Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Ficus carica

Fig (Ficus carica L.), a deciduous fruit tree that belongs to the Moraceae family, is cultivated worldwide as an important fruit crop for raw and processed foods. Quantitative real-time PCR (RT-qPCR) is a widely used method in F. carica to elucidate expression of genes related to various physiological responses. However, no studies have identified appropriate reference genes for RT-qPCR normalization in F. carica. In this study, 12 genes were selected from the F. carica genome as candidate reference genes for normalizing target gene expression. All candidate genes exhibited high amplification efficiency and specificity in the absence of primer dimers or extra PCR amplicons. The expression levels of the candidate genes were measured in three different plant tissues (fruit, leaf, and stem) under fungal pathogen infection using RT-qPCR. Their expression stabilities were evaluated using four computational algorithms: geNorm, Normfinder, delta-Ct, and BestKeeper. The RefFinder program was also used to calculate the geometric mean of the stability rankings obtained from these algorithms. The comprehensive ranking revealed that FcYLS8, FcPP2A, and FcAP2M were the most stable reference genes under biotic stress in the fruits, leaves, and stems, respectively. In contrast, traditional reference genes such as FcACT2, FcEF-1α, FcGAPDH, FcUBC21, and FcUBQ5 exhibited relatively low expression stability in all tested tissues. This study identified and validated stable reference genes for RT-qPCR normalization in F. carica, thus providing a valuable resource for accurate gene expression studies under biotic stress and highlighting the importance of validating reference genes to ensure reliable and reproducible RT-qPCR analysis.