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

Area of Science:

• Plant biology
• Molecular biology
• Bioinformatics

Background:

• Accurate gene expression quantification relies on stably expressed housekeeping genes (HKGs) for normalization.
• HKG expression can vary with developmental stage, tissue, and treatment, potentially introducing bias.
• Limited research exists on stable reference genes in invasive Reynoutria species, hindering specialized metabolism studies.

Purpose of the Study:

• To identify the most stable reference genes for accurate gene expression analysis in three common Reynoutria species (R. japonica, R. sachalinensis, and R. × bohemica).
• To enable reliable transcriptomic and functional studies of specialized metabolism and natural product biosynthesis in these species.

Main Methods:

• Evaluated twelve candidate HKGs across rhizome, leaf, and flower tissues from three Reynoutria species.
• Confirmed primer specificity and amplification efficiency using standard-curve analysis.
• Assessed gene expression stability using ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder algorithms.

Main Results:

• Identified significant organ- and species-dependent differences in HKG stability.
• Determined that up to three reference genes are suitable for interspecific normalization within the Reynoutria genus.
• This is the first systematic validation of HKGs across closely related knotweed species.

Conclusions:

• Provides a robust foundation for future transcriptomic and functional studies in Reynoutria.
• Enables accurate gene expression analysis for understanding specialized metabolism and natural product biosynthesis.
• Highlights the importance of validating reference genes for specific experimental conditions and species.