Identification of reference genes for real-time quantitative PCR in different development stage in chili pepper fruits(Capsicum annuum and Capsicum chinense)

Abstract The selection of appropriate reference genes is a fundamental prerequisite for reliable gene expression analysis using real-time quantitative polymerase chain reaction (RT-qPCR). To date, no systematic study has identified reference genes for normalizing RT-qPCR data in pepper fruits across different developmental stages. In this study, we evaluated the expression stability of seven commonly used internal control genes and nine additional candidates derived from available transcriptomic datasets in two widely cultivated chili pepper species (Capsicum annuum and Capsicum chinense) at various fruit developmental stages. The expression stability of the 16 candidate genes was assessed using four established algorithms: NormFinder, ΔCt, BestKeeper, and GeNorm. Our results demonstrated that CT8 and EIF were the most stably expressed genes during fruit development in Capsicum annuum ‘TJ’ and Capsicum chinense ‘7054’, respectively. This study provides a foundation for accurate normalization of RT-qPCR analyses in pepper fruits at different developmental stages and will help ensure the reliability of subsequent gene expression studies in these species.