Evaluation and validation of reference genes for RT-qPCR normalization in different sweet potato tissues

Sweet potato (Ipomoea batatas) is one of the most important crops of the world, displaying a significant economic importance in several countries. However, few studies exploring biotechnological tools, such as RT-qPCR, used for gene expression analysis, have been conducted so far, slowing down crop breeding programs. Here, a detailed analysis of previous validated reference genes, essential for normalization of RT-qPCR studies, was conducted, and new candidate genes were evaluated, addressing existing gaps for the development of molecular studies via RT-qPCR in sweet potato. To this end, five sweet potato reference genes studies were evaluated, and the six best-classified genes (IbCYC, IbARF, IbTUB, IbUBI, IbCOX and IbEF1α) were selected for further analysis. Additionally, four commonly used reference genes (IbPLD, IbACT, IbRPL and IbGAP) were also included in the study. The ten reference genes were analyzed across four different tissues (fibrous root, tuberous root, stem and leaf) from sweet potato plants grown under normal conditions. IbACT, IbARF and IbCYC were the most stable genes, displaying the lowest variation in expression levels across the tissues studied, while IbGAP, IbRPL and IbCOX were classified as the least stable genes according to the RefFinder algorithm. The results obtained here highlight the relevance of this type of investigation to ensure the reliability of relative gene expressions analyzed in sweet potato. In addition, it directly contributes for a better understanding of the biological processes associated with the performance of this crop, aiding future research addressing transcriptome analyses in this species, which displays high agricultural and industrial potential.