Selection and validation of suitable reference genes for RT-qPCR analysis in dove tree (Davidia involucrata Baill.)

Abstract

Fifteen candidate reference genes were selected from the dove tree genome. Their performance as internal controls for RT-qPCR analysis was assessed in bracts at different developmental stages, normal and abortive seeds, and leaves of different colors. The applicability of the candidate reference genes was validated through detection of the expression levels of three target genes normalized by different reference genes. Dove tree (Davidia) is a relic species and has some unique traits in its reproductive organs. The molecular mechanisms underlying the development of these organs have attracted increasing attention. RT-qPCR is a powerful tool for gene expression pattern detection, which is critical for studies on gene function or regulation. However, information regarding suitable reference genes, which ensures reliable RT-qPCR results, is absent in dove tree. Based on our previously obtained Davidia transcriptome data, 15 reference genes, including eight traditional and seven novel reference genes, were selected for this study. Using geNorm, NormFinder and BestKeeper software, the stability and applicability of these candidate reference genes were assessed with regard to different aspects including bract development, seed abortion and leaf color. The candidate reference genes were ranked by the software programs, and the most suitable and unsuitable genes for different situations were determined. In addition, the expression levels of three Davidia genes of interest involved in bract development, seed abortion and leaf color, respectively, were normalized with high-ranking and low-ranking reference genes. The comparison results demonstrated that the selection of “good” or “bad” reference genes led to differing conclusions.