Validation of reference genes for quantitative RT-PCR normalization inSuaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy

Abstract

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization.Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds ofS. aralocaspicaunder different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds,β-TUB andGAPDHappeared to be the most suitable references under different developmental stages and tissues.GAPDHwas the appropriate reference gene under different germination time points and salt stress conditions, andACTINwas suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools,β-TUB served as the most stable reference gene, whereas18S rRNAand28S rRNAperformed poorly and presented as the least stable genes in our study.UBQseemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK) of C4 pathway and a salt tolerance-related gene (SAT) ofS. aralocaspicawere used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work inS. aralocaspicaand these data will facilitate further studies on gene expression in this species and other euhalophytes.