BACKGROUND
it has been widely documented that major food crops associate naturally with beneficial soil microbes, including the arbuscular mycorrhizal fungi , an ancient group of root-inhabiting fungi belonging to the phylum glomeromycota  <cit> .
these ecologically crucial symbionts enhance the uptake of water and nutrients of plants they colonize, especially phosphate  <cit> , as well as protect them against pathogens  <cit>  and play a key role in soil structure  <cit> .
the benefits to plants provided by this association with amf could be enhanced by the manipulation of the fungal partner genetics, as demonstrated by angelard et al.
<cit>  and colard et al.
<cit> , using the model organism rhizophagus irregularis.
in both studies, through the exchange of nuclear genetic information via hyphal fusion, in vitro crossed-culture lines and segregated culture lines were generated.
this process influenced differentially the transcription of symbiosis-specific genes in rice, resulting in an increase in rice growth by a factor of five.
during the last decade numerous studies have been devoted to decipher the nuclear  <cit>  and mitochondrial genome organization patterns  <cit> , the sexual/asexual conundrum  and nuclear segregation process in amf  <cit> .
these data allowed new insights into the nuclear inheritance processes and segregation mechanisms that could occur in these fungi.
despite this progress, little is known about mitochondrial inheritance in amf.
since the mitochondrial genome encodes essential components of the cellular energy-producing apparatus, understanding mitochondrial dna  organization and inheritance in amf is of paramount importance, in order to manage more efficiently mycorrhizal associations at large scale  <cit> .
a previous study showed that amf mtdnas and nuclei migrate massively into spores during their formation  <cit> , but the fate of each parental haplotype following crosses needs to be investigated.
the publication of  <dig> complete amf mtdna  in the last few years allowed demonstrating that these sequences were all homoplasmic within isolates  <cit> .
these studies also showed that genomes are variable in defined intergenic regions between isolates, thus offering an unparalleled opportunity to design isolate-specific markers  <cit> .
recently, de la providencia et al.
<cit>  demonstrated length-heteroplasmy in spores formed near anastomosis regions between geographically distant r. irregularis in vitro isolates, using isolate-specific mitochondrial markers.
however, no information is available regarding the persistence of this length-heteroplasmic state in in vitro monosporal culture lines established from crossed parental isolates.
such a study is needed not only to understand mtdna inheritance processes and the persistence of mitochondrial haplotypes in stable culture lines, but also to use mtdna as a criterion to define a reliable amf taxonomic unit  <cit> , either at a genus, species or isolate level.
the mitochondrial inheritance process requires an active transport of organelles along the cytoskeleton and relies on membrane fission and fusion events.
it plays a crucial role in the adaptation of the organism in energy requirements.
most of the molecular machinery and cellular mechanisms mediating these processes have been conserved along the fungal and animal kingdom evolution  <cit> .
in filamentous fungi, sexual crosses lead to uniparental transmission of mitochondria  <cit> , whereas mitochondria are biparentally inherited in budding yeast  <cit> .
in yeast, microfilaments, such as actin, play an important role in the positioning and motility of mitochondria, but microtubules are the principal mitochondrial transporter in many other fungi  <cit> .
for many years, the study of mitochondrial dynamics in fungi relied mainly on microscopic observations.
however, the sequencing of fungal genomes is an important step towards a better understanding of the molecular machinery mediating mitochondrial inheritance.
in that regard, the recently available r. irregularis genomic sequence data  <cit>  provide new tools to identify molecular components involved in mitochondrial dynamics in amf.
no study has yet investigated potential mitochondrial segregation and/or inheritance mechanisms occurring in amf.
in the model organism saccharomyces cerevisiae, mitochondrial inheritance is controlled by the mitochondrial segregation apparatus , which ensures a reliable transmission of mitochondrial organelles and their genomes to the progeny.
searches of sequence databases reveal evolutionarily conserved proteins for all known budding yeast msa components in schizosaccharomyces pombe and in filamentous fungi such as neurospora crassa and aspergillus nidulans, notably  <cit> .
this suggests that the core machinery of mitochondrial inheritance has been conserved during evolution, even in organisms that use different cytoskeletal systems for organellar transport  <cit> .
the msa in s. cerevisiae is a trans-membrane proteic complex consisting of three mitochondrial membrane proteins , forming the core component of the apparatus, along with mmm <dig>  mdm <dig> and mdm <dig>  which interact with the core component .
this proteic complex links the mitochondrial outer membrane to the actin cytoskeleton and the mitochondrial inner membrane to mitochondrial nucleoids, which are clusters of similar mtdnas packaged by proteins abf <dig>  aco <dig> and ilv <dig> and maintained together by holliday junctions .
however, the molecular machinery of mtdna inheritance remains largely unknown in many other organisms  <cit> .
in this study, we tested the hypothesis that monosporal crossed-culture lines would maintain the mitochondrial length-heteroplasmy  <cit>  previously observed in r. irregularis in vitro spore progenies arising from crossed-cultures of genetically divergent isolates.
in addition, we investigated nuclear inheritance following anastomosis in spore progenies and monosporal crossed-culture lines.
furthermore, we searched for the existence of a putative msa in glomeromycota, by finding the best reciprocal blast of yeast msa proteins in the published genome of r. irregularis  <cit>  and its transcriptome  <cit> , and tested their orthology with all available sequences on the database.
RESULTS
germination and fungal development of monosporal culture lines
cultures from each combination  were performed.
after identifying interaction zones between mycelia from different isolates, which were characterized by the formation of hyphal contacts, randomly chosen spores  were harvested from the interaction zone of each combination, individually cut out from the mycelium and placed in a new petri dish in association with ri t-dna transformed chicory  roots as described in the methods section .
for each combination, approximately half of the inoculated spores germinated.
the observed germination rate corresponds to rates previously observed in four in vitro cultured glomeraceae species  <cit> .
following root contact and colonisation, some cultures stopped their growth, without producing any spores, and therefore could not be used in this study.
the rate of mycelium development and spore production greatly varied among cultures, the combination of isolates daom- <dig> and daom- <dig> being the most successful, resulting in  <dig> colonized dishes containing between  <dig> and  <dig> newly formed spores.
the combination of isolates daom- <dig> and daom- <dig> produced seven colonized petri dishes with few, newly formed spores .
we obtained  <dig> poorly developed cultures from isolates combination daom-234328/daom- <dig>  and only nine produced sufficient amount of fungal material required for further analysis.
in total,  <dig> monosporal crossed-cultures lines were genotyped.fig.
<dig> schematic drawing of the experimental design.
a observation of hyphal contacts between two clusters of spores from different isolates b identification of spores produced in anastomosis regions and inoculation of a single spore on a new petri dish with ri t-dna transformed chicory roots i.e.
monosporal cultures.
after germination and colonisation of the petri dish, spores are extracted and their dna is used for genotyping of the monosporal culture
genotyping analysis
all mitochondrial markers showed to be isolate-specific when challenged against other rhizophagus isolates .
the  <dig> monosporal crossed-culture lines genotyped by qpcr approach presented only one parental mtdna haplotype.
in all nine progenies of the combination daom-197198/daom- <dig>  we only detected the daom- <dig> haplotype, while in the combination daom-240415/daom- <dig>  all seven cultures exhibited the daom- <dig> haplotype.
however, among the  <dig> monosporal cultures of the combination daom-197198/daom- <dig>  two cultures showed the daom- <dig> haplotype and the other  <dig> cultures were generated with the daom- <dig> haplotype.
genotyping results are summarized in table  <dig>  the qpcr data are shown in the additional file  <dig>  interestingly, our results showed an apparent selection and/or segregation bias towards a given haplotype in each combination.
indeed, in combinations where the daom- <dig> haplotype was present, it seemed to be preferentially selected over the two other haplotypes.
also, the daom- <dig> haplotype dominated when it was combined with the daom- <dig> haplotype, the latter was not detected in any monosporal culture lines.
we also corroborated the heteroplasmy detected in an earlier study by de la providencia et al.
<cit> , although at a lower rate, using the same genetic material from single spores, thus confirming non-self fusion between genetically-close isolates as an important mechanism shaping genetic exchange .
two nuclear markers, bg <dig> and bg <dig>  previously described and suggested for r. irregularis genotyping , were used to determine the nuclear inheritance in single spores  <cit>  as well as in monosporal crossed-cultures lines.
for nuclear markers tested in single spore progenies, we observed different scenarios:  only one parental mitochondrial haplotype, with the corresponding parental nuclear genotype  one parental mitochondrial haplotype and both parental nuclear genotype  both parental mitochondrial haplotypes and only one parental nuclear genotype.
interestingly, out of  <dig> successfully analysed single spores, four showed the presence of both parental markers.
missing data correspond to incomplete pcr reactions due low amount of genomic dna remaining from previous experiments published by de la providencia  <cit> .
in crossed-culture lines, no biparental nuclear inheritance was detected .table  <dig> specificity test of qpcr markers.
the marker for each isolate is, at least, specific to the other two isolates used in this study
g. cerebriforme daom227022
adequate controls were carried, in triplicate, both in singleplex and multiplex.
multiplex reaction could not be performed with the combination of  <dig> and  <dig>  because of limitations of the calibrated fluorophores and filter-limitations of the instrument.
for each positive assay, in parenthesis is the mean ct value from triplicates with 2 ng of dna per essay
fig.
<dig> amplification curve of qpcr assay.
a amplification curves of a heteroplasmic spore showing two amplification curves corresponding to each parental haplotype r. irregularis daom <dig>  and r. irregularis daom <dig>  b amplification curve of a homoplasmic monosporal culture assay containing both markers but showing the presence of only one parental haplotype; r. irregularis daom <dig>  and r. irregularis daom <dig>
mitochondrial segregation machinery and nucleoid genes orthology in r. irregularis
the genome of r. irregularis daom- <dig>  <cit>  possesses a nearly full set of genes that exhibited a high degree of similarity to genes coding for the core structure of the msa and involved in the formation of nucleoids in s. cerevisiae and other filamentous fungi .
orthologous candidates in r. irregularis corresponded to the best reciprocal blast hit with the s. cerevisiae msa protein .
to confirm orthology, phylogenetic trees were constructed using all known orthologous genes in fungi and other organisms.
all seven phylogenies performed with clusters of orthologous genes  supported the idea that r. irregularis proteins are putative orthologs of fungal segregation apparatus and nucleoid proteins .
it is noteworthy that in most cases the r. irregularis proteins come out basal in our analyses with low bootstrap support, probably given the lack of sequences availability belonging to lower fungal taxa.
the presence of one s. cerevisiae paralog was observed in the aco <dig> phylogeny, as well as two yeast homologs and one paralog in abf <dig> cog phylogeny.
however, in both cases, the r. irregularis protein grouped with the expected s. cerevisiae protein, playing a role in the mitochondrial segregation processes.
we did not find any paralogs in r. irregularis.
only two genes were missing in r. irregularis, i.e.
mdm <dig> and mdm <dig>  however, their role in the mitochondrial inheritance process remains unclear in yeast.
they are speculated to play an interactive role with the segregation apparatus core components  <cit> .
furthermore, for these two proteins, the search for cogs recovered only a few yeast sequences, which leads us to believe they might not be necessary to the segregation process in filamentous fungi.
all putative orthologs found in r. irregularis are likely functional since they are also found in expressed sequence tags   <cit> .table  <dig> evidence for rhizophagus irregularis protein orthology with the saccharomyces cerevisiae mitochondrial segregation and nucleoid proteic machinery
proteina
adescribed proteins and known functions in saccharomyces cerevisiae , along with the accession number of the r. irregularis ortholog gene candidates
bas shown in additional file 3
cbased on the r. irregularis transcriptome data
dtwo proteins in r. irregularis and s. cerevisiae showed close similarity to each other because they share the same high mobility hmg-box proteic domains
fig.
<dig> representation of the putative mitochondrial segregation apparatus and nucleoid structure in rhizophagus irregularis.
based on protein orthology analysis with s. cerevisiae and other filamentous fungi.
the core components of this machinery consist of a complex of three mitochondrial membrane proteins, which are mmm <dig>  mdm <dig> and mdm <dig> .
three proteins are thought to interact with the main complex, mmm <dig>  mdm <dig> and mdm <dig> .
the proteins responsible for mtdna packaging and nucleoid formation are abf <dig>  aco <dig> and ilv <dig> .
unknown proteins are thought to link the outer membrane complex to the cytoskeleton  and the nucleoids to the inner membrane by interacting with mmm <dig>  the yellow outlining shows proteins for which no putative orthologs were found in r. irregularis
discussion
homoplasmy rather than length-heteroplasmy in monosporal cultures lines from crossed-cultures
previous results have shown that biparental mtdna inheritance leading to a heteroplasmic state occurred in the spore progeny from crossed-cultures of divergent r. irregularis isolates, however heteroplasmy was not detected in germinated spores  <cit> .
using taqman markers developed in the present study, we did not detect heteroplasmy in monosporal cultures from crossed parental isolates but confirmed the heteroplasmic status of the crossed-cultures spores observed in de la providencia et al.
<cit> , although at a lower rate.
several factors might explain why we obtained dissimilar results with markers designed in different intergenic regions.
the isolate-specific mitochondrial markers are designed in highly variable mobile elements rich regions, which have been shown to be recombination prone  <cit>  and might compromise their specificity.
also, the use of the whole genome amplification  technique  <cit>  can introduce significant bias regarding the insertions of snps or the formation of chimeras  <cit> .
the latter could induce an overestimation of the presence of a given haplotype in a sample, especially since the occurrence of allelic drop-out and preferential amplification is well documented in single cell analysis using a pcr based approach  <cit> .
for these reasons, taqman qpcr assay is more reliable to assess mtdna inheritance, given the nature of the highly variable/dynamic regions in which amf isolate specific markers are designed.
earlier studies have demonstrated that genetic exchanges occur via vegetative hyphal fusion , resulting in nuclei coexisting in a common cytoplasm, altering both the plant and fungal phenotypes  <cit> .
based on the formation of anastomosis between genetically-close r. irregularis isolates, several studies have shown that heterogeneous populations of nuclei  <cit>  and mtdna  <cit>  are randomly inherited at different frequencies   <cit>  into the progeny  <cit> .
these findings support the paradigm that based on the coenocytic nature of the amf fungal mycelium, nuclei, mitochondria and other organelles can migrate between close or distant regions of genetically-close fungal colonies and form the so-called common mycorrhizal networks   <cit> .
however, studies on nuclear dynamics along the symbiotic extraradical mycelium challenged this point of view and brought a somewhat discordant note to the supposed continuous stream of mycelial cytoplasm/protoplasm  <cit> .
using in vivo two-photon microscopy techniques, these authors revealed a patchy distribution of nuclei throughout the mycelia and also demonstrated that nuclear flow occurs in pulses, being independent from the cytoplasmic streaming.
these studies suggest that mitochondria, like nuclei, might not be equally distributed along hyphae.
therefore, through anastomosis originating from different individuals, the inheritance of mitochondria into the spore progeny could be strongly related to the number, type and frequency of the organelle at the interaction zone .
the latter could explain the lack of detection of a heteroplasmic state in the progeny spore used to start the monosporal culture.
in the event that one haplotype would be crucially underrepresented and therefore the heteroplasmy undetectable by a qpcr assay, such a small amount of a mitochondrial haplotype could easily be lost through stochastic drift in subsequent subcultures, and consequently homoplasmy would persist at the vegetative phase in these fungi.
the dominance of some haplotypes we observed would still imply a selection and/or segregation bias of one haplotype over the other.
together with our observations of a dominant homoplasmic state in the monosporal crossed-culture lines, these studies strongly support a plausible mechanism of mtdna segregation.
this in turn offers an explanation for the low frequency, revised in this study , of heteroplasmic spores resulting from crossed, geographically and genetically distant isolates  <cit>  and the non-detection of heteroplasmic monosporal crossed-cultures lines arising from these spores.
moreover, results obtained with nuclear markers show that spores can have both parental nuclear markers, whereas only one mitochondrial parental haplotype.
this suggests that both nuclear and mitochondrial inheritance are governed by apparently independent segregation mechanisms.
evidence for a mitochondrial segregation mechanism in rhizophagus irregularis
although orthology analyses were not entirely conclusive given the basal positioning of the r. irregularis sequences in most cases, the identification of putative orthologs in r. irregularis genome and transcriptome to the msa and nucleoid proteins found in other fungi offers a hypothesis to explain the mitochondrial segregation we observed in this study.
the mmm1-like, mdm10-like and mdm12-like proteins are an integral part of the mitochondrial membranes and constitute the core components of the msa.
this proteic complex connects the mitochondrial outer membrane to the cytoskeleton in other fungi  <cit> , while mmm <dig> is thought to link mitochondrial nucleoids to the inner membrane of the mitochondria  <cit> .
proteins involved in the msa have not been reported to play a role in other molecular pathways in s. cerevisiae or filamentous fungi and are well-conserved through fungal evolution  <cit> .
if indeed mitochondria were linked to the cytoskeleton , along with their mtdna bound to the inner membrane, their movement through the cytoplasm would not be as free as actually alleged.
this hypothesis could potentially explain why the fungal protoplasm moves slower after anastomosis between two divergent isolates as observed earlier  <cit> .
also, three proteins of the mitochondrial membrane are also proposed to interact with the core components of the msa, which are mdm <dig>  mdm <dig> and mmm <dig>  they are thought to play a role in nucleoid stability, mitochondrial morphology and distribution in yeast  <cit> .
however, we only found a putative ortholog for the mmm <dig> protein, which might suggest that other proteins could play the same function as mdm <dig> and mdm <dig> or that they are not essential in the amf mitochondrial segregation pathway.
given the phylogenetic divergences between glomeromycota and higher fungi  further phylogenetic analysis will need to include putative orthologous and paralogous genes belonging to basal fungi.
currently ongoing sequencing efforts, such as the jgi  <dig> fungal genome projects, will greatly help to unravel these evolutionary relationships  <cit> .
furthermore, we identified putative orthologs for the three proteic constituents of mt-nucleoids in amf.
the abf <dig> protein is the core packaging element of mt-nucleoids in yeast.
it is a non-histone dna binding high mobility group  protein, which shows homology to nuclear chromatin proteins  <cit> .
this homology explains the difficulty of obtaining the best reciprocal blast .
the cog used to perform the abf <dig> phylogeny  included other proteins that were homologs, and also paralogs, since s. cerevisiae underwent an ancient genome duplication  <cit> .
the two other essential components of mt-nucleoid are the bi-functional proteins aco <dig> and ilv <dig>  <cit> .
they are both highly conserved in amf, probably due to their metabolic role in the citric acid cycle and amino acid biosynthesis, respectively  <cit> .
their expression is metabolically regulated and they play a role in packaging mtdna into favourable conformations, while protecting dna against oxidative stress.
the detection in r. irregularis genome and transcriptome of well conserved putatively orthologous proteins involved exclusively in the msa, suggest that they are functional and that they might also be involved in a similar process in amf.
the existence of mt-nucleoids in amf is important because it accelerates the rate at which mtdna would segregate, since it is directly correlated to the effective population size  <cit> .
nucleoids were also shown to create a genetic bottleneck and to be responsible for the rapid mitochondrial segregation observed in yeast  <cit> .
interspecific differences in the morphology, size and distribution of mitochondrial nucleoids have been visualized using different microscopic methods in yeast  <cit> .
to further investigate these findings in glomeromycota, it would be interesting to perform immunofluorescent experiments targeting amf putative nucleoid proteins, since green fluorescent protein  tagging has been shown to be transient and render unstable transformants in these multinucleated coenocytic organisms  <cit> .
CONCLUSIONS
although mitochondrial homoplasmy seems to be the rule rather than the exception in monosporal progeny originating from in vitro crossed-cultures, the coexistence of numerous mtdna haplotypes in the same cytoplasm and the occurrence of homologous mitochondrial recombination might be common mechanisms in natural populations.
moreover, the observation that mitochondrial and nuclear exchanges are not necessarily correlated suggests the presence of independent mitochondrial and nuclear segregation mechanisms.
this supports the presence of a mitochondrial segregation apparatus in r. irregularis and provides a better understanding of the mitochondrial inheritance process and segregation in these fungi.
much remains to be learned about how proteins potentially implicated in this segregation mechanism interact with other elements and influence both mtdna and nuclear inheritance mechanisms in amf.
it would be interesting to study the dynamics of genetic exchanges in natural and/or disturbed environments in order to unravel the underlying segregation processes to better understand  further application of mitochondrial markers in population genetics studies and  their effects on both the amf and the host plant fitness.

 it has been widely documented that major food crops associate naturally with beneficial soil microbes, including the arbuscular mycorrhizal fungi, an ancient group of root-inhabiting fungi belonging to the phylum glomeromycota. these ecologically crucial symbionts enhance the uptake of water and nutrients of plants they colonize, especially phosphate, as well as protect them against pathogens and play a key role in soil structure. the benefits to plants provided by this association with amf could be enhanced by the manipulation of the fungal partner genetics, as demonstrated by angelard et al. and colard et al., using the model organism rhizophagus irregularis. in both studies, through the exchange of nuclear genetic information via hyphal fusion, in vitro crossed-culture lines and segregated culture lines were generated. this process influenced differentially the transcription of symbiosis-specific genes in rice, resulting in an increase in rice growth by a factor of five. during the last decade numerous studies have been devoted to decipher the nuclear and mitochondrial genome organization patterns, the sexual/asexual conundrum and nuclear segregation process in amf. these data allowed new insights into the nuclear inheritance processes and segregation mechanisms that could occur in these fungi. despite this progress, little is known about mitochondrial inheritance in amf. since the mitochondrial genome encodes essential components of the cellular energy-producing apparatus, understanding mitochondrial dna organization and inheritance in amf is of paramount importance, in order to manage more efficiently mycorrhizal associations at large scale. a previous study showed that amf mtdnas and nuclei migrate massively into spores during their formation, but the fate of each parental haplotype following crosses needs to be investigated. the publication of complete amf mtdna in the last few years allowed demonstrating that these sequences were all homoplasmic within isolates. these studies also showed that genomes are variable in defined intergenic regions between isolates, thus offering an unparalleled opportunity to design isolate-specific markers. recently, de la providencia et al. demonstrated length-heteroplasmy in spores formed near anastomosis regions between geographically distant r. irregularis in vitro isolates, using isolate-specific mitochondrial markers. however, no information is available regarding the persistence of this length-heteroplasmic state in in vitro monosporal culture lines established from crossed parental isolates. such a study is needed not only to understand mtdna inheritance processes and the persistence of mitochondrial haplotypes in stable culture lines, but also to use mtdna as a criterion to define a reliable amf taxonomic unit, either at a genus, species or isolate level. the mitochondrial inheritance process requires an active transport of organelles along the cytoskeleton and relies on membrane fission and fusion events. it plays a crucial role in the adaptation of the organism in energy requirements. most of the molecular machinery and cellular mechanisms mediating these processes have been conserved along the fungal and animal kingdom evolution. in filamentous fungi, sexual crosses lead to uniparental transmission of mitochondria, whereas mitochondria are biparentally inherited in budding yeast. in yeast, microfilaments, such as actin, play an important role in the positioning and motility of mitochondria, but microtubules are the principal mitochondrial transporter in many other fungi. for many years, the study of mitochondrial dynamics in fungi relied mainly on microscopic observations. however, the sequencing of fungal genomes is an important step towards a better understanding of the molecular machinery mediating mitochondrial inheritance. in that regard, the recently available r. irregularis genomic sequence data provide new tools to identify molecular components involved in mitochondrial dynamics in amf. no study has yet investigated potential mitochondrial segregation and/or inheritance mechanisms occurring in amf. in the model organism saccharomyces cerevisiae, mitochondrial inheritance is controlled by the mitochondrial segregation apparatus, which ensures a reliable transmission of mitochondrial organelles and their genomes to the progeny. searches of sequence databases reveal evolutionarily conserved proteins for all known budding yeast msa components in schizosaccharomyces pombe and in filamentous fungi such as neurospora crassa and aspergillus nidulans, notably. this suggests that the core machinery of mitochondrial inheritance has been conserved during evolution, even in organisms that use different cytoskeletal systems for organellar transport. the msa in s. cerevisiae is a trans-membrane proteic complex consisting of three mitochondrial membrane proteins, forming the core component of the apparatus, along with mmm mdm and mdm which interact with the core component. this proteic complex links the mitochondrial outer membrane to the actin cytoskeleton and the mitochondrial inner membrane to mitochondrial nucleoids, which are clusters of similar mtdnas packaged by proteins abf aco and ilv and maintained together by holliday junctions. however, the molecular machinery of mtdna inheritance remains largely unknown in many other organisms. in this study, we tested the hypothesis that monosporal crossed-culture lines would maintain the mitochondrial length-heteroplasmy previously observed in r. irregularis in vitro spore progenies arising from crossed-cultures of genetically divergent isolates. in addition, we investigated nuclear inheritance following anastomosis in spore progenies and monosporal crossed-culture lines. furthermore, we searched for the existence of a putative msa in glomeromycota, by finding the best reciprocal blast of yeast msa proteins in the published genome of r. irregularis and its transcriptome, and tested their orthology with all available sequences on the database.  germination and fungal development of monosporal culture lines cultures from each combination were performed. after identifying interaction zones between mycelia from different isolates, which were characterized by the formation of hyphal contacts, randomly chosen spores were harvested from the interaction zone of each combination, individually cut out from the mycelium and placed in a new petri dish in association with ri t-dna transformed chicory roots as described in the methods section. for each combination, approximately half of the inoculated spores germinated. the observed germination rate corresponds to rates previously observed in four in vitro cultured glomeraceae species. following root contact and colonisation, some cultures stopped their growth, without producing any spores, and therefore could not be used in this study. the rate of mycelium development and spore production greatly varied among cultures, the combination of isolates daom- and daom- being the most successful, resulting in colonized dishes containing between and newly formed spores. the combination of isolates daom- and daom- produced seven colonized petri dishes with few, newly formed spores. we obtained poorly developed cultures from isolates combination daom-234328/daom- and only nine produced sufficient amount of fungal material required for further analysis. in total, monosporal crossed-cultures lines were genotyped.fig. schematic drawing of the experimental design. a observation of hyphal contacts between two clusters of spores from different isolates b identification of spores produced in anastomosis regions and inoculation of a single spore on a new petri dish with ri t-dna transformed chicory roots i.e. monosporal cultures. after germination and colonisation of the petri dish, spores are extracted and their dna is used for genotyping of the monosporal culture genotyping analysis all mitochondrial markers showed to be isolate-specific when challenged against other rhizophagus isolates. the monosporal crossed-culture lines genotyped by qpcr approach presented only one parental mtdna haplotype. in all nine progenies of the combination daom-197198/daom- we only detected the daom- haplotype, while in the combination daom-240415/daom- all seven cultures exhibited the daom- haplotype. however, among the monosporal cultures of the combination daom-197198/daom- two cultures showed the daom- haplotype and the other cultures were generated with the daom- haplotype. genotyping results are summarized in table  the qpcr data are shown in the additional file interestingly, our results showed an apparent selection and/or segregation bias towards a given haplotype in each combination. indeed, in combinations where the daom- haplotype was present, it seemed to be preferentially selected over the two other haplotypes. also, the daom- haplotype dominated when it was combined with the daom- haplotype, the latter was not detected in any monosporal culture lines. we also corroborated the heteroplasmy detected in an earlier study by de la providencia et al., although at a lower rate, using the same genetic material from single spores, thus confirming non-self fusion between genetically-close isolates as an important mechanism shaping genetic exchange. two nuclear markers, bg and bg previously described and suggested for r. irregularis genotyping, were used to determine the nuclear inheritance in single spores as well as in monosporal crossed-cultures lines. for nuclear markers tested in single spore progenies, we observed different scenarios: only one parental mitochondrial haplotype, with the corresponding parental nuclear genotype one parental mitochondrial haplotype and both parental nuclear genotype both parental mitochondrial haplotypes and only one parental nuclear genotype. interestingly, out of successfully analysed single spores, four showed the presence of both parental markers. missing data correspond to incomplete pcr reactions due low amount of genomic dna remaining from previous experiments published by de la providencia. in crossed-culture lines, no biparental nuclear inheritance was detected.table specificity test of qpcr markers. the marker for each isolate is, at least, specific to the other two isolates used in this study g. cerebriforme daom227022 adequate controls were carried, in triplicate, both in singleplex and multiplex. multiplex reaction could not be performed with the combination of and because of limitations of the calibrated fluorophores and filter-limitations of the instrument. for each positive assay, in parenthesis is the mean ct value from triplicates with 2 ng of dna per essay fig. amplification curve of qpcr assay. a amplification curves of a heteroplasmic spore showing two amplification curves corresponding to each parental haplotype r. irregularis daom and r. irregularis daom b amplification curve of a homoplasmic monosporal culture assay containing both markers but showing the presence of only one parental haplotype; r. irregularis daom and r. irregularis daom mitochondrial segregation machinery and nucleoid genes orthology in r. irregularis the genome of r. irregularis daom- possesses a nearly full set of genes that exhibited a high degree of similarity to genes coding for the core structure of the msa and involved in the formation of nucleoids in s. cerevisiae and other filamentous fungi. orthologous candidates in r. irregularis corresponded to the best reciprocal blast hit with the s. cerevisiae msa protein. to confirm orthology, phylogenetic trees were constructed using all known orthologous genes in fungi and other organisms. all seven phylogenies performed with clusters of orthologous genes supported the idea that r. irregularis proteins are putative orthologs of fungal segregation apparatus and nucleoid proteins. it is noteworthy that in most cases the r. irregularis proteins come out basal in our analyses with low bootstrap support, probably given the lack of sequences availability belonging to lower fungal taxa. the presence of one s. cerevisiae paralog was observed in the aco phylogeny, as well as two yeast homologs and one paralog in abf cog phylogeny. however, in both cases, the r. irregularis protein grouped with the expected s. cerevisiae protein, playing a role in the mitochondrial segregation processes. we did not find any paralogs in r. irregularis. only two genes were missing in r. irregularis, i.e. mdm and mdm however, their role in the mitochondrial inheritance process remains unclear in yeast. they are speculated to play an interactive role with the segregation apparatus core components. furthermore, for these two proteins, the search for cogs recovered only a few yeast sequences, which leads us to believe they might not be necessary to the segregation process in filamentous fungi. all putative orthologs found in r. irregularis are likely functional since they are also found in expressed sequence tags.table evidence for rhizophagus irregularis protein orthology with the saccharomyces cerevisiae mitochondrial segregation and nucleoid proteic machinery proteina adescribed proteins and known functions in saccharomyces cerevisiae, along with the accession number of the r. irregularis ortholog gene candidates bas shown in additional file 3 cbased on the r. irregularis transcriptome data dtwo proteins in r. irregularis and s. cerevisiae showed close similarity to each other because they share the same high mobility hmg-box proteic domains fig. representation of the putative mitochondrial segregation apparatus and nucleoid structure in rhizophagus irregularis. based on protein orthology analysis with s. cerevisiae and other filamentous fungi. the core components of this machinery consist of a complex of three mitochondrial membrane proteins, which are mmm mdm and mdm. three proteins are thought to interact with the main complex, mmm mdm and mdm. the proteins responsible for mtdna packaging and nucleoid formation are abf aco and ilv. unknown proteins are thought to link the outer membrane complex to the cytoskeleton and the nucleoids to the inner membrane by interacting with mmm the yellow outlining shows proteins for which no putative orthologs were found in r. irregularis discussion homoplasmy rather than length-heteroplasmy in monosporal cultures lines from crossed-cultures previous results have shown that biparental mtdna inheritance leading to a heteroplasmic state occurred in the spore progeny from crossed-cultures of divergent r. irregularis isolates, however heteroplasmy was not detected in germinated spores. using taqman markers developed in the present study, we did not detect heteroplasmy in monosporal cultures from crossed parental isolates but confirmed the heteroplasmic status of the crossed-cultures spores observed in de la providencia et al., although at a lower rate. several factors might explain why we obtained dissimilar results with markers designed in different intergenic regions. the isolate-specific mitochondrial markers are designed in highly variable mobile elements rich regions, which have been shown to be recombination prone and might compromise their specificity. also, the use of the whole genome amplification technique can introduce significant bias regarding the insertions of snps or the formation of chimeras. the latter could induce an overestimation of the presence of a given haplotype in a sample, especially since the occurrence of allelic drop-out and preferential amplification is well documented in single cell analysis using a pcr based approach. for these reasons, taqman qpcr assay is more reliable to assess mtdna inheritance, given the nature of the highly variable/dynamic regions in which amf isolate specific markers are designed. earlier studies have demonstrated that genetic exchanges occur via vegetative hyphal fusion, resulting in nuclei coexisting in a common cytoplasm, altering both the plant and fungal phenotypes. based on the formation of anastomosis between genetically-close r. irregularis isolates, several studies have shown that heterogeneous populations of nuclei and mtdna are randomly inherited at different frequencies into the progeny. these findings support the paradigm that based on the coenocytic nature of the amf fungal mycelium, nuclei, mitochondria and other organelles can migrate between close or distant regions of genetically-close fungal colonies and form the so-called common mycorrhizal networks. however, studies on nuclear dynamics along the symbiotic extraradical mycelium challenged this point of view and brought a somewhat discordant note to the supposed continuous stream of mycelial cytoplasm/protoplasm. using in vivo two-photon microscopy techniques, these authors revealed a patchy distribution of nuclei throughout the mycelia and also demonstrated that nuclear flow occurs in pulses, being independent from the cytoplasmic streaming. these studies suggest that mitochondria, like nuclei, might not be equally distributed along hyphae. therefore, through anastomosis originating from different individuals, the inheritance of mitochondria into the spore progeny could be strongly related to the number, type and frequency of the organelle at the interaction zone. the latter could explain the lack of detection of a heteroplasmic state in the progeny spore used to start the monosporal culture. in the event that one haplotype would be crucially underrepresented and therefore the heteroplasmy undetectable by a qpcr assay, such a small amount of a mitochondrial haplotype could easily be lost through stochastic drift in subsequent subcultures, and consequently homoplasmy would persist at the vegetative phase in these fungi. the dominance of some haplotypes we observed would still imply a selection and/or segregation bias of one haplotype over the other. together with our observations of a dominant homoplasmic state in the monosporal crossed-culture lines, these studies strongly support a plausible mechanism of mtdna segregation. this in turn offers an explanation for the low frequency, revised in this study, of heteroplasmic spores resulting from crossed, geographically and genetically distant isolates and the non-detection of heteroplasmic monosporal crossed-cultures lines arising from these spores. moreover, results obtained with nuclear markers show that spores can have both parental nuclear markers, whereas only one mitochondrial parental haplotype. this suggests that both nuclear and mitochondrial inheritance are governed by apparently independent segregation mechanisms. evidence for a mitochondrial segregation mechanism in rhizophagus irregularis although orthology analyses were not entirely conclusive given the basal positioning of the r. irregularis sequences in most cases, the identification of putative orthologs in r. irregularis genome and transcriptome to the msa and nucleoid proteins found in other fungi offers a hypothesis to explain the mitochondrial segregation we observed in this study. the mmm1-like, mdm10-like and mdm12-like proteins are an integral part of the mitochondrial membranes and constitute the core components of the msa. this proteic complex connects the mitochondrial outer membrane to the cytoskeleton in other fungi, while mmm is thought to link mitochondrial nucleoids to the inner membrane of the mitochondria. proteins involved in the msa have not been reported to play a role in other molecular pathways in s. cerevisiae or filamentous fungi and are well-conserved through fungal evolution. if indeed mitochondria were linked to the cytoskeleton, along with their mtdna bound to the inner membrane, their movement through the cytoplasm would not be as free as actually alleged. this hypothesis could potentially explain why the fungal protoplasm moves slower after anastomosis between two divergent isolates as observed earlier. also, three proteins of the mitochondrial membrane are also proposed to interact with the core components of the msa, which are mdm mdm and mmm they are thought to play a role in nucleoid stability, mitochondrial morphology and distribution in yeast. however, we only found a putative ortholog for the mmm protein, which might suggest that other proteins could play the same function as mdm and mdm or that they are not essential in the amf mitochondrial segregation pathway. given the phylogenetic divergences between glomeromycota and higher fungi further phylogenetic analysis will need to include putative orthologous and paralogous genes belonging to basal fungi. currently ongoing sequencing efforts, such as the jgi fungal genome projects, will greatly help to unravel these evolutionary relationships. furthermore, we identified putative orthologs for the three proteic constituents of mt-nucleoids in amf. the abf protein is the core packaging element of mt-nucleoids in yeast. it is a non-histone dna binding high mobility group protein, which shows homology to nuclear chromatin proteins. this homology explains the difficulty of obtaining the best reciprocal blast. the cog used to perform the abf phylogeny included other proteins that were homologs, and also paralogs, since s. cerevisiae underwent an ancient genome duplication. the two other essential components of mt-nucleoid are the bi-functional proteins aco and ilv. they are both highly conserved in amf, probably due to their metabolic role in the citric acid cycle and amino acid biosynthesis, respectively. their expression is metabolically regulated and they play a role in packaging mtdna into favourable conformations, while protecting dna against oxidative stress. the detection in r. irregularis genome and transcriptome of well conserved putatively orthologous proteins involved exclusively in the msa, suggest that they are functional and that they might also be involved in a similar process in amf. the existence of mt-nucleoids in amf is important because it accelerates the rate at which mtdna would segregate, since it is directly correlated to the effective population size. nucleoids were also shown to create a genetic bottleneck and to be responsible for the rapid mitochondrial segregation observed in yeast. interspecific differences in the morphology, size and distribution of mitochondrial nucleoids have been visualized using different microscopic methods in yeast. to further investigate these findings in glomeromycota, it would be interesting to perform immunofluorescent experiments targeting amf putative nucleoid proteins, since green fluorescent protein tagging has been shown to be transient and render unstable transformants in these multinucleated coenocytic organisms.  although mitochondrial homoplasmy seems to be the rule rather than the exception in monosporal progeny originating from in vitro crossed-cultures, the coexistence of numerous mtdna haplotypes in the same cytoplasm and the occurrence of homologous mitochondrial recombination might be common mechanisms in natural populations. moreover, the observation that mitochondrial and nuclear exchanges are not necessarily correlated suggests the presence of independent mitochondrial and nuclear segregation mechanisms. this supports the presence of a mitochondrial segregation apparatus in r. irregularis and provides a better understanding of the mitochondrial inheritance process and segregation in these fungi. much remains to be learned about how proteins potentially implicated in this segregation mechanism interact with other elements and influence both mtdna and nuclear inheritance mechanisms in amf. it would be interesting to study the dynamics of genetic exchanges in natural and/or disturbed environments in order to unravel the underlying segregation processes to better understand further application of mitochondrial markers in population genetics studies and their effects on both the amf and the host plant fitness. 
however, the persistence of this phenomenon in monosporal culture lines from crossed parental isolates, its correlation with nuclear exchanges and the potential mechanisms responsible for mitochondrial inheritance are still unknown. we present the basic building blocks for a better understanding of the mitochondrial inheritance process and segregation in these important symbiotic fungi. we also investigated the presence of a putative mitochondrial segregation apparatus in glomeromycota by identifying proteins similar to those found in other fungal groups. these results strongly support the presence of a nuclear-independent mitochondrial segregation mechanism in r. irregularis. furthermore, a nearly complete set of genes was identified with putative orthology to those found in other fungi and known to be associated with the mitochondrial segregation in saccharomyces cerevisiae and filamentous fungi. our findings suggest that mitochondrial segregation might take place either during spore formation or colony development and that it might be independent of the nuclear segregation machinery. arbuscular mycorrhizal fungimitochondrial segregationanastomosismitochondrial nucleoidsprotein orthologymonosporal culturesissue-copyright-statement© the author 2016 using the amf model organism r. irregularis, we tested whether the presence of a heteroplasmic state in progeny spores was linked to the occurrence of nuclear exchanges and whether the previously observed heteroplasmic state persisted in monosporal in vitro crossed-culture lines. however, only one parental mitochondrial dna and nuclear genotype were recovered in each monosporal crossed-cultures, with an overrepresentation of certain mitochondrial haplotypes. arbuscular mycorrhizal fungi are members of the phylum glomeromycota, an early divergent fungal lineage that forms symbiotic associations with the large majority of land plants. we observed the occurrence of biparental inheritance both for mitochondrial and nuclear markers tested in single spores obtained from crossed-isolates.
 arbuscular mycorrhizal fungi are members of the phylum glomeromycota, an early divergent fungal lineage that forms symbiotic associations with the large majority of land plants. these organisms are asexual obligate biotrophs, meaning that they cannot complete their life cycle in the absence of a suitable host. these fungi can exchange genetic information through hyphal fusions with genetically compatible isolates belonging to the same species. the occurrence of transient mitochondrial length-heteroplasmy through anastomosis between geographically distant rhizophagus irregularis isolates was previously demonstrated in single spores resulting from crossing experiments. however, the persistence of this phenomenon in monosporal culture lines from crossed parental isolates, its correlation with nuclear exchanges and the potential mechanisms responsible for mitochondrial inheritance are still unknown. using the amf model organism r. irregularis, we tested whether the presence of a heteroplasmic state in progeny spores was linked to the occurrence of nuclear exchanges and whether the previously observed heteroplasmic state persisted in monosporal in vitro crossed-culture lines. we also investigated the presence of a putative mitochondrial segregation apparatus in glomeromycota by identifying proteins similar to those found in other fungal groups.  we observed the occurrence of biparental inheritance both for mitochondrial and nuclear markers tested in single spores obtained from crossed-isolates. however, only one parental mitochondrial dna and nuclear genotype were recovered in each monosporal crossed-cultures, with an overrepresentation of certain mitochondrial haplotypes. these results strongly support the presence of a nuclear-independent mitochondrial segregation mechanism in r. irregularis. furthermore, a nearly complete set of genes was identified with putative orthology to those found in other fungi and known to be associated with the mitochondrial segregation in saccharomyces cerevisiae and filamentous fungi.  our findings suggest that mitochondrial segregation might take place either during spore formation or colony development and that it might be independent of the nuclear segregation machinery. we present the basic building blocks for a better understanding of the mitochondrial inheritance process and segregation in these important symbiotic fungi. the comprehension of these processes is of great importance since it has been shown that different segregated lines of the same isolate can have variable effects on the host plant. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arbuscular mycorrhizal fungimitochondrial segregationanastomosismitochondrial nucleoidsprotein orthologymonosporal culturesissue-copyright-statement© the author 2016 