Background: Due to its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a widely used method to study expression of genes responsive to stress. It is crucial to have a suitable set of reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt, heavy metal and low temperature) and hormone (SA and ABA) conditions as well as across different organ types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the expression stabilities of reference genes and the comprehensive rankings of gene stability were generated.

Results: The results indicated that EFL1B and YLS8 were the most stable reference genes under PEG simulated drought treatment. For high-salt treatment by NaCl, YLS8 and GAPDH were the most stable genes. Under CdCl₂ treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3 and ACTIN11 were sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference genes showed relative high stability under hormone treatments. For organs subset, UBI1, GAPDH and EFL1B were with the maximum stability. UBI1 and ADH3 were the top two genes which could be used reliably in all of the stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed by the expression pattern of AnnAhs.

Conclusions: The results perfect the selection of stable reference genes for future gene expression studies in peanut and provide a list of reference genes may be used in the future.