Dig nick translation mix

Flower buds were sampled from 5- to 6-week-old plants and fixed in Farmer’s solution (acetic acid:ethanol, 1:3) for 1 h at 25 °C. Fixed buds were washed with 70% ethanol and then with distilled water for 5 min. The buds were treated with an enzyme solution (2% w/v cellulose Onozuka RS, 0.5% w/v pectolyase Y-23, 10 mM citrate buffer, pH 4.5) at 37 °C for 1 h. The buds were broken by pipetting and then filtered through a 100-µm nylon mesh. The filtered nuclear solution was centrifuged at 5000 × g for 1 min and the pellet was resuspended in Farmer’s solution. A drop of the nuclear solution was placed on a glass slide and allowed to dry. Then, FISH was performed as previously described^{46 (link)}. A centromere probe was amplified by PCR and labeled by nick translation using DIG-Nick Translation Mix (Sigma-Aldrich, St. Louis, MO, USA). Hybridized probes were visualized using a rhodamine-conjugated anti-digoxigenin antibody (Sigma-Aldrich). The number, size, and fluorescence intensity of FISH signals were measured using ImageJ 1.51 g. A binary image was generated from the fluorescent image and “Analyze Particles” in ImageJ was used to determine the signal size. To measure the fluorescence intensity, one pericentromeric signal was clipped from the raw image and “Measure” was used to determine the signal intensity. Subsequently, the background was subtracted from the signal intensity.

To analyze metaphase chromosomes, cells in metaphase were harvested using standard procedures. Briefly, cells were swollen in 75 mM KCl (at 37°C), fixed in methanol:acetic acid (3:1) and then dropped onto glass slides. To analyze anaphase and cytokinesis-blocked cells, cells were seeded on poly-L-Lysine slides (Sigma-Aldrich) and fixed in methanol:acetic acid (3:1) for fluorescence in situ hybridization (FISH) analysis. BAC clones were used to prepare FISH probes targeting the regions of interests: RP11-439L14 (GenBank: AC012454.8) for FOLD1, RP11-464P18 (GenBank: AC073105.1) for Chr2Q, RP11-799C6 (GenBank: AC068519.1) for Chr1CCEN, RP11-383P16 (GenBank: AC233288.1) for FRAXA (Supplementary Figure 1A). Probes were labeled using the BioNick labeling system (Thermo Fisher Scientific) or DIG-nick translation mix (Sigma Aldrich). FISH was carried out using standard procedures, as previously published (Bjerregaard et al., 2018 (link)). Slides were mounted using Vectashield mounting medium with DAPI (Vector Labs). Images were captured using an Olympus BX63 microscope. Images were analyzed using CellSens (Olympus) or Fiji/ImageJ software. In the analysis of “bent” shape of human chromosome 2 (Chr2), all of the images were analyzed independently by two researchers.

The DNA probe used for mapping the rDNA genes was pTa71. This plasmid contains a 9-kb EcoRI fragment from Triticum aestivum that includes the 45S rDNA region^{69 (link)}. pTa71 was labeled with digoxigenin-11-dUTP using a kit from Sigma-Aldrich (Dig-nick translation mix).
Telomeric repeats at both ends were detected using the single-strand oligonucleotide (CCCTAAA)₃, synthesized with Dy547 (red) (Isogen Life Science).
Four oligonucleotides, (AG)₁₀, (AC)₁₀, (GATA)₄ and (GACA)₄, supplied with biotin incorporated at both ends (Isogen Life Science), were used to analyze the microsatellites. They are representative of the most abundant microsatellite motifs clustered at the chromosomal level in the species so far analyzed^{47 (link)}.