Whole genome sequencing and the comparative analysis of housekeeping locuses and virulence genes from the commercial strains of Bacillus thuringiensis with insecticidal activity

Russian collection of agricultural microorganisms (RCAM) supports 37 strains of Bacillus thuringiensis with pronounced insecticidal activities, which are used for the production of phytoprotective biopreparations. The effectiveness of biopreparations depends on the quality of the microbial material. Monitoring of the purity and authenticity of the commercial strains can be accomplished by their molecular-genetic certification. Currently, the most effective technology for detailed genetic characterization of strains is a whole genome sequencing. However, despite the obvious demand for whole genome sequencing for the certification of commercial strains, its widespread use is limited because of considerable labour-intensive and cost. Therefore, the developing of rapid methods for high-throughput sequencing is necessary to reduce the cost of analysis and the terms of its implementation. One possible solution might be to analyze the part of genome, which includes the most taxonomically and functionally important genetic loci (referent-complexes). The goal of this work was to detect the referent-complexes of 10 B. thuringiensis strains having different serotypes. After receiving whole genome sequences the comparative analysis was carried out to search for the most divergent genetic loci that determine the uniqueness of strains. To identify most divergent loci of housekeeping genes and virulence genes in the strains, we used BioNumerics («Applied Maths», USA) program and MEGA v. 5.0 program for clustering according to Neighbour-Joining method. As a result, 10 housekeeping genes ( glpT, glpF, pyrE, purF, purH, pta, gyrB, ftsA, panC and isd ), as well as 8 virulence genes ( hblA, hblC, hblD, nheA, nheC, capA, capC and inA ) were selected. For each strain two referent complexes were constructed, representing the concatenated sequences of selected housekeeping and virulence loci taken separately. Total length of the referent-complexes was 11809 and 10094 bp respectively. In the future the sets of primers will be created for multiplex analysis of referent complexes in the high-throughput DNA sequencing and the rapid method for genetic certification of the commercial B. thuringiensis strains will be developed.