Repeat-mediated Recombination Research Articles

Repeat-mediated recombination results in Complex DNA structure of the mitochondrial genome of Trachelospermum jasminoides

BackgroundTrachelospermum jasminoides has medicinal and ornamental value and is widely distributed in China. Although the chloroplast genome has been documented, the mitochondrial genome has not yet been studied.ResultsThe mitochondrial genome of T. jasminoides was assembled and functionally annotated using Illumina and nanopore reads. The mitochondrial genome comprises a master circular molecular structure of 605,764 bp and encodes 65 genes: 39 protein-coding genes, 23 transfer RNA (tRNA) genes and 3 ribosomal RNA genes. In addition to the single circular conformation, we found many alternative conformations of the T. jasminoides mitochondrial genome mediated by 42 repetitive sequences. Six repetitive sequences (DRS01–DRS06) were supported by nanopore long reads, polymerase chain reaction (PCR) amplifications, and Sanger sequencing of the PCR products. Eleven homologous fragments were identified by comparing the mitochondrial and chloroplast genome sequences, including three complete tRNA genes. Moreover, 531 edited RNA sites were identified in the protein-coding sequences based on RNA sequencing data, with nad4 having the highest number of sites (54).ConclusionTo our knowledge, this is the first description of the mitochondrial genome of T. jasminoides. Our results demonstrate the existence of multiple conformations. These findings lay a foundation for understanding the genetics and evolutionary dynamics of Apocynaceae.

The de novo assembly and characterization of the complete mitochondrial genome of bottle gourd (Lagenaria siceraria) reveals the presence of homologous conformations produced by repeat-mediated recombination.

Bottle gourd is an annual herbaceous plant that not only has high nutritional value and many medicinal applications but is also used as a rootstock for the grafting of cucurbit crops such as watermelon, cucumber and melon. Organellar genomes provide valuable resources for genetic breeding. A hybrid strategy with Illumina and Oxford Nanopore Technology sequencing data was used to assemble bottle gourd mitochondrial and chloroplast genomes. The length of the bottle gourd mitochondrial genome was 357547 bp, and that of the chloroplast genome was 157121 bp. These genomes had 27 homologous fragments, accounting for 6.50% of the total length of the bottle gourd mitochondrial genome. In the mitochondrial genome, 101 simple sequence repeats (SSRs) and 10 tandem repeats were identified. Moreover, 1 pair of repeats was shown to mediate homologous recombination into 1 major conformation and 1 minor conformation. The existence of these conformations was verified via PCR amplification and Sanger sequencing. Evolutionary analysis revealed that the mitochondrial genome sequence of bottle gourd was highly conserved. Furthermore, collinearity analysis revealed many rearrangements between the homologous fragments of Cucurbita and its relatives. The Ka/Ks values for most genes were between 0.3~0.9, which means that most of the genes in the bottle gourd mitochondrial genome are under purifying selection. We also identified a total of 589 potential RNA editing sites on 38 mitochondrial protein-coding genes (PCGs) on the basis of long noncoding RNA (lncRNA)-seq data. The RNA editing sites of nad1-2, nad4L-2, atp6-718, atp9-223 and rps10-391 were successfully verified via PCR amplification and Sanger sequencing. In conclusion, we assembled and annotated bottle gourd mitochondrial and chloroplast genomes to provide a theoretical basis for similar organelle genomic studies.

Highly active repeat-mediated recombination in the mitogenome of the aquatic grass Hygroryza aristata

BackgroundFloating bamboo (Hygroryza aristata) is an endangered species with a narrow native distribution and is renowned for its unique aesthetic qualities, which holds significant ecological and ornamental value. However, the lack of genetic information research, with only one complete plastome available, significantly hampers conservation efforts and further research for this species.ResultsIn this research, we sequenced and assembled the organelle genomes of floating bamboo, including the mitogenome (587,847 bp) and plastome (135,675 bp). The mitogenome can recombine into various configurations, which are mediated by 25 repeat pairs (13 SRs, 6 MRs, 1 LR, and 5 CRs). LR1 and SR5 are particularly notable as they have the ability to combine with other contigs, forming complex repeat units that facilitate further homologous recombination. The rate of homologous recombination varies significantly among species, yet there is still a pronounced positive correlation observed between the length of these repeat pairs and the rate of recombination they mediate. The mitogenome integrates seven intact protein-coding genes from the chloroplast. The codon usage patterns in both organelles are similar, with a noticeable bias towards C and T on the third codon. The gene map of Poales shows the entire loss of rpl6, succinate dehydrogenase subunits (sdh3 and sdh4). Additionally, the BOP clade retained more variable genes compared to the PACMAD clade.ConclusionsWe provided a high-quality and well-annotated mitogenome for floating bamboo and demonstrated the presence of diverse configurations. Our study has revealed the correlation between repeat length and their corresponding recombination rate despite variations among species. Although the mitogenome can potentially exist in the form of a unicircular in vivo, this occurrence is rare and may not be stable.

Analysis of the Rhodomyrtus tomentosa mitochondrial genome: Insights into repeat-mediated recombination and intra-cellular DNA transfer

Plant mitochondrial genomes participate in encoding proteins crucial to the major producers of ATP in the cell and replication and heredity of their own DNA. The sequences and structure of the plant mitochondrial genomes profoundly impact these fundamental processes, and studies of plant mitochondrial genomes are needed. We reported the complete sequences of the Rhodomyrtus tomentosa mitochondrial genome here, totaling 400,482 bp. Nanopore ONT reads and PCR amplification provided evidence for recombination mediated by the eight repeat pairs for the R. tomentosa mitochondrial genome. Thirty-eight genes were identified in the R. tomentosa mitochondrial genome. Comparative analyses of the mitochondrial genome and plastome and PCR amplification suggest that five fragments of mitochondrial plastid DNA were unfunctional sequences resulting from intracellular gene transfer. Phylogenetic analysis based on each and all of the 27 mitochondrial protein-coding genes of nine Myrtales species revealed that R. tomentosa always clustered with other species of Myrtaceae. This study uncovered the enormous complexity of the R. tomentosa mitochondrial genome, the active repeat-mediated recombinations, the presence of mitochondrial plastid DNAs, and the topological incongruence of Myrtales among the single-gene trees.

Comprehensive analysis of the mitochondrial genome of Ziziphus mauritiana Lam.: Investigating sequence structure, repeat-mediated recombination, chloroplast-derived transfer, RNA editing, and evolution

Ziziphus mauritiana Lam. is a valuable semi-deciduous tropical fruit tree with economic, medicinal, nutritional, and environmental significance. While the chloroplast genome of Z. mauritiana has been studied, the decoding of its mitochondrial genome (mtGenome) is crucial for various research areas, including molecular phylogeny and functional genomics. In this study, we extracted high-quality genomic DNA from the green leaves of Z. mauritiana and assembled the mtGenome by integrating sequencing data from both long and short reads. The Z. mauritiana mtGenome spans 324,124 bp with a 45.43 % GC content and exhibits six distinct conformations alongside the main circular sequence due to three pairs of repetitive fragments that facilitate recombination. RNA editing events resulted in changes in the physicochemical properties of amino acids, with genes expressed at higher levels showing an increased frequency of editing sites. Chloroplast-to-mitochondria horizontal gene transfers occurred in Z. mauritiana; however, these transferred fragments had few mutations, posing a challenge in determining the exact timing of these transfers. Phylogenetic analysis highlights a slower evolution rate of mitochondrial genes compared to chloroplast genes. These insights greatly enhance our understanding of the dynamics of the Z. mauritiana mtGenome, including recombination, gene expression, evolutionary processes, and more. The data and results of this study will serve as valuable mtGenome data resources within the Rhamnaceae family, thereby facilitating further genomics-based studies on Z. mauritiana.

The mitochondrial genome of the holoparasitic plant Thonningia sanguinea provides insights into the evolution of multi-chromosomal structure.

Multi-chromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multi-chromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multi-chromosomal structure of 18 circular chromosomes of 8.7 - 19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome, however no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multi-chromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination and repeat degradation.

Characterisation of the complete mitochondrial genome of Taraxacum mongolicum revealed five repeat-mediated recombinations.

We reported the mitochondrial genome of Taraxacum mongolicum for the first time. Five pairs of repeats that can mediate recombination were validated, leading to multiple conformations of genome. Taraxacum mongolicum belongs to the Asteraceae family and has important pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled the complete mitochondrial genome (mitogenome) of T. mongolicum using Illumina and Oxford Nanopore sequencing data. This genome corresponded to a circular molecule 304,467bp long. It encodes 52 unique genes including 31 protein-coding, 3 ribosomal RNA (rRNA) and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, the existence of alternative conformations mediated by five repetitive sequences in the mitogenome was identified and validated. Recombination mediated by the inverted repeats resulted in two conformations. Conversely, recombination mediated by the two direct repeats broke one large circular molecule into two subgenomic circular molecules. Furthermore, we identified 12 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. Lastly, we identified a total of 278 RNA-editing sites in protein-coding sequences based on RNA-seq data. Among them, cox1 and nad5 gene has the most sites (21), followed by the nad2 gene with 19 sites. We successfully validated 213 predicted RNA-editing sites using PCR amplification and Sanger sequencing. This project reported the first mitogenome of T. mongolicum and demonstrated its multiple conformations generated by repeat-mediated recombination. This genome could provide critical information for the molecular breeding of T. mongolicum, and also be used as a reference genome for other species of the genus Taraxacum.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

Assembly of the Complete Mitochondrial Genome of Gelsemium elegans Revealed the Existence of Homologous Conformations Generated by a Repeat Mediated Recombination.

Gelsemium elegans (G. elegans) is a Chinese medicinal plant with substantial economic and feeding values. There is a lack of detailed studies on the mitochondrial genome of G. elegans. In this study, the mitochondrial genome of G. elegans was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of G. elegans is represented by two circular chromosomes of 406,009 bp in length with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected 145 pairs of repeats and found that four pairs of repeats could mediate the homologous recombination into one major conformation and five minor conformations, and the presence of conformations was verified by PCR amplification and Sanger sequencing. A total of 124 SSRs were identified in the G. elegans mitochondrial genome. The homologous segments between the chloroplast and mitochondrial genomes accounted for 5.85% of the mitochondrial genome. We also predicted 477 RNA potential editing sites and found that the nad4 gene was edited 38 times, which was the most frequent occurrence. Taken together, the mitochondrial genome of G. elegans was assembled and annotated. We gained a more comprehensive understanding on the genome of this medicinal plant, which is vital for its effective utilization and genetic improvement, especially for cytoplasmic male sterility breeding and evolution analysis in G. elegans.

Highly active repeat-mediated recombination in the mitogenome of the holoparasitic plant Aeginetia indica.

Mitogenomes of most flowering plants evolve slowly in sequence, but rapidly in structure. The rearrangements in structure are mainly caused by repeat-mediated recombination. However, patterns of repeat-mediated recombination vary substantially among plants, and to provide a comprehensive picture, characterization of repeat-mediated recombination should extend to more plant species, including parasitic plants with a distinct heterotrophic lifestyle. Here we assembled the mitogenome of the holoparasitic plant Aeginetia indica (Orobanchaceae) using Illumina sequencing reads. The mitogenome was assembled into a circular chromosome of 420,362 bp, 18,734 bp longer than that of another individual of A. indica which was assembled before as a linear molecule. Synteny analysis between the two mitogenomes revealed numerous rearrangements, unique regions of each individual and 0.2% sequence divergence in their syntenic regions. The A. indica mitogenome contains a gene content typical of flowering plants (33 protein-coding, 3 rRNA, and 17 tRNA genes). Repetitive sequences >30 bp in size totals 57,060 bp, representing 13.6% of the mitogenome. We examined recombination mediated by repeats >100 bp in size and found highly active recombination for all the repeats, including a very large repeat of ~16 kb. Recombination between these repeats can form much smaller subgenomic circular chromosomes, which may lead to rapid replication of mitochondrial DNA and thus be advantageous for A. indica with a parasitic lifestyle. In addition, unlike some other parasitic plants, A. indica shows no evidence for horizontal gene transfer of protein-coding genes in its mitogenome.

Long-read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsis msh1 mutants.

The abundant repeats in plant mitochondrial genomes can cause rapid genome rearrangements and are also a major obstacle in short-read sequencing studies. Nuclear-encoded proteins such as MSH1 are known to suppress the generation of repeat-associated mitochondrial genome variants, but our understanding of these mechanisms has been constrained by the limitations of short-read technologies. Here, we used highly accurate long-read sequencing (PacBio HiFi) to characterize mitochondrial and plastid genome variants in Arabidopsis thaliana msh1 mutant individuals. The HiFi reads provided a global view of recombination dynamics with detailed quantification of parental and crossover recombination products for both large and small repeats. We found that recombination breakpoints were distributed relatively evenly across the length of repeated sequences and detected widespread internal exchanges of sequence variants between pairs of imperfect repeats in the mitochondrial genome of msh1 mutants. Long-read assemblies of mitochondrial genomes from seven other A. thaliana wild-type accessions differed by repeat-mediated structural rearrangements similar to those observed in msh1 mutants, but they were all in a simple low-heteroplasmy state. The Arabidopsis plastid genome generally lacks small repeats and exhibited a very different pattern of variant accumulation in msh1 mutants compared with the mitochondrial genome. Our data illustrate the power of HiFi technology in studying repeat-mediated recombination in plant organellar genomes and improved the sequence resolution for recombinational processes suppressed by MSH1. Plant organellar genomes can undergo rapid rearrangements. Long-read sequencing provides a detailed and quantitative view of mitochondrial and plastid genome variants normally suppressed by MSH1, advancing our understanding of plant organellar genome dynamics.

De novo assembly of the complete mitochondrial genome of sweet potato (Ipomoea batatas [L.] Lam) revealed the existence of homologous conformations generated by the repeat-mediated recombination

Sweet potato (Ipomoea batatas [L.] Lam) is an important food crop, an excellent fodder crop, and a new type of industrial raw material crop. The lack of genomic resources could affect the process of industrialization of sweet potato. Few detailed reports have been completed on the mitochondrial genome of sweet potato. In this research, we sequenced and assembled the mitochondrial genome of sweet potato and investigated its substructure. The mitochondrial genome of sweet potato is 270,304 bp with 23 unique core genes and 12 variable genes. We detected 279 pairs of repeat sequences and found that three pairs of direct repeats could mediate the homologous recombination into four independent circular molecules. We identified 70 SSRs in the whole mitochondrial genome of sweet potato. The longest dispersed repeat in mitochondrial genome was a palindromic repeat with a length of 915 bp. The homologous fragments between the chloroplast and mitochondrial genome account for 7.35% of the mitochondrial genome. We also predicted 597 RNA editing sites and found that the rps3 gene was edited 54 times, which occurred most frequently. This study further demonstrates the existence of multiple conformations in sweet potato mitochondrial genomes and provides a theoretical basis for the evolution of higher plants and cytoplasmic male sterility breeding.

Assembly of the complete mitochondrial genome of an endemic plant, Scutellaria tsinyunensis, revealed the existence of two conformations generated by arepeat-mediated recombination.

We assembled the complete mitochondrial genome of Scutellaria tsinyunensis in this study. Repeat-mediated recombination resulted in the formation of two conformations of the mitochondrial genome in S. tsinyunensis. Scutellaria tsinyunensis belongs to the family Lamiaceae, distributed only in the Jinyun Mountain, Chongqing, China. As a valuable endemic and small population species, it is regarded as a natural resource potentially with significant economic and ecological importance. In this study, we assembled a complete and gap-free mitochondrial genome of S. tsinyunensis. This genome had a length of 354,073bp and the base composition of the genome was A (27.44%), T (27.30%), C (22.58%), and G (22.68%). This genome encodes 59 genes, including 32 protein-coding genes, 24 tRNA genes, and 3 rRNA genes. The Sanger sequencing and Oxford Nanopore sequencing confirmed a pair of direct repeats had mediated genome recombination, resulting in the formation of two conformations. The gene conversation between plastome and mitochondrial genome was also observed in S. tsinyunensis by detecting gene migration, including six tRNA genes (namely, trnW-CCA, trnI-CAU, trnH-UUU, trnD-GUC, trnN-GUU, and trnM-CAU), five protein-coding gene fragments, and the fragments from 2 rRNA genes. Moreover, the dN/dS analysis revealed the atp9 gene had undergone strong negative selection, and four genes (atp4, mttB, ccmFc, and ccmB) probably had undergone positive selection during evolution in Lamiales. This work reported the first mitochondrial genome of S. tsinyunensis, which could be used as a reference genome for the important medicinal plants of the genus Scutellaria, and also provide much-desired information for molecular breeding.

Cytoplasmic MSH2 Related to Genomic Deletions in the MSH2/EPCAM Genes in Colorectal Cancer Patients With Suspected Lynch Syndrome.

BackgroundA large proportion of patients with Lynch syndrome (LS) have MSH2 abnormalities, but genotype-phenotype studies of MSH2 mutations in LS are still lacking. The aim of this study was to comprehensively analyze the clinicopathological characteristics and molecular basis of colorectal cancer (CRC) in patients with uncommon MSH2 cytoplasmic expression.MethodsWe retrospectively reviewed 4195 consecutive cases of CRC patients diagnosed between January 2015 and December 2017 at the Cancer Hospital Chinese Academy of Medical Sciences. Of the 4195 patients with CRC, 69 were indicated to have abnormal MSH2 expression through tumor immunohistochemical staining. Genetic tests, such as next-generation sequencing, large genomic rearrangement (LGR) analysis, microsatellite instability status analysis and genomic breakpoint analysis, were performed. Clinicopathological and molecular characteristics and clinical immunotherapy response were analyzed.ResultsForty-five of 69 patients were identified to have LS with pathogenic germline mutations in MSH2 and/or EPCAM. Of these LS patients, 26.7% were confirmed to harbor large genomic rearrangements (LGRs). Of note, three tumors from two unrelated family pedigrees exhibited a rare cytoplasmic MSH2 staining pattern that was found in LS patients with EPCAM/MSH2 deletions. RNA analysis showed that two novel mRNA fusions of EPCAM and MSH2 resulted in the predicted protein fusion with MSH2 cytoplasmic localization. Analyses of genomic breakpoints indicated that two novel deletions of EPCAM and MSH2 originated from Alu repeat-mediated recombination events. Our study also provides clinical evidence for the beneficial effect of the PD-1 inhibitor pembrolizumab for CRC patients that exhibit cytoplasmic MSH2 staining.ConclusionOur study demonstrates that the rare cytoplasmic MSH2 staining pattern should be fully recognized by pathologists and geneticists. Given the specific genotype-phenotype correlation in LS screening, we advocate that all CRC patients with cytoplasmic MSH2 staining in histology should be screened for LGRs of EPCAM and MSH2.

Repeats in mitochondrial and chloroplast genomes characterize the ecotypes of the Oryza.

The online version contains supplementary material available at 10.1007/s11032-020-01198-6.

Wide crossing diversify mitogenomes of rice

BackgroundIn most angiosperms, the inheritance of the mitochondria takes place in a typical maternal manner. However, very less information is available about if the existence of structural variations or not in mitochondrial genomes (mitogenomes) between maternal parents and their progenies.ResultsIn order to find the answer, a stable rice backcross inbred line (BIL) population was derived from the crosses of Oryza glaberrima/Oryza sativa//Oryza sativa. The current study presents a comparative analysis of the mitogenomes between maternal parents and five BILs. There were recorded universal structural variations such as reversal, translocation, fusion, and fission among the BILs. The repeat-mediated recombination and non-homologous end-joining contributed virtually equal to the rearrangement of mitogenomes. Similarly, the relative order, copy-number, expression level, and RNA-editing rate of mitochondrial genes were also extensively varied among BILs.ConclusionsThese novel findings unraveled an unusual mystery of the maternal inheritance and possible cause for heterogeneity of mitogenomes in rice population. The current piece of work will greatly develop our understanding of the plant nucleo-cytoplasmic interaction and their potential role in plant growth and developmental processes.

Genetic variation of FUT2 in a Peruvian population: identification of a novel LTR-mediated deletion and characterization of 4 nonsynonymous single-nucleotide polymorphisms.

The human FUT2 gene, which encodes a secretor type α(1,2)fucosyltransferase, is reported to have several population-specific single-nucleotide polymorphisms (SNPs) and copy number variations. However, little is known about genetic variation of FUT2 in Native Americans. To detect SNPs and copy number variations of the FUT2 gene in Peruvians, direct sequencing and digital polymerase chain reaction were performed. Haplotypes of observed SNPs were estimated by PHASE software or cloning into plasmids. The functional significance of nonsynonymous SNPs was examined by transient transfection assay. We identified three novel nonfunctional alleles (se178,357 , se841 , and sedel4 ) due to two nonsynonymous SNPs (178C > T and 841G > A) and a novel long terminal repeat-mediated recombination with a 4.3-kb deletion in 70 Peruvians. The frequency of nonfunctional alleles was relative low (20.7%). Because se841 has a relatively high frequency (5.7%), it might be a suitable genetic marker for Peruvians. We identified three novel nonfunctional alleles in 70 Peruvians. To our knowledge, this is the first time a long terminal repeat-mediated gene recombination event at the FUT2 locus has been detected.

High and Variable Rates of Repeat-Mediated Mitochondrial Genome Rearrangement in a Genus of Plants.

For 30 years, it has been clear that angiosperm mitochondrial genomes evolve rapidly in sequence arrangement (i.e., synteny), yet absolute rates of rearrangement have not been measured in any plant group, nor is it known how much these rates vary. To investigate these issues, we sequenced and reconstructed the rearrangement history of seven mitochondrial genomes in Monsonia (Geraniaceae). We show that rearrangements (occurring mostly as inversions) not only take place at generally high rates in these genomes but also uncover significant variation in rearrangement rates. For example, the hyperactive mitochondrial genome of Monsonia ciliata has accumulated at least 30 rearrangements over the last million years, whereas the branch leading to M. ciliata and its sister species has sustained rearrangement at a rate that is at least ten times lower. Furthermore, our analysis of published data shows that rates of mitochondrial genome rearrangement in seed plants vary by at least 600-fold. We find that sites of rearrangement are highly preferentially located in very close proximity to repeated sequences in Monsonia. This provides strong support for the hypothesis that rearrangement in angiosperm mitochondrial genomes occurs largely through repeat-mediated recombination. Because there is little variation in the amount of repeat sequence among Monsonia genomes, the variable rates of rearrangement in Monsonia probably reflect variable rates of mitochondrial recombination itself. Finally, we show that mitochondrial synonymous substitutions occur in a clock-like manner in Monsonia; rates of mitochondrial substitutions and rearrangements are therefore highly uncoupled in this group.

Mgm101 Is a Rad52-related Protein Required for Mitochondrial DNA Recombination

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

Differential DNA Rearrangements of Plastid Genes, psbA and psbD, in Two Species of the Dinoflagellate Alexandrium

Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes, which are carried individually on small circular molecules, termed 'minicircles'. Although the prevalent plastid chromosome of most algae and plants has only a single copy of each gene, our previous study showed that low copy numbers of multiple variants of the gene psbA co-exist with the 'ordinary' gene encoding the D1 protein in minicircles of Alexandrium tamarense. Although none of the psbA variants encoded the entire protein, they persisted in culture. In this study, we compared the distribution and structure of psbA and psbD variants in two species of Alexandrium to characterize DNA rearrangement within these genes. In addition to four previously reported psbA variants, three psbD variants were found in A. tamarense minicircles. The ordinary psbA and psbD genes also co-existed with variants in another species, A. catenella. The sequences of the ordinary genes were virtually identical in the two species. All the variants comprised insertion or deletion mutations, with no base substitutions being identified. Duplicated parts of the coding sequences were contained in most of the insertions. Short direct repeats (4-14 bp) and/or adenine + thymine-rich motifs were present in all mutation regions, although the position and/or the sequence of each DNA rearrangement was unique to each variant. The results indicated that replication-based repeat-mediated recombination was responsible for generation of the variants.