Evaluation of Oryza coarctata candidate reference genes under different abiotic stresses

Oryza coarctata Roxb. is a wild halophytic species found mostly on South Asian seashore. It can tolerate salinity up to 4 S m(-1), which is equivalent to 479 mM NaCl. This species has unique morphological, anatomical, as well as physiological adaptations, which helps it to survive under high salinity and submerged conditions. Thus, it is considered to be a reservoir of salinity and submergence tolerance genes. For quantification of gene expressions, it is necessary to identify stable reference genes and validate stress responsive genes by real time quantitative PCR. The present study deals with the expression analyses of eight candidate reference genes, namely, ubiquitin 2, histone 3, beta-tubulin, alpha-tubulin, glyceraldehyde-3-phosphate dehydrogenase, eukaryotic initiation factor 4-alpha (eIF4-alpha), cyclophilin, and actin (ACT) under different abiotic stresses such as salinity, cold, drought, heat, submergence, submergence with saline water (dual stress), and abscisic acid treatment. The four different statistical algorithms, namely, Bestkeeper, NormFinder, geNorm, and RefFinder were used to rank the genes in order of stability. The analysis revealed that ACT and eIF4-alpha were the two most stable reference genes, which was further validated by determining the expressions of two well-known stress responsive genes alcohol dehydrogenase and dehydration responsive element binding protein 2A under submergence and drought stress, respectively. This study is the first report to analyse the reference genes for O. coarctata.