Integrative chromosome-scale analysis of cupuassu reveals structural and regulatory innovations shaping witches' broom disease resistance and provides a resource for functional studies in Theobroma

Cupuassu ( Theobroma grandiflorum) is a fruit tree native to the Brazilian Amazon, increasingly integrated into regional bioeconomies. Its cultivation is severely threatened by the witches' broom disease (WBD), caused by Moniliophthora perniciosa. A telomere-to-telomere (T2T) genome is available for the WBD-susceptible genotype C1074; however, the lack of a resistant reference has hindered a detailed understanding of the mechanistic basis of host resistance. Here, we present the first T2T assembly of the resistant genotype C174 (415.8 Mb) and a comparative analysis with C1074, which integrates structural variant detection, gene duplication profiling, transposable element (TE) analysis, and time-resolved host–pathogen transcriptomics. C174 displays distinctive tandem and dispersed duplications, lineage-specific TE insertions, and phased defense-gene expression, together with higher heterozygosity that suggests broader allelic diversity. Although C1074 encodes more NLR receptors (541 vs. 434), most are transcriptionally silent; by contrast, C174 shows sequential induction of pattern-recognition receptors, MAP-kinase components, diverse transcription factors, and pathogenesis-related proteins. Within the major WBD-resistance QTL, localized on chromosome 6, we identified two duplicated DUF4220/ DUF594 genes unique to C174 that are induced after infection and exhibit episodic positive selection, consistent with recent neofunctionalization. Genome-wide, TEs are differentially expressed and colocalized with immune loci in a genotype-specific manner, indicating TE-associated regulatory variation. Collectively, these results suggest that structural, duplicative, and regulatory innovations—rather than simple gene-count expansion—shape WBD resistance in cupuassu. The C174 T2T assembly, immune-related variants catalogue, and DUF4220/ DUF594 candidates constitute a valuable resource for molecular breeding, genome editing, and the sustainable improvement of this tropical perennial crop.