Comprehensive Analysis of Agronomic Traits, Saponin Accumulation, and SNP-Based Genetic Diversity in Different Cultivars of Panax notoginseng

Background: Given the need to optimize Panax notoginseng cultivation, screen high-quality germplasm, and clarify its insufficiently elucidated genetic–phenotype–quality associations (e.g., saponin accumulation), this study was conducted. Methods: Agronomic traits were measured, saponin accumulation was determined via high-performance liquid chromatography (HPLC), and comprehensive performance was evaluated through integrated cluster analysis and fuzzy membership function assessment; additionally, single-nucleotide polymorphism (SNP)-based genetic diversity analysis was conducted to explore the genetic basis of trait variations. Results: Agronomic traits exhibited coefficients of variation (CVs) of 2.95–18.12%, with primary root length showing the highest variability. Phenotypic cluster analysis divided the materials into three groups. Group I (“Miaoxiang No.1”, “Dianqi No.1”, “Miaoxiang Kangqi No.1”) was characterized by tall plants, sturdy stems, heavy roots, and long/large leaves. Saponin determination results revealed significant differences in notoginsenoside R1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rd, and total saponins among cultivars (order: “Zijing” > “Dianqi No.1” > original cultivar > “Miaoxiang Kangqi No.1” > “Miaoxiang No.1” > “Miaoxiang No.2”), with “Zijing” having the highest total saponin accumulation (18.13%); no significant difference was observed in ginsenoside Rg1 accumulation. The GATK initially identified 16,329,600 SNPs, and 115,930 high-quality SNPs were retained after Samtools filtration. SNP-based Neighbor-joining (NJ) clustering grouped the cultivars into three categories, with the original cultivar clustered alone as one category. Through comprehensive evaluation, three superior germplasm lines (“Miaoxiang Kangqi No.1”, “Miaoxiang No.1”, “Dianqi No.1”) were identified. A significant negative correlation (p < 0.05) was found between compound leaf petiole length and saponin accumulation. Conclusions: This integrated analytical strategy clarifies the links between genetics, phenotype, and quality, providing a scientific foundation for P. notoginseng germplasm screening and facilitating future molecular breeding efforts.