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Traditionally globe artichoke and leafy cardoon have been cultivated for use as vegetables but these crops are now finding multiple new roles in applications ranging from paper production to cheese preparation and biofuel use, with interest in their functional food potential. So far, their chromosome complements have been poorly investigated and a well-defined karyotype was not available. In this paper, a detailed karyo-morphological analysis and molecular cytogenetic studies were conducted on globe artichoke (Cynara cardunculus Linnaeus, 1753 var. scolymus Fiori, 1904) and leafy cardoon (C. cardunculus Linneaus, 1753 var. altilis De Candolle, 1838). Fluorescent In Situ Hybridization In Suspension (FISHIS) was applied to nuclei suspensions as a fast method for screening of labelling probes, before metaphase spread hybridization. Classic Fluorescent In Situ Hybridization (FISH) on slide, using repetitive telomeric and ribosomal sequences and Simple Sequence Repeats (SSRs) oligonucleotide as probes, identified homologous chromosome relationships and allowed development of molecular karyotypes for both varieties. The close phylogenetic relationship between globe artichoke and cardoon was supported by the very similar karyotypes but clear chromosomal structural variation was detected. In the light of the recent release of the globe artichoke genome sequencing, these results are relevant for future anchoring of the pseudomolecule sequence assemblies to specific chromosomes. In addition, the DNA content of the two crops has been determined by flow cytometry and a fast method for standard FISH on slide and methodological improvements for nuclei isolation are described. [ABSTRACT FROM AUTHOR]