Disruption of recombination machinery alters the mutational landscape in plant organellar genomes.

Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targeted mutS homolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats in Arabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes of A. thaliana mutant lines lacking radA, recA1, or recA3. In contrast, osb2 and why2 mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data from A. thaliana organellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants and msh1 mutants. Finally, using long-read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.

UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with anti-damage antibodies and sequenced (XR-seq) to produce high resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.

Investigating low frequency somatic mutations in Arabidopsis with Duplex Sequencing.

Mutations are the source of novel genetic diversity but can also lead to disease and maladaptation. The conventional view is that mutations occur randomly with respect to their environment-specific fitness consequences. However, intragenomic mutation rates can vary dramatically due to transcription coupled repair and based on local epigenomic modifications, which are non-uniformly distributed across genomes. One sequence feature associated with decreased mutation is higher expression level, which can vary depending on environmental cues. To understand whether the association between expression level and mutation rate creates a systematic relationship with environment-specific fitness effects, we perturbed expression through a heat treatment in Arabidopsis thaliana. We quantified gene expression to identify differentially expressed genes, which we then targeted for mutation detection using Duplex Sequencing. This approach provided a highly accurate measurement of the frequency of rare somatic mutations in vegetative plant tissues, which has been a recent source of uncertainty in plant mutation research. We included mutant lines lacking mismatch repair (MMR) and base excision repair (BER) capabilities to understand how repair mechanisms may drive biased mutation accumulation. We found wild type (WT) and BER mutant mutation frequencies to be very low (mean variant frequency 1.8×10-8 and 2.6×10-8, respectively), while MMR mutant frequencies were significantly elevated (1.13×10-6). These results show that somatic variant frequencies are extremely low in WT plants, indicating that larger datasets will be needed to address the fundamental evolutionary question as to whether environmental change leads to gene-specific changes in mutation rate.

UV damage induces production of mitochondrial DNA fragments with specific length profiles.

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). In eukaryotic cells, photodamage and other bulky lesions occurring in nuclear genomes (nucDNAs) can be repaired through nucleotide excision repair (NER), where dual incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current views hold that the only way to eliminate bulky DNA damage in mtDNAs is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with anti-damage antibodies and sequenced (XR-seq) to produce high resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in 2-nt (S. cerevisiae) or 4-nt (A. thaliana) intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. We hypothesize that these fragments may reflect the outcome of a previously uncharacterized mechanism of NER-like repair in mitochondria or a programmed mtDNA degradation pathway.

Chromosome-Level Genome Assembly for the Angiosperm Silene conica.

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on Pacific Bioscience HiFi, Hi-C, and Bionano technologies. The assembly produced 10 scaffolds (1 per chromosome) with a total length of 862 Mb and only ∼1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genus's ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes.

Chromosome-level genome assembly for the angiosperm Silene conica.

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on PacBio HiFi, Hi-C and Bionano technologies. The assembly produced 10 scaffolds (one per chromosome) with a total length of 862 Mb and only ~1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genus's ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes. Significance: Whole-genome sequences have been largely lacking for species in the genus Silene even though these flowering plants have been used for studying ecology, evolution, and genetics for over a century. Here, we address this gap by providing a high-quality nuclear genome assembly for S. conica, a species known to have greatly accelerated rates of sequence and structural divergence in its mitochondrial and plastid genomes. This resource will be valuable in understanding the coevolutionary interactions between nuclear and cytoplasmic genomes and in comparative analyses across this highly diverse genus.

Complete Sequence of a 641-kb Insertion of Mitochondrial DNA in the Arabidopsis thaliana Nuclear Genome.

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 data sets 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.

Mitochondrial mutations in Caenorhabditis elegans show signatures of oxidative damage and an AT-bias.

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. Furthermore, 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.

Detecting de novo mitochondrial mutations in angiosperms with highly divergent evolutionary rates.

Although plant mitochondrial genomes typically show low rates of sequence evolution, levels of divergence in certain angiosperm lineages suggest anomalously high mitochondrial mutation rates. However, de novo mutations have never been directly analyzed in such lineages. Recent advances in high-fidelity DNA sequencing technologies have enabled detection of mitochondrial mutations when still present at low heteroplasmic frequencies. To date, these approaches have only been performed on a single plant species (Arabidopsis thaliana). Here, we apply a high-fidelity technique (Duplex Sequencing) to multiple angiosperms from the genus Silene, which exhibits extreme heterogeneity in rates of mitochondrial sequence evolution among close relatives. Consistent with phylogenetic evidence, we found that Silene latifolia maintains low mitochondrial variant frequencies that are comparable with previous measurements in Arabidopsis. Silene noctiflora also exhibited low variant frequencies despite high levels of historical sequence divergence, which supports other lines of evidence that this species has reverted to lower mitochondrial mutation rates after a past episode of acceleration. In contrast, S. conica showed much higher variant frequencies in mitochondrial (but not in plastid) DNA, consistent with an ongoing bout of elevated mitochondrial mutation rates. Moreover, we found an altered mutational spectrum in S. conica heavily biased towards AT→GC transitions. We also observed an unusually low number of mitochondrial genome copies per cell in S. conica, potentially pointing to reduced opportunities for homologous recombination to accurately repair mismatches in this species. Overall, these results suggest that historical fluctuations in mutation rates are driving extreme variation in rates of plant mitochondrial sequence evolution.

Mutational Pressure Drives Differential Genome Conservation in Two Bacterial Endosymbionts of Sap-Feeding Insects.

Compared with free-living bacteria, endosymbionts of sap-feeding insects have tiny and rapidly evolving genomes. Increased genetic drift, high mutation rates, and relaxed selection associated with host control of key cellular functions all likely contribute to genome decay. Phylogenetic comparisons have revealed massive variation in endosymbiont evolutionary rate, but such methods make it difficult to partition the effects of mutation versus selection. For example, the ancestor of Auchenorrhynchan insects contained two obligate endosymbionts, Sulcia and a betaproteobacterium (BetaSymb; called Nasuia in leafhoppers) that exhibit divergent rates of sequence evolution and different propensities for loss and replacement in the ensuing ∼300 Ma. Here, we use the auchenorrhynchan leafhopper Macrosteles sp. nr. severini, which retains both of the ancestral endosymbionts, to test the hypothesis that differences in evolutionary rate are driven by differential mutagenesis. We used a high-fidelity technique known as duplex sequencing to measure and compare low-frequency variants in each endosymbiont. Our direct detection of de novode novo mutations reveals that the rapidly evolving endosymbiont (Nasuia) has a much higher frequency of single-nucleotide variants than the more stable endosymbiont (Sulcia) and a mutation spectrum that is potentially even more AT-biased than implied by the 83.1% AT content of its genome. We show that indels are common in both endosymbionts but differ substantially in length and distribution around repetitive regions. Our results suggest that differences in long-term rates of sequence evolution in Sulcia versus BetaSymb, and perhaps the contrasting degrees of stability of their relationships with the host, are driven by differences in mutagenesis.

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes.

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, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by 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 disrupting MSH1 (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 in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch 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. MSH1 has 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.

The Tempo and Mode of Angiosperm Mitochondrial Genome Divergence Inferred from Intraspecific Variation in Arabidopsis thaliana.

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.