Validation of reference genes for real-time quantitative PCR in Brachiaria grass under salt stress

Abstract

Salinity is one of the most important abiotic stresses that affect plant yield. Quantitative gene expression using real-time PCR (RT-qPCR) is a powerful tool to measure the transcriptional changes that occur in plant tissues under stress. For valid RT-qPCR analysis, normalization against the appropriate reference genes is essential for data accuracy. Despite the importance of Brachiaria grass (syn. Urochloa) as a tropical forage; there are no studies on the stability of reference genes under salt stress in this species. This study aimed to evaluate the stability of seven candidate reference genes: Actin 12; eukaryotic initiation factor 4A; elongation factor 1- α; tubulin α-5; tubulin β-6; ubiquitin-conjugating enzyme and glucose-6-phosphate dehydrogenase for quantitative real-time PCR assays in Urochola brizantha under salt stress. Total RNA was extracted from shoots and roots of plants cultivated in a hydroponic system containing 200 mM NaCl during 0, 6, 12, and 24 h for RT-qPCR analysis. We used the RefFinder web tool to establish a comprehensive rank for gene stability. Eukaryotic initiation factor 4A ranked as the most stable gene for both tissues followed by tubulin α-5 (shoots) and actin12 (roots) while tubulin beta-6 and glucose-6-phosphate dehydrogenase were the least stable genes for shoots and roots, respectively. The most and the least stable genes were then used to normalize the relative expression of a Na+/H+ antiporter (NHX) gene. The relative quantification of this gene varied according to the internal controls (most stable, least stable housekeeping genes), confirming the choice of the reference genes.