fungal amplicons of soil conditioned by Eucalyptus dalrympleana
To determine whether plant lineages differ in the way they condition soil fungal communities, we characterized fungal communities in the 15 species-level soils collected from Tasmania using next-generation sequencing. We extracted DNA from 0.25 g of each species-level soil using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) following the manufacturers instructions and assessed the quality and purity of the extracts using a Nanodrop 1000 Spectrophotometer (Thermoscientific). We then amplified the extracts with polymerase chain reaction (PCR) using a set of two primers5.8S-Fun and ITS4-Fun (Taylor 2014, Taylor et al., 2016)that was made to target the ITS2 region with the broadest possible coverage of fungi. The primers contained the Illumina overhang adapter sequences on their 5 ends, following Illuminas 16S Metagenomic Sequencing Library Preparation methods. The initial PCR consisted of 2.5 uL of a 1:10 dilution of each template, 10 uM each primer, and 12.5 uL of 2X KAPA HiFi Hot Start Ready Mix. A negative control of water only was included in the PCR and carried throughout the process. The PCR cycling parameters consisted of 95 for 3 minutes, then 35 cycles of 95 for 1 minute, 54 for 1 minute, 72 for 1 minute, followed by a final extension of 72 for 10 minutes. PCR products were cleaned with Agencourt AMPure XP beads (Beckman Coulter Genomics), then given individual indexes with the Nextera XT (Illumina) index PCR. The resulting products were quantified and pooled at equal molar concentrations, then purified again with AMPure XP beads. The samples were then checked for quality and quantity on a High Sensitivity 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on the Illumina MiSeq platform at the University of Tennessee Genomics Core. The amplicons were sequenced 300 bases, paired-end, using version 3 reagents, taking up approximately 20% of a run.