Stabilizing the Baseline: Reference Gene Evaluation in Three Invasive Reynoutria Species

Accurate normalization is crucial for reliable gene expression quantification and depends on stably expressed housekeeping genes (HKGs) as internal controls. However, HKGs expression varies with developmental stage, tissue type, and treatments, potentially introducing bias and compromising data accuracy. Thus, validating candidate reference genes under defined conditions is essential. <i>Reynoutria</i>, also known as giant Asian knotweeds, is a Polygonaceae family genus of several medicinal plants producing a diverse array of specialized metabolites of pharmacological interest. Outside their native range, these plants are also noxious invasive weeds, causing significant environmental and economic threats. Research on stable reference genes in these species is limited, with a primary focus on <i>R. japonica</i>. To enable accurate gene expression analysis related to specialized metabolism and natural product biosynthesis, we aimed to identify the most stable reference genes across the most common species: <i>R. japonica</i> Houtt., <i>R. sachalinensis</i> (F. Schmidt) Nakai, and their hybrid-<i>R.</i> × <i>bohemica</i> Chrtek & Chrtková. In this study, we evaluated twelve candidate HKGs (<i>ACT</i>, <i>TUA</i>, <i>TUB</i>, <i>GAPDH</i>, <i>EF-1γ</i>, <i>UBQ</i>, <i>UBC</i>, <i>60SrRNA</i>, <i>eIF6A</i>, <i>SKD1</i>, <i>YLS8</i>, and <i>NDUFA13</i>) across three tissue types (rhizomes, leaves, and flowers) from three <i>Reynoutria</i> species sampled at peak flowering. Primer specificity and amplification efficiency were confirmed through standard-curve analysis. We assessed expression stability using ΔCt, geNorm, NormFinder, and BestKeeper, and generated comprehensive rankings with RefFinder. Our integrated analysis revealed organ- and species-dependent stability differences, yet identified up to three reference genes suitable for interspecific normalization in <i>Reynoutria</i>. This represents the first systematic, comparative validation of HKGs across closely related knotweed species, providing a robust foundation for future transcriptomic and functional studies of their specialized metabolism and other biological processes.