Validation of reference genes for quantifying changes in physiological gene expression in apple tree under cold stress and virus infection

Quantitative reverse transcription PCR is used for gene expression analysis as the accurate and sensitive method. To analyze quantification of gene expression changes in apple plants, 10 housekeeping genes (ACT, CKL, EF-1α, GAPDH, MDH, PDI, THFs, UBC, UBC10, and WD40) were evaluated for their stability of expression during infection by Apple stem grooving virus (ASGV) or in cold-stress apple plant buds. Five reference-gene validation programs were used to establish the order of the most stable genes for ASGV as CKL>THFs>GAPDH>ACT, and the least stable genes WD40<UBC10<EF-1α for infection by ASGV. For treatment by three different temperatures, the most stable genes were WD40>CKL>UBC10, and the least stable genes were ACT<UBC<THFs. To validate our findings, using two of the most stable and the one least stable validated reference genes, one defense responsive gene and one cold-stress-responsive gene were examined to compare their relative changes in gene expression. In conclusion, our results provide a useful framework for choice of suitable reference genes according to different biotic or abiotic stress conditions in plant species.