Evaluation of reference genes for quantitative analysis of gene expression in Lippia alba under abiotic stress

Key messageExperimental conditions affect the expression of reference genes. NADHis the most stable gene under saline, exogenous hormones, wounding, and cold stress;whereas TUB is most stable under osmotic stress. Lippia alba (Mill.) N.E. Brown (Verbenaceae) is a medicinal plant of commercial interest because it is a source of essential oils, such as citral and linalool. Abiotic stress can modulate biosynthetic pathways and regulate the production of secondary metabolites found in essential oils. RT-qPCR is a useful tool for investigating the expression of biosynthetic genes and select cultivars with improved essential oil composition. However, RT-qPCR requires normalization to a stable reference genes, appropriate for each experimental condition. Here, we describe the evaluation of reference genes for RT-qPCR analysis in L. alba. Six constitutive genes (NADH, CIT, G6i, TUB, RNApol, and ELONG) were analyzed using BestKeeper, GeNorm, NormFinder, and RefFinder software. TUB emerged as the most stable gene under osmotic stress; whereas NADH was most stable under saline, wounding, cold, and exogenous abscisic acid and salicylic acid stress. Therefore, TUB and NADH are recommended for use as reference genes in expression studies involving L. alba.

Automated summary

Experimental conditions affect the expression of reference genes. NADH is the most stable gene under saline, exogenous hormones, wounding, and cold stress; whereas TUB is most stable under osmotic stress. Lippia alba is a medicinal plant with commercial value as a source of essential oils. RT-qPCR is a useful tool for studying gene expression and selecting improved essential oil composition. TUB and NADH are recommended as reference genes for expression studies in L. alba.