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Pankin A.,Institute of Agricultural Biotechnology | Sokolova E.,Institute of Agricultural Biotechnology | Rogozina E.,Institute of Plant Industry | Kuznetsova M.,Institute of Phytopathology | And 3 more authors.
Plant Genetic Resources: Characterisation and Utilisation | Year: 2011

A coiled coil-nucleotide binding site-leucine-rich repeat gene RB/Rpi-blb1 isolated from Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans and is currently employed in potato breeding for durable late blight (LB) resistance. RB homologues were reported in several Solanum species; some of them retained defence function. Here, we report additional evidence on RB-like sequences in 21 Solanum species of the section Petota. The panel of Solanum species was screened with three RB-related PCR markers. RB-like sequences were found in every tested Solanum accession, suggesting universal distribution of RB structural homologues among Solanum genomes, while locus-specific RB-629 was found only in 15 species. Phylogenetic analysis of RB-629 sequences suggested a highly conservative pattern of polymorphisms that was neither species-nor series-specific. Apparently, duplication and evolution of RB-like loci preceded Solanum speciation. Marker presence and particular haplotypes were not immediately associated with high LB resistance. © 2011 NIAB.


Sokolova E.,Institute of Agricultural Biotechnology | Pankin A.,Institute of Agricultural Biotechnology | Beketova M.,Institute of Agricultural Biotechnology | Kuznetsova M.,Institute of Phytopathology | And 4 more authors.
Plant Genetic Resources: Characterisation and Utilisation | Year: 2011

New races of Phytophthora infestans rapidly defeat potato late blight (LB) resistance based on Solanum demissum germplasm, and breeders search for new sources of durable LB resistance. We developed and verified six sequence characterized amplified region markers recognizing the race-specific genes R1 and R3 of S. demissum and the broad-spectrum resistance gene RB of S. bulbocastanum and the germplasms of these species and used them to screen 209 accessions of 21 wild Solanum species. In addition to S. demissum, homologues of R1 and R3 were found in several species of series Demissa, Longipedicellata and diploid Tuberosa; R3 homologues were also detected in S. bulbocastanum, S. cardiophyllum and S. ehrenbergii. The RB homologues were found in a wider range of Solanum species. The markers of R1 and R3 genes reliably discerned between germplasms of S. tuberosum ssp. tuberosum and wild sources of LB resistance. Following introgression, the species-specific markers of demissum and bulbocastanum germplasm were rapidly lost, whereas the markers of R1 and R3 genes lasted through several meiotic generations and were maintained at high frequencies in modern potato cultivars. The presence of these markers in demissoid potato cultivars was significantly associated with LB resistance, presuming that both genes contribute to overall defence response. © 2011 NIAB.


Ryazanova A.Y.,Moscow State University | Kubareva E.A.,Moscow State University | Grman I.,Comenius University | Lavrova N.V.,Gamaleya Institute of Epidemiology and Microbiology | And 3 more authors.
Analyst | Year: 2011

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5′-CCNGG-3′ (N = A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5′-AGGACAAATTGTCCT-3′ in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, KD, of the M.SsoII-DNA complexes decreased in the order 60oct > 60reg > 60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, ka, was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell. © 2011 The Royal Society of Chemistry.


Kuznetsova M.A.,Institute of Phytopathology | Kozlovsky B.E.,Institute of Phytopathology | Beketova M.P.,Institute of Agricultural Biotechnology | Sokolova E.A.,Institute of Agricultural Biotechnology | And 4 more authors.
Mikologiya I Fitopatologiya | Year: 2016

Recently new aggressive lineages have emerged in the West European populations of Phytophthora in-festans and have overcome many elite potato varieties known for high late blight resistance. These lineages have rapidly moved eastward displacing the previously known strains of the pathogen. To evaluate the changes in Ph. infestans populations which spread across the North-Western Russia, we hawe realized in 2013-2014 two pilot experiments with isolates obtained from leaves collected in the field potato plots of the Pushkin laboratories of the N. I. Vavilov Institute of Plant Genetic Resources (VIR) in the Leningrad Region of Russia. In this study the phytopathologic characteristics of Pushkin isolates of Ph. infestans were supplemented, for the first time, with the molecular evidence. Potato leaves naturally infected with Ph. infestans were collected from variety Sarpo Mira, which manifested high level of late blight resistance in the European countries, and several interspecific hybrid clones from the VIR collection. The ability of the pathogen to damage various potato genotypes was evaluated in field and laboratory trials, Rx-gene virulence phenotypes, mating type, aggressiveness and response to fungicide metalaxyl were assessed. Mating type identification was based on the oospore development and the presence of CAPS marker. The microsatellite analysis of Ph. infestans isolates was conducted using the established set of primers for 12 SSR loci. We have confirmed the previously observed tendency for complexity of the race prof ile in the Pushkin population of Ph. infestans and enhanced proportion of A2 genotypes, which is consistent with the general trend observed in the Western and Central Europe up to 2013. The isolates with A2 mating type were sensitive to metalaxyl. Indices of aggressiveness of strains collected from the leaves of potato genotypes highly resistant to late blight were significantly lower than in the case of more susceptible varieties. The indices for aggressiveness did not expressly match the prof iles of virulence genes in the isolates. We presume that in 2013, isolates of Ph. infestans were collected late in the season when only resistant and moderately resistant potato plants stayed alive. In 2014, at the earlier stage of late blight progression, we also collected more aggressive isolates from susceptible potato genotypes. In the 2013 and 2014 sets of Pushkin isolates, we have not discerned the aggressive genotypes from the Western Europe.


Globa A.G.,Institute of Agricultural Biotechnology | Alekseev Y.I.,Institute of Agricultural Biotechnology | Arzumanyan V.G.,Russian Academy of Medical Sciences | Zaborova V.A.,Moscow State University | Guridov A.A.,Russian Academy of Medical Sciences
Biomeditsinskaya Khimiya | Year: 2012

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Pmpionibacterium spccics: P. acnes, P. g)-anulosim and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen.


Drobyazina P.E.,Institute of Agricultural Biotechnology | Khavkin E.E.,Institute of Agricultural Biotechnology
Gene | Year: 2011

Day length controls development in many plants. In Arabidopsis thaliana, the CONSTANS (CO) gene has been firmly established as a key component in the photoperiodic pathway of floral transition; less is known about CONSTANS-LIKE1 (COL1) orthologues of this gene in Arabidopsis and several other species. The CONSTANS protein comprises two B-box-type zinc fingers, CCT domain, and a variable middle region (MR) which corresponds to exon 2 in the COL1 genes of Solanum species. Solanum COL1 proteins are over 85% identical within the genus and about 50% similar to Arabidopsis CO. Comparative COL1 analysis in several cultivated and wild Solanum species discerned two gene variants, which differed in the structures of exon 2 and introns 1 and 2. In exon 2, two variants were primarily discerned by the numbers of AAC/AAT and CAA/CAG repeats coding for polyasparagine and polyglutamine tracts in MR; therefore two variants were dubbed short and long COL1 genes (sCOL1 and lCOL1). However, intron 1 in lCOL1 was shorter than in sCOL1 due to three indels, whereas intron 2 in available COL1 sequences was represented by three different variants. The temporal profiles of sCOL1 and lCOL1 expression in tuberosum potato dramatically differed under short and long day, and the level of sCOL1 expression exceeded that of lSOL1 by an order of magnitude. Both sCOL1 and lCOL1 were found in each Solanum genome under study and in each individual plant, and the ratio of their copy numbers was not related to plant ploidy and photoperiodic response. Evidently the evolution of two COL1 genes preceded Solanum speciation, and the day-length response of diverse Solanum genotypes does not stem from the primary COL1 structure. © 2010 Elsevier B.V.


Pankin A.A.,Institute of Agricultural Biotechnology | Khavkin E.E.,Institute of Agricultural Biotechnology
American Journal of Botany | Year: 2011

Premise of the study: Traditional taxonomy and nomenclature of Brassiceae (Brassicaceae) species do not reflect their phylogeny. Revision of the species and generic limits supported by extensive molecular data seems crucial. Methods and Results: Genome-specific polymorphisms extracted from non-coding and coding sequences were used to develop 14 sequencec haracterized a mplified r egion (SCAR) markers specific for the Brassica B genome. These SCARs were verified against 77 accessions of six U-triangle Brassica species and used to screen 23 accessions of seven wild Brassiceae species to test for their cross-species amplification. SCARs were found in all B-genome Brassica species and also in Sinapisarvensis. Conclusions: SCAR markers can be employed for discerning B-genome chromosomes in Brassica species and S. arvensis to reliably identify B-genome species and their natural hybrids. The combined molecular evidence supports the suggestion to revise the generic limits of Brassica and Sinapis. © 2011 Botanical Society of America.


Kalle E.,Swedish University of Agricultural Sciences | Gulevich A.,Institute of Agricultural Biotechnology | Rensing C.,Copenhagen University
Journal of Microbiological Methods | Year: 2013

In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. © 2013 Elsevier B.V.


PubMed | Institute of Agricultural Biotechnology
Type: Journal Article | Journal: Gene | Year: 2010

Day length controls development in many plants. In Arabidopsis thaliana, the CONSTANS (CO) gene has been firmly established as a key component in the photoperiodic pathway of floral transition; less is known about CONSTANS-LIKE1 (COL1) orthologues of this gene in Arabidopsis and several other species. The CONSTANS protein comprises two B-box-type zinc fingers, CCT domain, and a variable middle region (MR) which corresponds to exon 2 in the COL1 genes of Solanum species. Solanum COL1 proteins are over 85% identical within the genus and about 50% similar to Arabidopsis CO. Comparative COL1 analysis in several cultivated and wild Solanum species discerned two gene variants, which differed in the structures of exon 2 and introns 1 and 2. In exon 2, two variants were primarily discerned by the numbers of AAC/AAT and CAA/CAG repeats coding for polyasparagine and polyglutamine tracts in MR; therefore two variants were dubbed short and long COL1 genes (sCOL1 and lCOL1). However, intron 1 in lCOL1 was shorter than in sCOL1 due to three indels, whereas intron 2 in available COL1 sequences was represented by three different variants. The temporal profiles of sCOL1 and lCOL1 expression in tuberosum potato dramatically differed under short and long day, and the level of sCOL1 expression exceeded that of lSOL1 by an order of magnitude. Both sCOL1 and lCOL1 were found in each Solanum genome under study and in each individual plant, and the ratio of their copy numbers was not related to plant ploidy and photoperiodic response. Evidently the evolution of two COL1 genes preceded Solanum speciation, and the day-length response of diverse Solanum genotypes does not stem from the primary COL1 structure.


PubMed | Institute of Agricultural Biotechnology
Type: Journal Article | Journal: Planta | Year: 2013

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at -20 C decreased both the number of the low-affinity sites and their dissociation constant (KD); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [(3)H]dihydroFC and the competition between [(3)H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H.

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