Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR

To facilitate gene expression studies in pearl millet (Pennisetum glaucum (L.) R. Br.), the key reference genes including ACP, ACT, TUB, CYP, EF-1α, EIF4A, GAPDH, MDH, PP2C, UBC and S24 were selected based on the available literature, and their expression stabilities were studied to determine their suitability for normalizing gene expression in pearl millet. Sequence information of the reference genes were obtained from the closely related species and cloned from pearl millet using homology based cloning strategy. Further, expression stabilities were validated for their accurate expression in different tissues, genotypes and abiotic stress treatments using three statistical algorithms including geNorm, NormFinder and RefFinder. Analysis showed that while the expression of EF-1α and EIF4A was most stable in different plant tissues, MDH and EIF4A were stable under different abiotic stress conditions. Amongst the different genotypes of pearl millet tested, while UBC and MDH genes exhibited most stable expression, MDH and ACP showed greater stability in all samples set. Interestingly, the widely used reference genes S24 and TUB were found to be least stable across all the tested samples. Pair-wise analysis showed that two reference genes were sufficient for proper normalization, except when analyzing the gene expression studies in all samples set. Results of this study can help in the selection of reference genes for quantitative real time PCR (qRT-PCR) normalization in pearl millet that will contribute towards more accurate and reliable quantification of transcripts in this important crop of the drylands