Southern blotting

Genomic DNA (~2.5 to 5 μg) was digested with Bgl II and Pst I, electrophoresed on an 0.8% agarose gel at 25 V for ~18 hours, blotted onto a BrightStar-Plus Positively Charged Nylon Membrane (Thermo Fisher Scientific) in 0.4 M NaOH and 0.6 M NaCl transfer solution, and then baked at 80°C for 2 hours. The membranes were hybridized overnight at 42°C using an Eg fp fragment labeled by DIG DNA Labeling and Detection Kit (Roche) as the probe. The membranes were washed at 42°C in 0.1× saline sodium phosphate EDTA and 0.1% sodium dodecyl sulfate (Roche) until the background signal was eliminated. The signal was detected with the DIG DNA Labeling and Detection Kit (Roche), and images were captured using a Bio-Rad ChemiDoc XRS+ System.