Identification of optimal and novel reference genes for quantitative real‐time polymerase chain reaction analysis in grapevine

Background and Aims Quantitative real‐time polymerase chain reaction (qRT‐PCR) has the advantages of sensitivity, specificity and repeatability, and is commonly used to investigate the expression levels of genes. The selection of appropriate endogenous reference genes is the pre‐condition for acquiring the desired results of qRT‐PCR. This study aimed to identify and screen the most suitable reference genes for grapevines. Methods and Results Three novel genes from previous transcriptomic data were selected for their stable expression either at the developmental stages of the grape berry or under the various treatments. The expression stability of these three candidate genes and six commonly used reference genes were evaluated with geNorm, NormFinder and BestKeeper algorithms in several grapevine samples. The geNorm analysis indicated that two reference genes were sufficient to normalise expression. The optimal reference gene combinations were: VvEF1‐γ and PPR2 in fruit; RRM1 and EF1‐α in leaf; EF1‐α and Actin in tendril of several cultivars. The combination of EF1‐α and VvEF1‐α was the most appropriate reference gene for fruits at different developmental stages. When all the samples were considered, the combination of RRM1 and Ubiquitin was optimal. Conclusions Different reference genes should be employed when qRT‐PCR is performed in different grapevine samples. The gene RRM1 is a new appropriate reference gene for grapevine. Significance of the Study This study provided alternatives for the optimal reference genes in qRT‐PCR, and identified one novel and appropriate reference gene (RRM1) for grapevine.