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Validation of reference genes for quantitative gene expression in the Lippia alba polyploid complex (Verbenaceae)

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Abstract

Lippia alba (Verbenaceae) is one of the most studied species of the genus Lippia, mainly due to its medicinal properties. The species was described as a polyploid complex with five cytotypes. The comparison of gene expression in species with several ploidal levels needs to be conducted carefully due to possible changes in gene regulation. Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Besides being an extremely powerful technique, relative quantification by Real-Time quantitative PCR (RT-qPCR) needs the normalization with a stable reference gene. We evaluated the stability of nine candidate reference genes in Lippia alba with different ploidal levels using NormFinder, geNorm, and RefFinder software. The product of each primer showed a single peak in the melting curve. The R2 value ranged from 0.998 to 1000 and primers efficiency ranged from 98.95% to 129%. The CIT gene came up as a stable housekeeping gene, being appropriate for studies in polyploid accessions of Lippia alba. Considering that polyploidy is widely documented in Angiosperms, the results can be used not only for further gene expression studies in L. alba but also as a possible reference gene for other polyploid complexes. Differential stability among different genes highlights the importance of the validation of reference genes used for RT-qPCR approach in polyploid studies.

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Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank D. S. Batista (UFPB) for data analysis help and V. C. Souza (UFJF) for providing the RNAseq data. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (432412-2016-6 and 313740-2017-8), Fundação de Amparo à Pesquisa do Estado de Minas Gerais-Fapemig (CRA RED 0053-16), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Capes.

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Authors and Affiliations

  1. Departamento de Biologia, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, Minas Gerais, 36036-900, Brazil

    Juliana Mainenti Leal Lopes, Elyabe Monteiro de Matos, Laís Stehling de Queiroz Nascimento & Lyderson Facio Viccini

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  1. Juliana Mainenti Leal Lopes
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  2. Elyabe Monteiro de Matos
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  3. Laís Stehling de Queiroz Nascimento
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EM, JL, and LN performed experiments, analyzed the data and wrote the manuscript. LV revised the manuscript and supervised the study. All authors read the paper and approved the final manuscript.

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Correspondence to Lyderson Facio Viccini.

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Lopes, J.M.L., de Matos, E.M., de Queiroz Nascimento, L.S. et al. Validation of reference genes for quantitative gene expression in the Lippia alba polyploid complex (Verbenaceae). Mol Biol Rep 48, 1037–1044 (2021). https://doi.org/10.1007/s11033-021-06183-6

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