Selection and Validation of qRT-PCR Reference Genes in Elsholtzia stauntonii

Selecting and validating appropriate reference genes is a prerequisite for accurate gene expression analysis by means of quantitative real-time PCR (qRT-PCR), particularly in plant species with limited molecular research. Elsholtzia stauntonii is an economically important ornamental, medicinal, and aromatic plant. Although it exhibits a good tolerance to cold and drought, it is notably susceptible to waterlogging stress. To date, systematic investigations into its gene expression in different tissues and under waterlogging stress have not been extensively performed. In the present study, eight candidate reference genes, including GAPDH, ACT, UBQ, UBC, TUA, TUB, EF1-α, and eIF3K, were investigated using qRT-PCR in 13 samples from two distinct groups. The expression stability of these candidate genes was comprehensively assessed using the most commonly adopted algorithms: geNorm, NormFinder, Comparative ΔCt, BestKeeper, and RefFinder. The results demonstrated that UBC and EF1-α were the most suitable reference genes for different tissue samples, whereas ACT, GAPDH, and UBQ exhibited optimal reference genes for leaves under waterlogging stress. In contrast, eIF3K and TUB were identified as the least stable reference genes in different tissue samples and waterlogged leaves, respectively. To validate the suitability of the selected reference genes, the expression patterns of phenylalanine ammonia-lyase gene 1 (ElstPAL1) were analyzed. Comprehensive consideration of reference gene performance and the validation results confirmed that GAPDH and ACT were applicable and accurate for qRT-PCR normalization for different tissues of E. stauntonii, while ACT, GAPDH, and UBQ were effective for waterlogging-stressed leaves. These findings establish a solid foundation for future gene expression studies in E. stauntonii samples from different tissues and subjected to waterlogging stress and will facilitate future molecular studies in this economically important species.