Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut

The reverse transcription quantitative real-time PCR (RT-qPCR) is becoming increasingly important for gene expression studies. However, the accuracy and reliability of RT-qPCR depend on normalizing expression to reference genes. In this study, ten candidate reference genes, including cyclophilin (CYP), elongation factor 1b (EF1b), α-tubulin (TUA5), β-tubulin (TUB4), ubiquitin10R (UBQ10R), 60S ribosomal RNA (60S), alcohol dehydrogenase (ADH3), metalloprotease (MTP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT2) were evaluated for the stability of expression in three tissues of two peanut cultivars [Zhonghua 2(ZH2) and 99-1507] under Al stress by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). The results suggested that the top-ranked reference genes under Al-induced programmed cell death (PCD) in peanut were UBQ10R, EF1b and CYP, with the most suitable combination of reference genes being [UBQ10R+ACT2]. The UBQ10R exhibited the most stable expression in all samples, while TUB4 was the least stable gene. The relative expression of AhMC1 (the caspase-like protease family gene, which played a significant role in Al-induced PCD) showed that there was no significant difference with the best reference gene and the best gene combination in RT-qPCR normalization, but there was significant difference with the least stable gene TUB4 as reference gene. This is the first study to evaluate the stability of reference genes in peanut under Al-induced PCD, and the results will provide guidance to identify appropriate reference genes for further RT-qPCR analyses under Al stress in peanut.