Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (Pisum sativum L.) Subjected to Abiotic and Biotic Stress

Dry pea (Pisum sativum L.) is grown as human and animal feed throughout the world. Large yield losses in pea due to biotic and abiotic stresses compel an improved understanding of mechanisms of stress tolerance and genetic determinants conditioning these tolerances. The availability of stably expressed reference genes is a prerequisite for examining differential gene expression. The objective of this study was to examine the expression profile of several candidate reference genes across a broad range of commercial pea cultivars. Expression profiles of five candidate reference genes; 18s rRNA, actin, TIF, β tubulin-2 and β tubulin-3 were examined. Relative quantifications of candidate reference genes were estimated from control plants, plants after 48 h of cold treatment, and plants 24 and 48 h after inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease of pea. RT-qPCR was performed on cDNA synthesized from three food grade spring peas, Ariel, Aragorn, and Sterling, and two spring yellow peas, Delta and Universal, which are used as animal feed. Analysis of variance (ANOVA) of CT values demonstrated significant variation between varieties and treatments under cold and disease conditions. The most abundant transcripts among tested reference genes were for 18s rRNA. Stability analysis indicated that TIF and β tubulin-3 genes were the most stably expressed candidate genes under both cold and disease stress and could serve as reference genes across a wide range of pea cultivars.