The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.chromosomal instability ͉ mitochondrion ͉ mutation rate ͉ mutational spectrum ͉ Saccharomyces cerevisiae D espite its relevance to every aspect of genetics and evolution, our understanding of the mutation process and its bearing on organismal fitness remains quite limited (1-4). Owing to the technical difficulties in directly observing very low-frequency events, most estimates of the per-nucleotide mutation rate are derived either from surveys of visible mutations at reporter loci (to enhance the detectability of mutations) or from nucleotide-sequence comparisons of silent sites in distantly related species (to magnify the accumulation of mutations). Neither approach is without problems, the first requiring assumptions about the fraction of mutations with observable phenotypic effects and the second relying on assumptions about interspecific divergence times, generation lengths, and neutrality of the monitored nucleotide sites.Long-term mutation-accumulation (MA) experiments, whereby replicate lines are taken through regular bottlenecks to minimize the efficiency of selection, have proven to be highly valuable resources for procuring spontaneous mutations in an essentially unbiased fashion (5-8). However, brute-force sequencing of PCR-amplified products constrains the number of mutations that can be detected in a reasonable amount of time. Here, we demonstrate the feasibility of whole-genome sequencing as a means to assay the complete spectrum of mutational effects in a moderately sized eukaryotic genome.Our analyses are based on an examination of parallel MA lines of a key model system, the yeast Saccharomyces cerevisiae. The initially isogenic lines were passed through 200 single-cell bottlenecks on a 3-to 4-day cycle of clonal growth for a total of Ϸ4,800 cell divisions per line [see supporting information (SI) Text]. Although there is some opportunity for the selective elimination of deleterious mutations during daily clonal amplification, this effect is quite small under the imposed bottlenecking procedure. For mutations with a relative selective disadvantage of s ϭ ...

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10−8 per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.