Strain-Specific Markers of Rhizobia according to Whole Genome Sequencing Data

The field studies of rhizobial inoculants require using simple and reliable methods for identification of the strains used to find out exactly which strain has formed a nitrogen-fixing nodule. This problem appears when testing the competitive ability of inoculant strains with respect to the local rhizobial strains, in order to follow the fate of inoculant strains in the long-term periods after introduction of the strains; finally, such methods may be in demand for protecting the rights of the owners and developers of the strains. The essence of the proposed identification method consists in the search of strain-specific DNA regions, which are absent in other genomes of the same species, and construction of a primer system for multiplex PCR, which allows simple, reliable, and rapid strain identification. The advantages of this approach relative to other identification techniques are, firstly, high reproducibility and, secondly, the fact that the method is based on detection of the structural variants, the contributions of which to the evolution of rhizobial genomes is rather high, whereas most of the genome fingerprinting techniques (AFLP, RAPD, REP, ERIC, etc.) are based on the detection of nucleotide polymorphisms in short genome fragments but miss a lot of events related to genome rearrangements and horizontal gene transfer. The method proposed can also be used for monitoring the evolutionary dynamics of rhizobial inoculant strains, particularly in unique fragments of the genome, which is very important for R. leguminosarum, where the proportion of unique sequences is much higher compared to other rhizobia.