Genome shuffling enables quantitative trait locus mapping in Bacillus subtilis

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion in Bacillus subtilis to generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size ([~]10 kb), and these associations were validated through targeted genetic swaps. This B. subtilis QTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria. One-Sentence SummaryBacterial genome shuffling mimics sexual recombination and enables genetic mapping of complex phenotypes to causal alleles.