Random Spore Analysis and Tetrad Analysis

For random spore analysis samples of the cultures of the isolates grown on plates of sporulation medium for 5 days at room temperature were suspended in Zymolyase-T20 (0.05 mg ml⁻¹) solution to dissolve the vegetative cells and lyse the walls of the asci. After incubation at 37°C for 1 h, the suspension was sonicated and aliquots were then spread on YEA plates where the viable spores formed colonies. For tetrad analysis samples of the sporulating cultures were suspended in the Zymolyase-T20 solution and incubated at 37°C for 15 min. Aliquots were then streaked on YEA plates, and four-spored asci were pulled from the streaks by micromanipulation. The asci were dissected and their spores were separated from each other on YEA plates to let them form individual colonies. The colonies produced by the spores (spore clones) were tested for the presence of the auxotrophic markers of the parental strains by replica-plating on SMA plates and SMA plates supplemented with the corresponding compounds.