Identification of Suitable Reference Genes for Expression Studies in Pomegranate Under Different Biotic and Abiotic Stress Conditions

Abstract Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate