Woodland wildfire enables fungal colonization of encroaching Douglas-fir
DNA was extracted from a 0.1g subsample of roots showing visible fungal colonization using a Qiagen DNEasy PlantPro kit according to the manufacturers instructions (Qiagen; Hilden, Germany). Root samples from plants without visible colonization were not sequenced. The fungal internal transcribed spacer (ITS) region of the extracted DNA was amplified in 96-well plates with modified ITS1F-KYO1 and ITS2-KYO2 primers using a first polymerase chain reaction (PCR; Toju, Yamamoto, Tanabe, Hayakawa, & Ishii, 2016). Molecular tags for multiplexing were attached with a second PCR (Toju et al., 2016). PCR product was verified using gel electrophoresis, then cleaned with a magnetic bead purification method (Talbot et al., 2014). We quantified the product fluorometrically using a Qubit fluorometer (Thermo Fisher Scientific; Waltham, MA, United States), and then pooled all samples to equimolar concentration before submitting the library for 2 x 300 Illumina MiSeq sequencing at the Stanford Functional Genomics Facility (SFGF).