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).