Selection of suitable reference gene for gene expression studies during groundnut seed germination

Understanding the mechanism of seed germination requires a sturdy selection of reference genes for expression analysis using qRT-PCR. The accurate normalization of genes becomes necessary to circumvent erroneous result, as housekeeping genes does not remain stable over the different experimental condition in various tissue or species. Reliable and stable reference gene during ethrel induced germination of groundnut seeds is not reported yet. In this study, seven candidate reference genes were selected based on previous reports in groundnut under different experimental conditions. Seeds of NRCG 14380 (dormant) and TAG 24 (non-dormant) genotypes were treated with ethrel and sampled at 0, 6, 12 and 24 hours after incubation. The stability of reference genes such as actin1 (ACT1), actin11 (ACT11), alcohol dehydrogenase (ADH3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), ubiquitin C (UBC) and alpha-Tubulin (TUA) was analyzed through a different statistical algorithm such as BestKeeper, NormFinder, geNorm and its consensus stability ranking was retrieved using RefFinder. Among all reference genes studied, ACT1 was found most stable reference gene in our study. Stability and reliability of ACT1 and ADH3 (most stable) reference genes were further validated through qRT- PCR with relative quantification of two germination related genes ACC oxidase 1 (ACO1) and gibberellic acid regulated protein which showed consistency in their expression level. Our result revealed a stable reference gene that could be used as an internal control gene for gene expression study of ethrel treated groundnut seed germination.