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.     

Length variation in the internal transcribed spacers of ribosomal DNA in Picea abies and related species

The structure and variation of nuclear ribosomal DNA (rDNA) units of Picea abies, (L.) Karst. was studied by restriction mapping and Southern hybridization. Conspicuous length variation was found in the internal transcribed spacer (ITS) region of P. abies, although the length of this region is highly conserved both within and among most of the plant species. Two types of ITS variants (A and B), displaying a size difference of 0.5 kb in the ITS2 region, were present within individuals of P. abies from Sweden, Central Europe and Siberia. A preliminary survey of 14 additional Eurasian and North American species of Picea suggested that length variation in the ITS region is widespread in this genus. Alltogether three length variants (A, B and C) were identified. Within individuals of eight Picea species, two length variants were present within the genome (combinations of A and B variants in P. glehnii, P. maximowiczii, P. omorika, P. polita and P. sitchensis and variants B and C in P. jezoensis, P. likiangensis and P. spinulosa). Within individuals from five species, however only one rDNA variant was present in their genome (variant A in P. aurantiaca, P. engelmannii, P. glauca, P. koraiensis and P. koyamai; variant B in P. bicolor). The ITS length variation will be useful as a molecular marker in evolutionary studies of the Picea species complex, whose phylogeny is controversial. The presence of intraindividual variation in, and shared polymorphism of the, ITS length variants raises questions about the occurrence of interspecific hybridization during the evolutionary history of Picea.     

Origin of tetraploid cultivated asparagus landraces inferred from nuclear ribosomal DNA internal transcribed spacers’ polymorphisms

The genus Asparagus includes a group of wild species that are closely related to the cultivated Asparagus officinalis (2n = 2× = 20). The narrow genetic background present in the asparagus cultivars shows the importance of asparagus landraces and the wild related species. The study of both genetic resources becomes necessary to facilitate their effective use in the breeding programmes. ‘Morado de Huetor’ (MH) and ‘Violetto d’Albenga’ (VA) are tetraploid asparagus landraces (2n = 4× = 40) cultivated in Spain and Italy, respectively, and whose origin remains unknown. To discover the origin of these landraces, a phylogenetic study was conducted based on restriction fragment length polymorphism (RFLP) of nuclear ribosomal DNA (nrDNA). The sequence of the two internal transcribed spacers (ITS) flanking the nrDNA5.8S gene (ITS1‐5.8S‐ITS2) were analysed for RFLP in 11 populations including both landraces (MH and VA), A. officinalis (wild and cultivated) and a group of closely related wild species (Asparagus maritimus, Asparagus prostratus, Asparagus pseudoscaber and Asparagus tenuifolius) with a European distribution. Restriction fragment patterns of both cultivated asparagus (2×) and two populations of A. maritimus (6×) from the Adriatic Sea area were present in the MH landrace. However, VA showed a similar pattern to A. officinalis. This study revealed that MH seems to be a hybrid between A. officinalis and A. maritimus that may have occurred in the Adriatic Sea region where hybridisations between cultivated diploid and wild species may have taken place. The origin of another tetraploid landrace (VA) might have had a similar origin but followed a different evolutionary path. Therefore, these landraces constitute a valuable genetic resource that could be used to enlarge the genetic background of modern cultivars. The ploidy levels of the populations employed in this study were analysed and levels not described previously were detected: A. maritimus (12×), A. tenuifolius (6×) and A. pseudoscaber (2×).     

Phylogeny of 12 species of genusGlycine Willd. reconstructed with internal transcribed region in nuclear ribosomal DNA

The ITS-Is of 24 accessions belong to 10 species of subgenusGlycine, and 2 species of subgenusSoja of genusGlycine were amplified, cloned and sequenced. According to the homology of the sequences, the phylogeny of the 24 accessions were reconstructed. The reconstructed dendrogram showed that there were some divergent genomic types found in the previously classified species, such asG. tomentella, G. canescens andG. tabacina, and they might be some cryptic species by morphologic analysis.     

Strain and species identification by restriction fragment length polymorphisms in the ribosomal DNA repeat of Candida species

Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.     

Phytogeny of 12 species of genus Glycine Willd. reconstructed with internal transcribed region in nuclear ribosomal DNA

The ITS-Is of 24 accessions belong to 10 species of subgenus Glycine, and 2 species of subgenus Soja of genus Glycine were amplified, cloned and sequenced. According to the homology of the sequences, the phy-logeny of the 24 accessions were reconstructed. The reconstructed dendrogram showed that there were some divergent genomic types found in the previously classified species, such as G . tomentella, G. canescens and G. tabacina, and they might be some cryptic species by morphologic analysis.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA.

Ribosomal DNA variation was used to study evolutionary relationships among five fungal-like protoctistan Phytophthora species. On the basis of morphological and ecological characteristics, four of these species--P. palmivora, P. megakarya, P. capsici, and P. citrophthora--were once thought to be related. Variation within a species was extensively studied in a fifth, outgroup species--P. cinnamomi--known, on the basis of ecological, isozyme, and mitochondrial DNA studies, to be variable. Internal transcribed spacer regions (ITS I, between the 18S and 5.8S rDNAs; and ITS II, between the 5.8S and 25S rDNAs) from 27 isolates of these five species were analyzed by polymerase chain reaction amplification and direct sequencing. Intraspecific variability was undetected or low. Interspecific nucleotide difference was 0.3%-14.6%, and comparisons of variable regions permitted the evaluation of phylogenetic relationships among species. Both neighbor-joining and parsimony analysis of ITS variability support a close relationship between cacao isolates of P. capsici and P. citrophthora and a common lineage for P. palmivora and P. megakarya. Large distance values were estimated between P. cinnamomi and the other species. Inferred relationships based on ITS variability were compared with those based on other characters. The catalog of sequences provides the information necessary to design taxon-specific probes potentially useful in taxonomic, ecological, and population-level studies.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Identification of four scallop species using PCR and restriction analysis of the ribosomal DNA internal transcribed spacer region

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA region spanning the 5.8S RNA gene and the 2 flanking internal transcribed spacers (ITSs) was performed to establish DNA-based molecular markers for the identification of the scallops Aequipecten opercularis, Chlamys distorta, Mimachlamys varia, and Pecten maximus. Chlamys distorta was distinguished simply by ITS size. Species-specific restriction patterns were found with the restriction enzyme AluI, and also with SmaI for A. opercularis and M. varia. When ITS sizes and the RFLPs obtained with SmaI were combined, the 4 scallops were also differentiated. Additional species-specific RFLPs were revealed after ITS-2 PCR amplification and subsequent digestion with Hsp92II. Using this marker, canned scallops were identified. Thus this work provides a simple, reliable, and rapid method for the identification of scallops that can be used when species-specific morphologic characteristics are removed or when specimens are small in size.     

Phylogeny of 12 species of genusGlycine Willd. reconstructed with internal transcribed region in nuclear ribosomal DNA

The ITS-Is of 24 accessions belong to 10 species of subgenusGlycine, and 2 species of subgenusSoja of genusGlycine were amplified, cloned and sequenced. According to the homology of the sequences, the phylogeny of the 24 accessions were reconstructed. The reconstructed dendrogram showed that there were some divergent genomic types found in the previously classified species, such asG. tomentella, G. canescens andG. tabacina, and they might be some cryptic species by morphologic analysis. Project supported by the Chinese Academy of Sciences     

Molecular phylogeny of JapaneseAmanita species based on nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal DNA

The molecular phylogeny of 36 specimens of JapaneseAmanita species was studied based on nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The phylogenetic tree obtained supported the traditional classification systems of Bas (1969) and Singer (1986), which are based on morphological characters, in the division of the genusAmanita is divided into subgeneraAmanita andLepidella by the amyloidity of basidiospores. However, at section-level, we suggest that subgenusAmanita should be divided into three sections (Amanita, Vaginatae, andCaesareae). Our results also showed the necessity to modify the taxonomic treatments at section-level in the subgenusLepidella. It appears that the establishment ofA. muscaria andA. pantherina from a common ancestral species might be a very recent event, or these might be lower taxa of same species. As for three subspecies ofA. hemibapha and three varieties ofA. vaginata, it is necessary to grade up their taxonomical ranks from subspecies/variety to species. A new combination,A. javanica, is proposed forA. hemibapha subsp.javanica.     

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.     

Sequence heterogeneity in the internal transcribed spacers of two rat ribosomal DNA clones

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.     

The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma

The usefulness of the internally transcribed spacer 2 (ITS2) of the nuclear ribosomal gene complex is tested for providing taxonomic characters to identify Trichogramma species. The ITS2 sequences of a group of sibling species of the T. deion/T. pretiosum complexes were determined. A simple and precise identification key to the species of these assemblages was constructed using as taxonomic characters the size of the ITS2 and the difference in restriction length polymorphism of species with similarly sized ITS2. Individual wasps can be identified by amplification of their ITS2 with general primers, determining the size of the PCR product using standard agarose electrophoresis, followed in some species by a DNA-digestion with a restriction enzyme. Because this system works well for a number of closely related species we are hopeful that similar PCR-based identification can be extended to all species of the genus once their ITS2 sequences have been determined. The advantage of this identification system over the morphology-based system is that non-specialists are able to quickly and cheaply identify individual specimens. In addition, species specific primers were tested for the two most common species of these groups (i.e. T. pretiosum and T. deion). These primers can be used either as a direct identification tool or as a method to confirm the identification using the general key. The phylogeny of this group of wasps was also analyzed based on the ITS2 sequence.