S. uvarum strain YJF2600 (MATa hoΔ::NatMX trp1Δ::hphMX4) and YJF2601 (MATα hoΔ::NatMX trp1Δ::hphMX4) were crossed to previously constructed S. cerevisiae mitochondrial knockout strains (4348). S. cerevisiae strains with wild-type mtDNA were crossed in parallel as control. MATa and MATα strains were mixed on YPD and incubated at room temperature overnight. The mating mixtures were either replica-plated (initial trial) or resuspended in sterile water and plated (second trial) onto YPGly. The YPGly plates were incubated at 37°C for 7 to 10 days to select for 37°C-respiring recombinants. The mating mixtures of cox2Δ and cox3Δ crosses were also plated to CM-trp-his-leu-lys-ura at room temperature to select for diploid hybrids, which allowed us to estimate the recombination rate to be about 0.05 to 0.1%. The 37°C-respiring colonies were picked and streaked on YPD at room temperature for single colonies. For the initial trial, the 37°C-respiring cells were streaked on YPD twice. For the cox1Δ and atp6Δ crosses, the plates were left at room temperature for 3 days after 7 days at 37°C incubation, and colonies growing from the recovery period were also picked and streaked. We also tried selecting for recombinants at 33°C and 35°C for the crosses with cobΔ, atp6Δ, and atp8Δ strains, from which we isolated few recombinants at 37°C. However, selection at 35°C did not significantly increase either the number or the size of the recombinant colonies compared to 37°C, and 33°C is too low a temperature to distinguish any heat-tolerant recombinants from nonrecombinant S. uvarum mtDNA; we thus did not sequence colonies from these selections. As a result, 3 + 12, 4 + 48, 3 + 25, 2 + 3, 0 + 7, and 0 + 1 strains (initial trial + second trial) from the cox2Δ, cox3Δ, cox1Δ, cobΔ, atp6Δ, and wild-type D273-10B control crosses, respectively, were generated. A total of 102 strains were subjected to whole-genome sequencing and phenotyping.

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