Abstract

(1) Reverse transcription quantitative real-time PCR (RT-qPCR) requires reliable reference genes for accurate data normalization; however, optimal reference genes for the economically and ecologically valuable timber species Phoebe zhennan remain uncharacterized; (2) Here, we selected nine candidate reference genes derived from full-length transcriptome sequencing to evaluate their expression stability across abiotic (drought) and biotic (Colletotrichum fructicola infection) stresses. Transcript abundance was analyzed via RT-qPCR using four distinct algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), with RefFinder used to reconcile analytical discrepancies and generate a definitive consensus ranking; (3) Our analysis showed that expression stability is highly context-dependent: CYP20-1 and HSP70-1 were the most stable reference genes under drought stress, whereas Actin-101 and Actin constituted the optimal pair under disease stress. For cross-condition assessments, Actin-101 and β-Tubulin served as the most reliable baseline combination. Subsequent empirical validation quantifying stress-responsive transcripts demonstrated a significant positive correlation between RT-qPCR relative expression and corresponding RNA-seq data (drought: R = 0.80; disease: R = 0.76); (4) This study identifies and validates the first set of reference genes for P. zhennan, providing a foundation for accurate gene expression analysis in this species, which is crucial for understanding its response to environmental stresses.