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

Abstract

Radish (Raphanus sativus) is an important cruciferous root crop with a close relationship to Chinese cabbage (Brassica rapa). 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. GAPDH, DSS1, and UP2 were optimal reference genes for gene expression analysis in all organs and conditions in radish. UPR, GSNOR1, and ACTIN2/7 were the most stable reference genes in different radish organs. UP2 and GAPDH were suitable reference genes for radish pistil development studies. RPII, UBC9, and GAPDH had the most stable expression in radish under various stresses. DSS1, UP2, and TEF2 were the optimal reference genes for Chinese cabbage organs, whereas TUA was optimal for the distant hybrid. UP2, and TEF2 were appropriate reference genes for all of the samples together. The optimal reference genes we identified, UP2, GAPDH, UPR, and GSNOR1 were verified by normalizing the expression patterns of YAB3, RPL, and FUL. 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.

Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data

“…Fortunately, BestKeeper still showed some conformance in the top five most stable reference genes with geNorm and Normfinder (Figure 4; Tables 2 and 3). Finally, a widely used web-based tool RefFinder was used to obtain a consensus stability ranking of each candidate gene under the given conditions according to the geometric mean of the attributed weights of each gene [45,47]. For RefFinder analysis, hnRNP, RPL5, TBP, and EF-1α were ranked as the top four most stable reference genes similar with the results from geNorm, Normfinder, and BestKeeper.…”

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plants under differential biotic stresses

“…Similarly, EF1-a gene was found to perform well for aphid-infested chrysanthemum 39 , and EF1A 2a, EF1A 1a1 and EF1A 2b were also identified as the best RG in JA-treated leaves of soybean 40 . GAPDH, ACTIN and UBC are the commonly used RGs for qRT-PCR analysis in varied plant, whose function is maintaining cell survival irrespective of physiological conditions [41][42][43] . In this study, we found that ACTIN, UBC and GAPDH were the top three appropriate RGs for the whole samples of T. aurantii-infested leaves ( Table 4), but GAPDH and ACTIN were less stable in peach 44 .…”

The 2b protein and C-terminal region of the 2a protein indispensably facilitate systemic movement of cucumber mosaic virus in radish with supplementary function by either the 3a or the coat protein

“…Primers used for quantitative RT-PCR amplification of RNA2 were selected to anneal to corresponding identical sequences between CMV-Y and CMV-D8. ACTIN2/7 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes [9] were used as reference genes for relative quantification (Additional file 5: Table S3).…”