Ancestral karyotype reconstruction provides insights into stepwise chromosome evolution and bract coloration in the banana family.

The banana family (Musaceae) exhibits remarkable diversity in karyotype structure and bract coloration, yet the evolutionary dynamics of chromosome rearrangements and the regulatory basis underlying color diversification remain poorly understood. Here, we present a telomere-to-telomere (T2T), gap-free genome assembly of Musa exotica, an ornamental species with brightly colored bracts, representing an early-branching lineage within sect. Callimusa (Musa L.). By integrating this high-quality genome with available Musaceae genomes, we reconstruct the ancestral Musaceae karyotype (AMK) with a haploid chromosome number of n = 17. Comparative genomic analyses reveal recurrent, lineage-specific inter-chromosomal rearrangements across extant Musaceae lineages, resulting in stepwise chromosome number reductions to n = 11, 10, and 9. This karyotype trajectory is consistent with DNA-based phylogenetic relationships and suggests that chromosomal reorganization contributed to early lineage diversification within the family. Notably, rearrangement-associated regions are enriched for functionally important genes, particularly structural genes (chalcone synthase [CHS] and flavanone 3-hydroxylase [F3H]) and regulatory transcription factors (MYB and basic-helix-loop-helix [bHLH]) involved in anthocyanin biosynthesis. Integrative transcriptomic and regulatory analyses demonstrate coordinated activation of anthocyanin biosynthetic genes (CHS, CHI, F3'5'H, and ANS) in brightly colored bracts, with expression divergence largely decoupled from gene dosage and predominantly driven by transcriptional regulation. Co-expression analyses reveal extensive MYB- and bHLH-enzyme interactions, underscoring their central role in modulating pathway activity and color diversification. Collectively, our results provide a comprehensive evolutionary framework for Musaceae genomic evolution and lineage divergence and link chromosome structural evolution to regulatory rewiring and phenotypic diversification.