Identification and selection of reference genes for real-time quantitative PCR in Dianthus spiculifolius subjected to five abiotic stresses

• D. spiculifolius : Northeast China's dual-purpose landscaping-restoration plant. • 14 candidate reference genes were selected from D. spiculifolius transcriptome data. • Stability of 14 reference genes of D. spiculifolius were assessed. • DsPTBT1 proved the most stable RG for RT-qPCR across drought, cold, ABA and Pb stresses. • DseIP-4a was identified as the most stable RG for RT-qPCR under Cd stresses. Dianthus spiculifolius is a highly valuable landscaping plant and ecological restoration material in northeastern China owing to its low-growing, clumped, and aesthetically pleasing morphology, exceptional resilience to adverse conditions, and robust adaptability to nutrient-deficient soil environments. However, the molecular mechanisms governing D. spiculifolius growth and stress responses remain largely unknown. Consequently, identifying stable reference genes, a critical prerequisite for achieving reliable gene expression normalization in RT-qPCR studies, is essential to facilitate the mechanistic investigation of this species. In this study, we analyzed the expression levels of 14 candidate reference genes in D. spiculifolius seedlings under abiotic stress conditions using real-time quantitative PCR (RT-qPCR). Three software programs were used to evaluate the stability of the expression of these 14 genes. A comprehensive analysis showed that DsPTBT1 was the most stable reference gene under drought, cold, abscisic acid (ABA), and lead (Pb) stresses, whereas DseIF-4a was the most suitable reference gene under cadmium (Cd) stress. DsTBP2 was the least stable gene in all treatments, except Cd stress. In addition, we used two optimal and one suboptimal reference gene to calibrate the expression pattern of the target gene DsCBL2 , thereby validating this perspective. This study identified and validated the first stable reference genes for D. spiculifolius , thereby establishing a critical foundation for gene expression analysis in this and related Dianthus species.