SNPs and their flanking sequences were determined by Sanger sequencing of PCR products amplified from randomly picked Japanese individuals (in most cases, eight individuals).

SNPs were identified by Phred/Phrap/Polyphred (http://www.phrap.org/; http://droog.gs.washington.edu/polyphred/) and visual inspection. Insertions/deletions were detected by software developed by us (Miura et al. JP-4009481-B2).

Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H⁺-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H⁺-ATPase, suggesting that vacuolar H⁺-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analyzed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analyzed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance.

Yeast cells exposed to the air-drying stress were inoculated to YPD medium. After grown for 25 h, OD₆₃₀ was measured using microtitre platereader Elx800. OD₆₃₀ of non-stressed control cells was measured after grown for 16 h.