1. Spontaneous Changes in Ploidy Are Common in Yeast
Yaniv Harari, Yoav Ram, Nimrod Rappoport, Lilach Hadany, Martin Kupiec 
Current Biology 
Volume 28, Issue 6, Pages e4 (March 2018)
DOI: /j.cub
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2. Current Biology 2018 28, 825-835.e4DOI: (10.1016/j.cub.2018.01.062)
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3. Figure 1
Ploidy FC Profiles for the Haploid Yeast Strain BY4741 Exposed to Various Stress Conditions for 100 Generations
The A, B, and C rows represent biological triplicates for each stress condition. The 4C peaks observed are marked with arrows. Haploid (BY4741; orange) and diploid (BY4743; blue) controls are also shown, with 1C, 2C, and 4C peaks marked. All cultures were grown in 5 mL YPD liquid media containing the relevant stressing agent at the stated concentration. After reaching stationary phase, cultures were diluted 1,000-fold (5 μL inoculated into 5 mL of fresh liquid media), ten times.
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4. Figure 2
Cell-Type Incidence after 100 Generations within the Different Cultures
(A) The average diploid cell percentage within the population after 100 generations in various stress conditions, identified by single-colony analysis by FC.
(B) The pie charts present the percentage of three different cell types (haploids MATa [orange], MAT-hom diploids MATa/MATa [green], and MAT-het diploids MATa/MATalpha [blue]) within the different cell cultures after 100 generations in specific stress conditions (biological triplicates are marked with roman numerals). For each pie chart, between 28 and 32 single colonies were tested for mating ability and by FC for DNA content. Diploids were also tested by PCR to validate their MAT locus alleles.
(C) A schematic one-step model for the formation of MAT-het diploids (blue) and MAT-hom diploids (green) from a heterothallic haploid cell (orange).
(D) A schematic two-step model for the formation of MAT-het diploids. In this model, endoreduplication is followed by a homologous recombination event to produce MATa/MATalpha (blue) diploids.
See also Figures S1–S3.
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5. Figure 3
Heterothallic BY4741 MATa and BY4742 MATalpha Haploid Yeast Cells Spontaneously Become Diploids upon Long-Term Exposure to 5% Ethanol in the Medium
(A) FC shows that a 4C peak appears after 60–80 generations in medium containing 5% ethanol. The 1C peak disappears at generation 110/130, respectively, in the BY4741 and BY4742 strains.
(B) The abundance of MAT-het diploid cells within the BY4741 and BY4742 cell cultures during continuous growth in the presence of 5% ethanol for 300 generations. From each time point, 24 random candidates (single cells within the population) were chosen and tested for their mating capability. Up to generation 150, each new circle represents a ten-generation interval; between generations 150 and 300, each new circle represents a 50-generation interval.
(C) A schematic graph showing the abundance of diploid cells within the population in the BY4741 and BY4742 cell cultures, as can be seen by the FC analysis results. The estimation for the diploid abundance within the population (gray) was calculated by the ratio of the 4C peak to the 1C + 4C peaks together. At the top of this graph, we plotted the abundance of MATa/MATalpha diploid cells (blue) present at each time point.
(D) The abundance of haploid cells (MATa or MATalpha) and MAT-hom and MAT-het diploids within the BY4741 and BY4742 cell cultures at specific time points, during continuous growth in the presence of 5% ethanol. For each time point, 24 single colonies were tested by FC for DNA content and by PCR and mating test for the identification of their mating alleles.
(E) PCR results showing the kinetics for the appearance and complete takeover of the MAT-het diploids within the BY4741 and BY4742 cell cultures. The upper band (∼500 bp) is a specific MATa PCR product, and the lower band (∼400 bp) is a specific MATalpha PCR product.
See also Figures S4 and S5.
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6. Figure 4
Fluctuation Test for the Frequency of Mating-Type Switching and Heterozygous Diploid Formation
(A) A schematic chart for the fluctuation assay. From each 5-FOA fluctuation plate, eight random colonies were tested for DNA content using FC analysis and for mating-type identity using MAT multiplex PCR.
(B) Fluctuation assay for the quantification of the rate of MAT-het cell formation within different haploid MATa strains and in MAT-hom diploid cells. A MATa haploid strain of the W303 background was also tested. ND, not detected.
(C) The effect of ethanol and hydroxyurea exposure on the rate of MAT-het diploid formation within a MATa haploid cell population.
(D) The effect of ethanol and hydroxyurea exposure on the rate of mating-type switching events in a haploid MATa Δste2 Δste3 cell population.
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7. Figure 5 Cell-Type Fitness
(A) Drop assay for the sensitivity of haploid cells and MAT-hom and MAT-het diploid cells at the end of a long-term growth experiment involving caffeine.
(B) Similar drop assay for hydroxyurea.
(C) The relative growth rate (cell-division time) of haploid and MAT-hom and MAT-het diploid cells in YPD with different ethanol concentrations and in the presence of 1 M KCl. The results are normalized to the growth rate of haploid cells in YPD medium.
(D) Competition assay to determine the relative fitness of haploid and MAT-hom and MAT-het diploid cells in YPD with different ethanol concentrations and in the presence of 1 M KCl. Each bar in the graph shows the specific cell-type prevalence within the culture after eight passages in the relevant liquid medium (each time 10 μL was inoculated into fresh 160 μL stress medium in a 96-well plate and grown to stationary phase before the next passage). Each competition was performed in five biological repeats, The abundance of each culture at time 0 (before the competition started) is also shown for comparison.
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8. Figure 6
Inference of Endoreduplication Rate Using Approximate Bayesian Computation
(A) The mean absolute error of our Wright-Fisher model compared to experimental data (Figure 3D). Each red mark represents the mean absolute error in 100 model simulations, for five genotypes (MATa, MATalpha, MATa/MATa, MATa/MATalpha, MATalpha/MATalpha) and six time points, corresponding to a single endoreduplication rate. The best estimate is denoted by the black arrow. Blue markers represent the near-best estimates, for which the error was higher than the minimum by <10%.
(B) Change in genotype frequency over the generations. Markers represent empirical results with 95% confidence intervals (estimated with bootstrap; 10,000 resamples). The bold lines represent the average genotype frequencies in 100 simulations with the best estimate of the endoreduplication rate; similarly, the other lines represent simulations with the near-best estimates (denoted by blue markers in A). Parameters: population size after each dilution, 106; mating-type switching rate, 10−7; ten generations per day.
(C) Probability of diploidization by mating or endoreduplication as a function of the population size and the mating-type switching rate. The red marker marks the value combination relevant for our experiments. The dashed line marks value combinations for which the average number of switches per generation in the population is 1/100, which happens to fall on the area where the probability for diploidization by either mechanism is 50%–50%. Parameters: endoreduplication rate, 5.3 × 10−5; ten generations per day.
(D) Schematic model for the evolution of diploids. Diploids can evolve by endoreduplication, resulting in MAT-hom diploids, or by mating-type switching, which converts haploids from one mating type to another, followed by mating, which results in a MAT-het diploid. Diploids can transition between the heterozygous and the two homozygous genotypes by mating-type switches and loss of heterozygosity events. The rates at which each of these steps takes place were experimentally determined (in black) or estimated (in red) according to the model in (A).
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