DNA extraction and PCR analysis

Four hundred µl of DNA extraction buffer (160 mM Tris, 56 mM EDTA, 30 mM sodium metabisulfite and 1.6 M sodium chloride) was added into tissue powder (approximately 100 mg) and centrifuged at 13,000 × g for 5 min. Three hundred µl of supernatant was taken and mixed with 300 µl of 100% isopropanol. The mixture was incubated at room temperature for 10 min with occasionally tube-inverting and then centrifuged at 13,000 × g for 5 min. The pellet was washed with 300 µl of 70% ethanol and air-dried overnight. The dried DNA pellet was dissolved in 50 µl of nuclease-free water (Qiagen, Germany). DNA concentration was measured using a UV5Nano spectrophotometer (Mettler-Toledo, US).

Six C. sativa genes (encoding actin, ubiquitin, EF-1α, chitinase 5, chitinase 2 and chitinase 4-like) were predicted from the C. sativa draft genome [55]. Gene and primer details are described in Additional file 7. A 100 ng of DNA template was added to 25 µl of PCR reaction mixture, consisted of 1 × MyTaq Red buffer, 0.5 U MyTaq DNA polymerase (Bioline, US) and 0.4 µM forward and reverse primers each. The PCR amplification was performed using a T100 thermal cycler (Bio-Rad, US) with an initial denaturation of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1.15 min at 72 °C, and a final extension of 5 min at 72 °C. A 10 µl of the amplification product was resolved in 1% agarose gel electrophoresis at 85 V for 50 min. The gel was stained with ethidium bromide and analyzed using Gel Doc EZ imager equipped with ImageLab software (Bio-Rad, US).