Genetic homogenization due to chronic clonal propagation poses a notable challenge to the sustainable utilization of Coptis chinensis Franch. We developed a novel elite cultivar, ‘Chulian No. 1’, with superior yield (+34%), disease resistance, and pharmacopoeia-standard alkaloid profiles through comprehensive agronomic evaluation. To understand the genetic basis of these traits, we constructed a high-quality chromosome-level genome assembly, revealing long terminal repeat retrotransposon (comprising 41.92% of the genome) shows significant enrichment near expanded stress/alkaloid biosynthesis genes. Whole-genome resequencing of 235 accessions indicated extreme genetic uniformity (π < 0.0018), but our novel geospatial-autoencoder model successfully identified three eco-geographic groups that could not be discriminated using traditional approaches. Genome-wide association analysis identified the leaf glossiness-associated gene CcHAB1 on chromosome 1. Comparative transcriptomics revealed correlations between CcHAB1 expression and cuticular wax biosynthesis gene regulation via ABA and phenylpropanoid pathways, potentially explaining enhanced disease resistance in glossy-leaf accessions. This work enables molecular breeding for medicinal plants with limited genetic diversity and provides insights into trait maintenance despite genetic homogenization.

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