Selection of reference genes suitable for qRT-PCR expression profiling of biotic stress, nutrient deficiency and plant hormone responsive genes in bread wheat

Abstract

Quantitative real-time PCR (qRT-PCR) is the most widely used and reliable method for gene expression analysis. The accuracy of qRT-PCR relies on normalization of gene expression based on reference genes, however recent studies have shown that none of the reference genes can be used universally across different experimental conditions. In the present study, four most commonly used reference genes, viz., ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, actin (ACT) and 18S ribosomal RNA were considered. Using 32 wheat samples representing nutrient deficiency (deficiency of nitrogen, phosphorus and potassium), salinity, biotic stress (leaf rust, yellow rust, stem rust, wounding) hormone (salicylic acid, jasmonic acid, abscisic acid, auxin and gibberellin) treatments at two time points after treatment (24 and 48 h) were used to study the expression stability of reference genes. The four reference genes displayed huge variation in cycle threshold (Ct) values in the selected RNA samples. The expression stability was further quantified using three most widely used statistical software tools, viz., BestKeeper, NormFinder and geNorm. The most stable among the candidate reference genes was ACT, as indicated by the statistical analysis by different tools. Our results may assist in selecting most desirable reference gene for increasing precision of gene expression studies in bread wheat.