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Old World lupins constitute an interesting model for evolutionary research due to diversity in genome size and chromosome number, indicating evolutionary genome reorganization. It has been hypothesized that the polyploidization event which occurred in the common ancestor of the Fabaceae family was followed by a lineage-specific whole genome triplication (WGT) in the lupin clade, driving chromosome rearrangements. In this study, chromosome-specific markers were used as probes for heterologous fluorescence in situ hybridization (FISH) to