A method for the identification and evaluation of Glycyrrhiza germplasm based on DNA barcodes and leaf micromorphology

Abstract

Large-scale artificial cultivation of medicinal Glycyrrhiza plants is imperative due to the short supply and the dramatic decline in wild populations. Xinjiang is the main Glycyrrhiza production area of China, but the quality of cultivated Glycyrrhiza plants is low due to the mixed cultivation of multiple seeds of unknown origins. To explore the identification method and efficient cultivation of elite Glycyrrhiza germplasms in Xinjiang, China, in this study, the differences in leaf micromorphological characteristics and main active ingredients of 6 Glycyrrhiza species and 2 hybrids in Xinjiang were clarified, and then identification on the basis of DNA barcode was conducted. The results showed that: (1) The combination of nuclear gene ITS2, chloroplast nucleic acid sequences trnH-psbA and trnV-ndhC could accurately identify Glycyrrhiza species and hybrids, and the calculation of genetic distance could preliminarily determine the maternal species of hybrids. ITS2 and trnH-psbA results indicated that maternal parent of hybrid I was Glycyrrhiza inflata. The ITS2 and trnV-ndhC results showed that the maternal parent of hybrid II was either Glycyrrhiza uralensis or Glycyrrhiza aspera. (2) Seventeen leaf micromorphological traits were utilized to compare and differentiate the tested Glycyrrhiza species. Traits such as glandular trichome density (GTDe), shape (GTS), diameter (GTDi), and wax layer ornamentation (WLo) exhibited significant interspecific variations. Specifically, G. prostrata lacked glandular trichomes entirely, G. aspera displayed the lowest GTDe, the glandular trichomes of G. inflata were partially scaly, while those of G. uralensis were cylindrical with a notably larger diameter compared to G. glabra and G. glabra var. glandulosa. The upper epidermis granules of G. glabra var. glandulosa were less abundant and their surfaces smoother than in other tested Glycyrrhiza species. Cluster analysis based on 11 micromorphological traits with absolute weight values exceeding 0.7 in the first four principal components, including GTDi, revealed that all tested Glycyrrhiza species clustered into a single category. Hybrid types showed close affinity to G. uralensis and G. inflata, findings consistent with DNA barcode results. (3) The comprehensive evaluation of germplasms in Xinjiang found that Glycyrrhiza glabra was superior to other germplasms (Glycyrrhiza glabra > G. glabra var. Glandulosa > G. uralensis > G. inflata > G. aspera > hybrid I > G. prostrata > hybrid II), indicating that the hybridization reduced the medicinal value of Glycyrrhiza species. This study is of great significance for the identification of Glycyrrhiza species, the conservation of elite Glycyrrhiza germplasms, and the healthy development of the medicinal plant market.