DNA Extraction Methods

Four DNA extraction methods (rapid DNA extraction, automatic magnetic bead-based DNA extraction, spin column-based DNA extraction, and CTAB/phenol/chloroform-based DNA extraction) were used in this study for comparisons. For rapid DNA extraction, the artificially Rs-inoculated and field-infected rice samples were washed, surface-sterilized with 1% sodium hypochlorite (NaHClO), rinsed in sterile water, and dried under a laminar flow hood to eliminate epiphytic microbes. The surface-sterilized rice leaf sheaths were cut into 1 cm² sections (300 mg), put into a mortar and mill in 1.4 mL of lysis buffer (25 mM NaOH, 2 mM EDTA). After centrifugation at 6,000 × g for 1 min, the supernatant with gDNA was subjected to further molecular detection. Each rice sheath leaf (300 mg) was frozen in liquid nitrogen and finely ground using a mortar and mill. The other three DNA extraction protocols, which were based on the automatic silica-coated magnetic bead-based DNA extraction (taco mini Automatic Nucleic Acid Extraction System, GeneReach, United States), spin column-based DNA extraction (Viogene genomic mini kit, Viogene-BioTek, Taipei, Taiwan), and CTAB/phenol/chloroform-based DNA extraction methods (Porebski et al., 1997), were further carried out according to the manufacturers’ instructions and the protocol described by Porebski et al. (1997), respectively. Genomic DNA was dissolved in a 0.1 × TE buffer (1 mM Tris–HCl and 0.1 mM EDTA, pH 8.0) and stored at −20°C for further molecular detection assays.