Selection and validation of reference genes for the normalization of RT-qPCR gene expression data in Rheum tanguticum...

Rheum tanguticum, an endemic species from the Qinghai-Xizang Plateau, is a significant perennial and medicinal plant recognized for its robust resistance to abiotic stresses, including drought, cold, and salinity. To advance the understanding of stress-response mechanisms in R. tanguticum, this study aimed to establish a reliable set of housekeeping genes as references for normalizing RT-qPCR gene expression analyses. Ten candidate genes were selected based on prior studies of related species and transcriptomic data for R. tanguticum. Their expression stability was evaluated across three tissue types (leaf, stem, and root) under four abiotic stress treatments using a comprehensive suite of bioinformatics tools. Our findings revealed that RtEF1α was the most stable reference gene in salt- and drought-stressed roots and drought-stressed stems, while RtTUB was most stable in salt-stressed stems. RtSAND was identified as the most stable reference gene in salt-, drought-, heavy metal-, and hormone-stressed leaves. For heavy metal- and hormone-stressed roots and heavy metal-stressed stems, RtUBC demonstrated the highest stability, whereas RteIF6A was most stable in hormone-stressed stems. Furthermore, the utility of these reference genes was validated by assessing the expression pattern of the drought-related gene RtNAC. This study is the first report on selecting and validating reference genes of R. tanguticum under various stress conditions, which will benefit future investigations of the genomic mechanisms involved on stress resistance in this species.