Megabase agarose

All 124 S. uberis isolates were genotyped by PFGE. DNA was extracted with Bio-Rad CHEF genomic DNA plug kit (BioRad, Segrate, Italy) according to the manufacturer’s instructions. Each plug was digested with 20 U of SmaI (Roche) for 3 h at 25 °C and washed with 5 mL of TE buffer for 10 min at room temperature before being loaded on a 1% certified Megabase agarose (BioRad) gel. The lambda PFG ladder (New England Biolabs, Ipswich, MA, USA) was used as a molecular size standard. Electrophoresis was carried out in a contour-clamped homogeneous electric field (CHEF)- Mapper system (BioRad) at 14 °C in 0.5× TBE buffer. DNA fragments were separated after 18 h migration with 6 V/cm, 120° at pulse times of 10–45 sec. Gels were stained with ethidium bromide and photographed with Alliance LD2 gel documentation system (UVITEC, Cambridge, UK) in TIFF format. The S. uberis digitalized PFGE patterns were analyzed with the Gel Compar II software (Applied Maths, Sint-Martens-Latem, Belgium). Dendrograms were generated by the unweighted pair group method with arithmetic averages (UPMGA) using the Dice correlation coefficient with a position tolerance of 1.5%. Isolates with ≥90% similar profiles were considered to represent the same clone [10 (link)].

The electrophoretic mobility shift of λ-DNA nucleosome array reconstituted with histone octamers was assessed by agarose gel (Fig. S3A). The equivalent of 200 ng DNA with 5% sucrose was loaded into 0.65% Megabase agarose (BioRad) in 0.2× Tris–borate buffer and separated at 20 V for 24 h. The gel was stained with SYBR™ Gold Nucleic Acid Gel Stain (ThermoFisher Scientific, USA).

Bloodstream parasites at 2×10⁸ cells/ml were purified using DEAE cellulose (DE52) chromatography, and subsequently used to prepare chromosome blocks as previous described [30] (link). DNA from T. b. evansi and T. b. brucei cells was embedded in low-melt agarose blocks (final concentration of 5×10⁷ cells/ml) according to [31] (link), and was resolved using a 1% Megabase agarose (Bio-Rad) gel with 0.5X TBE buffer in the CHEF-DRIII system (Bio-Rad). S. cerevisiae DNA was used as a size marker. Pulse field gel electrophoresis (PFGE) was run at 14°C under the following conditions: switch time A increased from 28.6 s to 228 s for 24 hrs, followed by switch time B, with increase from 28.6 s to 1,000 s for another 24 hrs. The angle was set to 120° and voltage gradient to 3 V/cm. The PFGE gel was stained with ethidium bromide after the run.

Yeast cells harvested from YPD cultures were treated with zymolyase (Sigma‐Aldrich) for 2 hr at 30 °C. The resulting spheroplasts were used to prepare chromosomal DNA agarose blocks and treated with proteinase K. Taphrina chromosomal DNA was separated on 0.7% Megabase agarose (Bio‐Rad) gels with a Bio‐Rad DR III system with switching intervals of 60 s for 2 hr at 160 V/cm, and 60–20 s for 40 hr at 200 V/cm as previously described (Xu et al., 1995). Chromosomal DNA of Saccharomyces cerevisiae YPH80 (New England BioLabs) was used as the molecular weight marker.

Our pulsed‐field gel electrophoresis (PFGE) assay conditions have been described previously (Ahmed et al., 2019). Briefly, 0.4 × 10⁶ cells were seeded in a six‐well tissue culture plate and treated with indicated doses of Etop (Sigma) for 24 h and Merb (Santa Cruz Biotechnology, Dallas, TX, USA) for 48 h, respectively. 48 h drug treatment involved media replacement with fresh drug added every 24 h. DMSO‐treated cells were used as control. Harvested cells were embedded in 2% low melting agarose (Sigma) plugs followed by incubation in lysis buffer (0.2% sodium deoxycholate, 1% sodium lauroyl sarcosine, 100 mm EDTA, and 1 mg·mL⁻¹ proteinase K) at 50 °C for 24 h. Plugs were washed four times in TE buffer for 1 h each and electrophoresed through 1% megabase agarose (Bio‐Rad) on CHEF DR II (Bio‐Rad). Lambda PFG ladder (NEB, Ipswich, MA, USA) that ranges from 48.5 to 1018 kb was used as a marker. Subsequently, PFGE gels were stained with ethidium bromide, and quantification was performed using imagej software, as described in detail in Figure legends. Following PFGE, telomeric DNA was detected by Southern blotting using TeloTAGGG Telomere Length Assay kit (Roche, 12209136001) as per the manufacturer's protocol and previously detailed in (Ahmed et al., 2019).

All Chemicals were purchased from Sigma-Aldrich (Saint-Quentin-en-Yvelines, France), except for Megabase agarose^® (Biorad, Marnes-la-Coquette, France) and dihydrorhodamine 123 (Cayman Chemical Company, Ann Arbor, MI, USA).

Spheroplasts with intact chromosomal DNA were generated in plugs, as previously described [179 (link)], for Kwoniella strains CBS10118, CBS8507 and PYCC6329 with minor modifications: i) cells were grown in Yeast Nitrogen Base (YNB) liquid minimal medium supplemented with NaCl (1 M), and ii) zymolase (25 mg/mL), or Trichoderma harzianum lysing enzymes (50 mg/mL for K. mangrovensis) were used to lyse the cells embedded in the agarose, with overnight reactions at 37°C. Gels were prepared with 0.8 or 0.9% of Megabase agarose (BioRad) and chromosomal separation was conducted with the CHEF DR-II System and the CHEF Mapper XA System (Bio-Rad, Richmond CA) using different running and buffer conditions, and different size markers selected based on the chromosome size range of each assay (details in S2 Fig). Following electrophoresis, gels were stained with ethidium bromide, unstained using the running buffer, and photographed under a UV transillumination imaging system.