BACKGROUND
while producing a circular genetic map, plant mitochondrial chromosomes comprise a heterogeneous population of highly branched, circularly permuted linear molecules  <cit> .
in plants, large-sized mitochondrial repeats  conduct high-frequency reciprocal recombination to subdivide the genome  <cit> .
given the linear structure of the genome, this recombination activity is suspected to participate in recombination-mediated replication initiation  <cit> .
mitochondrial genome plasticity is evident in extensive dna polymorphism among closely related lines of a given plant species  <cit> , rapid accumulation of polymorphisms within a line under cell culture conditions  <cit>  and novel insertions, deletions and sequence chimeras  <cit> .
these types of genomic alterations are causative in cytoplasmic male sterility  in a wide range of plant species  <cit> , implying an adaptive rationale for the genomic phenomena  <cit> .
still, questions remain about the processes underlying mitochondrial genome variation, since most previous studies have addressed existing variations without the benefit of de novo events.
mitochondrial dna replication in plants is not well-detailed, but both rolling circle and recombination-mediated replication have been suggested  <cit> .
likewise, the dna repair mechanisms accounting for unusually low nucleotide substitution rates in plants are not known  <cit> .
sufficient mitochondrial dna has been extracted from plant tissues to suggest the presence of multiple copies of the genome per cell, but available data suggest that the genome is organized into a heterogeneous population of dna molecules maintained at vastly different relative copy numbers.
some substoichiometric dna configurations are stably maintained in the genome and transmitted to progeny at levels approximating  <dig> copy per  <dig> cells  <cit> .
the term "substoichiometric shifting"  refers to the reversible interconversion of these low-level forms to high copy numbers, influencing gene expression in the process  <cit> .
this intragenomic copy number shifting is rapid, likely occurs during plant reproduction  <cit>  and involves differential recombination at intermediate-sized repeats within the genome  <cit> .
asymmetrical recombination is characterized by the accumulation of only one of the two predicted dna exchange products.
intermediate repeats support asymmetrical recombination in the plant mitochondrial genome at extremely low levels.
this recombination activity accounts for faint "ghost" band polymorphisms commonly reported in dna gel blot hybridization experiments involving long film exposure times  <cit> .
disruption of the arabidopsis nuclear gene msh <dig> , a component of mitochondrial recombination surveillance  <cit> , allows increased recombination activity at these repeats.
recent studies of mitochondrial genomic changes in the arabidopsis msh <dig> mutant, performed using pcr and dna gel blot analysis, permitted functional identification of  <dig> intermediate repeat pairs  <dig> to  <dig> bp in size  <cit> , implying a potential for massive rearrangement of the mitochondrial genome by this type of recombination activity.
in "first-generation" msh <dig> mutants created by pollination of a wild-type line with mutant msh <dig> pollen and selection of the f <dig> msh1/msh <dig> segregant, low-level recombination activity is initiated at each repeat.
by the "advanced generation," derived by recurrent self-pollination of the msh <dig> mutant, recombination is extensive and recombinants are present at high copy numbers  <cit> .
most previously acquired information about plant mitochondrial genome variation has relied on studies of cms mutants and dna polymorphism in natural populations.
discovery of msh <dig> permits direct investigation of mitochondrial de novo recombination and its relationship to high levels of structural variation and unusually low nucleotide substitution rates unique to plants.
in our present study, next-generation sequencing provided sufficient sensitivity for detailed investigation of the asymmetrical strand invasion and gene conversion process as well as sufficient throughputfor cataloging genomic alterations associated with rapid, intraspecific mitochondrial genome evolution.
RESULTS
next-generation sequence analysis reveals additional intermediate repeats not previously detected
mitochondrial asymmetric dna exchange in the msh <dig> mutant involves  <dig> repeat pairs ranging from  <dig> to  <dig> bp in size .
this was deduced by identification of paired adjacent sequences that were known to map distal to one another.
such paired-end sequences were clustered in msh <dig> first- and advanced-generation mutants in a manner distinct from the wild type .
all identified clusters could be explained by recombination events involving intermediate repeats.
while confirming previously identified repeats  <cit> , deep sequencing revealed  <dig> new repeat pairs ranging from  <dig> to  <dig> bp in size, with sequence homology within each pair ranging from 85% to 98% and the smallest repeats displaying the highest identity .
all recombination detected at intermediate repeats showed asymmetrical exchange activity.
intermediate repeats reveal essential features of the mitochondrial dna exchange process
the size range of active repeats reflects the length of homology required to permit dna exchange.
while repeated sequences smaller than  <dig> bp were detected in the genome, they showed no evidence of recombination activity, suggesting that  <dig> bp approximates a lower size limit for msh1-associated dna exchange.
recombination patterns at intermediate repeats imply a process of break-induced replication on the basis of the uniformly asymmetrical nature of dna exchange  <cit> .
each repeat showed a conserved polarity for strand invasion, which was suggested by the accumulation of only one of the recombinant products and, in some cases, concomitant loss of one of the parental forms  <cit> .
the observed polarity was confirmed by comparing high-throughput sequence frequencies at sites flanking repeats in first- and advanced-generation msh <dig> mutants with the wild type .
analysis of clustered intermediate repeats allowed us to compare relative strand invasion polarities and showed both opposite and same-strand polarities at neighboring repeats, with no obvious association with local direction of transcription .
it is possible that these sites of active, bidirectional strand invasion might represent replication origins .
sequence analysis of the recombination product for nonidentical repeats, composing about 50% of all active intermediate repeats, provided evidence of heteroduplex formation followed by mismatch repair .
according to our data, nucleotide mismatches are repaired using the donor strand as a template.
in figure 3a, the nucleotide insertions in the donor strand are cleaved, giving the appearance of partial gene conversion.
however, a more extensive survey of repeats showed no clear pattern of donor or recipient strand cleavage for correction of nucleotide insertions.
these results reveal the presence of a mitochondrial mismatch repair mechanism in plants that is independent of msh <dig>
to assess the influence of msh <dig> recombination on mitochondrial genome copy number, we compared relative copy number at  <dig> sites within the mitochondrial genome of the wild type  and the msh <dig> mutant .
in each case, copy number was higher in the mutant, implying that overall mitochondrial dna copy number increases with enhanced recombination.
one possible explanation for this observation might be enhanced break-induced replication of the genome in the absence of msh1
mitochondrial recombination in the msh <dig> mutant produces two patterns of genomic rearrangement
we compared the read frequency for parental forms in different generations of the msh <dig> mutant against wild type.
this allowed us to trace the fate of parental products over time in the msh <dig> mutant.
most repeats appeared to fall into one of two general categories .
class i repeats lost one parental form as they acquired the recombinant, resulting in the appearance of both deletion and gain of genomic forms.
class ii repeats retained the parental forms as they acquired the recombinant, resulting in the appearance of a novel genomic form.
the parental form that was reduced was consistently that which donates the invading strand.
this "gain" and "loss" of genetic environments is the consequence of sss, and pcr-based analysis has revealed that these genomic environments change in relative copy number merely as a consequence of recombination  <cit> .
from a genomic perspective, sequences residing in proximity to these intermediate repeats have the greatest likelihood of undergoing sss, although we have identified sequences some distance from an identifiable repeat that also undergo sss.
we observed several essential genes residing in proximity to class ii repeats , but none residing adjacent to class i repeats .
presumably, the relative proximity of a repeat to essential genetic information could impose selection for the parental form, the class ii pattern.
in msh <dig> advanced- versus first-generation mutants, we investigated the reversibility of recombination activity upon replacement of the msh1allele.
while first-generation msh <dig> rearrangements appeared to be fully reversible by crossing the msh <dig> line with the wild type and deriving an f <dig> progeny, advanced-generation rearrangements were not reversible, based on analysis of  <dig> individual f <dig> plants from both crosses and assaying three repeats  for recombinant configurations .
we attribute these results to cytoplasmic sorting of the least prevalent forms once msh <dig> is reintroduced.
thus, in the first generation, where the quantity of recombinant molecule is low relative to nonrecombinant, recombinant forms are diluted and nonrecombinant forms predominate.
in advanced generations, where recombinant forms have accumulated to higher levels, their loss by sorting is precluded.
the arabidopsis mitochondrial genome undergoes rapid and extensive rearrangement
in our investigation of the influence of double-strand break -mediated changes on genomic variation, we sequenced and assembled the mitochondrial genomes of ecotypes col- <dig> and landsberg erecta  and compared these with the publicly available c <dig> sequence .
among the three ecotypes, we found virtually identical genomic coding capacity within genomes that had undergone extensive rearrangements.
the majority of the structural polymorphisms found can be attributed to dsb repair activity by either nonhomologous end-joining  or intermediate-sized repeat-mediated recombination .
stoichiometric variation among ecotypes includes a repeat-containing interval of  <dig>  kb, originally reported by forner et al.
<cit> , that is present in col- <dig> but substoichiometric in c <dig> and ler  <cit> .
the corresponding "junction" configuration that lacks this  <dig> -kb insertion is present in c <dig> and ler, but is substoichiometric in col- <dig>  likewise, an unrelated interval of  <dig>  kb present in c <dig> and ler was found to be substoichiometric in col- <dig>  the corresponding "junction" lacking this  <dig> -kb interval is present in col- <dig> but substoichiometric in c <dig> and ler .
while cross-ecotype genomic comparisons gave evidence of ecotype-specific divergence by insertion and deletion of distinct genetic information, this variation was largely accounted for by asymmetrical recombination-mediated sss.
researchers in earlier cross-genotype studies in maize and sugar beet, involving multiple lines, reported similar mitochondrial variation  <cit> .
while the researchers in neither of the earlier studies investigated substoichiometric forms in the genome, we suggest that some of the reported insertions and deletions distinguishing the closely related genotypes in those species likely derive from sss activity.
the extensive mitochondrial dna polymorphism among lines within a species was not surprising, given what has been reported in other plant studies  <cit> .
what was significant in our investigation was the discovery that nearly all of the variation observed could be accounted for by dsb repair activity or nucleotide substitutions.
this observation prompted us to investigate the extent to which dsb repair processes have participated in plant mitochondrial genome evolution.
dsb repair occurs widely in arabidopsis mitochondrial genomes
to assess the role of dsb repair in mitochondrial genome evolution, we assembled and compared mitochondrial sequences from  <dig> different arabidopsis ecotypes.
we collaborated with the arabidopsis  <dig> genomes project  to obtain mitochondrial sequences for subsequent identification of polymorphisms.
the mitochondrial genome sequences were scanned for snps to assess phylogenetic relationships among the ecotypes.
cross-ecotype snp comparisons suggested that the  <dig> ecotypes comprised six phylogenetic groups .
the derived groupings appeared to correspond generally with the geographic origin of each line, and the variation measured is likely to reflect divergence over a span of  <dig>  years  <cit> .
this observation would be consistent with the observed sequence divergence across ecotypes, which was approximately one-twentieth that of arabidopsis lyrata, with an estimated divergence time of four to five million years.
mitochondrial structural polymorphisms revealed extensive genomic variation distinguishing each line.
the majority of structural changes represented nonhomologous, recombination or small deletion activity .
the small deletions were a small proportion  of the total structural polymorphisms detected.
cross-ecotype cataloging of observed structural variation revealed remarkable correspondence with the six snp-based phylogenetic groups .
these observations would be expected if dsb repair served as a prominent contributor to the rapid evolution rate of plant mitochondrial genomes.
discussion
while we have not yet identified the genomic features that condition asymmetrical recombination in the arabidopsis mitochondrial genome, we have shown in the present study that the process comprises the primary pattern of dna exchange at intermediate-sized repeat sequences in the absence of msh <dig>  it is possible that the observed asymmetrical exchange might be a consequence of differential maintenance of recombination products rather than the consequence of dominant strand invasion polarity.
while it has been suggested that analysis of low-frequency recombination events can sometimes be confused with pcr-generated artefacts  <cit> , our analysis of de novo recombination in msh <dig> confirms prolific dna exchange activity.
data from our studies suggest that strand invasion occurs throughout the genome, a likely consequence of random dsbs, but is prevented by msh <dig> from producing successful dna exchange in stretches of sequence homology under about  <dig> bp.
in the absence of msh <dig>  strand invasion proceeds at smaller lengths of homology to a lower limit of approximately  <dig> bp.
in the wild-type line, mitochondrial dna exchange at intermediate repeats does occur, but at very low levels, presumably because it is prevented by msh <dig> surveillance.
this infrequent recombination is detected as faint ghost bands by dna gel blot analysis  <cit> .
msh <dig> encodes a carboxy-terminal giy-yig endonuclease domain, although direct participation in double-strand cleavage to abort dna exchange has not yet been formally tested.
msh <dig> presumably permits repair of a dsb at stretches of homology greater than  <dig> bp following heteroduplex formation, assuming a break-induced replication model .
in the msh <dig> mutant, heteroduplex formation occurs within the intermediate repeat stretches of homology.
the observation of heteroduplex-mediated mismatch repair in this study provides important evidence relevant to the low mitochondrial nucleotide substitution rates reported in plants  <cit> .
our study also shows that this mismatch repair activity is independent of msh <dig>  interestingly, nucleotide divergence within intermediate repeat intervals, estimated across  <dig> arabidopsis ecotypes at  <dig>  per  <dig>  bp, approximates the average for the genome as a whole, which is  <dig> per  <dig>  bp.
however, in regions of the genome where high-frequency reciprocal dna exchange occurs, within large  repeats, levels of polymorphism were  <dig> times lower, averaging  <dig>  per mbp for the  <dig> -kb repeat across  <dig> ecotypes.
these observations imply that the low-frequency exchanges observed at intermediate repeats in the wild-type line may be sufficient to confer the low nucleotide substitution rate observed generally in the arabidopsis mitochondrial genome.
in animal lineages, nucleotide substitution rates in the mitochondrial genome are much higher, with the notable exception of corals and their relatives  <cit> .
intriguingly, the corals appear to be the only known animal lineage encoding a mitochondrial msh <dig>  with similarities in structure to msh <dig> in plants  <cit> .
analysis of mitochondrial genomic rearrangements differentiating the col- <dig>  ler and c <dig> ecotypes revealed three primary classes of genomic changes as recombination, nhej and deletion, all of which may represent consequences of dsb repair .
an important aspect of nhej may be the formation of sequence chimeras, some of which encode transcriptionally active open reading frames that could condition phenotypic variations, such as cms.
proximity of a cms sequence chimera to a class i repeat can allow changes in copy number of the region by recombination at the repeat.
cms induction was shown to occur by msh <dig> suppression in various plant species  <cit> .
the resulting mitochondrial dna rearrangements are presumed to result in copy number amplification of a novel, preexistent, cms-inducing genomic environment.
sss-associated copy number reduction of male sterility sequences has been associated with spontaneous reversion to fertility in common bean, brassica, and maize s-type cytoplasm  <cit> .
whether the frequency of reversion responds to altered msh <dig> expression in the plant, and whether msh <dig> expression is influenced by environmental cues such as unsuccessful pollination, is an intriguing possibility.
msh <dig> transcription is environmentally responsive  <cit> , but it is not clear whether carbon flux acts as a signal.
an isolated male-sterile plant, following a period of unsuccessful pollination, might affect carbon signaling to suppress msh <dig>  amplifying mitochondrial recombination for sss and reversion to fertility.
such a process, if it occurred late in the flowering cycle, would ensure a successful seed set.
CONCLUSIONS
in our present study, we conducted a detailed analysis of de novo recombination and genome rearrangement processes that characterize plant mitochondrial genomes.
the rapid rate of plant mitochondrial intraspecific structural variation and low nucleotide substitution rates can both be accounted for by unusual dsb repair within the genome.
these observations consequently implicate dsb repair processes as a driver in the evolutionary divergence of plant and animal mitochondria.
the dynamic nature of plant mitochondrial genomes, and their ability to retain genetic variation substoichiometrically, presumably enhance environmental responsiveness and increase reproductive versatility.

 while producing a circular genetic map, plant mitochondrial chromosomes comprise a heterogeneous population of highly branched, circularly permuted linear molecules. in plants, large-sized mitochondrial repeats conduct high-frequency reciprocal recombination to subdivide the genome. given the linear structure of the genome, this recombination activity is suspected to participate in recombination-mediated replication initiation. mitochondrial genome plasticity is evident in extensive dna polymorphism among closely related lines of a given plant species, rapid accumulation of polymorphisms within a line under cell culture conditions and novel insertions, deletions and sequence chimeras. these types of genomic alterations are causative in cytoplasmic male sterility in a wide range of plant species, implying an adaptive rationale for the genomic phenomena. still, questions remain about the processes underlying mitochondrial genome variation, since most previous studies have addressed existing variations without the benefit of de novo events. mitochondrial dna replication in plants is not well-detailed, but both rolling circle and recombination-mediated replication have been suggested. likewise, the dna repair mechanisms accounting for unusually low nucleotide substitution rates in plants are not known. sufficient mitochondrial dna has been extracted from plant tissues to suggest the presence of multiple copies of the genome per cell, but available data suggest that the genome is organized into a heterogeneous population of dna molecules maintained at vastly different relative copy numbers. some substoichiometric dna configurations are stably maintained in the genome and transmitted to progeny at levels approximating copy per cells. the term "substoichiometric shifting" refers to the reversible interconversion of these low-level forms to high copy numbers, influencing gene expression in the process. this intragenomic copy number shifting is rapid, likely occurs during plant reproduction and involves differential recombination at intermediate-sized repeats within the genome. asymmetrical recombination is characterized by the accumulation of only one of the two predicted dna exchange products. intermediate repeats support asymmetrical recombination in the plant mitochondrial genome at extremely low levels. this recombination activity accounts for faint "ghost" band polymorphisms commonly reported in dna gel blot hybridization experiments involving long film exposure times. disruption of the arabidopsis nuclear gene msh, a component of mitochondrial recombination surveillance, allows increased recombination activity at these repeats. recent studies of mitochondrial genomic changes in the arabidopsis msh mutant, performed using pcr and dna gel blot analysis, permitted functional identification of intermediate repeat pairs to bp in size, implying a potential for massive rearrangement of the mitochondrial genome by this type of recombination activity. in "first-generation" msh mutants created by pollination of a wild-type line with mutant msh pollen and selection of the f msh1/msh segregant, low-level recombination activity is initiated at each repeat. by the "advanced generation," derived by recurrent self-pollination of the msh mutant, recombination is extensive and recombinants are present at high copy numbers. most previously acquired information about plant mitochondrial genome variation has relied on studies of cms mutants and dna polymorphism in natural populations. discovery of msh permits direct investigation of mitochondrial de novo recombination and its relationship to high levels of structural variation and unusually low nucleotide substitution rates unique to plants. in our present study, next-generation sequencing provided sufficient sensitivity for detailed investigation of the asymmetrical strand invasion and gene conversion process as well as sufficient throughputfor cataloging genomic alterations associated with rapid, intraspecific mitochondrial genome evolution.  next-generation sequence analysis reveals additional intermediate repeats not previously detected mitochondrial asymmetric dna exchange in the msh mutant involves repeat pairs ranging from to bp in size. this was deduced by identification of paired adjacent sequences that were known to map distal to one another. such paired-end sequences were clustered in msh first- and advanced-generation mutants in a manner distinct from the wild type. all identified clusters could be explained by recombination events involving intermediate repeats. while confirming previously identified repeats, deep sequencing revealed new repeat pairs ranging from to bp in size, with sequence homology within each pair ranging from 85% to 98% and the smallest repeats displaying the highest identity. all recombination detected at intermediate repeats showed asymmetrical exchange activity. intermediate repeats reveal essential features of the mitochondrial dna exchange process the size range of active repeats reflects the length of homology required to permit dna exchange. while repeated sequences smaller than bp were detected in the genome, they showed no evidence of recombination activity, suggesting that bp approximates a lower size limit for msh1-associated dna exchange. recombination patterns at intermediate repeats imply a process of break-induced replication on the basis of the uniformly asymmetrical nature of dna exchange. each repeat showed a conserved polarity for strand invasion, which was suggested by the accumulation of only one of the recombinant products and, in some cases, concomitant loss of one of the parental forms. the observed polarity was confirmed by comparing high-throughput sequence frequencies at sites flanking repeats in first- and advanced-generation msh mutants with the wild type. analysis of clustered intermediate repeats allowed us to compare relative strand invasion polarities and showed both opposite and same-strand polarities at neighboring repeats, with no obvious association with local direction of transcription. it is possible that these sites of active, bidirectional strand invasion might represent replication origins. sequence analysis of the recombination product for nonidentical repeats, composing about 50% of all active intermediate repeats, provided evidence of heteroduplex formation followed by mismatch repair. according to our data, nucleotide mismatches are repaired using the donor strand as a template. in figure 3a, the nucleotide insertions in the donor strand are cleaved, giving the appearance of partial gene conversion. however, a more extensive survey of repeats showed no clear pattern of donor or recipient strand cleavage for correction of nucleotide insertions. these results reveal the presence of a mitochondrial mismatch repair mechanism in plants that is independent of msh to assess the influence of msh recombination on mitochondrial genome copy number, we compared relative copy number at sites within the mitochondrial genome of the wild type and the msh mutant. in each case, copy number was higher in the mutant, implying that overall mitochondrial dna copy number increases with enhanced recombination. one possible explanation for this observation might be enhanced break-induced replication of the genome in the absence of msh1 mitochondrial recombination in the msh mutant produces two patterns of genomic rearrangement we compared the read frequency for parental forms in different generations of the msh mutant against wild type. this allowed us to trace the fate of parental products over time in the msh mutant. most repeats appeared to fall into one of two general categories. class i repeats lost one parental form as they acquired the recombinant, resulting in the appearance of both deletion and gain of genomic forms. class ii repeats retained the parental forms as they acquired the recombinant, resulting in the appearance of a novel genomic form. the parental form that was reduced was consistently that which donates the invading strand. this "gain" and "loss" of genetic environments is the consequence of sss, and pcr-based analysis has revealed that these genomic environments change in relative copy number merely as a consequence of recombination. from a genomic perspective, sequences residing in proximity to these intermediate repeats have the greatest likelihood of undergoing sss, although we have identified sequences some distance from an identifiable repeat that also undergo sss. we observed several essential genes residing in proximity to class ii repeats, but none residing adjacent to class i repeats. presumably, the relative proximity of a repeat to essential genetic information could impose selection for the parental form, the class ii pattern. in msh advanced- versus first-generation mutants, we investigated the reversibility of recombination activity upon replacement of the msh1allele. while first-generation msh rearrangements appeared to be fully reversible by crossing the msh line with the wild type and deriving an f progeny, advanced-generation rearrangements were not reversible, based on analysis of individual f plants from both crosses and assaying three repeats for recombinant configurations. we attribute these results to cytoplasmic sorting of the least prevalent forms once msh is reintroduced. thus, in the first generation, where the quantity of recombinant molecule is low relative to nonrecombinant, recombinant forms are diluted and nonrecombinant forms predominate. in advanced generations, where recombinant forms have accumulated to higher levels, their loss by sorting is precluded. the arabidopsis mitochondrial genome undergoes rapid and extensive rearrangement in our investigation of the influence of double-strand break -mediated changes on genomic variation, we sequenced and assembled the mitochondrial genomes of ecotypes col- and landsberg erecta and compared these with the publicly available c sequence. among the three ecotypes, we found virtually identical genomic coding capacity within genomes that had undergone extensive rearrangements. the majority of the structural polymorphisms found can be attributed to dsb repair activity by either nonhomologous end-joining or intermediate-sized repeat-mediated recombination. stoichiometric variation among ecotypes includes a repeat-containing interval of kb, originally reported by forner et al., that is present in col- but substoichiometric in c and ler. the corresponding "junction" configuration that lacks this -kb insertion is present in c and ler, but is substoichiometric in col- likewise, an unrelated interval of kb present in c and ler was found to be substoichiometric in col- the corresponding "junction" lacking this -kb interval is present in col- but substoichiometric in c and ler. while cross-ecotype genomic comparisons gave evidence of ecotype-specific divergence by insertion and deletion of distinct genetic information, this variation was largely accounted for by asymmetrical recombination-mediated sss. researchers in earlier cross-genotype studies in maize and sugar beet, involving multiple lines, reported similar mitochondrial variation. while the researchers in neither of the earlier studies investigated substoichiometric forms in the genome, we suggest that some of the reported insertions and deletions distinguishing the closely related genotypes in those species likely derive from sss activity. the extensive mitochondrial dna polymorphism among lines within a species was not surprising, given what has been reported in other plant studies. what was significant in our investigation was the discovery that nearly all of the variation observed could be accounted for by dsb repair activity or nucleotide substitutions. this observation prompted us to investigate the extent to which dsb repair processes have participated in plant mitochondrial genome evolution. dsb repair occurs widely in arabidopsis mitochondrial genomes to assess the role of dsb repair in mitochondrial genome evolution, we assembled and compared mitochondrial sequences from different arabidopsis ecotypes. we collaborated with the arabidopsis genomes project to obtain mitochondrial sequences for subsequent identification of polymorphisms. the mitochondrial genome sequences were scanned for snps to assess phylogenetic relationships among the ecotypes. cross-ecotype snp comparisons suggested that the ecotypes comprised six phylogenetic groups. the derived groupings appeared to correspond generally with the geographic origin of each line, and the variation measured is likely to reflect divergence over a span of years. this observation would be consistent with the observed sequence divergence across ecotypes, which was approximately one-twentieth that of arabidopsis lyrata, with an estimated divergence time of four to five million years. mitochondrial structural polymorphisms revealed extensive genomic variation distinguishing each line. the majority of structural changes represented nonhomologous, recombination or small deletion activity. the small deletions were a small proportion of the total structural polymorphisms detected. cross-ecotype cataloging of observed structural variation revealed remarkable correspondence with the six snp-based phylogenetic groups. these observations would be expected if dsb repair served as a prominent contributor to the rapid evolution rate of plant mitochondrial genomes. discussion while we have not yet identified the genomic features that condition asymmetrical recombination in the arabidopsis mitochondrial genome, we have shown in the present study that the process comprises the primary pattern of dna exchange at intermediate-sized repeat sequences in the absence of msh it is possible that the observed asymmetrical exchange might be a consequence of differential maintenance of recombination products rather than the consequence of dominant strand invasion polarity. while it has been suggested that analysis of low-frequency recombination events can sometimes be confused with pcr-generated artefacts, our analysis of de novo recombination in msh confirms prolific dna exchange activity. data from our studies suggest that strand invasion occurs throughout the genome, a likely consequence of random dsbs, but is prevented by msh from producing successful dna exchange in stretches of sequence homology under about bp. in the absence of msh strand invasion proceeds at smaller lengths of homology to a lower limit of approximately bp. in the wild-type line, mitochondrial dna exchange at intermediate repeats does occur, but at very low levels, presumably because it is prevented by msh surveillance. this infrequent recombination is detected as faint ghost bands by dna gel blot analysis. msh encodes a carboxy-terminal giy-yig endonuclease domain, although direct participation in double-strand cleavage to abort dna exchange has not yet been formally tested. msh presumably permits repair of a dsb at stretches of homology greater than bp following heteroduplex formation, assuming a break-induced replication model. in the msh mutant, heteroduplex formation occurs within the intermediate repeat stretches of homology. the observation of heteroduplex-mediated mismatch repair in this study provides important evidence relevant to the low mitochondrial nucleotide substitution rates reported in plants. our study also shows that this mismatch repair activity is independent of msh interestingly, nucleotide divergence within intermediate repeat intervals, estimated across arabidopsis ecotypes at per bp, approximates the average for the genome as a whole, which is per bp. however, in regions of the genome where high-frequency reciprocal dna exchange occurs, within large repeats, levels of polymorphism were times lower, averaging per mbp for the -kb repeat across ecotypes. these observations imply that the low-frequency exchanges observed at intermediate repeats in the wild-type line may be sufficient to confer the low nucleotide substitution rate observed generally in the arabidopsis mitochondrial genome. in animal lineages, nucleotide substitution rates in the mitochondrial genome are much higher, with the notable exception of corals and their relatives. intriguingly, the corals appear to be the only known animal lineage encoding a mitochondrial msh with similarities in structure to msh in plants. analysis of mitochondrial genomic rearrangements differentiating the col- ler and c ecotypes revealed three primary classes of genomic changes as recombination, nhej and deletion, all of which may represent consequences of dsb repair. an important aspect of nhej may be the formation of sequence chimeras, some of which encode transcriptionally active open reading frames that could condition phenotypic variations, such as cms. proximity of a cms sequence chimera to a class i repeat can allow changes in copy number of the region by recombination at the repeat. cms induction was shown to occur by msh suppression in various plant species. the resulting mitochondrial dna rearrangements are presumed to result in copy number amplification of a novel, preexistent, cms-inducing genomic environment. sss-associated copy number reduction of male sterility sequences has been associated with spontaneous reversion to fertility in common bean, brassica, and maize s-type cytoplasm. whether the frequency of reversion responds to altered msh expression in the plant, and whether msh expression is influenced by environmental cues such as unsuccessful pollination, is an intriguing possibility. msh transcription is environmentally responsive, but it is not clear whether carbon flux acts as a signal. an isolated male-sterile plant, following a period of unsuccessful pollination, might affect carbon signaling to suppress msh amplifying mitochondrial recombination for sss and reversion to fertility. such a process, if it occurred late in the flowering cycle, would ensure a successful seed set.  in our present study, we conducted a detailed analysis of de novo recombination and genome rearrangement processes that characterize plant mitochondrial genomes. the rapid rate of plant mitochondrial intraspecific structural variation and low nucleotide substitution rates can both be accounted for by unusual dsb repair within the genome. these observations consequently implicate dsb repair processes as a driver in the evolutionary divergence of plant and animal mitochondria. the dynamic nature of plant mitochondrial genomes, and their ability to retain genetic variation substoichiometrically, presumably enhance environmental responsiveness and increase reproductive versatility. 
in the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution. the mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining activities producing variability in size and organization. arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene msh, lending itself to detailed study of de novo mitochondrial genome activity. substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes. de novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. forty-seven repeat pairs were active in dna exchange in the msh mutant. recombination sites showed asymmetrical dna exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. plant mitochondrial dna also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. we obtained evidence of double-strand break repair, including nhej, sequence deletions and mitochondrial asymmetric recombination activity in arabidopsis wild-type and msh mutants on the basis of data generated by illumina deep sequencing and confirmed by dna gel blot analysis. on a larger scale, with mitochondrial comparisons across arabidopsis ecotypes, similar evidence of dsb repair activity differentiated ecotypes.
 the mitochondrial genome of higher plants is unusually dynamic, with recombination and nonhomologous end-joining activities producing variability in size and organization. plant mitochondrial dna also generally displays much lower nucleotide substitution rates than mammalian or yeast systems. arabidopsis displays these features and expedites characterization of the mitochondrial recombination surveillance gene msh, lending itself to detailed study of de novo mitochondrial genome activity. in the present study, we investigated the underlying basis for unusual plant features as they contribute to rapid mitochondrial genome evolution.  we obtained evidence of double-strand break repair, including nhej, sequence deletions and mitochondrial asymmetric recombination activity in arabidopsis wild-type and msh mutants on the basis of data generated by illumina deep sequencing and confirmed by dna gel blot analysis. on a larger scale, with mitochondrial comparisons across arabidopsis ecotypes, similar evidence of dsb repair activity differentiated ecotypes. forty-seven repeat pairs were active in dna exchange in the msh mutant. recombination sites showed asymmetrical dna exchange within lengths of 50- to 556-bp sharing sequence identity as low as 85%. de novo asymmetrical recombination involved heteroduplex formation, gene conversion and mismatch repair activities. substoichiometric shifting by asymmetrical exchange created the appearance of rapid sequence gain and loss in association with particular repeat classes.  extensive mitochondrial genomic variation within a single plant species derives largely from dsb activity and its repair. observed gene conversion and mismatch repair activity contribute to the low nucleotide substitution rates seen in these genomes. on a phenotypic level, these patterns of rearrangement likely contribute to the reproductive versatility of higher plants. 