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Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.)

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Abstract

Quantitative reverse transcriptase PCR is a powerful tool for precise gene expression quantification. Using suitable reference genes is crucial for a proper normalization and interpretation of results, particularly in dynamic transcriptional networks with the involvement of several miRNAs and their targets, such as somatic embryogenesis (SE). Here, we aimed to identify genes to be used as internal controls for quantitative detection of miRNAs and mRNAs during the SE process in Solanum betaceum. The expression of EF1α, GAPDH, ACT, UBQ10, Fe-SOD, U6, snoR14, miR159a, miR162 and miR166a was tested in nine time-points of the SE process, particularly during induction, embryo development and conversion. Analyses of transcriptional stability were performed using RefFinder tool that integrates geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among these candidates, Fe-SOD, ACT and U6 emerged consistently as the most suitable to normalize gene expression, whereas miR166a was the most appropriate for normalization in calli samples. In order to validate these results, expression of miR396 and GRF1 was quantified and normalized with the most and the least stable genes and also with the most stable miRNA. Fe-SOD + ACT revealed to be the optimal combination for precise quantification of miRNA-mediated regulation of gene expression during SE in tamarillo.

Key message

Fe-SOD + ACT is the most suitable combination of reference genes for RT-qPCR normalization and accurate quantification of miRNAs and their targets during tamarillo somatic embryogenesis induction, embryo development and conversion stages.

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Funding

This work was supported by the Fundação para a Ciência e a Tecnologia (Portugal) [doctoral research grant number SFRH/BD/136925/2018 awarded to Daniela Cordeiro; P2020|COMPETE grant number PTDC/BAA-AGR/32265/2017, BP4BP – Tamarillo breeding: better plants for better products]; and the Centre for Functional Ecology of the University of Coimbra.

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Authors and Affiliations

  1. Department of Life Sciences, Centre for Functional Ecology, University of Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal

    Daniela Cordeiro, Miguel Rito, Jorge Canhoto & Sandra Correia

  2. Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, 78000, Versailles, France

    Filipe Borges

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  1. Daniela Cordeiro
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  2. Miguel Rito
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Contributions

DC: Conceptualization, Methodology, Investigation, Formal analysis, Writing—original draft. MR: Investigation, Writing—Reviewing and Editing. FB: Resources, Writing- Reviewing and Editing. JC: Supervision, Writing—Reviewing and Editing, Funding acquisition. SC: Conceptualization, Supervision, Writing—Reviewing and Editing, Funding acquisition.

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Correspondence to Sandra Correia.

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Communicated by Goetz Hensel.

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Cordeiro, D., Rito, M., Borges, F. et al. Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.). Plant Cell Tiss Organ Cult 143, 109–120 (2020). https://doi.org/10.1007/s11240-020-01901-7

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