Fungal ITS amplicon seq of Fusarium xyrophilum pseudoflowers
Samples were lyophilized and genomic DNA extractions were performed using the DNeasy Plant Mini Kit (QIAGEN) following the manufacturers protocol. Amplicons targeting the ITS1 region of the fungal internal transcribed spacer were generated using a two-step approach. Fungal ITS primers ITS1-F (5-CTTGGTCATTTAGAGGAAGTAA-3) and 58A2R (5-CTGCGTTCTTCATCGAT-3) (Gardes and Bruns 1993; Martin and Rygiewicz 2005) and Platinum II HotStart Mastermix (Invitrogen) were used. PCR products were checked using 1% agarose gel. PCR products were purified with Mag-Bind TotalPure NGS clean up beads (Omega Bio-Tek). The second PCR step used forward and reverse primers designed with overhangs to permit barcode attachment and standard Illumina overhang adaptors needed for Illumina amplicon sequencing. The second PCR reaction included 5 L of cleaned PCR product. Barcoded amplicons were normalized using the SequalPrep Normalization Plate Kit (Invitrogen) and pooled together. The pool was concentrated using a Savant SpeedVac at 55C for 3 hours and then run on a 1.2% agarose gel. Expected bands were excised and purified using the PureLink Quick Gel Extraction kit (Invitrogen). Sequencing was performed on an Illumina MiSeq using the 2  300 cycle v2 kit.