Paris polyphylla is an important medicinal plant that can biosynthesize polyphyllins with multiple effective therapies, ranging from anti-inflammation to antitumor; however, the genetic diversity of Paris polyphylla is still unclear. To explore the genetic characteristics of cultivation populations in primary planting areas, we developed 10 expressed sequence tag simple sequence repeat (EST-SSR) markers related to polyphyllin backbone biosynthesis and utilized them in 136 individuals from 10 cultivated populations of P. polyphylla var. yunnanensis. The genetic diversity index showed that ten loci had relatively high genetic polymorphism levels. Shannon information of loci suggested that more information occurred within population and less information occurred among population. In addition, the overall populations exhibited a low degree of differentiation among populations, but maintained a high degree of genetic diversity among individuals, resulting in high gene flow and general hybridization. The genetic structure analysis revealed that 10 populations possibly derived from two ancestral groups and all individuals were found with different levels of admixture. The two groups were different from the cultivation groups at population level, suggesting the cross-pollination among cultivars. These findings will provide insights into the genetic diversity of the germplasm resources and facilitate marker-assisted breeding for this medicinal herb.

Nitrogen (N) sources critically influence both agronomic performance and secondary metabolism in medicinal plants. Understanding how different forms of nitrogen affect plant growth and the biosynthesis of valuable secondary metabolites is essential for optimizing cultivation practices and enhancing crop medicinal quality. In this study, Erigeron breviscapus (Vant.) Hand.–Mazz., a medicinal herb renowned in traditional Chinese medicine for its bioactive flavonoids such as scutellarin with neuroprotective and cardiovascular therapeutic effects, was cultivated under various N treatments—nitrate (NO₃⁻–N), ammonium (NH₄⁺–N), and urea [CO(NH₂)₂]—and compared to an N-free control. All N treatments significantly enhanced plant height, leaf area, biomass, and overall yield, with nitrate-N providing the most pronounced growth benefits. Metabolomic profiling identified 387 known metabolites, primarily flavonoids, exhibiting distinct accumulation patterns under each N form. Transcriptomic analyses revealed substantial differences in gene expression, with nitrate-N inducing the greatest number of differentially expressed genes (DEGs). Integration of metabolomic and transcriptomic data uncovered co-expression modules linking candidate regulatory genes, such as cytochrome P450s, MYB transcription factors, and glycosyltransferases, to specific flavonoids, including quercetin-3-O-glucoside and apigenin. These findings elucidate how different N sources modulate flavonoid biosynthesis in E. breviscapus, revealing molecular mechanisms underlying N-mediated flavonoid biosynthesis, which can contribute to optimized fertilizer strategies. This research enhances both the medicinal quality and yield of this important medicinal plant by revealing key gene–metabolite networks, thereby offering valuable insights for metabolic engineering and sustainable cultivation practices.