Two different genome shuffling strategies used to generate hybrids starting from the eight parental strains. (A) Conceptual outline of targeted and random genome shuffling. Genome shuffling based on random mating (left) allows random mating between spores form the eight parental strains (F1) and between spores derived from genome-shuffled hybrid populations (F2 and F3). Genome shuffling based on targeted mating (right) exploits the selection of true outcrossed hybrids using plasmid-based markers at each stage, ensuring the presence of the eight initial genomes in the final F3 hybrids. For simplicity, homologous chromosomes of the parental strains have been given the same color, although parental strains were heterozygous diploids. (B) Details of the experimental procedures used for genome shuffling strategies. Genome shuffling based on random mating (left) was performed using two different types of selection after each round of mating. First, hybrids were selected for their capacity to grow in the presence of ethanol by first inoculating them into medium containing 5% (v/v) ethanol followed by growth in the presence of 10 to 12% (v/v) ethanol (referred to as ‘growth selection,’ similar to the selection applied for targeted mating). Alternatively, the hybrids were selected for survival in medium containing very high (18 to 22% (v/v)) ethanol levels (‘survival selection’). In parallel to these two approaches, we also carried out shuffling without any selection in between the different rounds of hybridization (‘no selection’). For targeted mating (right), a robot is used to perform specific crosses between the eight parental strains in all pairwise combinations, followed by screening for ethanol tolerance (growth capacity in the presence of ethanol). The best-performing hybrids were used as parental strains for the next round of robot-based targeted mating, and in parallel similar breeding schemes were carried out without applying any selection after each round of shuffling. See ‘Methods’ and Additional file 1: Figure S5 for more details about these procedures.