Fungal ITS2, isolated from Eucalyptus tereticornis leaf litter
DNA was extracted from approximately 200 mg of each of the samples that were analysed for litter chemistry. The samples were ground into a fine powder with 5 mm steel beads in a TissueLyser II (Qiagen), then 1 ml of CTAB buffer (0.1 M Tris-HCL, 1.4 M NaCl, 0.02M EDTA, 20 g.l-1 of cetyltrimethyl ammonium bromide with 4% (w/v) polyvinylpyrrolidone) was added to each sample. The samples were digested at 65  with mixing at 1000 rpm for 1 hour. The samples were then spun for 7 minutes at 16 000 rpm, following which 500 l of the supernatant was transferred to a new tube and 500 l of chloroform:isoamyl alcohol (24:1) added. The samples were mixed by inversion for 5 minutes and spun at 16 000 g for 7 minutes, after which up to 450 l of the upper aqueous phase was then transferred to a new tube. The DNA was precipitated by addition of 0.08 volumes of cold 7.5M ammonium acetate and 0.54 volumes of cold isopropanol, followed by 30 minutes at -20 . The samples were spun at 16 000 g for 3 minutes and the supernatant removed. The DNA was then washed with 700 l of 70 % cold ethanol, followed by 700 l of cold 95% ethanol. The samples were spun at 16 000 g for 1.5 minutes after each wash and the ethanol removed. The samples were dried for 30 minutes at 65  and then re-suspended in 100 l of TE buffer (10 mM Tris-HCL, 1 mM EDTA). For sequencing of clear DNA samples, concentrated samples were generally diluted to 10 ng/l while samples with concentrations of less than 15 ng/l were left neat. The darkly colours samples were diluted to 1 ng/l prior to sequencing. DNA samples were submitted to the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, NSW, Australia). Amplicons were generated using fITS7 (5-GTGARTCATCGAATCTTTG-3; Ihrmark et al. 2012) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'; White et al. 1990). All amplicons were purified using the Agencourt AMpure XP system (Beckman Coulter, Lane Cove, NSW, Australia) and genomic libraries were prepared using the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Paired-end (2 x 251 bases) sequencing was performed on the Illumina MiSeq platform.