RESUMO
The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya-Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA-trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P-distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia-recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well-supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.
RESUMO
In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.
