Arbuscular mycorrhizal fungal ITS2, isolated from soil and amplified using nested PCR
(Reference list can be found at https://doi.org/10.1111/mec.14536) DNA was extracted from each root sample using a PowerSoil DNA isolation kit (MO BIO, Carlsbad, USA) following the manufacturer_s instructions. An additional step involving root tissue disruption was conducted prior to the extraction, using 5 mm stainless steel beads for 1 min at 30 Hz in a TissueLyser (Qiagen, Germany). Extracted DNA was quantified using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific, Wilmington, USA), and then diluted to 2 ng/_L in 10_L prior to polymerase chain reaction (PCR) amplification. All PCRs were performed in PTC-200 thermal cyclers (Bio-Rad, California, USA). DNA samples were amplified using PCR primers described in Kr_ger et al. (2009), which target specifically AM fungi and provide coverage of all AM fungal lineages. Each reaction used 5 _L KAPA mix (Kapa Biosystem, Wilmington, USA), 0.4_L LSU_Ar (10_M), 0.4_L SSU_Af (10_M) and 4.2_L of diluted DNA template. Thermocycling conditions for this first step were: 95_C for 3 min, 30 cycles of 98_C for 20 s, 60_C for 30 s and 72_C for 50 s, followed by 72_C for 2 min. In the second PCR round, each reaction contained 9 _L of master mix and 1 _L diluted PCR product (2 _L DNA / 98 _L water). The master mix contained: 5 _L KAPA mix, 0.4 _L LSU_Br (10_M), 0.4 _L SSU_Cf (10_M), 3.2 _L water. Thermocycling conditions for this first step were: 95_C for 3 min, 30 cycles of 98_C for 20 s, 60_C for 30 s and 72_C for 50 s, followed by 72_C for 2 min. The PCR products were purified using Agencourt AMPure XP system (Beckman Coulter, Lane Cove, NSW, Australia) and diluted with PCR-grade water to 5 ng/_L in 20 _L. Samples were pooled by plant individual by mixing 10_L of each sample. The ITS2 region was sequenced by Illumina MiSeq at the Ramaciotti Centre for Genomics (NSW, Australia), using fITS7 (5_-GTGARTCATCGAATCTTTG-3_; (Ihrmark et al. 2012) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; White et al. 1990), and genomic libraries were prepared using the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Paired-end (2 x 251 bases) sequencing was performed on the Illumina MiSeq platform at the Ramaciotti Centre for Genomics (Sydney, NSW).