Optimization of the Aflp-Fingerprinting Method for a Molecular-Genetic Certification of Agricultural Microorganisms

Abstract

S u m m a r yThe study involved 16 strains of the Russian collection of agricultural microorganisms (RCAM) related to the genera Bacillus, Lactobacillus, Rhizobium, and Bradyrhizobium. The aim of the work was to develop a procedure of the genotypic certification of strains with AFLP-fingerprinting (amplified fragment length polymorphism), providing detection of nucleotide polymorphisms and small rearrangements. As a result the protocol of AFLP-fingerprinting, ranging from the isolation of DNA from microbial cells and ending with computer data processing is developed. It is shown that the method of AFLP-fingerprinting has a high specificity, resolution and reproducibility of results. This method can be used to obtain individual genetic passports of microorganisms deposited in the RCAM, in order to protect the copyright of the commercial strains.Keywords: microbial collections, genotypic certification of microorganisms, AFLP-fin-gerprinting.Scientific work on microbial culture collections involves many research programs, one of which is identification and certification of microbial cultures necessary for a safe use of microbial resources in biotechnology (including plant growing, animal husbandry, and food industry), and the copyright protection of commercial strains. Today, such studies use the methods based on physiological and biochemical properties of bacterial strains and modern molecular genetic techniques, such as BOX, ERIC, REP-PCR and AFLP-fingerprinting (1, 2). Different variants of fingerprinting are based on the study of bacterial genomic DNA that can reveal their individual features important for documentation in strain-specific certificates. AFLP-fingerprinting (amplified fragment length polymorphism) -one of the most promising methods for molecular genetic certification of microorganisms. This method is based on the analysis of length polymorphism of restricted and amplified DNA fragments (3, 4). High sensitivity of AFLP allows using it for identification of individual profiles of bacterial strains and distinguishing them within one species. Previously, AFLPfingerprinting was successfully applied in studies of genetic differences between closely related strains of nodule bacteria belonging to different genera (5-7).In this regard, the purpose of this work was establishing the technique for genetic certification of bacterial strains maintained in Russian Collection of Agricultural Microorganisms based on AFLP-fingerprinting.Technique. The objects of study were 16 practically valuable bacterial strains from Russian Collection of Agricultural Microorganisms (RCAM): Rhizobium leguminosarum (348, 700, 261, and 712), Rhizobium galegae 912, Bradyrhizobium japonicum 630, Bradyrhizobium sp. 820, Bacillus thuringiensis (626, 634, 603, 640, and 642), as well as Lactobacillus plantarum (616, 621, 631, and 613). The strains of nodule bacteria were cultured on yeast-mannitol agar (YMA) at 28 C, Bacillus thuringiensis -on meatpeptone agar (MPA), lactobacteria -on liquid wort (8).Total DNA of nodule bacteria was ...