Development of selection method for Glycyrrhiza uralensis superior clones with high-glycyrrhizic acid contents using DNA sequence polymorphisms in glycyrrhizic acid biosynthetic genes

Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.