Pangenomes-identified singletons for designing specific primers to identify bacterial strains in a plant growth-promoting consortium.

BACKGROUND: The use of plant growth-promoting microorganisms represents a sustainable way to increase agricultural yields and plant health. Thus, the identification and tracking of these microorganisms are determinants for validating their positive effects on crops. Pangenomes allow the identification of singletons that can be used to design specific primers for the detection of the studied strains.
OBJECTIVE: This study aimed to establish a strategy based on the use of whole-genome sequencing and pangenomes for designing and validating primer sets for detecting Bacillus cabrialesii TE3T, Priestia megaterium TRQ8, and Bacillus paralicheniformis TRQ65, a promising beneficial bacterial consortium for wheat. METHODS AND
RESULTS: The identification of singletons of TE3T, TRQ8, and TRQ65 was performed by pangenomes using the Kbase platform and subsequently analyzed using BLAST®. The identified DNA regions were used for primer design in AlleleID version 7. Primers were validated by multiplex PCR using pure template DNA from each studied strain, combinations of two or three DNA from these strains, and DNA from agricultural soil samples enriched (and not) with the bacterial consortium. Here, we report the first design of primers capable of detecting and identifying the beneficial strains TE3T, TRQ8, and TRQ65.
CONCLUSIONS: The use of pangenomes allowed the distinction of unique sequences that enables the design of primers for specific identification of the studied bacterial strains. This strategy can be widely used for the design of primer sets to detect other strains of interest for combating biopiracy, and commercial protection of biological products, among other applications.