RAPD and ISSR analyses of species and populations of the genus Stachys

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus- and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.     

RAPD and ISSR analyses of species and populations of the genus Stachys

Molecular analysis of the genome was performed for 14 species of the genus Stachys. RAPD and ISSR analyses of the Stachys genome revealed 574 polymorphic fragments, including genus-and species-specific markers. Based on the patterns, UPGMA and the Jacquard coefficient were used to estimate the genetic distances between Stachys species and populations and to construct dendrograms reflecting the phylogenetic relationships among the Stachys species. Molecular analysis of the Stachys genome refined the phylogenetic positions of some species and revealed synonymous species.     

Thyroid abnormalities associated with protracted childhood exposure to 131I from atmospheric emissions from the Mayak weapons facility in Russia

Between 1948 and 1960, the Mayak nuclear weapons facility in Ozyorsk, Russia discharged relatively high levels of radionuclides, primarily (131)I, into the atmosphere, resulting in appreciable exposure to the residents of Ozyorsk. To evaluate the association between thyroid diseases and childhood exposure to radioiodines, we screened 894 Ozyorsk residents born between 1952 and 1953. The study population was comprised of 581 exposed individuals living in Ozyorsk during the years of heaviest exposure and 313 nonexposed individuals who moved to Ozyorsk when radiation exposure from Mayak largely had ended. The screening protocol included a patient interview, palpation of the thyroid, cervical lymph nodes and salivary glands, an ultrasound examination, and measurement of fT4, TSH and TPOAb. Twenty-eight percent of the study group was diagnosed with a thyroid abnormality. The prevalence of nodular disease was significantly higher in the exposed group (20.7%) compared with the nonexposed (14.4%) group (relative risk = 1.4, 95% CI = 1.1; 1.9). Risks were larger for solitary nodules and for nodules > or = 10 mm in diameter. Expansion of the study to increase the number of persons screened as well as detailed dose estimation would offer an unique opportunity to evaluate thyroid disease in relation to chronic exposure to radioiodines during childhood.     

Identification and characterization of intraspecific variability of the sucrose synthase gene Sus4 of potato (Solanum tuberosum)

Nucleotide and amino acid variability of fragments of the Sus4 gene encoding the sucrose synthase enzyme was studied in 24 potato cultivars selected in Russia and other countries and differing in starch content in tubers. Both SNPs and indels were detected in a chosen Sus4 gene fragment including the sequence from exon 3 to exon 6 and corresponding to the main part of the sucrose synthase domain. Four types of Sus4 sequences were revealed depending on the presence of an insertion in introns 4 and 5 and of the mononucleotide octamer (T)8 in intron 5. Differentiation of these sequences was confirmed by statistical methods. Sixteen amino acid substitutions were identified in the translated sequence, of which eleven were nonsynonymous. Specific varietal nucleotide and amino acid substitutions were also revealed, which can be used in future for marking potato cultivars/genotypes. No direct associations between the mutational changes and the starch content were found in the potato cultivars studied by us.     

AFLP marking of the genotypes of leek (Allium porrum) varieties

The results of the AFLP analysis of 16 leek (Allium porrum) accessions and related species of the sections of the genus Allium are presented. Restriction enzymes and primer combinations for the identification of the genotypes of the A. porrum accessions were chosen. As a result, 265 polymorphic AFLP fragments were amplified for 25 analyzed genotypes, and specific spectra of DNA fragments were obtained for each accession. A total of 24 fragments specific for the A. porrum genome was detected, of which only two characterized the genotypes of individual accessions. A wide range of genetic diversity (0.11-0.32) was revealed for the A. porrum varieties and lines used in the analysis. The highest level of similarity in the analyzed set of accessions was found between A. porrum and sand leek (A. scorodoprasum).     

Proteomic Screening for Amyloid Proteins

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.     

Molecular AFLP Analysis of the Genotypes of Pepper Capsicum annuum Cultivars

The results of AFLP study of 14 Capsicum annuum cultivars are presented. In spite of the known low genomic variation of large-fruited sweet pepper, AFLP analysis proved to be suitable for detecting polymorphism and genotyping pepper cultivars. Nine primer pairs were selected to allow identification of the cultivars under study. Among-cultivar polymorphism detectable with these primers was estimated at 16.5%. A characteristic AFLP pattern was obtained for each cultivar. Several cultivar-specific fragments were revealed for seven cultivars. On the basis of the AFLP data, genetic distances between cultivars were determined and a tree was constructed by means of hierarchic cluster analysis (UPGMA) with the Jacquard coefficient. It was assumed that this information is useful in breeding programs involving the cultivars examined.     

Genetic Diversity and Phylogenetic Relationships in the Genus Lycopersicon (Tourn.) Mill. as Revealed by Inter-Simple Sequence Repeat (ISSR) Analysis

Inter-simple sequence repeat (ISSR) analysis was for the first time used to study the genetic diversity and phylogenetic relationships in 54 wild accessions and cultivars of the genus Lycopersicon. Analysis involved 14 ISSR primers homologous to microsatellite repeats and containing additional selective anchor nucleotides. In total, 318 ISSR fragments were amplified for the wild and cultivated tomato genomes. The interspecific polymorphism revealed with the ISSR primers was 95.6%. Species-specific ISSR fragments were detected for each tomato species. The highest number (more than 20) of species-specific fragments were obtained for L. esculentum sensu lato, although the intraspecific variation of ISSR patterns was low. UPGMA cluster analysis was used to construct a dendrogram and to estimate the genetic distances between the species of the genus Lycopersicon; between populations ofL. peruvianum, L. pimpinellifolium, and L. esculentum; and between tomato cultivars. The ISSR-based phylogeny was generally consistent with Lycopersicon taxonomy based on morphological and molecular evidence, suggesting the applicability of ISSR analysis for genotyping and phylogenetic studies in tomato.     

RAPD analysis of genome polymorphism in the family Lemnaceae

The multilocus RAPD analysis of intergeneric, inter-and intraspecific nuclear genome polymorphism was used for the first time to assess intergeneric, interspecific, and intraspecific polymorphism in Lemnaceae growing on the territory of Russia. The origin of the chosen accessions overlapped with the natural range of duckweeds in Russia. Seventy-five Lemnaceae accessions representing eight species (L. minor, L. gibba, L. turionifera, L. japonica, L. trisulca, L. aequinoctialis, S. polyrhiza, and L. punctata) from three genera (Lemna, Spirodela, and Landoltia), were analyzed. The highest variability levels were revealed in L. minor accessions (0.03–0.20). Species L. trisulca and S. polyrhiza were characterized by values of genetic distance 0.01–0.18 and 0.03–0.16, respectively. The lowest polymorphism levels were detected for L. turionifera (0.01–0.11). The dendrogram based on RAPD data showed that L. aequinoctialis was the most genetically distant species of the genus Lemna. Accessions of species L. turionifera and L. japonica, as well as L. minor and L. gibba, did not form separate species-specific subclusters; rather, they fell into clusters with L. japonica/L. turionifera and L. minor/L. gibba. Accessions of the genera Spirodela and Landoltia formed two separate clusters combined into one group.