基于DNA条形码的紫花地丁、苦地丁及甜地丁鉴定OA

Objective To authenticate Viola philippica(Zihuadiding),Corydalis bungeana(Kudiding),and Gueldenstaedtia verna(Tiandiding)using the nucleotide sequence of the second internal transcribed spacer(ITS2),a DNA barcode marker.Methods Total genomic DNA was extracted from samples via a column-based plant ge-nomic DNA extraction kit.Fluorescent quantitative polymerase chain reaction(PCR)amplification was performed using universal ITS2 primers;following optimization of the annealing temperature,bidirectional sequencing was con-ducted on target bands.The obtained sequences were assembled with CodonCode Aligner 11.0.1,and primer and flanking sequences were trimmed.Genetic distances and parsimony-informative sites were calculated using MEGA 11.0 software,and a neighbor-joining(NJ)phylogenetic tree was constructed.Additionally,the secondary structure of the ITS2 sequence was predicted online.Results The intraspecific genetic distances of V.philippica,C.bun-geana,and G.verna were 0.01,0.00,and 0.00,respectively,while interspecific genetic distances ranged from 0.69 to 0.82.Specifically,the genetic distance(0.69)and number of parsimony-informative sites(93)between V.philippica and C.bungeana were lower than those between V.philippica and G.verna(0.82,102).The interspecific genetic dis-tance between G.verna and C.bungeana was 0.81,with 100 parsimony-informative sites.The NJ phylogenetic tree showed that each species formed a monophyletic clade;V.philippica and C.bungeana clustered into the same major branch,which was sister to the clade of G.verna.Analysis of the ITS2 secondary structure revealed that V.philippica lacked the typical"one-ring and four-arms"configuration,whereas both C.bungeana and G.verna possessed this structure—with distinct differences in the apical loop of arm Ⅲ and the middle loop of arm Ⅳ between the latter two.Conclusion ITS2 sequences combined with their secondary structures can effectively distinguish V.philippica,C.bungeana,and G.verna,and clarify their phylogenetic relationships:V.philippica and C.bungeana are closely rela-ted.This method provides a reliable and reproducible molecular basis for the accurate identification of Dididing-class medicinal materials.