Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR.

Vaccinium bracteatum Thunb. (VBT) is widely distributed in the mountainous areas in eastern and southern China. VBT leaves have great medical value and can be used to stain rice to produce "Wumifan". Its fruits also contain rich nutrients. However, there has been limited attention to exploring the molecular content of VBT. Previously, we performed RNA-seq on three typical VBT fruits that were at various stages of ripening, although a reliable reference gene was lost in validation.In this study, we selected ten candidate reference genes based on previous studies and transcriptomics analyses. Subsequently, these genes were evaluated using a combination of methods, including geNorm, NormFinder, and Bestkeeper, with a comprehensive ranking assessment. As a result, we found that the actin2, NADH, and ADK genes have high reliability for analysing the expression levels of genes involved in fruit development. Furthermore, the transcript levels of 15 DEGs from transcriptomic analysis were assessed using NADH as a reference gene, and RT-qPCR data were highly consistent with the transcriptomic data. These results provide reliable reference genes for further studying gene expression, which will be beneficial for comprehensively exploring VBT.