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Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress

  • Original Paper
  • Published:
Biologia Plantarum

Abstract

Licorice (Glycyrrhiza glabra L.) is an important medicinal plant accumulating high-value secondary metabolites. Real-time reverse transcription quantitative PCR (RT-qPCR) has become a common method for studying gene expression, and the availability of stable reference genes is a prerequisite to obtain accurate quantification of transcript abundance. Therefore, an experiment was designed to determine appropriate reference genes for gene expression studies in licorice. Based on reports in the literature and the availability of genomic sequences, eight putative reference genes were chosen. Further, the expression stabilities of these genes were evaluated in leaf and root tissues under normal and drought stress conditions using three distinct statistical algorithms including geNorm, NormFinder, and BestKeeper. Among the investigated genes, ubiquitin-conjugating enzyme E2 (UBC2), elongation factor 1 α (EF1), and actin (ACT) under normal conditions and ACT, β-tubulin (BTU), and UBC2 under drought stress conditions were the most stable genes in leaves, whereas BTU, ACT, and UBC2 under normal and drought stress conditions were identified as the most stable genes in roots. Nevertheless, the use of glyceraldehyde-3-phosphate dehydrogenase, F-box protein, and BTU have not been approved as reference genes for RT-qPCR data normalization. The findings in this study highlight the importance of the use of well-validated reference genes to the success of gene expression analysis using RT-qPCR.

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Abbreviations

ACT:

actin

BTU:

β-tubulin

Cq:

quantification cycle

CV:

coefficient of variation

EF1:

elongation factor 1 α

FBP:

F-box protein

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

HIS3:

histone H3

M:

expression stability

RT-qPCR:

reverse transcription quantitative polymerase chain reacion

UBC2:

ubiquitin-conjugating enzyme E2

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  1. Department of Plant Breeding, Faculty of Agriculture, University of Kurdistan, Sanandaj, 66177-15177, Iran

    A. Maroufi

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Acknowledgments: This study was financially supported by the University of Kurdistan, Sanandaj, Iran.

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Maroufi, A. Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress. Biol Plant 60, 645–654 (2016). https://doi.org/10.1007/s10535-016-0601-y

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  • DOI: https://doi.org/10.1007/s10535-016-0601-y

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