Hemsleya ellipsoidea (Xuedan) is a phylogenetically distinct medicinal species within the Cucurbitaceae family, notable for its ability to accumulate cucurbitacin IIa—a bioactive triterpenoid with potent anti-inflammatory and antibacterial activities. Here, we present a chromosome-scale reference genome for H. ellipsoidea, assembled using Oxford Nanopore, Illumina, and Hi-C sequencing technologies. The 535.68 Mb genome, with a contig N50 of 15.36 Mb, encodes 25 230 protein-coding genes across 14 pseudo-chromosomes, of which 63.85% comprise repetitive elements. Comparative genomic and phylogenomic analyses reveal that H. ellipsoidea diverged early (~84.7 MYA) from other cucurbits, maintaining several ancestral chromosomal segments but exhibiting lineage-specific rearrangements, reflecting an independent evolutionary trajectory without recent whole-genome duplication. Two conserved but functionally specialized biosynthetic gene clusters related to cucurbitacins formation were identified, suggesting coordinated regulation of triterpenoid metabolism. Integration of genomic and transcriptomic data enabled the reconstruction of the cucurbitacin IIa biosynthetic pathway and the identification of key structural enzymes and transcription factors. Distinct tissue-specific expression patterns further indicate root-localized synthesis and accumulation of cucurbitacin IIa. Collectively, this work provides the first high-quality genome of a medicinal Cucurbitaceae species and offers new insights into the chromosomal evolution, metabolic specialization, and adaptive diversification of H. ellipsoidea. The genomic resource also lays a foundation for functional genomics, metabolic engineering, and molecular breeding toward high-value triterpenoid production.

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