Validation of candidate reference genes in RT-qPCR studies of developing olive fruit and expression analysis of four genes involved in fatty acids metabolism

Olive (Olea europaea L.) is an evergreen Mediterranean oil fruit tree with great economic, cultural and historical importance. For accurate gene expression studies of specific genes, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is often the method of choice, using suitable reference genes (RGs). This study identified RGs for RT-qPCR studies of developing olive fruit from 29 RG candidates. We used 12 sampling points to cover the five stages of olive fruit development. According to the results of the geNorm algorithm, the two best RGs were TIP41-like family protein (TIP41) and TATA binding protein (TBP), while several classical RGs proved not to be suitable. Using the two new RGs, four genes involved in the metabolism of fatty acids were studied and showed distinct expression patterns associated with mesocarp development and ripening stages. In addition to identifying two RGs for future analysis of gene expression in olive fruit, our results also provide a list of potential RGs that can be easily tested in other studies of olive gene expression in different developmental stages or in biologically challenged olive tissues. The results are also valuable for future research on genes that influence the synthesis and accumulation of olive fruit metabolites.