Establishment of reference (housekeeping) genes via quantitative real-time PCR for investigation of the genomic basis of abiotic stress resistance in Psammochloa villosa (Poaceae).

Psammochloa villosa is a desert plant growing in Northwest China with considerable resistance to abiotic stress, including drought, cold, and salt. To facilitate future studies of stress resistance in Psammochloa villosa, we sought to establish a suite of reference (or housekeeping) genes for utilization within future gene expression studies. Specifically, we selected nine candidate genes based on prior studies and new transcriptomic data for P. villosa, and we evaluated their expression stability in three different tissues of P. villosa under different treatments simulating abiotic stress conditions using four different bioinformatics assessments. Our results showed that TIP41 (TIP41-like family protein) was the most stable reference gene in drought- and salt-stressed leaves and salt-stressed stems, ELF-1α (elongation factor 1-α) was the most stable in cold-stressed leaves and drought- and salt-stressed roots, ACT (actin) was the most stable in drought-stressed stems, TUA (α-tubulin) was the most stable in cold-stressed stems, and 18S rRNA (18S ribosomal RNA) was the most stable in cold-stressed roots. Additionally, we tested the utility of these candidate reference genes to detect the expression pattern of P5CS (Δ-pyrroline-5-carboxylate synthetase), which is a drought-related gene. This study is the first report on selecting and validating reference genes of P. villosa under various stress conditions and will benefit future investigations of the genomic mechanisms of stress resistance in this ecologically important species.