Validation of reference genes for qRT-PCR analysis in peel and flesh of six apple cultivars (Malus domestica) at diverse stages of fruit development

Apple (Malus domestica), as one of world’s significant fruits, color of fruit contributes a lot for its high economic benefits and edible values. Quantitative real-time polymerase chain reaction (qRT-PCR) is a technique in molecular biology commonly used for the analysis of gene expression. Actually, stability of reference genes used in data normalization affects the accuracy and reliability of qRT-PCR to a large extend. Therefore, the present study was designed to identify and validate the suitable reference genes during the fruit development. We evaluated the stability of 10 candidate reference genes in peel or flesh samples of six cultivars during different development conditions. The qRT-PCR results showed that amplification efficiency of all candidate genes ranged from 95 to 111.3%. Three software packages geNorm, NormFinder, and BestKeeper recommended different reference genes in one same sample sets. So, an online tool RefFinder was applied for integrating above three results and then these 10 reference genes were ranked comprehensively. According to RefFinder, for all fruit development conditions, WD40, ACT and GAPDH were identified as the most reliable reference genes for apple peel; EF-1α, CKL and WD40 for apple flesh, and MDH was the least stable reference gene for both samples. And, the expression patterns of target gene MdUFGT also validated this result. Further, we validated the suggested combination of different genes. Through the selection and validation of these internal reference genes, the accuracy and reliability of gene expression analysis in apple fruit tissues can be improved.