Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA,POLIB,MSH1,RECA3,UNG,FPG, andOGG1) for effects on mutation rates in the model angiospermArabidopsis thalianaby applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations even at low heteroplasmic frequencies. We find that disruptingMSH1(but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations inmsh1mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within themutSmismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses.MSH1has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

2Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence 3 evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct 4 mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested 5 seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, 6 POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model 7 angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex 8 sequencing) that can detect newly arisen mitochondrial and plastid mutations still at low 9 heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads 10 to massive increases in the frequency of point mutations and small indels and changes to the 11 mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show 12 transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a 13 contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within 14 the mutS mismatch repair family that we find is also present outside of green plants in multiple 15 eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain 16 bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant 17 cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in 18 plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates 19 perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous 20 recombination.It has been apparent for more than 30 years that rates of nucleotide substitution in plant 24 mitochondrial and plastid genomes are unusually low (1,2). In angiosperms, mitochondrial and 25 plastid genomes have synonymous substitution rates that are on average approximately 16-fold and 26 5-fold slower than the nucleus, respectively (3). The fact that these low rates are evident even at 27 sites that are subject to relatively small amounts of purifying selection (4, 5) suggests that they are 28 the result of very low underlying mutation rates -a surprising observation especially when 29 contrasted with the rapid accumulation of mitochondrial mutations in many eukaryotic lineages (6,7). 30Although the genetic mechanisms that enable plants to achieve such faithful replication and 31 transmission of cytoplasmic DNA sequences have not been determined, a number of hypotheses 32 can be envisioned. One possibility is that the DNA polymerases responsible for replicating 33 mitochondrial and plastid DNA (8) might have unusually high fidelity. However, in vitro assays with 34 the two partially redundant bacterial-like organellar DNA polymerases in Arabidopsis thaliana, PolIA 35(At1g50840) and PolIB (At3g20540), have indicated that they are highly error-prone (9), with 36 misincorpo...

Intracellular transfers of mitochondrial DNA continue to shape nuclear genomes. Chromosome 2 of the model plant Arabidopsis thaliana contains one of the largest known nuclear insertions of mitochondrial DNA (numts). Estimated at over 600 kb in size, this numt is larger than the entire Arabidopsis mitochondrial genome. The primary Arabidopsis nuclear reference genome contains less than half of the numt because of its structural complexity and repetitiveness. Recent datasets generated with improved long-read sequencing technologies (PacBio HiFi) provide an opportunity to finally determine the accurate sequence and structure of this numt. We performed a de novo assembly using sequencing data from recent initiatives to span the Arabidopsis centromeres, producing a gap-free sequence of the Chromosome 2 numt, which is 641-kb in length and has 99.933% nucleotide sequence identity with the actual mitochondrial genome. The numt assembly is consistent with the repetitive structure previously predicted from fiber-based fluorescent in situ hybridization. Nanopore sequencing data indicate that the numt has high levels of cytosine methylation, helping to explain its biased spectrum of nucleotide sequence divergence and supporting previous inferences that it is transcriptionally inactive. The original numt insertion appears to have involved multiple mitochondrial DNA copies with alternative structures that subsequently underwent an additional duplication event within the nuclear genome. This work provides insights into numt evolution, addresses one of the last unresolved regions of the Arabidopsis reference genome, and represents a resource for distinguishing between highly similar numt and mitochondrial sequences in studies of transcription, epigenetic modifications, and de novo mutations. Significance statement Nuclear genomes are riddled with insertions of mitochondrial DNA. The model plant Arabidopsis has one of largest of these insertions ever identified, which at over 600-kb in size represents one of the last unresolved regions in the Arabidopsis genome more than 20 years after the insertion was first identified. This study reports the complete sequence of this region, providing insights into the origins and subsequent evolution of the mitochondrial DNA insertion and a resource for distinguishing between the actual mitochondrial genome and this nuclear copy in functional studies.

The mechanisms of sequence divergence in angiosperm mitochondrial genomes have long been enigmatic. In particular, it is difficult to reconcile the rapid divergence of intergenic regions that can make non-coding sequences almost unrecognizable even among close relatives with the unusually high levels of sequence conservation found in genic regions. It has been hypothesized that different mutation and repair mechanisms act on genic and intergenic sequences or alternatively that mutational input is relatively constant but that selection has strikingly different effects on these respective regions. To test these alternative possibilities, we analyzed mtDNA divergence within Arabidopsis thaliana, including variants from the 1001 Genomes Project and changes accrued in published mutation accumulation (MA) lines. We found that base-substitution frequencies are relatively similar for intergenic regions and synonymous sites in coding regions, whereas indel and nonsynonymous substitutions rates are greatly depressed in coding regions, supporting a conventional model in which mutation/repair mechanisms are consistent throughout the genome but differentially filtered by selection. Most types of sequence and structural changes were undetectable in 10-generation MA lines, but we found significant shifts in relative copy number across mtDNA regions for lines grown under stressed vs. benign conditions. We confirmed quantitative variation in copy number across the A. thaliana mitogenome using both whole-genome sequencing and droplet digital PCR, further undermining the classic but oversimplified model of a circular angiosperm mtDNA structure. Our results suggest that copy number variation is one of the most fluid features of angiosperm mitochondrial genomes.

Rapid mutation rates are typical of mitochondrial genomes (mtDNAs) in animals, but it is not clear why. The difficulty of obtaining measurements of mtDNA mutation that are not biased by natural selection has stymied efforts to distinguish between competing hypotheses about the causes of high mtDNA mutation rates. Several studies which have measured mtDNA mutations in nematodes have yielded small datasets with conflicting conclusions about the relative abundance of different substitution classes (i.e. the mutation spectrum). We therefore leveraged Duplex Sequencing, a high-fidelity DNA sequencing technique, to characterize de novo mtDNA mutations in Caenorhabditis elegans. This approach detected nearly an order of magnitude more mtDNA mutations than documented in any previous nematode mutation study. Despite an existing extreme AT bias in the C. elegans mtDNA (75.6% AT), we found that a significant majority of mutations increase genomic AT content. Compared to some prior studies in nematodes and other animals, the mutation spectrum reported here contains an abundance of CG→AT transversions, supporting the hypothesis that oxidative damage may be a driver of mtDNA mutations in nematodes. Further, we found an excess of G→T and C→T changes on the coding DNA strand relative to the template strand, consistent with increased exposure to oxidative damage. Analysis of the distribution of mutations across the mtDNA revealed significant variation among protein-coding genes and as well as among neighboring nucleotides. This high-resolution view of mitochondrial mutations in C. elegans highlights the value of this system for understanding relationships among oxidative damage, replication error, and mtDNA mutation.