Correlations between CIN and growth rate, total genome content, or degree of aneuploidy across the aneuploid strains.

<p>(A–C and E–F) Normalized density distribution of the growth rate (A), calculated ploidy (B), total number of chromosomes in the genome (C), total number of chromosomes in aneuploidy (E), and total number of the megabases in aneuploidy (F) in the aneuploid strains in the three CIN classes. Density distribution functions were fitted on the data based on a Gaussian kernel and normalized to a maximum density of 1. The area of the three underlying curves was scaled to the relative proportion of strains in each category. (D) Distribution of the aneuploid strains based on basal ploidy. Green: S strains; blue: MU strains; red: HU strains. P-values at the top of each graph refer to the difference in means between the S and HU strains by using a Welch's t-test.</p

Association of chromosome copy number imbalance with CIN.

<p>(A) Enrichment of strains belonging to the combined MU and HU classes of CIN relative to the S class (left) or to the HU class of CIN relative to the combined S and MU classes (right) among aneuploid strains displaying a particular chromosome copy number imbalance, calculated for each non-redundant pair of chromosomes. The enrichment is color-coded based on p-values calculated by means of Hypergeometric tests. Darker colors indicate more significant enrichment of strains belonging to the S class of CIN (left) or HU class of CIN (right) among strains with a non-1 copy number ratio between a given pair of chromosomes. (B) Diagram illustrating the experimental design for the assessment of the relationship between CIN and <i>MAD2</i>:<i>MAD1</i> ratio in 56 freshly generated aneuploid strains from isogenic triploid sporulation. (C–D) Frequency of aneuploid strains with stable or unstable ploidy grouped by their <i>MAD2</i>:<i>MAD1</i> ratio. Ploidy-stable strains (black histograms) were identified on the basis of their low level of ploidy variation among single colonies analyzed by FACS; ploidy-unstable strains (white histograms) were identified on the basis of high ploidy variation among single colonies. <i>MAD2</i>:<i>MAD1</i> ratios were determined by qPCR and are indicated on the x-axis. P-values at the top of (C–D) graphs refer to statistical association between the stability category and the <i>MAD2</i>:<i>MAD1</i> ratio category by means of a Fisher's exact test. Aneuploid strains were divided into all four possible <i>MAD2</i>:<i>MAD1</i> ratio classes (C) or based on <i>MAD2</i>:<i>MAD1</i> ratio equal or not equal to 0.5 (D).</p

Determination of karyotype changes in aneuploid strain populations.

<p>(A) Classification of the CIN level of the 27 analyzed aneuploid strains as stable (S, no CIN event linked to g20 population karyotype), mildly unstable (MU, 1 CIN event linked to g20 population karyotype) or highly unstable (HU, 2 or more CIN events linked to g20 population karyotype). The number of strains belonging to each CIN class is shown in parenthesis. (B–D) Karyotypes of the g20 population sample and of the eleven g30 colonies (left) and the reconstructed karyotype network (right) are shown for a representative S strain (B), MU strain (C) and HU strain (D). For the karyotype network, the area of the circles is proportional to the frequency each karyotype was found among the karyotyped samples (g20 population and g30 colonies); the circle containing the g20 population sample is depicted in gray; white circles represent the karyotypes of the g20 or g30 colonies (11 total) which were divergent from the g20 population karyotype due to loss (minus sign) and/or gain (plus sign) of specific chromosomes (in Roman letters). The labels inside the circles indicate the specific samples whose karyotypes are shown in the heat map on the left. Karyotypic relationship was reconstructed by using a parsimony approach and represented by lines connecting the different karyotypes; thicker connectors refer to the CIN events directly linked to the g20 population karyotype and used for the classification of the strains into CIN categories. The same information for all analyzed aneuploid strains is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002719#pgen.1002719.s007" target="_blank">Figure S7</a>.</p

Model of discrete scaling of the functionality of the mitotic system.

<p>Schematic representation of the chromosome segregation workload (green), the capacity of the mitotic system for accurate chromosome segregation (red), and the overall functional deficit (blue) of the mitotic system (the difference between workload and mitotic capacity) as a function of increasing number of chromosome in the genome (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002719#s3" target="_blank">Discussion</a>). The model is based on the assumption that the mitotic system increases its functionality via discrete steps only when a full set of chromosomes is gained, whereas the segregation workload increases linearly with the number of chromosomes. The resulting functional deficit explains why hypo-diploid strains are in general more chromosomally unstable than hyper-haploid strains as observed. Further studies will be required to verify whether this trend extends also to cells with a ploidy between 2N and 3N.</p

Frequency of aneuploid chromosomes in the different CIN classes.

<p>The graph shows the frequency at which each of the 16 chromosomes was found in aneuploidy (either monosomic in a diploid background or disomic in a haploid background) in the analyzed aneuploid strains. The frequency histogram is stratified across the three different CIN classes. Red: HU strains; blue: MU strains; green: S strains. P-value refers to the global association between the 16 chromosomes and the three CIN classes according to a Fisher's exact test.</p

Low rDNA copy number confers advantage under DNA replication stress.

<p>(A) 3 independent isolates each with normal or low rDNA copy number were generated by subcultuing wild-type or <i>fob1Δ GAL-POL1</i> cells in medium containing high or low galactose (high or low levels of Pol1 respectively) for ~50–75 generations (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.s006" target="_blank">S2 Table</a>) and 5-fold serial dilutions spotted on to medium with DNA replication stress (low Pol1). (B) 10-fold serial dilutions of wild-type and <i>fob1Δ</i> cells (~200–250 rDNA copies) along with cells having 20–110 rDNA copies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.ref003" target="_blank">3</a>] were spotted on medium containing hydroxyurea (HU). rDNA copy number was confirmed by ddPCR. Error bars represent standard deviation for each individual reaction.</p

Design and validation of a ddPCR assay for rDNA copy number measurement.

<p>(A) Targets (red bars) within the rDNA and single copy reference (<i>TUB1</i>) loci in yeast. (B) rDNA copy number in 3 independent isolates each of 2 different wild-type laboratory yeast strains, BY4741 and W303, and 4 isogenic strains with varying rDNA copy number generated by Ide et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.ref003" target="_blank">3</a>]. (C) rDNA copy number in 8 technical replicates each of BY4741 and W303. (D) rDNA copy number in 3 independent isolates each of mutants with expanded rDNA arrays. Error bars represent standard deviation for each individual reaction.</p

Contraction of the rDNA array promotes timely completion of DNA replication and cell cycle progression.

<p>Wild-type <i>GAL-POL1</i> cells were subcultured in medium containing high or low levels of galactose for ~75 generations to generate 3 independent isolates each with normal or low rDNA copy number (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007006#pgen.1007006.s006" target="_blank">S2 Table</a>). (A) Representative DNA content profiles over time are shown for asynchronous cultures of isolates with normal (i) and low (ii) rDNA copy number following inoculation into the indicated medium which determines high or low levels of DNA polymerase α. (B) Fraction of cells in S-phase in each of the 4 conditions in (A). Error bars indicate standard deviation based on 3 independent isolates. Statistical significance of differences between fraction of cells in S-phase in high and low levels of DNA polymerase α was calculated using a standard 2-tailed t-test. *—p<0.05, ****—p<0.0001. (C) Increased rARS firing in nicotinamide exacerbates growth defects under conditions of DNA replication stress.</p

Screen for essential genes that maintain rDNA copy number.

<p>(A) Distribution of rDNA copy number across the yeast ts mutant collection. 175 and 113 strains had significantly lower (<70 copies) and higher copy number (>119 copies) respectively (p<0.05). (B) -log₁₀ transformed FDR q-values for significantly enriched (p<0.001) GO terms (sorted in order of increasing p-values from top to bottom) in low copy number hits.</p