Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

Abstract

Abstract Real-time qPCR is currently the most sensitive technique available for the detection of low-level mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1α, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginia-type peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra- and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.