Selection and Validation of Reference Genes for RT-qPCR in Protonemal Tissue of the Desiccation-Tolerant Moss Pseudocrossidium replicatum Under Multiple Abiotic Stress Conditions

Plant abiotic stresses are the main cause of significant crop losses worldwide. The moss <i>Pseudocrossidium replicatum</i> is highly tolerant to different types of abiotic stress, such as desiccation. Our group is interested in identifying and characterising differentially expressed genes in response to abiotic stress in this species. However, a collection of validated reference genes for RT-qPCR analysis is essential to normalise the expression of genes in response to the conditions of interest. Here, we assessed 13 candidate reference genes for <i>P. replicatum</i> based on their expression stability across transcriptomes from six abiotic stress-related conditions using the RefFinder, BestKeeper, geNorm, and NormFinder programs. The stability and reliability of the proposed reference genes were evaluated under six experimental conditions: control, dehydration, rehydration, abscisic acid (ABA), NaCl, and sorbitol. Interestingly, most proposed reference genes exhibited high stability (low M values) across all analysed abiotic stress conditions. A pairwise variation analysis indicated that only one pair is necessary to normalise RT-qPCR experiments. Each gene was confirmed to normalise the expression of both upregulated and downregulated genes. This represents the first report of validated reference genes for RT-qPCR gene expression studies under abiotic stress in the protonemal tissue of a fully desiccation-tolerant moss.