Background: The Citrus genus, renowned for its economic importance, possesses a complex evolutionary history shaped by widespread interspecific hybridization. A detailed understanding of its genome structure is essential for both evolutionary studies and crop improvement. However, high-resolution, comparative karyotype analyses across the genus are lacking, and the DNA sequences that define functional Citrus centromeres—critical for genomic stability—remain poorly understood.

Methods: We developed a set of chromosome-specific oligonucleotide (oligo) probes based on the sweet orange (Citrus sinensis) reference genome. These probes were used to perform multicolor oligo-fluorescence in situ hybridization (oligo-FISH), or chromosome painting, on mitotic chromosomes from several ancestral and cultivated Citrus species. Concurrently, we employed chromatin immunoprecipitation with an antibody against the centromeric histone CENH3, followed by sequencing (ChIP-seq), to identify the core DNA sequences of functional centromeres.

Results: The oligo-probes successfully painted and distinguished all nine chromosome pairs in the analyzed species. Comparative karyotyping revealed a remarkable degree of conservation in chromosome number (2n=18), size, and morphology across the genus, indicating profound karyotypic stasis despite extensive hybridization. Our ChIP-seq analysis identified a highly conserved 165-bp satellite repeat (CitCEN165) as the dominant CENH3-associated sequence. Subsequent FISH experiments using a CitCEN165 probe confirmed its exclusive localization to the primary constriction of every chromosome in all tested species.

Conclusion: Our findings demonstrate that Citrus evolution has proceeded with minimal large-scale chromosomal rearrangement, a feature that likely facilitated its reticulate evolution. The identification and validation of the conserved CitCEN165 repeat as the primary centromeric DNA element provides a fundamental insight into Citrus genome architecture. This study offers a powerful cytogenomic toolkit and foundational knowledge that will significantly benefit future genetic research and advanced breeding strategies in Citrus.