A Near Telomere‐to‐Telomere Genome of <scp> <i>Belamcanda chinensis</i> </scp> Provides Insights Into Genome Evolution and the Biosynthesis of Characteristic Isoflavones

Belamcanda chinensis is a non-leguminous medicinal plant rich in bioactive isoflavones; however, the lack of a high-quality reference genome has limited elucidation of its isoflavone biosynthetic and modification network. Here, we present the first near telomere-to-telomere genome assembly of B. chinensis (4.18 Gb), generated using Illumina survey reads, PacBio HiFi and Oxford Nanopore long reads, and Hi-C scaffolding, achieving high completeness and accuracy (assembly BUSCO: 98.70%; LAI: 17.2). Ks/synteny-depth analyses and fossil-calibrated dating, with calibration at four fossil nodes, indicate two lineage-specific WGD events (~54.6 and ~27.3 MYA). These events drove significant expansions of key gene families involved in stress response and secondary metabolism. Leveraging this genome, we identified two key O-methyltransferases (BcOMT03 and BcOMT33), which are responsible for catalysing the biosynthesis of quality-marker compound irisflorentin. Meanwhile, BcUGT009, BcUGT119, BcUGT124, and BcUGT032 were characterised as glycosyltransferases with 7-O catalytic activity. Structural modelling and site-directed mutagenesis further elucidated the catalytic mechanism of BcUGT009, and its K404A mutant exhibited a significant increase in relative activity. Cross-species comparative analyses further revealed that convergent expansion of these key enzyme families underlies isoflavone biosynthetic capacity in both leguminous and non-leguminous plants. This study not only reveals the ancient polyploidization events of B. chinensis, the amplification of lineage-specific gene families and the biosynthetic pathway of characteristic isoflavones, but also provides a reference genome and functionally validated tailoring enzymes that will facilitate future heterologous pathway reconstruction and metabolic engineering of these isoflavones.