## 3 Results and Discussion  
### Wine Yeast Strain Screening and Characterization  
Previously, we screened and characterized 17 industrial wine yeast strains for their response to generational and ethanol-mediated changes [24; 25], and we chose three of them for this study. These three strains were chosen because they were: (a) Widely used in winemaking, (b) well-characterized at a molecular level, as well as (c) being the most prone to generational changes [24; 25]. Array-CGH profiles of strains 1 and 2 revealed that the most significant diversity in the gene copy number was observed within sub-telomeric regions in almost all chromosomes investigated; there was also a generation-mediated gain of genes responsible for telomere maintenance (_YRF1_ genes) and copper and cadmium detoxification (_CUP1_ genes) [24]. Such copy number variations of _CUP1_ are domestication-related traits in wine yeasts [29; 30]. Similarly, plasticity of the telomeric regions, reflected by several chromosomal translocations, is often observed in industrial yeast strains [1; 2; 31; 4; 31]. Analysis of the strain 3 genome using next-generation sequencing revealed that this strain showed 47,575 high-quality variants, including single nucleotide variants and indels, compared to the reference S288C genome. Almost 60% of the affected genes were primarily involved in metabolic process, in particular protein metabolism and DNA/RNA metabolic processes [17]. In addition, GSH analysis showed generational and ethanol-mediated changes in strain 3, which were manifested by an overrepresentation of genes belonging to positive transcription regulators, when compared to the corresponding ancestral strain [25]. In this study, we subjected the abovementioned strains to KCl-induced osmotic stress for 250 generations. Although KCl is not present in grape must, it serves as an osmotic stressor that is widely used as a model for studies of osmotic stress in fungi [10; 19; 32; 33]. Because it is widely accepted that high osmotic pressure may affect the growth and viability of the yeast cells as well as the genome stability [20; 24; 25], these three characteristics were determined. Although, there were no changes in growth when yeast strains were grown in standard YPD medium, strain-dependent alterations in growth were observed under stress conditions. Accelerations of growth were observed for three evolved derivatives of strain 2 (including the control 2_YPD strain) and for two KCl-evolved derivatives of strain 3 in the presence of both 1.5 M KCl and a high glucose concentration. Such an effect was not observed in the case of strain 1 (Figure 1). Changes in the growth rate did not result from cell death as cell viability was not compromised (data not presented). Because genetic stability is one of the desirable characteristics of wine yeasts [34], we compared the chromosome patterns between evolved and the corresponding ancestral strains. Pulsed-field gel electrophoresis (PFGE) analysis revealed that adaptive evolution under both control and KCl-induced osmotic stress did not affect the karyotypes of the analyzed strains (data not presented). Amongst other desirable features of wine yeast strains are high tolerance to osmotic stress as well as enhanced vitality. The latter term refers to the metabolic activity, fitness, or vigor of a starter  
Figure 1: Generational and osmotic stress-induced changes in the growth rate of strains 1, 2, and 3. Ancestral and evolved yeast strains were grown in (**A**) YPD medium under standard conditions; (**B**) in the presence of 1.5 M KCl; or (**C**) high glucose concentration. Data are presented as the means \(\pm\) SD; n = 3. 1, 2, and 3-ancestral strains 1, 2, and 3, respectively; 1_YPD, 2_YPD, 3_YPD-VPD-evolved derivatives of strains 1, 2, and 3 (control), respectively; 1_KCL_a, 2_KCL_a, and 3_KCL_a-1.25 M KCl-evolved derivatives of strains 1, 2, and 3, respectively; 1_KCL_b, 2_KCL_b, and 3_KCL_b-1.5 M KCl-evolved derivatives of strains 1, 2, and 3, respectively.