Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR

Quantitative real-time polymerase chain reaction (qRT-PCR) has been extensively used in several plant species as an accurate technique for gene expression analysis. However, the expression level of a target gene may be misconstrued due to unstable expression of the reference genes under different experimental conditions. Therefore, it is necessary to systematically evaluate these reference genes before experiments are conducted. Recently, more and more studies have focused on gene expression in pepper (Capsicum annuum L.). In this study, ten putative reference genes were chosen to identify expression stability by using geNorm and NormFinder statistical algorithms in ten different pepper sample pools, including those from different plant tissues (root, stem, leaf and flower) and from plants treated with hormones (salicylic acid and gibberellic acid) and abiotic stresses (cold, heat, salt and drought). EF1 alpha and UEP exhibited the most stable expression across all of the tested pepper samples. For abiotic stress or different hormone treatment, the ranking of candidate reference genes was not completely consistent, except for EF1 alpha which showed a relatively stable expression level. For different tissues, the expression of Actin1 was stable and it was considered an appropriate reference gene. It is concluded that EF1 alpha, UEP and Actin1 are suitable reference genes for reliable qRT-PCR data normalization for the tissues and experimental conditions used in this experiment.