Metabarcoding GH
The ITS1 region of fungal DNA was amplified using primers BITS-ACCTGCGGARGGATCA and B58S3- GAGATCCRTTGYTRAAAGTT [31]. Inline barcodes (5 base pairs) were incorporated at the 5' end of each primer for sample identification [32]. PCR amplification was performed using SolarBio PCR master mix (SolarBio, China) with the following cycling conditions: 95C for 3 minutes, followed by 35 cycles of 95C for 15 seconds, 48C for 20 seconds, and 72C for 10 seconds. PCR amplification was considered successful only when samples exhibited a distinct band of the expected size, and the negative control remained negative. Amplicons were visualized on a gel, normalized based on intensity, and pooled. Subsequent purification using 0.8x volume of SPRI beads was followed by library preparation using the NEB Ultra II Library Prep Kit to incorporate Illumina adapters and dual-index barcodes. Library quantification was performed using a Denovix high-sensitivity assay, and sequencing was conducted on a NovaSeq6000 platform (Illumina, San Diego) using 2x150 paired-end reads.