Reference gene validation for qRT-PCR based gene expression studies in different developmental stages and under biotic stress in apple

- Ten putative reference genes for qRT-PCR data normalization have been identified in apple.
- Stages of bud to fruit development and samples under biotic stresses were analyzed.
- PCR efficiencies of all the genes were found to be ranging between 94% and 107.6%.
- PP2A, RPL2 and GAPDH were found to be stable reference genes across the samples.
- The 18S rRNA, β-tubulin and ubiquitin were found to be the least stable genes.

Apple is one of the most important fruit crops and the availability of recently sequenced genome facilitates research on gene-trait relationship. Quantitative real-time PCR (qRT-PCR) is a sensitive technique to study gene expression, but its accuracy largely depends on stability of reference gene used for data normalization. Therefore, present study was aimed to identify and validate suitable reference gene in apple. Expression stabilities of 10 housekeeping genes, which are commonly used as reference genes in qRT-PCR studies were evaluated in samples of different developmental stages, various tissue types and under diverse biotic stress conditions in apple. The PCR efficiency of all the genes was found to be ranging between 94-107.6%. Analysis using geNorm, NormFinder and Bestkeeper programs demonstrated that their expression stabilities vary among sample sets. However, protein phosphatase 2A (PP2A), ribosomal protein L2 (RPL2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were found to be suitable reference genes across all the tissue types and under different biotic stress conditions, while 18S ribosomal RNA (18S), β-tubulin (TUB) and ubiquitin (UBQ) were found to be the least stable genes. Moreover, a combination of different reference genes was suggested for different sample sets. These, results suggest that selection of suitable reference gene depends on the tissue type and development stage or disease condition. Further, use of more than one reference genes in respective tissue types of apple is suggested to accurately normalize qRT-PCR data, in future.