Mutation and recombination are the major sources of genetic diversity in all organisms. In the baker’s yeast, all mutation rate estimates are in homozygous background. We determined the extent of genetic change through mutation and loss of heterozygosity (LOH) in a heterozygous Saccharomyces cerevisiae genome during successive vegetative and meiotic divisions. We measured genome-wide LOH and base mutation rates during vegetative and meiotic divisions in a hybrid (S288c/YJM789) S. cerevisiae strain. The S288c/YJM789 hybrid showed nearly complete reduction in heterozygosity within 31 generations of meioses and improved spore viability. LOH in the meiotic lines was driven primarily by the mating of spores within the tetrad. The S288c/YJM789 hybrid lines propagated vegetatively for the same duration as the meiotic lines, showed variable LOH (from 2 to 3% and up to 35%). Two of the vegetative lines with extensive LOH showed frequent and large internal LOH tracts that suggest a high frequency of recombination repair. These results suggest significant LOH can occur in the S288c/YJM789 hybrid during vegetative propagation presumably due to return to growth events. The average base substitution rates for the vegetative lines (1.82 × 10−10 per base per division) and the meiotic lines (1.22 × 10−10 per base per division) are the first genome-wide mutation rate estimates for a hybrid yeast. This study therefore provides a novel context for the analysis of mutation rates (especially in the context of detecting LOH during vegetative divisions), compared to previous mutation accumulation studies in yeast that used homozygous backgrounds.

Meiotic crossover frequencies show wide variation among organisms. But most organisms maintain at least one crossover per homolog pair (obligate crossover). In Saccharomyces cerevisiae, previous studies have shown crossover frequencies are reduced in the mismatch repair related mutant mlh3D and enhanced in a meiotic checkpoint mutant pch2D by up to twofold at specific chromosomal loci, but both mutants maintain high spore viability. We analyzed meiotic recombination events genome-wide in mlh3D, pch2D, and mlh3D pch2D mutants to test the effect of variation in crossover frequency on obligate crossovers. mlh3D showed $30% genome-wide reduction in crossovers (64 crossovers per meiosis) and loss of the obligate crossover, but nonexchange chromosomes were efficiently segregated. pch2D showed $50% genome-wide increase in crossover frequency (137 crossovers per meiosis), elevated noncrossovers as well as loss of chromosome size dependent double-strand break formation. Meiotic defects associated with pch2Δ did not cause significant increase in nonexchange chromosome frequency. Crossovers were restored to wild-type frequency in the double mutant mlh3D pch2D (100 crossovers per meiosis), but obligate crossovers were compromised. Genetic interference was reduced in mlh3D, pch2D, and mlh3D pch2D. Triple mutant analysis of mlh3D pch2D with other resolvase mutants showed that most of the crossovers in mlh3D pch2D are made through the Mus81-Mms4 pathway. These results are consistent with a requirement for increased crossover frequencies in the absence of genetic interference for obligate crossovers. In conclusion, these data suggest crossover frequencies and the strength of genetic interference in an organism are mutually optimized to ensure obligate crossovers. Meiosis is a reductional division that produces haploid gametes or spores from diploid progenitor cells. Ploidy reduction is achieved by one round of DNA replication, followed by two consecutive nuclear divisions (Meiosis I and II), producing four daughter cells (Roeder 1997). Crossovers promote the formation of chiasma which serves as a physical linkage between two homologs and opposes the spindle generated forces that pull apart the homolog pairs. This opposing set of forces provides the tension necessary to promote proper disjunction of homolog pairs at Meiosis I (Petronczki et al. 2003). Failure to maintain at least one crossover per homolog pair increases the probability of nondisjunction, resulting in aneuploid gametes (Serrentino and Borde 2012). Although crossovers are important for chromosome segregation, nonexchange chromosomes have been observed to segregate accurately forming viable gametes (Hawley et al. 1992;Davis and Smith 2003;Kemp et al. 2004;Newnham et al. 2010;Krishnaprasad et al. 2015). Meiotic crossovers (and noncrossovers) are initiated in S. cerevisiae by 150-170 programmed double-strand breaks (DSBs) formed by an evolutionarily conserved topoisomerase-like protein Spo11 and other

The Saccharomyces cerevisiae strain JAY270/PE2 is a highly efficient biocatalyst used in the production of bioethanol from sugarcane feedstock. This strain is heterothallic and diploid, and its genome is characterized by abundant structural and nucleotide polymorphisms between homologous chromosomes. One of the reasons it is favored by many distilleries is that its cells do not normally aggregate, a trait that facilitates cell recycling during batch-fed fermentations. However, long-term propagation makes the yeast population vulnerable to the effects of genomic instability, which may trigger the appearance of undesirable phenotypes such as cellular aggregation. In pure cultures of JAY270, we identified the recurrent appearance of mutants displaying a mother-daughter cell separation defect resulting in rough colonies in agar media and fast sedimentation in liquid culture. We investigated the genetic basis of the colony morphology phenotype and found that JAY270 is heterozygous for a frameshift mutation in the ACE2 gene (ACE2/ace2-A7), which encodes a transcriptional regulator of mother-daughter cell separation. All spontaneous rough colony JAY270-derived isolates analyzed carried copy-neutral loss-of-heterozygosity (LOH) at the region of chromosome XII where ACE2 is located (ace2-A7/ace2-A7). We specifically measured LOH rates at the ACE2 locus, and at three additional chromosomal regions in JAY270 and in a conventional homozygous diploid laboratory strain. This direct comparison showed that LOH rates at all sites were quite similar between the two strain backgrounds. In this case study of genomic instability in an industrial strain, we showed that the JAY270 genome is dynamic and that structural changes to its chromosomes can lead to new phenotypes. However, our analysis also indicated that the inherent level of genomic instability in this industrial strain is normal relative to a laboratory strain. Our work provides an important frame of reference to contextualize the interpretation of instability processes observed in the complex genomes of industrial yeast strains.

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional nonindependent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole-genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14- to 150-fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation.

In the baker’s yeast Saccharomyces cerevisiae, most of the meiotic crossovers are generated through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex. To understand the role of Msh4-Msh5 in meiotic crossing over, we determined its genome wide in vivo binding sites in meiotic cells. We show that Msh5 specifically associates with DSB hotspots, chromosome axes, and centromeres on chromosomes. A basal level of Msh5 association with these chromosomal features is observed even in the absence of DSB formation (spo11Δ mutant) at the early stages of meiosis. But efficient binding to DSB hotspots and chromosome axes requires DSB formation and resection and is enhanced by double Holliday junction structures. Msh5 binding is also correlated to DSB frequency and enhanced on small chromosomes with higher DSB and crossover density. The axis protein Red1 is required for Msh5 association with the chromosome axes and DSB hotspots but not centromeres. Although binding sites of Msh5 and other pro-crossover factors like Zip3 show extensive overlap, Msh5 associates with centromeres independent of Zip3. These results on Msh5 localization in wild type and meiotic mutants have implications for how Msh4-Msh5 works with other pro-crossover factors to ensure crossover formation.