Reference gene selection and validation for gene expression studies in downy mildew infected pearl millet by quantitative real-time PCR

Pearl millet is considered as one of the future crops in terms of food security due to its drought tolerant nature. However, the plant's susceptibility to the evolving Sclerospora graminicola causing downy mildew disease has enforced researchers to identify genes in pearl millet associated with disease resistance. This could be achieved by analyzing the dynamics of gene expression with quantitative real-time PCR (qPCR). qPCR is a sensitive and reliable approach that requires suitable reference genes (RGs) as internal controls for normalizing transcript abundance of genes of interest (GOI). In the current study, six RGs i.e. ACT, TUB, UBQ, GAPDH, PP2A and EF-1 alpha were evaluated for their expression stability in two experimental datasets: treatment and time-point. The former dataset analyzed the effect of elicitor treatment while the latter analyzed the effect of time-point on the expression stability of RGs in pearl millet during post pathogen inoculation. Three statistical softwares-geNorm, NormFinder and BestKeeper facilitated identification of PP2A, TUB and UBQ as stably expressing RGs for treatment dataset and PP2A and EF-1 alpha for time-point dataset. Validation of these stable RGs for both the datasets was performed by comparative analysis of normalization strategies on the transcript abundance of GOI involved in host resistance against downy mildew. Altogether, the present study forms a basis for RG selection during qPCR analysis of pearl millet-downy mildew or other similar plant- pathogen interaction.