To address this knowledge gap, we focus on Bacillus velezensis, a spore-forming, Gram-positive rhizobacterium widely used in commercial biostimulants due to its robust plant growth-promoting abilities. Although B. velezensis is agriculturally significant, the genetic basis of its establishment in plant-associated environments remains unclear. Our studies demonstrate that different B. velezensis and B. amyloliquefaceins strains exhibit significant variation in their ability to colonize Populus trichocarpa, a biofuel feedstock species. Colony-forming unit (CFU) quantification per mg of leaf tissue confirmed distinct differences in colonization efficiency among strains, suggesting that genetic variation plays a crucial role in establishment success. Given this variation, we sought to systematically investigate the genetic factors influencing colonization through a quantitative trait loci (QTL) mapping approach.
QTL mapping, a powerful tool widely used in eukaryotic genetics used to identify genetic regions associated with the varying phenotypes. QTL mapping was further explored and adapted so it can be used in bacterial system to understand genetic determinants associated with phenotypes of interest. Our bacterial QTL mapping approach enables genome-wide association studies in non-model bacteria, allowing us to dissect the genetic basis of complex traits such as microbial establishment. This technique relies on recombination between parental strains to generate a diverse mapping population with shuffled alleles, facilitating the identification of genetic loci that influence colonization and survival in environmental settings. As we were working in non-model system, we adapted QTL mapping for B. velezensis and explored genome shuffling and natural competence as tools for generating recombinant progeny populations. Specifically, to construct QTL mapping population we used genome shuffling via protoplast fusion and natural competence to recombine B. velezensis GBO-3 with B. amyloliquefaciens DSM-7, B. velezensis S4, and B. velezensis FZB42. This approach aims to maximize genetic diversity, enabling fine-scale mapping of loci associated with microbial fitness and colonization potential.
A crucial factor influencing recombination success in genome shuffling is the restriction-modification (RMS) system, which acts as a genetic barrier between strains. Our preliminary findings suggest that if RMS-mediated interference is partially circumvented it can enhance genetic variability in the recombined population.
Moving forward, we aim to refine our QTL mapping population to systematically identify genetic determinants that contribute to microbial establishment in plant-associated environments. By integrating bacterial genetics with microbiome engineering, this work lays the foundation for the rational design of soil microbiomes to enhance plant resilience, improve agricultural productivity, and promote ecosystem sustainability.