Identification of Reference Genes and Their Application in Analysis of Ginsenoside Biosynthesis Genes in American Ginseng Under Abiotic Stresses and Hormone Treatments

Quantitative real-time PCR (qRT-PCR) is a dominant technique for gene expression analysis in medicinal plants, and accurate qRT-PCR data normalization strictly relies on stably expressed reference genes (RGs). However, systematically validated RGs for Panax quinquefolius L. across diverse tissues, abiotic stresses, and hormone treatments are still lacking. In this study, 15 candidate RGs with FPKM values ranging from 7.21 to 210.56 were selected from RNA-Seq datasets based on expression abundance, coefficient of variation, and homology to commonly used reference genes. The expression stability of candidate RGs was evaluated under tissue-specific, drought, waterlogging, gibberellin A3 (GA3), and melatonin (MT) treatments using ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder methods. UBC28 and EF1-α ranked as the top two most stable reference genes across all tested conditions, with stability values of 2.21 and 2.38, respectively. Subsequent qRT-PCR validation of eight ginsenoside biosynthesis-related genes using the UBC28 and EF1-α normalization system showed that drought and waterlogging stresses induced significant expression fold changes (1.5–4.8-fold) in most ginsenoside synthetic genes, while exogenous GA3 and melatonin treatments caused no significant transcriptional regulatory effects under the tested experimental conditions. This study provides a set of reliable, condition-adaptive RGs for P. quinquefolius and establishes a robust molecular tool for future studies on ginsenoside biosynthesis and stress response mechanisms in American ginseng.