Verification of Fungal Transformants by Spore PCR

Positive transformants were firstly screened by hygromycin B. Then transformants losing plasmids were identified by spore PCR with primer pairs of Am-F/Am-R. Genomic DNA was extracted using the Plant Genomic DNA Extraction Kit (Tiangen). The spore PCR were performed as described previously (Ding et al. 2021 (link)). Transformants without carrying plasmids were further used for genotypic verification. Primers used for PCR analysis are listed in Additional file 1: Table S1. The theoretical PCR products were 1208 bp (the insertional fragment for pFC000-MH using primers of Mi-F/Mi-R), 1153 bp (the insertional fragment for pFC000-RH using primers of Re-F/Re-R), 1553 bp (the insertional fragment for pFC-MD using primers of MD-F/MD-R), and 1153 bp (the insertional fragment for pFC-RD2 using primers of RD-F/RD-R), respectively.