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

International audience; Key message Fe-SOD + ACTis 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.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 inSolanum betaceum. The expression ofEF1 alpha,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,ACTandU6emerged consistently as the most suitable to normalize gene expression, whereas miR166a was the most appropriate for normalization incallisamples. In order to validate these results, expression of miR396 andGRF1was 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.