title: 18S rRNA amplicon of rhizosphere AMF community
design: To collect AM fungal communities, the sterilized soil core was pushed approximately 5-7cm into the rhizosphere microhabitat along the root base where the plant stem meets the soil. All rhizosphere samples were stored on dry ice until temporary storage (-80C) at the Southwest Research Station in Portal, Arizona, USA. Samples were then shipped on dry ice to the University of Tennessee, Knoxville and immediately stored at -80C upon arrival. Prior to DNA isolation, all rhizosphere soil samples were thawed for one hour at room temperature. DNA isolation proceeded with 250 mg of rhizosphere soil per sample and the DNeasy PowerSoil HTP 96 Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA). The final elution volume was modified so that 50 L of Solution C6 was used. Additionally, the solutions sat on the DNA membrane for 5 minutes at room temperature prior to centrifuging the concentrated DNA down into a sterile 96 well collection plate. Libraries of the 18S rRNA gene for AM fungal communities were amplified using a nested PCR protocol. The first reaction utilized the primer pair NS1/NS4, which amplified approximately a 1200 bp product (White et al.,1990; Raja et al., 2017). The forward NS1 primer sequence was 5-GTAGTCATATGCTTGTCTC-3 and the reverse NS4 primer sequence was 5- CTTCCGTCAATTCCTTTAAG-3. Each reaction used 2.5 L of the DNA template, 5 L of both the forward and reverse primers, and 12.5 L of KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, MA, USA). This reaction ran at 95C for 3 minutes, followed by 30 cycles of: 95C for 30 seconds, 40C for 1 minute, and 72C for 1 minute with a final elongation step of 72C for 10 minutes. These reactions produced 25 L of PCR product. The following nested reaction amplified ~400 bp with the mixed primer pair, NS31 (Simon et al., 1992) and AML2 (Lee et al., 2008), which contain the sequences 5-TTGGAGGGCAAGTCTGGTGCC-3 and 5-GAACCCAAACACTTTGGTTTC-3, respectively. These reactions required 2.5 L of product from the first reactions, 5 L of NS31 and AML2, and 12.5 L of KAPA HiFi HotStart Ready Mix. These reactions began with 95C for 5 minutes followed by 40 cycles of: 95C for 45 seconds, 63.1C for 1 minute, 72C for 1.5 minutes with a final elongation step of 72C for 10 minutes. This nested PCR produced 25 L of AM fungal specific 18S rRNA gene product per sample. All 18S PCR products were then stored at -20C until further use. These primers were obtained from the i5/i7 Nextera XT Index Kit. For the indexing PCR, 5 L of purified PCR product from the previous reaction is required, along with 5 L of the uniquely combined forward and reverse Nextera index primers, 25 L of KAPA HiFi HotStart Ready Mix, and 10L of sterile nuclease free water. These reactions ran for an initial cycle of 95C for 3 minutes followed by an 8-cycle sequence of 95C for 3 seconds, 55C for 30 seconds, and 72C for 30 seconds and held at 72C for 5 minutes. These reactions yielded 50 L of product, that was purified with 56 L of Agencourt AMPure XP magnetic beads, and two washes of 220 L freshly prepared 80% ethanol. Cleaned and purified product was then resuspended in 25L of Tris-HCl. All DNA libraries were then pooled by equal concentrations. The pooled solution was diluted to 4pM with Tris and loaded onto a V3 flow cell with a 20% PhiX spike. This flow cell was run on an Illumina MiSeq instrument at the University of Tennessee Genomics Core, Knoxville, Tennessee, USA set to a 2x300 paired end cycle.