Independent mitochondrial and nuclear exchanges arising in Rhizophagus irregularis crossed-isolates support the presence of a mitochondrial segregation mechanism

Growth conditions and maintenance of fungal cultures and roots

Monoxenically produced spores of Rhizophagus irregularis isolates DAOM-197198 (Pont-Rouge, Quebec, Canada), DAOM-234328 (Finland) and DAOM-240415
(Dufrost, Manitoba, Canada) were provided by the DAOM collection (Ottawa, Ontario,
Canada). These three isolates were selected because of their different geographical
origins and because their mitochondrial genomes have been fully sequenced 18], 20]. Spores were subcultured in association with Ri T-DNA transformed chicory (Cichorium intybus) roots on a modified minimal (MM) medium 62] solidified with 0.4 % (w/v) gellan gum (Sigma). Cultures of the three R. irregularis isolates were incubated in the dark in an inverted position at 25 °C. Several thousand
spores and extraradical mycelia were obtained over a period of 12 weeks. Ri T-DNA
transformed chicory roots were routinely propagated by placing actively growing root
apexes on MM medium with subsequent incubation at 25 °C in the dark.

Crossed cultures and monosporal culture lines

Twenty crossed cultures from each combination (DAOM-197198/DAOM-234328; DAOM-197198/DAOM-240415;
DAOM-234328/DAOM-240415) were performed by inoculating a hundred spores in close vicinity
of Ri T-DNA transformed chicory roots, opposing each other at the extreme side of
a Petri plate (Fig. 1). Both colonies were checked weekly and their growth was traced in order to identify
interaction zones between mycelia from different isolates, which were characterized
by the formation of hyphal contacts. Subsequently, these contacts were checked under
a Discovery V12 stereomicroscope (Carl Zeiss, Canada) at magnifications of 6.7–40×.
Bright-field microscopy (Axio Imager M1, Carl Zeiss) was also used to observe details
of the hyphal interactions at higher magnifications.

After 15 weeks of growth, randomly chosen spores (i.e. progenies) were harvested from
the interaction zone of each combination, individually cut out from the mycelium and
placed in a new Petri plate (90 mm) containing MM medium in the close vicinity of
a Ri-T transformed chicory root. For each combination, 50 replicates, consisting of
one single spore associated with a chicory root were prepared. Each plate was checked
weekly for germination, root colonization and colony development over the next 11 weeks.

DNA extraction

Spores and hyphae were harvested by dissolving the gellan gum matrix in which cultures
were grown in a solution containing 0.0083 M sodium citrate and 0.0017 M citric acid.
Extracted fungal material was observed under a binocular microscope in order to detect
and remove any root contaminants. Spores and hyphae were gently crushed in a 1.5 ml
microtube using a sterilized pestle. DNA was extracted using the DNeasy Plant Mini
kit (Qiagen, Toronto, ON), according to the manufacturer’s instructions.

Mitochondrial marker development, genotyping by real-time PCR and sequencing of progeny
spores

In order to efficiently detect low copy numbers of a given mitochondrial haplotype
in each monosporal culture line, TaqMan isolate-specific markers were developed for
each parental isolate and were used to genotype monosporal crossed culture lines (Table 1 and see Additional files 1 and 4). Genotyping of monosporal cultures lines resulting from each crossing experiment
were performed in three replicates with approximately 2.5 ng of DNA per replicate.
Reactions were carried in 20 ?l, using iTaq™ Universal Probes Supermix (Bio-Rad, Canada)
with final primers concentration at 0.5 ?M and final probes concentrations at 0.1 ?M.
Reactions were performed using 5?FAM, 5?VIC and 5?NED dyes and their corresponding
quencher. However, due to limited availability of calibrated fluorophores and filter
limitations of the instrument, we could only perform multiplex qPCR for combinations
DAOM-197198/DAOM-240415 and DAOM-197198/DAOM-234328 and perform singleplex qPCR reactions
for the combination DAOM-240415/DAOM234328. Real time PCR assays were performed on
a ViiAâ„¢ 7 Real-Time PCR System (LifeTechnologies, Canada). PCR amplicons were visualized
on a 2.5 % agarose gel stained with GelRed (Invitrogen, Canada). Successful PCR amplicons
were sequenced according to the conventional Sanger technique at the Genome Quebec
Innovation Center (Montreal, QC). As a control for marker specificity, the new TaqMan
markers developed were challenged against R. irregularis (DAOM 242422 and DAOM 234179) and R. clarus (MUCL 46238). In addition, these markers were also tested in the spore progeny issued
from the previous study of de la Providencia et al. 20], in order to confirm these earlier results.

Sequence-based nuclear markers and genotyping

In order to assess the nuclear inheritance in single spores and monosporal cultures
both arising from crossed-isolates, two previously designed and used nuclear markers
were tested, locus BG112 5], 38] and locus BG62 37] (see Additional file 4). PCR reactions were carried in 25 ?l, using Taq DNA Polymerase (Quiagen, Canada)
with final primers concentration at 0.5 ?M, dNTP concentration at 0.2 mM and approximately
2.5 ng of DNA. PCR products were separated in a 20 cm long, 7 % acrylamide gel using
DCode Universal Mutation Detection System (Bio-Rad, Canada). Gels were run at 100
vts, 18 h for marker BG112 and 15 h for marker BG62, then stained in a SybrSafe bath
for 40 min and visualized in a Gel Doc XR System (Bio-Rad, Canada) (see Additional
file 5).

Protein orthology and phylogenetic analysis

Each amino acid sequences of the Saccharomyces cerevisiae MSA and/or protein involved in nucleoid formation (i.e. MMM1, MDM10, MDM12, MMM2,
MDM31, MDM32, ABF2, ACO1 and ILV5) were searched across the R. irregularis DAOM-197198 genome assembly 7] and transcriptome 36], using TBLASTN. Orthologous candidates in R. irregularis genome were identified using the best-reciprocal BLAST hit to these proteins. Seven
putative orthologous sequences were retrieved in R. irregularis; Uniprot accession numbers U9U1X0, U9UEK3, U9UFF5, U9TTI3, U9UF16, U9UI83 and U9UJR1,
respectively. Furthermore, clusters of orthologous genes (COGs), gathering sequences
from numerous organisms, were determined using STRING version 9.05 63] for each protein candidate. The resulting COGs were aligned using COBALT version
2.01 64]. Phylogenetic analyses and finding the best evolutionary model was done using the
integrative software TOPALI version 2.5 65]. For each protein candidate, maximum likelihood phylogenetic analyses were done with
the closest orthologs found in fungi. Phylogenies were performed accordingly to its
predicted model: the MMM1 protein phylogeny was done using the JTT?+?I?+?G model,
MMM2 with JTT?+?I?+?G, MDM12 with JTT?+?G, MDM10 with WAG?+?I?+?G, ACO1 with WAG?+?I?+?G, ABF2 with WAG?+?I?+?G, and finally the ILV5 phylogeny was performed using the WAG?+?G
model. The robustness of internal branches was evaluated based on 1000 bootstrap replicates
(60 % cut-off). Tree figures were completed using TreeGraph version 2.0.47 66].