Schematic overview of targeted genome shuffling. For the first round of targeted genome shuffling (creating pools of F1 hybrids), the eight parental strains (numbered 1 to 8) were crossed in all pairwise combinations, including inbreeding. After screening and selection (see main text), isolated F1 hybrids were used to create three funnels (A, B, C). In each funnel, the F1 hybrids were crossed in all pairwise combinations to make F2 hybrids, followed by screening and selection for ethanol tolerance. For the last round of genome shuffling, F2 hybrids were crossed in such a way that F3 hybrids incorporated genetic material from all eight initial parental strains. Note that these funnel schemes were also carried out for F1 hybrids from the same parental strains that were not selected for growth in the presence of ethanol, and for which no selection was performed in between the different rounds of hybridization.