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

   https://doi.org/10.1093/genetics/iyab039  

   Broz, Amanda K ; Waneka, Gus ; Wu, Zhiqiang ; Fernandes Gyorfy, Matheus ; Sloan, Daniel B ( March 2021 , Genetics)

   Sekelsky, J (Ed.)

   Abstract 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. 

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2. Using the Microbiome Amplification Preference Tool (MAPT) to Reveal Medicago sativa -Associated Eukaryotic Microbes

   https://doi.org/10.1094/PBIOMES-02-20-0022-R  

   Moccia, Katherine ; Papoulis, Spiridon ; Willems, Andrew ; Marion, Zachary ; Fordyce, James A. ; Lebeis, Sarah L. ( January 2020 , Phytobiomes Journal)

   null (Ed.)

   Although our understanding of the microbial diversity found within a given system expands as amplicon sequencing improves, technical aspects still drastically affect which members can be detected. Compared with prokaryotic members, the eukaryotic microorganisms associated with a host are understudied due to their underrepresentation in ribosomal databases, lower abundance compared with bacterial sequences, and higher ribosomal gene identity to their eukaryotic host. Peptide nucleic acid (PNA) blockers are often designed to reduce amplification of host DNA. Here we present a tool for PNA design called the Microbiome Amplification Preference Tool (MAPT). We examine the effectiveness of a PNA designed to block genomic Medicago sativa DNA (gPNA) compared with unrelated surrounding plants from the same location. We applied mitochondrial PNA and plastid PNA to block the majority of DNA from plant mitochondria and plastid 16S ribosomal RNA genes, as well as the novel gPNA. Until now, amplifying both eukaryotic and prokaryotic reads using 515F-Y and 926R has not been applied to a host. We investigate the efficacy of this gPNA using three approaches: (i) in silico prediction of blocking potential in MAPT, (ii) amplicon sequencing with and without the addition of PNAs, and (iii) comparison with cultured fungal representatives. When gPNA is added during amplicon library preparation, the diversity of unique eukaryotic amplicon sequence variants present in M. sativa increases. We provide a layered examination of the costs and benefits of using PNAs during sequencing. The application of MAPT enables scientists to design PNAs specifically to enable capturing greater diversity in their system. 

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

   https://doi.org/10.1093/genetics/iyab116  

   Waneka, Gus ; Svendsen, Joshua M ; Havird, Justin C ; Sloan, Daniel B ( August 2021 , Genetics)

   Surtees, J A (Ed.)

   Abstract 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. 

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4. Mutation rate and spectrum in obligately outcrossing Caenorhabditis elegans mutation accumulation lines subjected to RNAi-induced knockdown of the mismatch repair gene msh-2

   https://doi.org/10.1093/g3journal/jkab364  

   Katju, Vaishali ; Konrad, Anke ; Deiss, Thaddeus C. ; Bergthorsson, Ulfar ; Félix, ed., M. -A ( October 2021 , G3 Genes|Genomes|Genetics)

   DNA mismatch repair (MMR), an evolutionarily conserved repair pathway shared by prokaryotic and eukaryotic species alike, influences molecular evolution by detecting and correcting mismatches, thereby protecting genetic fidelity, reducing the mutational load, and preventing lethality. Herein we conduct the first genome-wide evaluation of the alterations to the mutation rate and spectrum under impaired activity of the MutSα homolog, msh-2, in Caenorhabditis elegans male–female fog-2(lf) lines. We performed mutation accumulation (MA) under RNAi-induced knockdown of msh-2 for up to 50 generations, followed by next-generation sequencing of 19 MA lines and the ancestral control. msh-2 impairment in the male–female background substantially increased the frequency of nuclear base substitutions (∼23×) and small indels (∼328×) relative to wildtype hermaphrodites. However, we observed no increase in the mutation rates of mtDNA, and copy-number changes of single-copy genes. There was a marked increase in copy-number variation of rDNA genes under MMR impairment. In C. elegans, msh-2 repairs transitions more efficiently than transversions and increases the AT mutational bias relative to wildtype. The local sequence context, including sequence complexity, G + C-content, and flanking bases influenced the mutation rate. The X chromosome exhibited lower substitution and higher indel rates than autosomes, which can either result from sex-specific mutation rates or a nonrandom distribution of mutable sites between chromosomes. Provided the observed difference in mutational pattern is mostly due to MMR impairment, our results indicate that the specificity of MMR varies between taxa, and is more efficient in detecting and repairing small indels in eukaryotes relative to prokaryotes.

    

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5. The decoupled evolution of the organellar genomes of Silene nutans leads to distinct roles in the speciation process

   https://doi.org/10.1111/nph.18966  

   Postel, Zoé ; Sloan, Daniel B. ; Gallina, Sophie ; Godé, Cécile ; Schmitt, Eric ; Mangenot, Sophie ; Drouard, Laurence ; Varré, Jean‐Stéphane ; Touzet, Pascal ( July 2023 , New Phytologist)

   There is growing evidence that cytonuclear incompatibilities (i.e. disruption of cytonuclear coadaptation) might contribute to the speciation process. In a former study, we described the possible involvement of plastid–nuclear incompatibilities in the reproductive isolation between four lineages ofSilene nutans(Caryophyllaceae). Because organellar genomes are usually cotransmitted, we assessed whether the mitochondrial genome could also be involved in the speciation process, knowing that the gynodioecious breeding system ofS. nutansis expected to impact the evolutionary dynamics of this genome.

   Using hybrid capture and high‐throughput DNA sequencing, we analyzed diversity patterns in the genic content of the organellar genomes in the fourS. nutanslineages.

   Contrary to the plastid genome, which exhibited a large number of fixed substitutions between lineages, extensive sharing of polymorphisms between lineages was found in the mitochondrial genome. In addition, numerous recombination‐like events were detected in the mitochondrial genome, loosening the linkage disequilibrium between the organellar genomes and leading to decoupled evolution.

   These results suggest that gynodioecy shaped mitochondrial diversity through balancing selection, maintaining ancestral polymorphism and, thus, limiting the involvement of the mitochondrial genome in evolution of hybrid inviability betweenS. nutanslineages.

    

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