Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs-geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder. The results showed that alpha-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that alpha-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions.