Selection of reference genes for quantitative real-time PCR analysis in halophytic plantRhizophora apiculata

Abstract

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.