Selection of reference genes for expression studies in Cicer arietinum L.: analysis of cyp81E3 gene expression against Ascochyta rabiei

Normalisation to a reference gene is the most common method of internally controlling for error in quantitative PCR (qPCR) experiments. Studies based on qPCR in chickpea have been carried out using potential reference genes exclusively. Inappropriate normalisation may result in the acquisition of biologically irrelevant data. We have tested the expression of 12 candidate internal control genes in 36 samples representing different organs/developmental stages, genotypes and stress conditions. The most stably expressed genes were PUBQ, GAPDH, UBQ and bHLH, whereas 18S rRNA and EF-1a showed considerable regulation. The most suitable combination of reference genes for the particular experimental sets tested is provided. To illustrate the use of chickpea reference genes, we checked the expression of a putative defence gene in two different genotypes infected with Ascochyta rabiei (Pass.) Lab. The set of reference genes presented here will enable the more accurate and reliable normalisation of qPCR results for gene expression studies in this important legume crop. Our findings can be used as a starting point for reference gene selection in experimental conditions different from those tested here.