Abstract
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.
Key message
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.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Fundação de Amparo à Pesquisa do Estado de Minas Gerais-Fapemig, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Capes for their financial support. We would like to thank Editage (www.editage.com) for English language editing.
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This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (31443/2021–5, INCT Plant Stress Physiology: 406455/2022–8), Fundação de Amparo à Pesquisa do Estado de Minas Gerais-Fapemig (RED 0053–16), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Capes.
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LN performed the in vitro experiments. EM, JL, LN, and VS performed the experiments; analyzed the data; and wrote the manuscript. DB helped with the experimental design and statistical analysis. MS, WO, and LV revised the manuscript. LV supervised the study. All authors have read and approved the final manuscript.
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Nascimento, L.S.Q., Lopes, J.M.L., de Matos, E.M. et al. Evaluation of reference genes for quantitative analysis of gene expression in Lippia alba under abiotic stress. Plant Cell Tiss Organ Cult 154, 433–442 (2023). https://doi.org/10.1007/s11240-023-02564-w
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DOI: https://doi.org/10.1007/s11240-023-02564-w