title: Arbuscular mycorrhizal fungal ITS2, isolated from soil sampled adjacent to a Eucalyptus seedling and amplified using nested PCR
design: (Reference list can be found at https://doi.org/10.1111/mec.14536) DNA was extracted from each sample using a PowerSoil DNA isolation kit (MO BIO, Carlsbad, USA) following the manufacturer's instructions, except DNA was eluted with 50 ul of autoclaved miliQ water. An additional step involving sample disruption was conducted prior to the extraction, using 5 mm stainless steel beads for 30 s (soil), 1 min (roots) or 3 min (litter) 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/uL in 10uL 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 Krueger et al. (2009), which target specifically AM fungi and provide coverage of all AM fungal lineages. Each reaction used 5 uL KAPA mix (Kapa Biosystem, Wilmington, USA), 0.4uL LSU_Ar (10uM), 0.4uL SSU_Af (10uM) and 4.2uL of diluted DNA template. Thermocycling conditions for this first step were: 95degC for 3 min, 30 cycles of 98degC for 20 s, 60degC for 30 s and 72degC for 50 s, followed by 72degC for 2 min. In the second PCR round, each reaction contained 9 uL of master mix and 1 uL diluted PCR product (2 uL DNA / 98 uL water). The master mix contained: 5 uL KAPA mix, 0.4 uL LSU_Br (10uM), 0.4 uL SSU_Cf (10uM), 3.2 uL water. Thermocycling conditions for this first step were: 95degC for 3 min, 30 cycles of 98degC for 20 s, 60degC for 30 s and 72degC for 50 s, followed by 72degC 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/uL in 20 uL. The ITS2 region was targeted for sequencing using fITS7 (5'-GTGARTCATCGAATCTTTG-3'; Ihrmark et al. 2012) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; White et al. 1990); genomic libraries were prepared using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) and paired-end (2 x 251 bases) sequencing was performed on the Illumina MiSeq platform at the Ramaciotti Centre for Genomics (Sydney, NSW) or the Western Sydney University Next Generation Sequencing Facility (Richmond, NSW).