Identification and validation of reference genes for quantitative real-time PCR in Corydalis saxicola under various abiotic stresses.

Quantitative real-time PCR (qRT-PCR) is a commonly used method for measuring gene expression, but its accuracy depends on the use of stable reference genes for data normalization. In this study, we evaluated the expression stability of 11 candidate reference genes in Corydalis saxicola across different tissues and under three abiotic stress conditions (Ca²+ stress, Se²+ stress, and drought stress). Using four analytical algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder), we identified the optimal reference genes: GAPDH2 and UBCE2-17 for different tissues; RPAP3 and UBCE2-5 A under Ca²+ stress; GAPDH8 and α-TUB under Se²+ stress; and ACT4 and RPAP3 under drought stress. Additionally, GAPDH2, TBP, and UBCE2-17 were recognized as the most suitable comprehensive reference genes applicable to both different tissues and various abiotic stress conditions. These results were validated by analyzing the expression patterns of CsBBEL9 and CsOMT9 (key genes involved in benzylisoquinoline alkaloid biosynthesis) as well as CsANN1 and CsANN9 (core genes regulating stress response). This study provides the first validated reference genes for C. saxicola, offering a more accurate and reproducible method for gene expression analysis in this rare medicinal plant. It will facilitate future research on secondary metabolism regulation and stress response mechanisms in C. saxicola.