Fungal TEF1 amplicon seq of Xyris sp. inflorescences
Samples were lyophilized and genomic DNA extractions were performed using the DNeasy Plant Mini Kit (QIAGEN) following the manufacturers protocol. Amplicons targeting the second and third exon and second intron of the translation elongation factor 1-alpha (TEF1) gene were generated using a two-step approach. Primers Fa_150 (5-CCGGTCACTTGATCTACCAG-3) and Ra-2 (5-ATGACGGTGACATAGTAGCG-3) (Cobo-Daz et al. 2019) 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.