Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis i
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ORIGINAL ARTICLE
Selection and validation of reference genes for qPCR analysis of miRNAs and their targets during somatic embryogenesis in tamarillo (Solanum betaceum Cav.) Daniela Cordeiro1 · Miguel Rito1 · Filipe Borges2 · Jorge Canhoto1 · Sandra Correia1 Received: 5 May 2020 / Accepted: 30 July 2020 / Published online: 14 August 2020 © Springer Nature B.V. 2020
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 DeltaCt algorithms. Among these candidates, Fe-SOD, ACTand 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 + ACTrevealed 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. Keywords Fruit tree · Gene expression · miRNAs expression · Normalization · RT-qPCR · Solanaceae
Introduction Somatic embryogenesis (SE) is a developmental process by which embryogenic cells express totipotency, developing into embryos and later on into plantlets (Méndez-Hernández et al. 2019). As a micropropagation tool, SE has been Communicated by Goetz Hensel. * Sandra Correia [email protected] 1
Department of Life Sciences, Centre for Functional Ecology, University of Coimbra, Calçada Martim de Freitas, 3000‑456 Coimbra, Portugal
Institut Jean‑Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, 78000 Versailles, France
2
extensively used as an efficient regeneration system and for the rapid and large-scale cloning of elite genotypes of numerous species under in vitro conditions (Iliev et al. 2010; Corredoira et al. 2019). Furthermore, this technique enables the study of plant embryology and other morphogenic events (Garcia-Gonzales et al. 2010; Vries and Weijers 2017). Tamarillo (Solanum betaceum Cav.), also named tree tomato, i
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