Selection and Validation of Appropriate Reference Genes for <scp>qRT</scp>‐<scp>PCR</scp> Analysis of <i>Iris germanica</i> L. Under Various Abiotic Stresses

Choosing the appropriate reference genes for quantitative real-time PCR (qRT-PCR) is very important for accurately evaluating expression of target genes. <i>Iris germanica</i> L. is a widely used horticultural plant with high ornamental value, which also shows a strong ability to tolerate abiotic stresses. No comprehensive research has been carried out on optimal reference genes in <i>Iris germanica</i> L. under abiotic stress. In this study, nine reference genes were selected as candidates based on the transcriptome sequencing data of <i>Iris germanica</i> L. The assessment of expression stability under various abiotic stress was conducted using four distinct methods (GeNorm, NormFinder, BestKeeper, and RefFinder). It was found that the optimal reference genes were <i>ACT</i> and <i>F3H</i> for drought and different temperature stresses. <i>EF1α</i> and <i>ACT</i> exhibited superior performance under salt stress. The expression of the <i>IgP5CS</i> gene was evaluated to provide additional validation for the accuracy of the selected optimal reference genes, indicating that inappropriate may lead to significant deviations in the results. This research identified reliable reference genes in <i>I. germanica</i> L. across various abiotic stress conditions, thereby facilitating the investigation into the molecular mechanisms responsible for stress tolerance in <i>I. germanica</i> L.