Identification and validation of reference genes for quantitative real-time PCR under salt stress in a halophyte, Sesuvium portulacastrum

Abstract

Sesuvium portulacastrum L. is a facultative halophyte with potential applications in phytoremediation and phytodesalination and offers as a system to understand mechanisms of salt adaptation. However, studies on gene identification, characterization and transcriptomics of this halophyte are limited due the paucity of genome sequence information and validation of gene expression data through quantitative real time-PCR (qRT-PCR). qRT-PCR is a reliable method for exact quantification of gene expression, but is dependent upon the selection of suitable reference gene(s). In the present study, using Sesuvium RNA-Seq data, we identified ten reference genes such as elongation factor 1-α, ubiquitin-conjugating enzyme E2, TATA-box binding protein, DnaJ-like protein, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase, eukaryotic initiation factor 4a, RNA-dependent RNA polymerase 1, α-tubulin and pentatricopeptide repeat and validated their stable expression in root and shoot tissues under 0, 100 and 250mM NaCl stress conditions. Five different statistical methods viz. Delta Ct, NormFinder, BestKeeper, geNorm and ReFinder were used to establish the most suitable reference genes for root (UCE 2, TBP, EF1-α) and shoot (α-TUB, EIF4a, EF1-α). Additionally, the expression of five salt responsive genes was calculated under salt stress using the best reference genes and compared with RNA-Seq data. The comparison between real time and RNA-Seq data showed a high correlation (r2=0.875), supporting the selection of the suitable reference genes. This study provides a good starting platform for successful gene quantification in S. portualcastrum.