Molecular Linkage Mapping in Rice

DNA was extracted using the young leaves following the method of Zheng et al. (1995) . PCR was performed in a 10-µL reaction system containing 2 µL of DNA template (approximately 50 ng), 0.1 µL of 100 mol/L each primer, 5 µL of 2 × Taq MasterMix (Shangya Biotech, Hangzhou, China), and 2.8 µL of ddH₂O. PCR program was performed with an initial denaturation at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 2 min. The PCR products were visualized on 2.5% agarose gels using GelRed staining (Biotium, Fremont, CA, USA). A total of 208 markers, including 121 simple sequence repeats and 87 InDel markers were used to construct the low-density PCR-based linkage map (Wu et al., 2020 (link)). A total of 18,194 single nucleotide polymorphism (SNP) markers spanned all the 12 rice chromosomes with an average genetic distance of 0.12 cM were used to construct the high-density linkage map (Ying et al., 2018 (link); Wu et al., 2020 (link)).