Selection and validation of reference genes for quantitative gene expression analyses in black locust (Robinia pseudoacacia L.) using real-time quantitative PCR

Black locust (Robinia pseudoacacia L.) is an easy to raise, fast growing, medium-sized deciduous tree species highly tolerant to harsh eco-conditions, i.e., drought and harsh winters, and it is widely adaptable to sandy, loamy, and marshy soils. The basis for this adaptability remains to be investigated at the transcriptomic level using real-time quantitative PCR (qPCR). Selection of a reliable gene for the normalization of qPCR data is important for obtaining accurate results in gene expression. The goal of this study was to identify an appropriate reference gene from 12 candidate genes for gene expression analysis in black locust exposed to various stressors such as abscisic acid (ABA), NaCl, polyethylene glycol (PEG) and varying temperatures. In GeNorm and NormFinder analyses, ACT (actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene expression were the most stable in all conditions except heat stress, but in BestKeeper analysis, GAPDH and helicase gene expression were the most stable under NaCl and heat stress. In contrast, ACT and GAPDH were highest under abscisic acid (ABA), GAPDH and beta TUB (beta tubulin) under cold stress, and helicase and EF1 alpha (elongation factor 1 alpha) under PEG stress. We found that the most stable reference gene combination for all conditions was ACT and GAPDH. Additionally, the expression pattern of NAC2 (a transcription factor) and BGL2 in different tissues and under different stress conditions was analyzed relative to ACT and GAPDH and UBQ (ubiquitin) the least stably expressed gene. NAC2 and BGL2 both had highest expression in flowers and pods under ABA stress at 48h. This study provides useful reference genes for future gene expression studies in black locust.