Meiotic recombination-mediated genome shuffling by recursive breeding for HWSSL tolerance. (A) A recursive mating methodology was used to create the HWSSL strains and populations used in this study. Large pools of UV mutants and genome-shuffled populations were screened on HWSSL gradient agar plates prior to each round of shuffling. (B) Portions of each population that showed more tolerance than the reference (WT) (black boxes) were scraped from gradient plates and used for genome shuffling (different rounds of genome shuffling are depicted - round 1 (R1), round 3 (R3) and round 5 (R5)). Initial UV mutant populations (UV) of each haploid mating type showing enhanced HWSSL tolerance were scraped and used to begin the recursive breeding methodology. Selection on HWSSL gradient plates was carried out between each round of GS in order to enrich the mating pool for strains showing the tolerance phenotype. A portion of each mutant pool (UV through five rounds of GS) was frozen for population sequencing (see ‘Results and discussion’). Individual colonies showing the highest tolerance to HWSSL were isolated from the frontier of growth. HWSSL, hardwood spent sulphite liquor.