Reference Gene Stability in Agrostemma githago Using Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) remains a cornerstone method for analyzing gene expression due to its high sensitivity, specificity, and reproducibility. However, for reliable results in relative quantification studies, the choice of an appropriate reference gene is critical to ensure accurate normalization. The expression of commonly used reference genes can vary depending on developmental stage and experimental conditions, making their validation essential. To date, no validated reference genes have been reported for Agrostemma githago L. (corn cockle, Caryophyllaceae). To facilitate research on genes involved in natural product biosynthesis and specialized metabolism regulation, we aimed to identify the most stable reference genes across various plant organs and cultivation conditions of this species. Drawing on previous literature, we have selected seven housekeeping genes widely used for evaluation: actin, β-tubulin, elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, histone H3, translation elongation factor 1, and eukaryotic translation initiation factor 5A1 (for which two primer sets were tested). The nucleotide sequences of these potential reference genes were identified from the A. githago transcriptome. Using qRT-PCR, transcript levels of seven potential reference genes were estimated in 40 different A. githago samples, including 25 in vitro samples under various treatment conditions and 15 soil-grown samples representing A. githago organs in different developmental stages. Expression stability of candidate reference genes was assessed using the RefFinder platform, which combines four commonly applied statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ-Ct method. The results revealed that the selection of optimal reference genes varied based on the particular organ, developmental stage and condition being examined. TIF5A1-2 (one of the two primer pairs tested) and GAPHD consistently exhibited the most stable expression under various conditions in vitro. EF1α and H3 exhibited superior performance across different organs of soil-grown plants. Moreover, our integrated analysis enabled the identification of the two most stable, universal reference genes suitable for normalization in A. githago under all tested conditions—H3 and TIF5A1-2. Our work provides a robust foundation for future transcriptomic and functional studies of the specialized metabolism of A. githago and other related species.