Molecular organization of 5S rDNA intergenic spacer in Gentiana pneumonanthe L. and G. punctata L.

Aim. The study was aimed at cloning and analysis of molecular organization of 5S rDNA intergenic spacer (IGS) in two Gentiana species of Ukrainian flora, G. pneumonanthe L. and G. punctata L. Methods. 5S rDNA IGS sequence was amplified using polymerase chain reaction (PCR) with a pair of primers specific for the gene coding region. The produced PCR products were fractionated by gel-electrophoresis, isolated, ligated into plasmid pUC18, cloned into E. coli, and then sequenced. Nucleotide sequences were aligned using the Muscle algorithm and analyzed in the Unipro UGENE software. Results. The intergenic spacer region of the 5S rRNA genes was cloned and sequenced for two Gentiana species of Ukrainian flora, G. pneumonanthe and G. punctata. Based on the analysis of the alignment of the IGS sequences of five Gentiana species from three sections, some features of molecular organization of IGS of 5S rRNA genes in the studied species were established. In particular, motifs typical for other angiosperm families were identified, such as conservative oligo-dT motif at the IGS 3'-end that served as a transcription termination site and AT-rich region preceding the coding region of 5S rRNA gene. However, in the region of transcription initiation, conservative GC-element in position -13 is changed to AC. Conclusions. The interspecific variation of molecular organization of 5S rDNA IGS was identified among Gentiana species that can be used to clarify the phylogenetic relationships between members of this genus.Keywords: Gentiana species, 5S rDNA intergenic spacer, molecular organization, phylogeny.

Organization of 5S ribosomal DNA of Poa pratensis L.

5S rDNA, which belongs to the class of repeated sequences, represents a convenient model for studying the molecular evolution of plants. The 5S rDNA repeated unit consists of a conserved region encoding 5S rRNA and variable intergenic spacer (IGS) that contains the motifs required for initiation and termination of transcription. The IGS sequences can be used as a molecular marker for elucidation of the phylogenetic relationships of low-ranking taxa. Today, the molecular organization of 5S rDNA in species of the Poaceae family, which includes many economically important crops, is still poorly understood. Therefore, the aim of the study was to investigate the organization and polymorphism of 5S rDNA IGS in the genome of Poa pratensis L., a member of one of the largest genera of the Poaceae family. Using PCR amplification, cloning, sequencing and analysis of the SRA database, two variants of the 5S rDNA repeated units were found in the genome of P. pratensis. The two variants possess 119 bp-long coding regions, whereas the length of IGS ranges from 169 to 185 bp. At the beginning of IGS, the oligo-T sequence of the RNA polymerase III transcription terminator is present. In members of the Poaceae family, the putative external elements of the 5S rDNA promoter differ from those in previously studied groups of plants.

Subrepeats in 5s rDNAs as a molecular marker in Acer platanoides L. populations

Aim. To study the genetic diversity of tree species it is necessary to use only those regions of genome, which evolve at the highest rate, such as 5S rDNA. To estimate the potential of 5S rDNA to be used as a molecular marker for genogeographic studies, the molecular organization of this genomic region was compared between samples from two geogra-phically remote Bulgarian and Ukrainian populations of Norway maple, Acer platanoides. Methods. PCR amplification, cloning and sequencing. Results. It was shown that in the genome of A. platanoides the 5S rDNA sequences are highly similar. However, in the 5S rDNA intergenic spacer (IGS) of A. platanoides from the Bulgarian population three copies of GTCCGTT subrepeats are present, whereas only one copy of this sequence occurs in plants from the Ukrainian population. Except for different number of subrepeats, the 5S rDNA sequences of the Bulgarian and Ukrainian samples of A. platanoides are identical. In two taxonomically distant species, A. platanoides and A. pseudoplatanus, the region of 5S rDNA IGS, which contains the potential external elements of the promoter of RNA polymerase III, shows no difference, and therefore evolves at a lower rate than other parts of the IGS. Conclusions. The identity of the potential external promoter elements within the genus Acer supports the idea that this part of the IGS could be involved in the initiation of 5S rDNA transcription. The presence of different numbers of sub-repeats in the 5S rDNA IGS in various populations of A. platanoides makes them suitable for identification of intraspecific forms and for evaluation of the intraspecific genetic diversity of A. platanoides. Keywords: 5S rDNA, molecular evolution, sub-repetitions, intraspecific variability, Acer.

Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01–0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed.

Competitiveness among Rhizobia for Nodulation of Introduced Leguminous Tree Gliricidia sepium in a Sub-Saharan Sandy Soil

Agroforestry systems are progressively integrated by small farmer holder to mitigate agricultural production charge and contribute to sustainable agriculture by restoring and maintaining sandy soil fertility. A greenhouse experiment was carried out to test the nodulation level of introduced Gliricidia sepium tree with foreign rhizobial reference strains TAL1769 and TAL1770 against native rhizobial community using PCR/RFLP techniques. Restriction patterns of the two inoculated reference strains obtained for 16S – 23S rDNA - IGS were different to those of indigenous rhizobia detected in all remained nodules collected from root plants regarding the restriction enzymes HaeIII and MspI. Nodule occupancy rates of the reference strains and native rhizobial strains of profile F ranged from 18.36 to 22.45 and were higher compared to others rhizobial strains detected in gliricidia rhizosphere. Therefore, regarding its high competitiveness level, the native rhizobial strain may be considered as a good candidate to inoculate the introduced legume tree gliricidia.

Pectobacterium carotovorum subsp. brasiliense Causes Soft Rot and Death of Neobuxbaumia tetetzo in Zapotitlan Salinas Valley, Puebla, Mexico

Neobuxbaumia tetetzo (Coulter) Backeberg (tetecho) is a columnar cactus endemic to Mexico. Tetecho plants, flowers, fruits, and seeds play an important role in the semiarid ecosystem, as they serve as a refuge and food for insects, bats, and birds, and are widely used by ethnic groups since pre-Hispanic times. Tetecho is affected by a soft rot that damages the whole plant and causes its fall and disintegration. Eight bacterial colonies of similar morphology were isolated from plants showing soft rot and inoculated in healthy tetecho plants, reproducing typical symptoms of soft rot 9 days after inoculation. Ten representative isolates were selected for phenotypic and genetic identification using 16s rDNA, IGS 16S-23S rDNA, and rpoS genes and for pathogenicity tests on several members of the cactus family and other plants. Based on the results, these bacterial isolates were identified as Pectobacterium carotovorum subsp. brasiliense. Inoculation of this bacteria caused soft rot in different cacti, fruits, leaves, and roots of other plants. This is the first report of the subspecies brasiliense of P. carotovorum causing soft rot and death in cacti in the world and the first report of this subspecies in Mexico.

Molecular organization of 5S rDNA in two ukrainian populations of sycamore (Acer pseudoplatanus)

Aim. The genetic variability of tree species has still not been studied enough at the molecular level. Considering the relatively low rate of molecular evolution in perennial tree species, it is necessary to use only those regions of genome, which demonstrate a high level of variability, such as 5S rDNA. Accordingly, to estimate the potential of 5S rDNA as a molecular marker for tree species, the organization of this genomic region was compared between samples from two geographically remote Ukrainian populations of sycamore, Acer pseudoplatanus. Methods. PCR amplification, cloning and sequencing of the 5S rDNA IGS of A.pseudoplatanus. Results. It was shown that only one variant of 5S rDNA repeat with a length of 475 bp is present in the genome of A. pseudoplatanus. Also, it was found that the elements typical for the angiosperm RNA polymerase III promoter, which are localized in IGS, appear to be different from those previously described for species of other families. The level of IGS sequence similarity within the populations exceeds 99%, while the level of IGS sequence similarity between various populations is only 93,3-94,3%. Conclusions. High rate of molecular evolution of the 5S rDNA IGS makes them a convenient molecular marker for evaluation of intraspecific variation in populations of A. pseudoplatanus.Key words: 5S rDNA, molecular markers, intraspecific variability, Acer

The phenotypic features used for distinguishing species within theCladonia furcatacomplex are highly homoplasious

TheCladonia furcatacomplex treated here comprisesC. farinacea,C. furcata,C. multiformis,C. scabriuscula,C. stereoclada, andC. subrangiformis. The well-known taxonomic complexity of this group is caused by wide phenotypic variation and high morphological similarity among the species, for which reason we investigated the distribution in the phylogeny of the phenotypic characters traditionally used to distinguish the species in this complex. A phylogenetic analysis of theC. furcatacomplex is presented here, based on three loci (ITS rDNA, IGS rDNA andRPB2), representing specimens from a broad geographical range (Europe, North America and New Zealand). The phylogenetic reconstructions were performed using Maximum Likelihood and Bayesian analyses. In addition, 14 features traditionally used for species delimitation within this complex were mapped onto the Bayesian phylogeny. All the species currently accepted, with the exception ofC. stereoclada, turned out to be polyphyletic. Most of the phenotypic characters studied are highly homoplasious with the exception of the podetium type. The solid podetia represent a diagnostic character ofC. stereoclada.