CHEF gel plugs were made by resuspending protoplasts in STE (1 M sorbitol, 25 mM Tris-HCl pH 7.5, 50 mM EDTA). Protoplast concentration was adjusted to 4 × 108 cells/ml and added to the same amount of 1.2% low melting agarose gel (Bio-Rad) solution. Protoplast suspensions (2 × 108 cells/ml) containing 0.6% low melting agarose gel were added to 50-well dispensable mold plates (Bio-Rad). Plugs were immersed in 10 ml of NDS (1% N-lauroyl sarcosinate sodium salt solution, 0.01 M Tris-HCl, 0.5 M EDTA) and incubated at 65 spm for 14 to 20 h at 37 °C. NDS was replaced with 0.05 M EDTA three times every 30 min. Plugs in 0.05 M EDTA were stored at 4 °C until use.
CHEF gel electrophoresis was done according to Inami et al.58 . Briefly, chromosomes were separated on 1% SeaKem® Gold Agarose (Lonza) in 0.5×TBE buffer at 4 to 7 °C for 260 h using a CHEF Mapper System (Bio-Rad). Switching time was 1200 to 4800 s at 1.5 V/cm with an included angle of 120°. The running buffer was exchanged every two or three days. Chromosomes of Hansenula wingei (Bio-Rad) were used as a DNA size marker. Gels were stained with 3×GelGreen (Biotium) to visualize chromosomes.
CHEF gel electrophoresis was done according to Inami et al.58 . Briefly, chromosomes were separated on 1% SeaKem® Gold Agarose (Lonza) in 0.5×TBE buffer at 4 to 7 °C for 260 h using a CHEF Mapper System (Bio-Rad). Switching time was 1200 to 4800 s at 1.5 V/cm with an included angle of 120°. The running buffer was exchanged every two or three days. Chromosomes of Hansenula wingei (Bio-Rad) were used as a DNA size marker. Gels were stained with 3×GelGreen (Biotium) to visualize chromosomes.