We constructed a diploid (WS82, legend) homozygous for the rad52
mutation; the haploid strains used in the construction (WS30-3 and WS53) differed by >25,000 single-nucleotide polymorphisms (SNPs). Four independent isolates of this diploid were subcultured on plates from a single cell to a colony at least 18 times, representing ~450 cell divisions. Samples were taken for analysis from the strain before subculturing and after various numbers of subculturing events. DNA was isolated from each isolate, and the chromosome compositions were examined by comparative genome hybridization (CGH) microarrays. For two of the four isolates, we observed progressive chromosome loss, culminating in haploidization for two of these isolates (). For example, in WS82-1, although the starting strain was a normal diploid (), by the fifth subcloning (SC5), the isolate had lost chromosomes IV, V, X, XII, and XIII (). Continued subcloning resulted in further chromosome loss ().
Number of each homolog (I–XVI) per cell in two derivatives of the rad52/rad52 diploid WS82 (WS82-1 and WS82-2) that show progressive chromosome loss during subculturing
Figure 1 CGH microarray analysis of aneuploidy in the subcultured rad52/rad52 diploid strain WS82. To examine the effects of the rad52 mutation on chromosome loss, we subcultured independent isolates of WS82 18–22 times. Each subculturing involved growth (more ...)
By the 18th subcloning, WS82-1 had the same gene dosage for all 16 chromosomes (). This hybridization pattern, by itself, cannot distinguish between haploids and diploids. To determine whether the strain was a haploid or a diploid, we crossed WS82-1 from SC18 with a RAD52 MATa
haploid strain (EAS18). The resulting strain would be a diploid or a triploid, depending on whether the strain shown in was a haploid or a diploid, respectively. When induced to undergo meiosis, diploid strains have good spore viability (>80%) whereas triploids have poor spore viability (<50%) (St. Charles et al. 2010
). We found that the strain produced by the cross had excellent spore viability (143 viable spores of 160 total, or 89%), indicating that the subcultured derivative of WS82-1 shown in was a haploid rather than a diploid. Similarly, by the same criteria described above, WS82-2 underwent haploidization. The WS82-3 and WS82-4 isolates will be described further below.
In addition to detecting changes in gene dosage, oligonucleotide-containing microarrays can also be used to determine whether a diploid strain is heterozygous or homozygous for a SNP (Gresham et al. 2008
). We used SNP arrays to confirm haploidy in the subcultured derivatives of WS82-1 and WS82-2 and to determine whether the chromosomes were preferentially lost from one of the two haploid parental strains (WS30-3 and WS53). illustrates that genomic DNA isolated from subculture 0 of WS82-1 hybridized equally well to WS30-3-specific and WS53-specific oligonucleotides; although all chromosomes were examined, only the data for chromosome VII are shown in . In contrast, genomic DNA isolated from SC18 of WS82-1 (the presumptive haploid strain) preferentially hybridized to the WS30-3-specific oligonucleotides for chromosome VII () and to the WS53-specific oligonucleotides for chromosome XIV (). As shown in (SC18 for WS82-1 and SC22 for WS82-2), of 32 chromosome losses, 12 were losses of the WS30-3-derived chromosomes and 20 were losses of the WS53-derived chromosomes; this difference is not statistically significant. These results confirm that WS82-1 and WS82-2 are haploid strains and further show that, as expected, none of the retained chromosomes had undergone mitotic recombination.
Figure 2 Analysis of chromosome loss using SNP microarrays. WS82 was derived from a cross of the haploids WS30-3 (closely related to S288c, sequence in Saccharomyces Genome Database) and WS53 (closely related to YJM789, sequenced by Wei et al. 2007). Four 25-base (more ...)
In contrast to the progressive chromosome loss observed in WS82-1 and WS82-2, WS82-3 and WS82-4 underwent a different process. From the CGH analysis (samples labeled with “C” in ) by SC22, WS82-3 appeared to have lost 13 of 16 chromosomes (retaining two copies of III, VIII, and IX), and WS82-4 appeared to have lost one complete set of chromosomes by SC18. At SC5, by CGH arrays, WS82-4 had lost chromosomes VIII, X, and XIII. After SC10, however, genomic DNA isolated from WS82-4 had a pattern of hybridization by SNP arrays, indicating that it was trisomic for many chromosomes. For example, in , the pattern of hybridization at SC10 indicated that the strain had three copies of chromosome XI: two derived from the WS30-3 parent and one derived from the WS53 parent. Similarly, for WS82-3, by SC5, the SNP array indicated that most of the homologs were present in more than two copies (). The discrepancy between the number of chromosomes in these strains as determined by CGH and SNP microarrays reflects what is measured by the two different methods. The CGH analysis can detect only deviations in copy number from the average copy number of the experimental strain (see legend); although twofold differences are usually clear, smaller differences are not. In contrast, with the SNP arrays, the relative hybridization levels of the experimental strain for each homolog are measured independently (see legend). In this type of array, by examining the hybridization values to the SNP-specific oligonucleotides, it is simple to determine both copy number and whether the homologs are identical. Thus, for WS82-4 (SC10), it is clear that there is one copy of chromosome XI derived from WS53 because the normalized hybridization ratio is 1 and two copies of XI derived from WS30-3 because the normalized hybridization ratio is ~1.4. In summary, where there is a discrepancy between the number of chromosomes as determined with CGH and SNP arrays, the SNP arrays are more accurate. We point out that no discrepancies for the two types of arrays were observed for WS82-1 and WS82-2.
Number of each homolog (I–XVI) per cell in two derivatives of the rad52/rad52 diploid WS82 (WS82-3 and WS82-4) that underwent genome duplications during subculturing
There are two explanations of the apparent genome duplications observed in isolates WS82-3 and WS82-4. First, it is possible that, during subculturing within each of these isolates, two derivatives arose: one that had lost the MATa-containing copy of chromosome III and one that had lost the MATα-containing copy of III. Mating between these derivatives would result in a strain with two, three, or four copies of each homolog, consistent with the SNP array data. An alternative possibility is that, during subculturing, WS82-3 and WS82-4 undergo whole-genome duplication. We favor the second possibility for two reasons. First, in the strains observed immediately after the postulated genome duplication (SC5 for WS82-3 and SC10 for WS82-4), WS82-3 had two copies of both the MATa- and MATα-containing chromosomes, and WS82-4 had two copies of the MATα- and one copy of the MATa-containing chromosomes. If the diploidization reflected mating, we would expect that the resulting strain would have only two copies of chromosome III, one with each mating type. Second, we and others (J. McCusker, personal communication) have observed that haploid strains of the YJM789 genetic background spontaneously diploidize; consequently, as the WS82 diploid loses chromosomes derived from the other genetic background, the diploidization phenotype characteristic of the WS53/YJM789 haploid parent may emerge.
Although the rad52
mutation stimulates both chromosome loss and gain in the subcultured cells in our experiments, it is likely that the main effect at the cellular level is to increase the rate of chromosome loss, and the chromosome gain observed in two isolates reflects either mating or whole-genome duplication during subculturing. A strong argument that the chromosome gains and losses in rad52
strains are not a consequence of an elevated rate in nondisjunction is that the individual homologs in WS82-1 and WS82-2 become monosomic, rather than exhibiting a mixture of monosomic and trisomic chromosomes. It should also be pointed out that chromosome loss continued in the WS82-3 and WS82-4 isolates after mating/genome duplication. For example, the number of chromosomes in WS82-3 decreased from 44 at SC5 to 34 at SC22.