Comparative Oligo‐FISH Mapping Illuminates Chromosomal Evolution Among Rutaceae Species Diverged Over 50 Million Years

ABSTRACT Comparative fluorescence in situ hybridization (FISH) mapping, particularly chromosome painting, was first developed in mammalian systems and generated much of the foundational knowledge on chromosomal relationships among diverse mammalian species prior to the genome sequencing era. In plants, the advent of region‐ and chromosome‐specific FISH probes based on massively synthesized oligonucleotides (oligos) has greatly expanded the utility of FISH in cytogenetic mapping. We developed oligo‐based barcode‐FISH and chromosome painting probes in the model citrus species Citrus maxima and applied them to 13 species from the Aurantioideae subfamily of Rutaceae. All 13 species retained complete chromosomal synteny with C. maxima despite ∼20 million years of divergence. Remarkably, these probes were also successfully applied to Boenninghausenia albiflora (Rutoideae subfamily), which diverged from citrus species ∼52 million years ago. Comparative FISH mapping revealed the mechanism underlying its change in basic chromosome number from 9 to 10 and identified three distinct chromosomal translocation events in B. albiflora . Together, these results demonstrate that oligo‐based FISH probes developed in a model plant species can be effectively applied across deeply diverged lineages, enabling the rapid reconstruction of chromosomal evolutionary histories on timescales previously inaccessible to plant cytogenetics.