Strain-Specific Loci in Bacterial Genomes: Whole-Genome Discovery, Genomic Context, and Application for Multi-Strain...

Monitoring individual strains in complex microbial communities remains a fundamental challenge in microbial ecology and biotechnology. Here, we present an integrated pipeline for identifying and validating strain-specific loci (SSL) in four biotechnologically relevant plant growth promoting strains from three genera (Stenotrophomonas, Bacillus, and Pseudomonas). The pipeline applies a two-round specificity-filtering strategy combining whole-genome comparison and high-sensitivity BLASTn validation of revealed strain-specific loci (SSL) against the NCBI nucleotide database. SSL count decreased with increasing Average nucleotide identity (ANIb) of the strains used for the analysis, ranging from one locus in B. halotolerans (ANIb = 98.91%) to 15 loci in S. rhizophila (ANIb = 86.49%). All 25 SSL were universally AT-rich, mainly accessory-genome-associated, with flanking regions enriched in genes of unknown function (34.6%) and mobile genetic elements (19.2%). TaqMan qPCR assays targeting SSL demonstrated high specificity-no target sequences were detected across ten geographically distinct soil samples, nor in a native rhizosphere metagenome-and sensitivity, with limits of detection of 0.01-0.1 pg of genomic DNA. Spike-in experiments in soil yielded method detection limits (MDL) of 850-15,000 CFU/g. All four strains were detected in the wheat rhizosphere seven days after consortium application in a field experiment, validating the pipeline for multi-strain field monitoring.