The four mutant plant sequences obtained through Sanger sequencing were labeled S31-34. S31’s first quality base was the 50th base, while the last quality base was the 615th base. S32 had no quality bases. S33 and S34’s first quality bases were also the 50th base, while the last quality bases were the 600th and 620th bases respectively. The NCBI pairwise BLAST between the Sanger and reference genome sequences revealed one misalignment at the beginning of the sequence, but in between the beginning and end, the Sanger and reference sequences were perfectly aligned except at the 133rd position. This was confirmed to be the position of the NaN1898_Bd1_70884553_Het mutation, and it was located on the 133rd position of the chromatogram (Figure 5). On the chromatogram, there were two peaks, G and A, at the 133rd position, although the algorithm read it as A. This indicated a heterozygous mutation with a wild type G base and mutant A base, which is consistent with the NaN1898_Bd1_70884553_Het mutation. This mutation was at position 715 in the whole genome sequence.
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