# Selection and Validation of Stable Reference Genes for RT-qPCR Analyses of Rumex patientia (Polygonaceae) Under Four Abiotic Stresses

## TL;DR

This study identifies reliable reference genes for gene expression analysis in Rumex patientia under various environmental stresses.

## Contribution

The study provides the first validated reference gene framework for R. patientia under multiple abiotic stress conditions.

## Key findings

- ACT was the most stable reference gene in root/leaf tissues under cold stress and salt-treated roots.
- TUA showed the highest stability in cold- and salt-stressed stems, while SKD1 was most stable in drought and heavy-metal conditions.
- Validation using the drought-inducible MYB transcription factor confirmed the reliability of the selected reference genes.

## Abstract

Background: Rumex patientia (Polygonaceae), a perennial herbaceous species predominantly found in northern temperate regions, has been historically utilized in traditional Chinese medicine for its hematological regulatory properties, including blood cooling, hemostasis, and detoxification. Despite the pharmacological value of this species, unvalidated reference genes compromise precise gene expression profiling. Methods: We initially selected eight candidate genes (ACT, GAPDH, YLS, SKD1, UBQ, UBC, EF-1α, TUA) from R. patientia transcriptomes and then assessed their transcriptional stability using RT-qPCR across root, stem, and leaf tissues under four abiotic stresses: cold, drought, salinity, and heavy metal exposure. Results: ACT emerged as the most stable reference gene in three specific scenarios: root/leaf tissues under cold stress, stems during drought exposure, and roots subjected to salt treatment, revealing distinct tissue–stress response patterns. TUA emerged as the most stable reference in cold- and salt-challenged stems, while SKD1 exhibited superior stability in drought-affected roots/leaves, heavy-metal-stressed tissues, and salt-treated leaves. Validation via the drought-inducible MYB transcription factor confirmed reference gene reliability. Conclusions: This work pioneers a standardized reference gene framework for R. patientia under multi-stress conditions, offering essential methodological foundations for subsequent molecular research in this medicinal plant.