Chromosomal grade

Chromosomal DNA was prepared from the cells of overnight YEL cultures in agarose plugs, as described by Nguyen et al. [60 (link)] for Saccharomyces. The plugs were washed in TE (Tris/EDTA) and inserted into wells of 1.1% agarose (chromosomal grade, Bio-Rad, Hercules, California, USA) gel prepared in a 0.5× TBE (Tris/Borate/EDTA) buffer. The chromosome-sized DNA molecules were separated using pulse-field electrophoresis in 0.5× TBE with a CHEF-Mapper apparatus (Bio-Rad). The running parameters were as follows: ramping for 300 s over 48 h and ramping for 600 s over 48 h at 3 V/cm in a TBE 0.5× buffer at 14 °C. Gels were removed from the tray, stained in an ethidium bromide bath (0.5 mg/100 mL) and destained in sterile water. For MSP-PCR fingerprinting, genomic DNA was extracted from overnight YEL cultures. The PCR reaction was carried out with the microsatellite oligonucleotide primer (GAC)₅, as described by Baleiras-Couto et al. [61 (link)]. Z. rouxii CBS 732^T was used as the reference strain in both procedures.

For DNA break analysis, 10⁶ cells were mixed into melted agarose inserts. The agarose inserts were incubated in proteinase K buffer (0.5 M EDTA, 1% N-laurylsarcosyl, and 1 mg/mL proteinase K) at 50 °C for 48 h, and thereafter, washed four times in Tris-EDTA buffer prior to loading onto a 1% agarose gel (chromosomal grade; Bio-Rad Laboratories) and separated using a CHEF DR III pulsed field gel electrophoresis apparatus for 24 h (Bio-Rad Laboratories; 120° field angle; 240 s switch time; 4 V/cm at 14 °C). The gel was subsequently stained with ethidium bromide and analyzed with ImageJ.

Karyotype analysis was performed as described by Antunovics et al. (2005) (link). Chromosomal DNA was prepared in agarose plugs as described by Nguyen and Gaillardin (1997) . Plugs were washed in TE and inserted into wells of 1.1% agarose (Chromosomal grade, Bio-Rad) gel prepared in 0.5 × TBE buffer. The chromosomes were separated by electrophoresis in 0.5 × TBE with a CHEF-Mapper apparatus (Bio-Rad). The running parameters were: 200V, linear ramping from 40 to 120 s for 24 h at 14°C. The chromosomal bands were visualized by staining with ethidium-bromide and destaining in sterile water.