Selection and Validation of Stable Reference Genes for Accurate qRT-PCR Analysis of Flower Color Development in Rhododendron lapponicum

Rhododendron lapponicum (L.) Wahlenb., prized for its vibrant and diverse floral displays, holds significant ornamental and ecological value. However, advances in its molecular breeding have been constrained by the absence of reliable tools for accurate gene expression analysis. A fundamental requirement for such studies is the identification of stable reference genes for qRT-PCR. To date, no systematically validated reference genes exist for normalizing gene expression across R. lapponicum cultivars with diverse flower colors, representing a major technical obstacle to elucidating the molecular mechanisms of color formation. This study aimed to fill this gap by systematically identifying and validating optimal reference genes for petal tissues in six distinct R. lapponicum cultivars. We assessed the expression stability of 11 candidate genes using four independent algorithms and integrated the results via RefFinder. Our comprehensive analysis across multiple algorithms consistently identified RlaEF1-α and RlaACT as the most stably expressed reference genes. Their reliability was robustly validated by normalizing the expression of RlaMYB113, a key anthocyanin regulator; the normalized expression levels showed an extremely significant difference between rose-red and white cultivars (p < 0.001) and produced a coherent, phenotype-correlated profile, in contrast to the distorted patterns obtained with unstable references. This study establishes RlaEF1-α and RlaACT as a precise dual-gene internal control for qRT-PCR. By providing a validated normalization framework, our work enables accurate quantification of color-related genes and directly supports molecular breeding efforts aimed at the targeted development and selection of novel R. lapponicum cultivars with desirable and stable flower colors.