Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]

Finger millet (Eleusine coracana L.) is an annual herbaceous self-pollinating C-4 cereal crop of the arid and semi-arid regions of the world. Finger millet is a food security crop proven to have resilience to changing climate and scores very high in nutrition. In the current study, we have assessed sixteen candidate reference genes for their appropriateness for the normalization studies in finger millet subjected to experimental regimes and treatments. Ten candidate reference genes (GAPDH, beta-TUB, CYP, EIF4 alpha, TIP41, UBC, G6PD, S24, MACP and MDH) were cloned and six (ACT, ELF1 alpha, PP2A, PT, S21 and TFIID) were mined from the NCBI database as well as from the literature. Expression stability ranking of the finger millet reference genes was validated using four different statistical tools i.e., geNorm, Norm-Finder, BestKeeper, Delta Ct and RefFinder. From the study, we endorse MACP, CYP, EIF4 alpha to be most stable candidate reference genes in all 'tissues', whereas PT, TFIID, MACP ranked high across genotypes, beta-TUB, CYP, ELF1 alpha were found to be best under abiotic stresses and 'all samples set'. The study recommends using minimum of two reference genes for RT-qPCR data normalizations in finger millet. All in all, CYP, beta-TUB, and EF1 alpha, in combination were found to be best for robust normalizations under most experimental conditions. The best and the least stable genes were validated for confirmation by assessing their appropriateness for normalization studies using EcNAC1 gene. The report provides the first comprehensive list of suitable stable candidate reference genes for nutritional rich cereal finger millet that will be advantageous to gene expression studies in this crop.