Suitable reference genes for real-time quantitative PCR in Salsola laricifilia under five abiotic stresses

Salsola laricifolia, a typical C-3-C-4 intermediate desert plant, is an important for understanding gene evolution and mechanisms for drought resistance. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a preferred choice for gene expression studies, but it requires stable reference genes for normalization. Therefore, we tested the expression stability of five candidate reference genes in S. laricifolia: EF1 alpha (elongation factor 1-alpha), ACT (actin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (tubulin), and 18S (18S ribosomal RNA). The expressions were tested in different tissues and under five stresses caused by abscisic acid (ABA), NaCl, NaHCO3, darkness, and osmotic stress (polyethylene glycol 6000, PEG). Four commonly used software programs (geNorm, NormFinder, BestKeeper, and RefFinder) were used. The results show the following most stable reference genes: GAPDH for ABA and dark treatments; EF1a for NaCl, PEG; and all samples; TUB for NaHCO3; and 18S for the controls. The ACT was not ranked first in any group, and was the least stable reference gene under the dark, NaHCO3, and PEG. Moreover, pairwise analysis by the geNorm algorithm shows that two best reference genes were 18S and EF1a for the controls, GAPDH, and 18S for the ABA and dark treatments, EF1a and TUB for the NaCl treatment, TUB and 18S for the NaHCO3 treatment, EF1a and GAPDH for the PEG treatment, and EF1a and 18S for all samples. The reference genes for RT-qPCR in S. laricifolia identified in our study will facilitate future work on targeted gene expression.