Genome shuffling based on targeted mating yields hybrids with increased growth and fermentation capacity. (A) The eight parental strains were crossed in all pairwise combinations. Each horizontal line represents the average growth in medium with different concentrations of ethanol for all hybrid populations, as well as the parental strains. The data was normalized and converted to a heat map, and strains were ranked from low to high based on their growth in 13% (v/v) ethanol. (B) Outcrossed F1 hybrids display higher ethanol tolerance than F1 inbreds (Mann-Whitney test: ****P ≤ 0.0001). (C) Most isolated F1 outcrossed hybrids show heterosis for ethanol tolerance, whereas most inbreds perform poorer than their parents (‘inbreeding depression’). Each horizontal line represents the average performance of all single clones from a certain cross, inbred or parental strain on different concentrations of ethanol. Strains were ranked from low to high based on 12% (v/v) ethanol data. (D) F3 hybrids subjected to selection for ethanol tolerance (measured by the capacity to grow in the presence of ethanol) after each round of genome shuffling show higher ethanol tolerance than unselected F3 hybrids (Mann-Whitney test: ****P ≤ 0.0001). The dot plots show the relative growth of all F3 isolates, obtained with and without selection after each round of shuffling, respectively, on 12% (v/v) ethanol. The data from the strongest parental strains (P3, P2, and P7) are shown for comparison. (E) Individual hybrids show different maximal ethanol accumulation in VHG fermentations. F1 hybrids selected for their capacity to grow in the presence of high ethanol levels on average show the highest ethanol production. Next, F3 hybrids subjected to selection after each round of shuffling on average show higher ethanol production that unselected F3 hybrids. The dotted line indicates the ethanol production level of Ethanol Red. Unpaired t-test: *P ≤ 0.05; ****P ≤ 0.0001.