4.2. Screening of Polymorphic Markers in JHN and RD6

To find polymorphic markers in JHN and RD6, InDel and simple sequence repeat (SSR) markers were used. Genomic DNA samples of JHN and RD6 rice cultivars were used for a PCR reaction to screen for polymorphic markers. Each PCR reaction component was filled in the 96-well plates. Then, 50 ng/μL DNA solution of each rice cultivar was used as the DNA template. The PCR reaction was prepared by mixing 2 μL of 50 ng/μL DNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 µL of 2.5 mM dNTP, 1 μL of 10× Buffer A, 0.5 μL of 50mM MgCl₂, 0.15 μL of Taq DNA polymerase (Vivantis, CA, USA), and 4.35 μL of dH₂O per 1 reaction. The PCR reaction was conducted by PCR machine (Eppendorf, Hamburg, Germany). The conditions for each step were as follows: 94 °C for 3 min for the predenaturation step, 94 °C for 45 s for the denaturation, 55 °C for 45 s for the annealing step, 72 °C for 1 min for the extension step, and 10 °C for 10 min for the postextension step. The denaturation to extension step was conducted over 35 cycles.

The primer pairs of 133 InDel markers [26] were used as primers to amplify DNA targets from DNA templates of JHN and RD6. The primer sequence information of InDel markers is shown in Table S1. The PCR products were visualized using 1.5% agarose gel electrophoresis. The 100 bp DNA ladder (Vivantis, CA, USA) was used to determine the sizes of PCR products in base pair units. For SSR markers, the primer pairs of 230 SSR markers were selected to study the polymorphism between JHN and RD6 rice cultivars. The primer sequence information of SSR markers is shown in Table S2. The PCR products were visualized by polyacrylamide gel electrophoresis.