Homogenisation, DNA extraction and sequencing
Prior to homogenisation of each sample, a metal cylinder and ball bearing were sterilised in a 6% sodium hypochlorite (NaOCl) solution for three minutes, followed by immersion in 70% ethanol (EtOH) for one minute. All leaf disks from one sample were homogenised together in the metal cylinder fastened to a Retsch® MM2000 laboratory mixer mill (Retsch® GmbH, Haan, Germany) for one minute at 70% of the maximum oscillation frequency and subsequently stored at -20⁰C until DNA extraction. DNA of 60 mg dried homogenised leaf material per sample was extracted using the my-Budget plant DNA extraction kit (Bio-Budget Technologies GmbH, Krefeld, Germany) following manufacturer’s instructions. The ITS region of all extracted samples was amplified using the ITS1-F (Gardes and Bruns, 1993) and ITS4 (White et al. , 1990) primer combination and was subsequently visualised on a 0.8% agarose gel with a 100bp ladder, together with positive and negative controls to ensure that we had indeed extracted fungal DNA without contamination.
Illumina amplicon library preparation utilised a nested PCR approach (Unterseher et al. , 2016) (Supporting Information Figure 1, Methods S1). The ITS region was amplified using the ITS1-F (Gardes and Bruns, 1993) and ITS4 (White et al. , 1990) primer combination. The final amplicons were subsequently visualised on a 0.8% agarose gel with added ethidium bromide (Supporting Information Methods S1). Concentration of DNA for pooling was quantified from the band intensity of the gel images using ImageJ (Schneider et al. , 2012) (Supporting Information Methods S1). The amplicon pools were cleaned using the CleanPCR magnetic bead kit (CleanNA, Waddinxveen, Nertherlands) (Supporting Information Methods S2). In total three samples were lost during homogenisation or due to low DNA concentration resulting in 186 samples that were sent for sequencing.
The final pooled amplicon library was sequenced at the Genomics Service Unit of the Ludwig Maximilians University (LMU) Biocenter on an Illumina MiSeq® sequencer (Illumina Inc., San Diego, CA, USA) using the MiSeq® Reagent Kit v3 Chemistry, for 2 × 250 bp paired-end sequencing. All raw sequences were deposited on the NCBI portal under the following accession codes: BioProject – PRJNA674320; BioSample (SAMN16634781 – SAMN16634966).