DNA extraction
Fresh seedling (0.1 g) samples were cut, wrapped in tinfoil, and
quick-frozen in liquid nitrogen barrels. The quick-frozen samples were
then placed into a mortar and pestle and ground quickly and thoroughly.
Next, 650 µL of preheated cetyltrimethylammonium bromide buffer was
added and mixed with the ground samples; the same amount of chloroform
was then added, and the contents were mixed slowly. After centrifugation
at 12,000 rpm for 15 min, the supernatant of the liquid was transferred
to a 1.5 mL Eppendorf (EP) tube, the same amount of pre-cooled isopropyl
alcohol on ice was added, and the mixture was mixed by slowly inverting
the EP tube. The supernatant was removed after centrifugation at 12,000
rpm and 4ºC for 10 min. One mL of 70 % ethanol was added to the EP tube
to remove the liquid supernatant, and DNA was recovered by immersing the
EP tube in ethanol solution. Centrifugation was performed at 12,000 rpm
and 4ºC for 10 min, the supernatant was discarded, and this procedure
was repeated (i.e., a total of two rounds of centrifugation). The EP
tube was left open, and the ethanol was dried at room temperature until
it had completely volatilized; 50 µL of sterile water was then added to
dissolve the DNA. After dissolution, the DNA was stored at –20ºC.