Strain-specific markers of rhizobia from whole-genome sequencing data

Abstract. Field trials of rhizobial inoculants require simple and reliable methods for identifying the strains used to determine which strain has formed a nitrogen-fixing nodule. This task arises when testing the competitiveness of inoculant strains against local rhizobia strains, to track the fate of inoculant strains over long periods after the introduction of strains, and finally, such methods may be in demand when protecting the rights of strain owners and developers. The essence of the proposed identification method is to search for strain-specific DNA regions that are absent in other genomes of the same species and to construct a primer system for multiplex PCR, allowing simple, reliable and rapid identification of the strain. The advantages of this approach over other identification methods are, firstly, high reproducibility, and secondly, that the method is based on the detection of structural variants, the contribution of which to the evolution of rhizobia genomes is very high, while most genomic fingerprinting methods (AFLP, RAPD, REP, ERIC, etc.) are based on the detection of nucleotide polymorphisms in short fragments of the genome, but miss many events associated with genomic rearrangements and horizontal gene transfer. The use of the proposed method can also serve to monitor the evolutionary dynamics of rhizobial inoculant strains, especially in unique fragments of the genome, which is very important for R. leguminosarum, where the proportion of unique sequences is much higher than in other rhizobia.