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.本研究采用点板法（spot-plate method），对所有菌株及其所有亚克隆的各类胁迫耐受性（重点关注工业相关胁迫，Gibson等，2007；Qiu等，2019）以及在丰富培养基与基本培养基上的生长情况进行了评估。本研究使用以下基于SD（合成限定培养基，synthetic defined，含2%葡萄糖、0.67%无氨基酸酵母氮源）的胁迫培养基：针对生物乙醇、艾尔啤酒、拉格啤酒、葡萄酒酵母的乙醇与高糖渗透压胁迫培养基；针对ADY烘焙酵母（ADY_Baker）的氯化钠与高糖渗透压胁迫培养基；以及针对益生菌酵母的盐胁迫与氧化胁迫（H₂O₂）培养基。在预实验中，我们确定了可用于区分亚克隆谱系的胁迫因子最优浓度（补充表S1）。将在YPD平板（酵母浸出粉胨葡萄糖培养基，YPD）上于30℃过夜培养的菌液用双蒸水（ddH₂O）洗涤，经血细胞计数板计数后调整至相同细胞浓度，随后以10μL滴液的形式，按约50000、5000、500、50、5个细胞的梯度点种至各胁迫培养基平板上。源自初始分离的样品短暂储存于4℃环境，并如前文所述避免了单细胞瓶颈效应。平板于30℃培养2天后，使用数码单反相机（DSLR camera）进行拍照，随后通过目视法评估菌落生长情况。