Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine

1. Дендрограмма, построенная методом ближнего соседа (NJ) для 25 штаммов Bt, выделенных на территории Украины по суммарным результатам ERIC-, BOX-ПЦР и saAFLP для 36 полиморфных признаков. Значения бутстрепа расчитывались в 1000 повторениях. Genetic diversity of Rhizobium sp. in Russian Federation and Ukraine V.S. Zotov (1), N.V. Punina (1), S.A. Khapchaeva (1), S.V. Didovych (2), T.N. Melnichuk (2), A.F. Topunov (1) • (1) A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia, е-mail: hin-enkelte@yandex.ru; (2) Institute of Agriculture of Crimea of National Academy of Agriculture Sciences of Ukraine, Department of microbiology, Simferopol, Ukraine ABSTRACT • The new taxonomic marker (hin-region) has been proposed which gives possibility for Rhizobium bacteria study on “species - group of strains” level. Using this marker the groups of Rhizobium strains were determined which could not be distinguished with other methods, and these results correlated with evolutionary similarity of bacteria. The developed approach for creating of marker systems affords to carry out effective inventory of inter- and intra-species genetic diversity of nodulating bacteria and to evaluate perspectives of their use in agriculture. The proposed marker system was used for description of Rhizobium bacteria samples isolated from different ecological-geographical regions of Ukraine and Russia. INTRODUCTION Legume family (Fabaceae) includes over 19,000 described species of plants that are usually able to enter into a symbiotic relationships with nitrogen-fixing bacteria forming the nodules on the roots. Bacteria of the genus Rhizobium is one of the representative symbiont of valuable crops, such as Pisum, Lens, Vicia faba, Trifolium, Phaseolus, Galega. Study of the taxonomic structure and biodiversity of this genus is very important due to the fact that knowledge of their genetic structure and polymorphism varietal plant lines reveals the evolutionary dynamics of the variability, and thus predicts the couple of micro-macrosymbiont most adapted to each other. This coordinated selection will allow creating the combinations of symbiont bacteria-plant with a more efficient process of nitrogen fixation for full development and the formation of high yield. At present, as a rule, taxonomy and evolutionary relationships of the order Rhizobiales are studied by comparison of the nucleotide sequences of 16S rRNA gene and MLSA analysis. However, this assessment does not always correspond to the dates obtained by other loci. Using universal primers systems, especially when we are working with a mixture of different microorganisms in one sample, makes MLSA analysis more time-consuming and limited due to the high divergence of specific markers. The adequately rhizobia’s isolates classification and taxonomy can’t always be realizing due to frequent interspecies recombination between different alleles of taxonomically important genes (ribosomal rRNA operon), and the horizontal transfer (sym-genes), that’s why we need to obtain additional genetic data. The genus Rhizobium is particular interest as a model object of this Order, that has a complex taxonomic structure, which is difficult to study by current methods. Previously, in details we’ve studied the genetic diversity of phytopathogenic bacteria of the genus Xanthomonas, affecting Brassica napus in the Moscow region and the North Caucasus region of Russia using hin-region PCR and saAFLP (XbaI) techniques (Zotov et al., 2010). In this study we used these techniques to research the genetic diversity of rhizobia from different eco-geographical zones of Ukraine. MATERIALS AND METHODS Bacterial strains. 73 rhizobial isolates was obtained from root nodules of legume plants (Pisum satіvum, Trifolium repens, T. pratense, Vicia faba, Phaseolus vulgaris, Galega orientalis) growing in different regions of Russia and Ukraine. 12 reference strains of Rhizobium sp. was obtained from cultures collections of Department of microbiology Institute of Agriculture of Crimea of NAAS and All-Russia Research Institute for Agricultural Microbiology RAAS, was investigated. DNA isolation. Total genomic DNAs of all Rhizobium strains were obtained after lysozyme-sodium dodecyl sulfate lysis followed by phenol-chloroform extraction and ethanol precipitation. saAFLP. The AFLP procedure was performed as described by Valsangiacomo (1995), with some modifications. 80 ng of DNA was digested with XmaJI (AvrII) restriction enzyme and ligated with single adapter specific for restriction half-site. The restriction-ligation reaction was performed at 37°C for 2 h in a total volume of 20 μl. Primer (Pr.CTAG2: GGCCGTGGTTTTAG-ctag) and PCR conditions were as described previously (Zotov et al., 2012). PCR amplification 16S rRNA gene and 16S-23SrRNA (ITS). The primers fD1 and rD1 were used to amplify 16S rRNA genes (Weisburg et al., 1991). For ITS amplification, the primers FGPS1490-72 (Normand et al., 1992) and ITS_R (Zotov et al., 2012) were used. PCR products was amplified, purified and directly sequenced. Hin-region PCR. Hin-region was amplified by PCR with primers systems described earlier Zotov et al. (2012). PCR fragments were amplified, purified, directly sequencedand aligned using the Clustal Wv.1.75(Thompsonetal., 1994). Phylogenetic trees were constructed with Mega v. 3.1 (Kumar & Nei, 2006), using the neighbor-joining algorithms with bootstrap and genetic distances computed by Kimura’s 2-parameter model (Kimura, 1980). Schematic representation of the genomic DNA from rhizobia including reference (blue) and proposed by us (sand color) target markers: ribosomal gene cluster, restriction sites of endonuclease with an average cutting frequency, protein-coding genes, symbiotic genes and intergenic regions. Alternative phylogenetic marker - gyrB gene 16S rRNA The nucleotide sequences of 16S rRNA gene analysis showed that isolates belonging to three species: the majority of isolates belonged to R.leguminosarum, one isolate to R.giardinii and 6 isolates did to R.galegae. However, the intraspecific polymorphism in this marker wasn’t identified. ITS The study of nucleotide sequences intergenic region 16S-23SrRNA (ITS) resulted to a significant intraspecific polymorphism. However, due to frequent recombination of this locus and its several genomic copies using of this region for taxonomic purposes is limited. Hin-region Eight genotypes, 4 of which are the form of R.leguminosarum were identified on the basis of differences in the structure of the hin-regions nucleotide sequences. The level of genetic diversity within the identified genotypes was 96.2 ÷ 99.9%, and between them - 25.5 ÷ 89.9%. REFERENCES Weisburg W.G., Barns S.M., Pelletier D.A., Lane D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study // J. Bacteriol. Vol. 173. P. 697–703. Normand P., Cournoyer B., Nazaret S., Simonet P., 1992. Analysis of a ribosomal operon in the actinomyceteFrankia // Gene. Vol. 111. P. 119–124. Valsangiacomo C., Baggi F., Gaia V., Balmelli T., Peduzzi R., Piffaretti J.-C. 1995. Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies // J. Clin. Microbiol. 33(7):1716-1719. Zotov V.S., Punina N.V., Ignatov A.N.43. et al., 2010. Elaboration and use of new approach to species and strain identification of phytopatological and nitrogen-fixing bacteria // Adaptation to Climate Change in the Baltic Sea Region: Contributions from Plant and Microbial Biotechnology. Program and Abstracts, Finland, P. 87. Zotov V.S., Punina N.V., Khapchaeva S.A., Didovich S.V., Melnichuk T.N., Topunov A.F. 2012. The new taxonomic marker of nodulation bacteria of Rhizobium genus and its evolution // EkologicheskayaGenetika. St. St.Petersburg.10(2):49-62 (in russian). CONCLUSION • According to the results the collection of the nodule genus Rhizobium can be represented as follows: • analysis of nucleotide sequences of 16S rRNA gene reveals of genera and species belonging isolates; • test inoculation of host plants determines the specificity of the strains (i.e. biovars); • nucleotide polymorphism intergenic region 16S-23SrRNA reflects the degree of intra- and inter-specific recombination; • using technique saAFLP we can determine relationships between groups of strains within the proposed genotypes. • efficiency of the symbiotic system can be evaluated with hin-region marker; • The advantage of hin-region PCR analysis include: the ease and speed of execution, universality of marker system to study intra-generic relationships, high informativity and reproducibility, the ability to work with complex objects (such us plant tissue and soil). Hin-region PCR analysis can be used as a basis for fast and accurate screening of natural populations of the nodule genus Rhizobium by this features. I/A I/A I/A+I/B I/B I/B Previously identified genotypes of rhizobia biovar viciae had variety special specificity, that was established by studying separately all nodules per plant.