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Background  Although molecular analysis offers a wide range of options for species identification, a universal methodology for classifying and distinguishing closely related species remains elusive. This study validated the effectiveness of utilizing the entire chloroplast (cp) genome as a super-barcode to help identify and classify closely related species.    Methods  We here compared 26 complete cp genomes of ten  Fritillaria  species including 18 new sequences sequenced in this study. Each species had repeats and the cp genomes were used as a whole DNA barcode to test whether they can distinguish  Fritillaria  species .Results  The cp genomes of  Fritillaria  medicinal plants were conserved in genome structure, gene type, and gene content. Comparison analysis of the  Fritillaria  cp genomes revealed that the intergenic spacer regions were highly divergent compared with other regions. By constructing the phylogenetic tree by the maximum likelihood and maximum parsimony methods, we found that the entire cp genome showed a high discrimination power for  Fritillaria  species with individuals of each species in a monophyletic clade. These results indicate that cp genome can be used to effectively differentiate medicinal plants from the genus  Fritillaria  at the species level.    Conclusions  This study implies that cp genome can provide distinguishing differences to help identify closely related  Fritillaria  species, and has the potential to be served as a universal super-barcode for plant identification.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13020-021-00460-z.

Plant super-barcode: a case study on genome-based identification for closely related species of Fritillaria