Identification of Reference Genes for RT-qPCR Assays in Sambucus nigra L

Reverse transcription quantitative PCR (RT-qPCR) is one of the most widely used techniques for gene expression analysis in molecular biology. However, the accuracy of relative gene expression quantification largely depends on the stability of the reference genes used for normalization. Sambucus nigra L. (elderberry) is a valuable medicinal and edible plant rich in anthocyanins and other bioactive compounds. Despite its increasing research and application value, no reference genes have been validated for this species. In this study, we applied RT-qPCR alongside four algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder) to assess the expression stability of nine candidate reference genes across ten samples representing five tissue types (including stems, flowers, leaves, roots, fruits) at different developmental stages. The results showed that VAMP and Pol were the most suitable reference gene combination for normalization across different tissues of S. nigra. For studies involving only vegetative tissues (leaves and stems), our results recommend that RPB2 and RPB5 are the most stable and suitable reference genes. This study provides reliable reference genes for accurate RT-qPCR-based gene expression analysis in S. nigra and establishes a useful methodological basis for future functional genomics and molecular breeding studies in this species.