Supplementary Material S1: Flow cytometric measurements of genome size
For: Gerstein, A. C., R. M. McBride, and S. P. Otto (2007) Ploidy Reduction in
Saccharomyces cerevisiae.
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Flow cytometric analysis was performed weekly to scan for ploidy change using a
protocol (Nash 1998) modified as previously described (Gerstein et al. 2006). Flow cytometry
allows us to track genome-size change by comparing the ancestral genomic content to that of the
evolved cells. By staining with SYTOX green dye and measuring the DNA content of 30000
cells, we sample cells at all phases of the cell cycle (Figure S1a). As we are interested in the
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unreplicated DNA content, change in genome size of the population is apparent when the G1
peak of DNA content shifts or becomes bimodal (as compared to the founder colony; Figure
S1b), indicating genome-size polymorphism within the population. Note that the relative height
of G1 and G2 peaks reflects the number of cells in a particular cell cycle phase, but is likely
reflective of culture conditions. These plots were analyzed using Flow-Jo (Tree Star, Inc.) with
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the Watson Pragmatic (Watson 1987) cell cycle analysis feature.
Flow cytometric analysis of an entire population of cells provides a good overall
picture of genomic-size variation, but it does not allow an accurate assessment of the relative
frequency of diploid-sized and triploid-sized cells, nor does it allow us to detect minor variants in
genome size (e.g., due to aneuploidy). We thus isolated twenty colonies from each of ten
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populations and performed flow cytometric analyses on each colony. The ten populations
consisted of two lines evolved from each of the five founder colonies, focusing on lines where
the population-level analyses indicated shifts in genome size. Flow cytometry was carried out on
two days. To control for potential day effects, the same t = 0 strains were measured on both
days, and all measurements from the second day were standardized so that the mean of the t = 0
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strains was the same on both days.
References
Gerstein, A.C., H.E. Chun, A. Grant, S.P. Otto (2006). Genomic convergence toward diploidy in
Saccharomyces cerevisiae. PLoS Genetics 2: e145
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Watson, J.V., S.H. Chambers, P.J. Smith (1987) A pragmatic approach to the analysis of DNA
histograms with a definable G1 peak. Cytometry 8:1-8
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Figure S1. Flow cytometric analysis is used to indicate approximate genome size. The
horizontal axis (FL1-H) in all graphs is a measure of dye fluorescence and is a proxy for genome
size. (a) A monomorphic population of triploid cells from line R6 3N
500 at time point 0. The first
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peak indicates the unreplicated DNA content of the population (G1 phase) while the second peak
indicates the replicated DNA content (G2 phase), with S phase (DNA synthesis) in between. (b)
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After 186 generations the population is polymorphic for genome size, as indicated by bimodal
G1 and G2 peaks. (c) Diploid and (d) triploid colonies isolated from the polymorphic
population.
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Figure S2. Flow cytometric population scans on ancestral colonies of known ploidy ((a)
haploid, (b) diploid, and (c) tetraploid) and the five founder colonies used to initiate this
experiement (d-h)
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Figure
S3. Flow cytometric population scans on lines initiated from a tetraploid founder colony after
186 generations of evolution. Dashed lines indicate population scans of founder colonies.
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Figure S4. Flow cytometric population scans on lines initiated from triploid founder colonies
and evolved for 186 generations. Dashed lines indicate population scans of founder colonies.
Additional peaks in panels (s), (u), and (x) indicate populations polymorphic for genome size.
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