponding to Heliantheae s.l. (sensu lato) and Eupatorieae. Most subtribes of helenioid Heliantheae as circumscribed by Robinson (1981) correspond closely with ITS clades. Polygeneric subtribes of helenioid Heliantheae that appear to be monophyletic based on ITS data include Flaveriinae sensu Turner and Powell (1977), Madiinae sensu Carlquist (1959), and Peritylinae sensu Robinson (1981). Chaenactidinae sensu Robinson (1981) is polyphyletic, but most members of the group are encompassed within only four ITS clades. Based on the ITS data and results of combined analyses of ITS variation plus previously published morphological and chloroplast DNA data, we conclude that Heliantheae s.s. and Eupatori...

Nuclear ribosomal DNA diversity of a cotton pest (Rotylenchulus reniformis) in the United States

The reniform nematode (Rotylenchulus reniformis) has emerged as a major cotton pest in the United States. A recent analysis of over 20 amphimictic populations of this pest from the US and three other countries has shown no sequence variation at the nuclear ribosomal internal transcribed spacer (ITS) despite the region’s usual variability. We investigated this unexpected outcome by amplifying, cloning and sequencing two regions of the nuclear ribosomal DNA (18S, ITS1) to ascertain whether any variation occurred within and among populations of reniform nematodes in Alabama, US. Both the nrITS1 and the relatively conserved 18S region showed a fairly substantial amount of variation among populations. The identity among ITS sequences ranged from 1.00 to 0.86, while sequence identity at the 18S ranged from 1.00 to 0.948. We conclude that variation does exist in these sequences in reniform nematodes, and the earlier report showing no ribosomal ITS variation in this pest might have been caused by preferential amplification of a conserved ITS paralog. Current and future application towards resistance in cotton varieties to this pest requires reliable information on the molecular variability of the nematode in cotton-growing areas. Key words: Ribosomal DNA, ITS, 18S, reniform nematode.