Reliable reference gene selection for quantitative real-time PCR (qRT-PCR) in floral developmental phases of dioecious species Coccinia grandis

Abstract

The flowering process is intricate and regulated by a combination of external and internal factors. Delving into gene expression research has the potential to enhance our comprehension of the molecular foundations underlying floral development. Because of its accuracy, specificity, reproducibility, and efficiency, qRT-PCR is now a biological research tool for studying expression pattern of desired genes. The gene expression investigations using qRT-PCR required a reference gene with relatively uniform expression levels in multiple biological samples, including different developmental stages, tissues, and experimental conditions. In this study, experimental sets offloral and floral organ development in the male and female plants of C. grandis, a dioecious Cucurbitaceae species, qRT-PCR profiling was performed using six reference genes as internal control with B-class floral identity gene, PISTILLATA (PI). To analyse the data, algorithms such as geNorm, NormFinder, RefFinder, and BestKeeper were used to pick out the best internal controls from a group of candidates. The optimal reference gene for qRT-PCR studies with floral samples has been recommended as β-actin combined with β-tubulin. This is the first report on the validation of candidate reference genes across flower developmental stages in the dioecious species C. grandis, which will provide basic data for research on the molecular mechanism underlying flower development in this species and lay the groundwork for similar studies in other related species.