Validation of reference genes for gene expression analysis using quantitative polymerase chain reaction in pea lines (Pisum sativum) with different lodging susceptibility

Real-time PCR data from every gene expression analysis experiment have to be normalised with appropriate reference genes, which are uniformly expressed in a sample set being analysed. Reference genes have to be tested independently for each experimental set. We estimated the gene expression of five potential reference genes (Phosphoprotein phosphatase 2A, histone H3, beta-tubulin, transcription factor IIA and actin) in 33 pea (Pisum sativum L.) samples. Plant material comprised stem fragments from four pea accessions (three cultivars with different lodging susceptibility and stem stiffness and one wild-type accession). Different phenological development stages of plants and environmental conditions were tested under three sample subsets. Phosphoprotein phosphatase 2A and beta-tubulin were the most stable genes in the subset of 30-day plants, grown in the greenhouse. Plants at the beginning of flowering stage were grown in the greenhouse and in the field. Phosphoprotein phosphatase 2A and transcription factor IIA exhibited the most stable expression under field conditions, while histone H3 and beta-tubulin were the most stable under greenhouse conditions. Phosphoprotein phosphatase 2A and beta-tubulin were also the most stable genes among all the tested samples. We have identified stably expressed genes, which may be used as a reference for normalisation of real-time PCR data in our sample subset. Our research is a good starting point for other studies of gene expression in pea stem tissue. This is the first step to perform gene expression studies connected with lodging resistance of pea.