Spot plate assay

Tolerance to various stress factors with a focus on industrially relevant stresses (Gibson et al., 2007; Qiu et al., 2019) and growth on rich and minimal media were assessed using the spot-plate method for all strains and all subclones of the strains. The following stress media based on SD (synthetic defined, 2% glucose, 0.67% yeast nitrogen base without amino acids) were used: ethanol and high sugar osmotic stress (Bioethanol, Ale, Lager, Wine yeasts), NaCl and high sugar osmotic stress (ADY_Baker), and salt and oxidative stress (H2O2) media for the Probiotic yeast. In preliminary experiments, we determined the optimal concentrations of stressors that may enable differentiating between subclone lineages (Table S1.). Samples grown overnight (30°C) on YPD plates were washed in ddH2O, prepared in equal cell concentrations after cell counting with a haemocytometer, and spotted in 10 µl drops in a series of approx. 50,000; 5,000; 500; 50; and 5 cells to the various plates. The samples originating from the initial isolations were briefly stored at 4°C, single-cell bottlenecks were avoided as described above. Plates were incubated at 30°C for 2 days before photographing them using a DSLR camera. Growth was evaluated visually.