*4.6. FISH Procedure*

FISH analysis was performed to further confirm the physical existence of the HOR units in the genome. Root tips were pre-treated in 0,002 M 8-hydroxyquinolin for 3 h in dark and fixed in 3:1 (*v*/*v*) 100% ethanol:acetic acid. The fixed root meristems were thoroughly washed in water and enzyme buffer (10 mM citrate buffer at pH 4.6) and partially digested in 0,3% (*w*/*v*) cytohelicase, pectolyase and cellulase (Sigma) at 37 ◦C for 3 h followed by washes in water [27]. The material, in a water drop, was carefully transferred onto a grease-free microscope slide and the cells were spread according to the technique of Pijnacker and Ferwerda [59] with modifications as previously described [60].

FISH experiments were performed with clones CficCl-61-40 X-1 and CacuCl-1-117 C-2 as probes labelled with Cy3 (Amersham, Amersham, Buckinghamshire, UK) and biotin (Roche, Basel, Switzerland) according to a standard oligolabeling protocol [61]. For evaluation of probe-specific chromosomal pattern probes were hybridized simultaneously to chromosomes of *C. acuminatum*, *C. bryoniifolium*, *C. ficifolium*, *C. iljinii*, *C. pamiricum*, *C. suecicum* and *C. vulvaria* (Figure 5, supplementary data 5). FISH was performed on ThermoBrite programmable temperature-controlled slide processing system at 63 ◦C for 3 h. Slides were stained with DAPI and mounted in antifade mountant (Vector Laboratories, Burlingame, CA, USA) and were examined and photographed on Zeiss Axio Imager.Z2 microscope system. Chromosome measurements were obtained by the analysis of metaphase plates using the computer application MicroMeasure version 3.3 [62].
