DNA extraction

Genomic DNA was extracted from urediniospores following the method described by Aljanabi and Martinez (1997) with modifications. Approximately 50 mg urediniospores of an isolate were transferred into a 2 mL clean centrifuge tube with steel beads (4 mm in diameter), ground for 6 min to fine powder in a swing mill (TissueLyser II, QIAGEN, Hilden, Germany), and added with 500 μL of pre-heated CTAB extraction buffer (0.4 M NaCl; 10 mM Tris-HCl, pH 8; 2 mM EDTA, pH 8, 65°C). The mixture was blended gently, incubated at 65°C for 1 h, with gentle tipping every 10 min, added with 750 μL phenol-chloroform-isoamylalcohol solution (v:v:v = 25:24:1, pH 7.8) and centrifuged at 10,000 g for 10 min at 4°C. After the upper phase was transferred to a clean tube and added with 300 μL chloroform, the mixture was centrifuged at 10,000 g for 10 min at 4°C. The upper phase was transferred to a new tube, added with pre-cooled 800 μL isopropanol and kept at −20°C overnight. After centrifuging at 10,000 g for 30 min at 4°C and discarding the supernatant, the DNA pellet was washed first with 75% (v/v) and then 100% ethanol. After drying for 2 h at room temperature, the DNA pellet was dissolved in 50 μL of 1 × TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The solution was added with 1.0 mL 20 μg/mL RNase and incubated at 37°C for 30 min to digest RNA. DNA was re-precipitated and dissolved in TE buffer as described above. DNA concentration was measured using an ND-1000 spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) and diluted to 50 ng/μL with 1X TE buffer to be used as template in polymerase chain reaction (PCR).