The baker's yeast diploid genome is remarkably stable in vegetative growth and meiosis

Abstract

Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1-2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.

Figure 1. Outline of vegetative and meiotic bottlenecks.

EAY2531 (relevant genotype MATa/MATalpha, HO/HO) was struck to single cells and then grown for 20 generations on YPD media to form single colonies. 20 such independent colonies were split into pairs of vegetative and meiotic mutation accumulation lines (one representative line shown for each). For the vegetative lines, a colony for each line was struck to single cells. This process was repeated 87 times to achieve ∼1740 generations of growth. At the end of generation 1740, a colony for each of the 20 independent lines was sporulated, and four haploid spores derived from each line were germinated and grown on YPD media to isolate chromosomal DNA for whole-genome sequencing. The 20 starting independent colonies of EAY2531 described above were also sporulated. One tetrad from each line was isolated and then germinated on YPD media and grown for 20 generations to form a colony. Each colony contained almost exclusively diploid cells as the result of intra-spore (shown here) and self-mating. For each line, the colony was then sporulated and the bottleneck was repeated 50 times. This yielded lines that were maintained for ∼1,000 vegetative generations, with one round of meiosis every 20 vegetative generations.

Figure 2. Simulation to estimate the upper limit for the meiotic mutation rate.

The histograms show the distribution of the final number of homozygous (white) and heterozygous (grey) mutations occurred in 10,000 independent simulated lines after 1,000 mitotic divisions and 50 meiotic bottlenecks in each line. The putative meiotic mutation rate (μ) used for each of the simulation is shown relative to the mitotic mutation rate (m). The red vertical lines show the average number of SNPs (all homozygous) observed in the T-50 lines. The P-value denotes the frequency of simulations with equal or lower number of SNPs than the observed value. Panel A and B show simulations in which meiotic mutations were set to occur before DNA replication and therefore are present in two chromatids. Panels C and D show simulations in which meiotic mutations were set to occur during or after DNA replication and are therefore present in one single chromatid. See Material and Methods and Figure S1 for further details on the simulations.

Figure 3. Physical analysis of chromosomes in vegetative and meiotic lines.

A) High resolution PFGE of full length chromosomal DNA stained with ethidium bromide. The corresponding chromosome numbers for the parental strain are shown to the left, and the positions of BioRad S. cerevisiae CHEF size markers are indicated to the right (marker lane was cropped out for clarity). B) Southern blot of the PFGE in A using the Y′ sequence as probe. C) Southern blot of MluI digested genomic DNA separated in PFGE and probed with the same Y′ probe as in B. The positions of BioRad lambda CHEF size markers and NEB lambda mono-cut size markers are indicated to the right (marker lanes were cropped out for clarity).

Figure 4. Mating patterns in S. cerevisiae tetrads.

A) kanMX and natMX drug markers were inserted in the same site in ARS314, located between PHO87 and BUD5, 1.5 KB proximal to MAT. The insertions do not disrupt either of the two genes. B) Outcomes from inter-spore and self-mating. MATa/MATalpha diploids that showed resistance to both antibiotics were categorized as resulting from inter-spore mating; those that showed resistance to only one antibiotic were categorized as resulting from a self-mating.