Droplet digital PCR (ddPCR) analysis

DNA extracted from soil and root samples was tested for the presence of R. irregulare DAOM 197198 using a probe-based ddPCR assay on the Bio-Rad QX100 Droplet Digital PCR System (Bio-Rad Laboratories, Inc., Hercules, California). This technique allows accurate detection and quantification of target DNA by partitioning the sample into >10,000 droplets prior to the PCR reaction. Primers and probe for this assay were designed to target the cox3-rnl intergenic region (Kokkoris et al., 2019), and the sequences are as follows (see Table S2 for further details):

Forward: 5′-AGCAAATCTAAGTTCCTCAGAG-3′

Reverse: 5′-ACTTCTATGGCTTTGTACAGG-3′

Probe: 5′-FAM/CCCACCAGG/ZEN/GCAGATTAATCTTCCTT/3IABKFQ-3′

These primers amplified only DAOM 197198 of 23 R. irregulare strains and 12 AM fungal species tested and have no predicted specificity to other AM fungal isolates. They also did not amplify in environmental soil samples taken from natural areas in nearby Merritt, BC, that should contain a similar background AM fungal community with a minimal risk of agriculturally introduced AM fungi.

The reaction contains 10 µl Bio-Rad ddPCR Supermix for Probes, 2 µl of Integrated DNA Technologies PrimeTime qPCR Assay mixture with a final concentration of 500 nM each forward and reverse primer and 250 nM probe, 1 µl of template (extracted DNA from soil), and 7 µl of dH20 for a total of 20 µl. Positive controls contained amplicon of the target sequence which was produced from DNA extracted from onion roots colonized by the biofertilizer used, and negative controls received sterile water. Three negative control wells were included on each plate, and all three wells must have zero positive droplets for the results of the plate to be considered valid. The total volume was transferred to the Bio-Rad QX100 Droplet Generator with 70 µl of Bio-Rad Droplet Generator Oil for Probes to produce 40 µl of droplets. We required a minimum of 10,000 droplets in an individual well during measurement for the well to be considered valid. The droplets were cycled at: 95 °C for 10 min, 50 cycles of 94 °C for 30 s and 57.8 °C for 2 min, 98 °C for 10 min, and 4 °C hold. After cycling, the amount of fluorescence in the droplets was measured by the Bio-Rad QX100 Droplet Reader. The number of positive (fluorescent) droplets in the sample is converted to the estimated number of target copies per µl of template using QuantaSoft (Version 1.7.4.0917, Bio-Rad Laboratories Inc.). The threshold for a positive call was manually set just below the bottom of the cloud produced in the positive control well on each plate (see Fig. S1). A high threshold was chosen because droplets that do not reach the fluorescence of this level are more likely false positives, as droplets that contain at least one copy of target sequence should reach roughly the same final fluorescence. Further, the primer set alone does amplify non-target sequence from the site, and although the probe-binding region is not a good match on these non-target amplicons, it may bind occasionally and produce some fluorescence (Fig. S2). However, if this does occur, the fluorescence should be much lower than true positives and thus excluded by our high threshold.