Selection and Validation of Reference Genes for RT-qPCR Across Different Organs, Flower Bud Developmental Stages, and Hormone Treatments in Ilex verticillata

Gene expression profiling plays a pivotal role in elucidating molecular regulatory networks and identifying functionally critical genes. However, the accuracy and reliability of quantitative real-time PCR (qPCR) assays fundamentally depend on the selection of stable expressed reference genes. Ilex verticillata, an economically important ornamental shrub, remains poorly characterized at the molecular level. In this study, the expression stability of 15 candidate reference genes was systematically evaluated across four major tissue types—roots, stems, leaves, and fruits. Using raw Ct values, the stability of each candidate reference gene was rigorously assessed and ranked by four widely accepted algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Based on consensus rankings, six genes (18S, 28S, Actin2, EIF4A, PT, and PP2A) were preliminarily identified as the most stable candidates. These six genes were subsequently validated in two additional experimental contexts: flower buds collected at distinct developmental stages and leaves subjected to a panel of phytohormone treatments. Results revealed that Actin2 exhibited the highest overall expression stability across all tested conditions, with 28S serving as a robust alternative; in contrast, 18S displayed comparatively low stability. To further corroborate the suitability of the selected reference genes, three biologically relevant target genes —IvFT, IvAGL19, and IvWUS2—were chosen for validation. Normalization using either the top-ranked single reference gene Actin2 or the optimal multi-gene combination yielded highly consistent and biologically plausible expression profiles for all three targets. Conversely, normalization against the least stable reference gene produced markedly inconsistent and unreliable expression patterns. Collectively, these findings establish a rigorous, experimentally validated framework for reference gene selection in qPCR-based studies of flower bud development and hormonal responses in Ilex verticillata.