Taq master mix

Genomic DNA was extracted from leaves according to a modified CTAB method described by Murray and Thompson (1980) (link) and adjusted to a final concentration of 25 ng/μL after quantification using a Nanodrop 2000 spectrophotometry (Thermo Scientific, United States). The DNA quality was further confirmed by 1.5% agarose gel electrophoresis at 90 V for 30 min.
The mutants were characterized by triple-primer PCR (polymerase chain reaction) (Figure 1). Reaction system (20 μL) contained ∼50 ng template DNA and 0.4 μM of each primer in 2 × Taq Master Mix (TOYOBO, Japan). The PCR program used included a hot start of 5 min at 94°C followed by 30 cycles of 45 s denaturation at 94°C, 45 s at 60°C (ND6011 for 58°C) annealing, 1 min polymerization at 72°C, and concluded by 10 min at 72°C. PCR products were detected by 1.5% agarose gel electrophoresis. Forward primer (FP) and reverse primer (RP) (Supplementary Table 1) at upstream and downstream of the Tos17 insertion position were designed using the NCBI software³. Tos17 insertion position primer (TP) (Supplementary Table 1), a specific sequence primer located at the conjunction of rice genome with the inserted Tos17, was adopted from the Gramene data resource⁴.

Four independent lines of each mutant in T₆ generation were randomly selected for collection of leaf tissues. Leaf sample from eight plants in each line of mutants, as well as the control, were collected and mixed to extract DNA for SSR amplification by PCR. PCR reactions were carried out in a 15 μL volume containing ∼50 ng DNA and 0.4 μM of each primer in 2 × Taq Master Mix (TOYOBO, Japan). The PCR program used included a hot start of 5 min at 94°C followed by 30 cycles of 1 min denaturation at 94°C, 1 min at suitable temperature annealing, 1 min polymerization at 72°C, and concluded by 10 min at 72°C. Sixty microsatellites distributed on chromosomes 1, 3 6, 8, 9, and 10 were randomly selected and analyzed for stability. Sequences of the various primers used in present study were showed in Supplementary Table 2. The PCR products were then separated by 8% polyacrylamide gel electrophoresis (Figure 3) and visualized by silver-staining according to Liu et al. (2007) (link).