Sanger Sequencing Protocol for Detecting Mutations

The presence of mutations in each T0 line was determined by PCR and Sanger sequencing. Genomic DNA isolated from the T0 plants was used for the polymerase chain reaction (PCR) using primers spanning the target sites (Table S1). PCR products were resolved on the agarose gel and extracted using the GeneJET Gel Extraction Kit (Thermo Scientific, USA) for sequencing from both ends using the forward and the reverse primers by the Sanger Sequencing method (Psomagen, Rockville, MD, USA). The sequence traces (ABI files) were analyzed on the Sequence Scanner 2 software (Applied Biosystems Inc., Waltham, MA, USA) and aligned with the reference sequences using the CLUSTAL-Omega multiple sequence alignment tool. The overlapping sequences arising from heterozygous alleles were separated using the CRISP-ID or Polypeak Parser tools [43 (link),44 (link)].