Gene

Real-time polymerase chain reaction

Brachiaria

Biology

Abiotic stress

Botany

Housekeeping gene

Genetics

Shoot

Reference genes

Forage

Gene expression

Plant Gene

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.

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