Identification of Optimal Reference Genes for Expression Analysis in Radish ( L.) and Its Relatives Based on Expression Stability.

Radish () is an important cruciferous root crop with a close relationship to Chinese cabbage (). RT-qPCR is used extensively to evaluate the expression levels of target genes, and accurate measurement of target gene expression with this method is determined by the valid reference genes used for data nomalization in different experimental conditions. Screening for appropriate reference genes with stable expression based on RT-qPCR data is important for gene expression and functional analysis research in radish and its relatives. However, many researches have thought that almost no single reference gene is widely suitable for all experimental conditions, and few researchers have paid attention to the validation of reference genes in radish gene expression analysis. In the present study, 12 candidate reference genes were selected for analysis. Their expression in 28 samples, including 20 radish samples from different organs and conditions, four Chinese cabbage organs and four organs of their distant hybrid, was assessed by RT-qPCR and then five software tools-ΔCt, geNorm, NormFinder, BestKeeper and RefFinder-were used to compare their expression stability. The results showed that the most suitable reference genes were different in different organs and conditions. , and were optimal reference genes for gene expression analysis in all organs and conditions in radish. , and were the most stable reference genes in different radish organs. and were suitable reference genes for radish pistil development studies. , and had the most stable expression in radish under various stresses. , and were the optimal reference genes for Chinese cabbage organs, whereas was optimal for the distant hybrid. , and were appropriate reference genes for all of the samples together. The optimal reference genes we identified, , and were verified by normalizing the expression patterns of , and . These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.