karyotypes, we qualitatively divided the 27 aneuploid strains into three classes (Figure 2A): (i) ‘stable’ (S) strains (n = 8), referring to those in which karyotype changes were not observed during our experiments (e.g. strain 245 at g30, Figure 2B); (ii) ‘mildly unstable’ (MU) strains (n = 10), in which only one chromosome copy number change event (involving either a single or multiple chromosomes) was observed at later generations (e.g. strain 226 at g30 in Figure 2C); and (iii) ‘highly unstable’ (HU) strains (n = 9) in which more than one CIN event was observed at early generations (e.g. 225 at g20 in Figure 2D). Interestingly, strains belonging to each of the three CIN categories displayed a wide range of apparent growth rates, estimated by regressing the absolute cell counts measured at the three different time points (g20, g25, g30; Figure 3A). Even though S strains exhibited a slightly higher average growth rate than HU strains, this difference was not statistically significant (P = 0.498, Figure 3A). This observation is consistent with our previous finding that stable aneuploid strains exhibit a wide range of growth abilities [15] and a recent report showing a lack of correlation between cell cycle delay and yeast artificial chromosome loss rate in yeast disomic strains [23]. Thus, it is unlikely that the observed CIN in aneuploid strains is a consequence of aneuploidy-associated fitness impairment under standard laboratory growth conditions. Evidence of a genome-level determinant of CIN in aneuploid strains To ask if the aneuploidy-associated CIN might be a conse- quence of certain global karyotypic features, we examined the correlation between CIN and parameters such as the total number of chromosomes or base pairs in the genome, or the total number of chromosomes or base pairs in aneuploidy, etc. This analysis led to two observations. First, S strains tended to have a ploidy lower than 1.5, whereas MU or HU strains tend to have a ploidy around 1.5 or higher. Compared to HU strains, S strains showed a significantly lower base pair content (P = 3.1561022), a signifi- cantly smaller number of total chromosomes in the genome (P = 2.0761022) and a significantly lower basal ploidy (P = 8.5261023) (Figure 3B, 3C and 3D). Because the analyzed aneuploid strains had ploidy between 1N and 2N, these correlations suggest that haploid genomes with a few extra chromosomes tend to be more stable than diploids missing a few chromosomes. Second, CIN did not correlate with the number of aneuploid chromosomes: S and HU strains were significantly different neither in the total number of chromosomes in aneuploidy (P = 0.231) nor in the number of megabases in aneuploid chromosomes (P = 0.907) (Figure 3E and 3F). For this analysis, we defined basal ploidy as the integer number corresponding to the most frequently-appearing chromosome copy number in an aneuploid genome and aneuploid chromo- somes as those with a copy number deviating from the basal ploidy (be it gains or losses). These observations suggest that the genome does not necessarily become more unstable as it departs further from the euploid state, however, there is a genome-level impact on CIN related to the degree of departure of the aneuploid chromosome number from the lower euploid state (in this case, the true haploid state). Chromosome-specific determinants of CIN We next examined the correlation between CIN and the presence of specific chromosomes in aneuploidy using the karyotyping data of the 27 strains characterized. For this analysis we focused on 21 strains for which aneuploid chromosomes could be assigned unambiguously based on basal ploidy assignment as explained above, but excluded 6 strains that had eight chromo- Karyotypic Determinants of Chromosome Instability somes with a copy number of 1 and eight chromosomes with a copy number of 2 (thus impossible to assign which chromosomes are in euploidy and which in aneuploidy). As expected, ChrVI aneuploidy was rarely found across the 21 aneuploid strains (only a single strain with ChrVI monosomy and a basal ploidy of 2N), consistent with previous reports of low tolerance of copy number imbalances of this particular chromosome, most probably due to the presence of several major cytoskeletal genes (e.g. ACT1, TUB2) on this chromosome [23,24,25]. We calculated the frequency at which each chromosome was present in aneuploidy across the 21 strains and searched for over- or under-representation across the three different classes of CIN (S, MU and HU) (Figure 4). In general, the frequency at which each of the 16 chromosomes was found in aneuploidy was not uniformly distributed across the three different classes of CIN (Fisher test P = 2.7561022). In particular, ChrVII aneuploidy was significantly associated with S strains (Fisher test P = 4.8161022) and ChrV aneuploidy was significantly associated with HU strains (Fisher test P = 2.0361022). To gain molecular insights into the chromosome features associated with different levels of CIN, we analyzed enrichment of genes potentially linked to CIN on ChrV and ChrVII in comparison to other chromosomes. We used several published datasets obtained from different types of screens for chromosome instability genes, such as ‘‘genes causing increased colony sectoring when deleted’’ (source: Saccharomyces Genome Database, SGD), ‘‘genes causing increased colony sectoring when overexpressed’’ (SGD) or ‘‘genes causing decreased chromosome/plasmid main- tenance when deleted’’ (SGD), as well as more comprehensive datasets such as ‘‘genes associated with chromosome instability’’ [26] or genes annotated with gene ontology (GO) term ‘‘chromo- some segregation’’. As shown in Figure S8, ChrV and ChrVII do not show exceptional or consistent over or under-representation of genes in any of these datasets. A lack of clear insights from the above analysis based on individual aneuploid chromosomes led us to consider the possibility that it is the relative dosage of two or multiple chromosomes rather than any particular aneuploid chromosome per se that affects karyotype stability. We thus performed a systematic analysis of all pair-wise combinations of the 16 chromosomes to determine if any imbalanced pairs (copy number ratio to be either 0.5 or 2, but not 1) were significantly associated with CIN. Using a hypergeometric test between two groups, relatively stable (S+MU) vs. highly unstable (HU), the imbalance between three different pairs of chromosomes were observed to distinguish the two CIN groups: ChrII vs. ChrVIII, ChrIII vs. ChrIX, and ChrVII vs. ChrX (Figure 5A, right panel). The last pair, ChrVII vs. ChrX (Hypergeometric test P,0.02), was surprising as the single chromosome analysis found ChrVII aneuploidy to be associated exclusively with karytoypically stable strains (Figure 4). A closer scrutiny found that in 2 of the 3 cases where ChrVII was in aneuploidy (strains 220 and 230, having 1 extra ChrVII with 1N basal ploidy), ChrX was also in aneuploidy with 1 extra copy, and thus their numbers were balanced. On the other hand, of the 8 aneuploid strains where ChrVII and ChrX had unequal copy numbers, 5 were highly unstable, 1 mildly unstable, and only 2 stable. These findings suggest that the effect of an individual aneuploid chromosome on CIN is dependent on the karyotypic context in which the aneuploid chromosome is present. We note that if the hypergenometric test was performed between S vs (MU+HU) groups, different pairs of chromosomes were observed whose imbalance distinguished the two groups (Figure 5A left panel). This observation suggests that there are potentially many different pairs of chromosomes whose copy number imbalance could lead to CIN. PLoS Genetics | www.plosgenetics.org 5 May 2012 | Volume 8 | Issue 5 | e1002719