Genome integration analysis of transformed C. magnum

The transformants and the WT strains of C. magnum were transferred to 100 mL fresh liquid PDA and cultured for 20 d at 27°C in the dark. Genomic DNA of 40 transformants and the WT strains of C. magnum were extracted by CTAB (Sangon Biotech, China).
To analyze genome integration of the transferred genes, the hph gene was amplified using the primer pair hph-F + hph-R (Table 1) and sequenced. PCR was were performed according to the protocol described by Guo et al. with slight modifications (Guo et al., 2022 (link)). The annealing temperature of hph was 60°C. PCR amplicons were purified and sequenced by Sangon Biotech Company (Shanghai, China).
The digestion of genomic DNA from 40 transformants or WT strains with EcoRI and HindIII (NEB, Ipswich, United States), electrophoresised on 0.75% agarose, were depurated, and transferred to nylon membranes (Hybond-N+; General Electric Company, Boston, United States). In the hybridization experiments, the digoxigenin (DIG) labeled probe corresponded to 1,380 bp hph of T-DNA from plasmid pATMT1 (Figure 1), and the hph gene was amplified using the primers hph-F and hph-R. Hybridization and chemiluminescence detection of the hybridized Dig-labeled probes were performed using the DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Germany).