| Summary: | Best known as licorice, <i>Glycyrrhiza</i> Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in <i>Glycyrrhiza</i>. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in <i>Glycyrrhiza.</i> The expression profiles of 14 candidate reference genes, including <i>Actin1</i> (<i>ACT</i>)<i>, Clathrin complex AP1</i> (<i>CAC</i>), <i>Cyclophilin</i> (<i>CYP</i>), <i>Heat-shock protein 40</i> (<i>DNAJ</i>), <i>Dehydration responsive element binding gene</i> (<i>DREB</i>), <i>Translation elongation factor1</i> (<i>EF1</i>), <i>Ras related protein</i> (<i>RAN</i>), <i>Translation initiation factor</i> (<i>TIF1</i>), <i>β-Tubulin</i> (<i>TUB</i>), <i>Ubiquitin-conjugating enzyme E2</i> (<i>UBC2</i>), <i>ATP binding-box transpoter 2</i> (<i>ABCC2</i>), <i>COP9 signal compex subunit 3</i> (<i>COPS3</i>), <i>Citrate synthase</i> (<i>CS</i>), and <i>R3H domain protein 2</i> (<i>R3HDM2</i>) from two congeneric species, <i>Glycyrrhiza uralensis</i> F. and <i>Glycyrrhiza inflata</i> B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In <i>G. uralensis</i>, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were <i>TIF1</i>, <i>DNAJ</i>, <i>CS</i>, and <i>ABCC2</i> for leaves and <i>DNAJ</i>, <i>DREB</i>, <i>CAC</i>, and <i>CAC</i> for roots, respectively. In comparison, the top ranked genes were <i>TUB</i>, <i>CAC</i>, <i>UBC2</i>, and <i>RAN</i> for leaves and <i>TIF1</i>, <i>ABCC2</i>, <i>CAC</i>, and <i>UBC2</i> for roots, respectively, under stress and hormonal treatments in <i>G. inflata</i>. <i>ACT</i> and <i>TIF1</i>, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that <i>EF</i>, <i>ACT</i>, <i>UBC2</i>, and <i>TUB</i> were the top choices for the abiotic stresses while <i>EF</i>, <i>UBC2</i>, <i>CAC</i>, and <i>ABCC2</i> were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in <i>Glycyrrhiza</i> species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.
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