The Use of DNA Barcoding on Recently Diverged Species in the Genus <i>Gentiana</i> (Gentianaceae) in China

<div><p>DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus <i>Gentiana</i> (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (<i>rbcL</i> + <i>matK</i>) and three promising complementary barcodes (<i>trnH-psbA</i>, ITS and ITS2) in 30 <i>Gentiana</i> species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). <i>rbcL</i> had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in <i>trnH-psbA</i> in <i>Gentiana</i> led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, <i>matK</i> + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus <i>Gentiana</i>. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. <i>Cruciata</i> because of hybridization events. Our analysis suggests that the status of <i>G</i>. <i>pseudosquarrosa</i> needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ <i>Gentiana</i> medicinal plants (<i>G</i>. <i>macrophylla</i>, <i>G</i>. <i>crassicauli</i>s, <i>G</i>. <i>straminea</i>, and <i>G</i>. <i>dahurica</i>), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.</p>