ITS of Endophytic Fungi from below-ground parts of Ephedra sinica Stapf
Take 0.5g of below-ground tissues of E. sinica treated with above methods respectively, and subsequently extract genomic DNA form a pool of above- and below-ground tissues from three different plants utilizing Plant Genomic DNA Kit (TIANGEN Biotech, Beijing, China) with three replicates following the manufacturers instructions (Wang et al. 2020). Fungal DNA were barcoded and identified on the basis of internal transcribed spacer 2 (ITS2) DNA sequences in rRNA gene regions, and the PCR amplification was performed by primers ITS 3F (GCAT CGAT GAAG AACG CAGC) and ITS 4 (TCCT CCGC TTAT TGAT ATGC) complete with Illumina adapters and unique 7 bp barcode sequences (Zhang et al. 2020; Cui et al. 2018). The products from triplicate runs were mixed as one sample to avoid PCR amplification deviation and purified utilizing DiaSpin DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China) with following the manufacturers instructions. Subsequently, the amplification products were sent for high-throughput amplicon sequencing using the Illumina Miseq platform (USA) to Sangon Biotech company (Shanghai, China)