Wu et al. Chin Med (2021) 16:52 https://doi.org/10.1186/s13020-021-00460-z Chinese Medicine RESEARCH Open Access Plant super-barcode: a case study on genome-based identifcation for closely related species of Fritillaria Lan Wu1†, Mingli Wu1†, Ning Cui1, Li Xiang1, Ying Li2, Xiwen Li1* and Shilin Chen1* Abstract Background: Although molecular analysis ofers a wide range of options for species identifcation, a universal meth- odology for classifying and distinguishing closely related species remains elusive. This study validated the efective- ness 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 efectively diferentiate medicinal plants from the genus Fritillaria at the species level. Conclusions: This study implies that cp genome can provide distinguishing diferences to help identify closely related Fritillaria species, and has the potential to be served as a universal super-barcode for plant identifcation. Keywords: Species identifcation, Closely related species, Chloroplast genome, Super-barcode, Genome comparison, Fritillaria Background of plant species has remained elusive due to the scarcity Although many biological studies depend on accurate and ambiguity of diagnostic characters. Fortunately the species identifcation and delimitation, such as the imple- advent of molecular markers made an impact on species ment of biodiversity conservation, therapy of disease and identifcation, and undoubtedly has made a substantial the identifcation of invasive species, taxonomic exper- contribution to systematics. However, currently none tise is collapsing [1, 2]. Morphology-based identifcation of the available DNA loci work for all species, espe- cially for the closely related species. Moreover, multiple closely related species that occupy the same area have *Correspondence: [email protected]; [email protected] always posed insurmountable barriers to the goal of cur- †Lan Wu and Mingli Wu contributed equally to this work 1 Key Laboratory of Beijing for Identifcation and Safety Evaluation rent highly accurate identifcation [3]. Terefore, a new of Chinese Medicine, Institute of Chinese Materia Medica, China Academy method is required in the search for a universal marker of Chinese Medical Sciences, Beijing 100700, China for taxon recognition. Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wu et al. Chin Med (2021) 16:52 Page 2 of 11 Te chloroplast (cp) genome is a versatile tool for single-locus markers have low resolution for Fritillaria phylogenetics. During the past decade, there have due to high sequence similarities. been many analyses addressing phylogenetic questions Compared with the most frequently used and predicted at deep nodes based upon the complete sequences of genus-specifc DNA barcodes, cp genome contains cp genomes [4–6]. As plant biologists enter the era in more variations with a signifcantly higher resolution which comparative genomics promises to address in- of phylogenies, which is valuable to reveal phylogenetic depth questions, the inestimable efectiveness of cp relationships between closely related species [12]. Cp genome in systematic studies quickly become clear. genome has been widely applied in phylogenetic analyses Te entire cp genome contains approximately as much [21–24], plant population studies [25], and plant identi- information as does the COI gene used in animals and fcation [7]. Te phylogenetic tree constructed based on it has the potential to provide distinguishing diferences complete cp genomes has a higher supporting rate and that can help molecularly identify closely related spe- discrimination power [26]. Li et al. [11] therefore pro- cies [7]. With advances in high-throughput sequencing, posed to use the entire cp genome as a super-barcode to achieving cp genome is easily acquirable at a large-scale accurately identify closely related species. with lower costs. Tis has promoted studies of system- Here, we compared 26 complete cp genomes, includ- atics using cp genome in Epimedium [8], Paris [9] and ing 18 newly sequenced genome sequences for this study, Sanguisorba [10]. Because of the low discrimination from ten Fritillaria species that are included in the Chi- power of general molecular markers in plants and their nese Pharmacopoeia 2020. We performed a comprehen- closely related species, researchers have proposed the sive analysis of the complete cp genomes of the Fritillaria entire cp genome as a super-barcode to discriminate species, which are difcult to be identifed by morphol- closely related species [11]. ogy and taxonomy alone. Te aims of our study were Te bulbs of Fritillaria species (called BeiMu, BM) as follows: (1) to verify the hypothesis whether super- have been used medicinally for more than 2000 years, barcode can be used as a universal barcode to identify specifcally in the treatment of dry cough and blood- closely related species, (2) to present 18 new complete cp stained sputum. Due to the over exploitation of natural genomes from ten Fritillaria species and explore poly- resources, the availability of Chuan BeiMu continues to morphic regions within Fritillaria cp genomes, and (3) to decline [12]. Currently, most Fritillaria species used as evaluate the discrimination power of cp genomes in the Chuan BeiMu are in the list of wild protected species genus Fritillaria at the species level. Te results demon- (level 3) in China, and the price of high-quality Chuan strated that the cp genome could be used to identify Fri- BeiMu can be as high as ~ 500$/kg. With the decreased tillaria at species level. Te entire cp genome was found availability and high price, Chuan BeiMu is often adulter- to be a most promising universal DNA marker in identif- ated by other cheaper bulbs from other Fritillaria spe- cation of closely related species. cies, with a market survey reporting the adulteration rate of Chuan BeiMu to be as high as 20% [13]. Methods Presently, Fritillaria bulbs are identifed by morpho- DNA extraction logical features [14] and chemical properties [15]. Unfor- Twenty-six cp genomes from ten Fritillaria species were tunately, diferent species can be morphologically similar used in this study (see Additional fle 1: Table S1). Fresh and they always have the similar chemical constituents, leaves of 18 individuals from nine Fritillaria species were which make the identifcation of Fritillaria difcult at the collected. Te cp genomes of eight additional individuals species level using traditional methods. Although DNA were downloaded from GenBank. Total genomic DNA barcoding provided accurate identifcation for plants, of each sample was isolated from ~ 200 mg of fresh leaf it is insufcient in the authentication of Fritillaria spe- using the DNeasy Plant Mini Kit (QIAGEN, Germany), cies. Luo and Xiang et al. [16, 17] reported that ITS2 according to manufacturer’s instructions. To meet sequence could not provide monophyletic clades for the the quality requirements for sequencing, we assessed genus Fritillaria at the species level. Meanwhile, Sharif the quality and quantity of each DNA sample using a [18] and TÜRKTAŞ et al. [19] constructed the phyloge- Qubit2.0 Fluorometer (Termo Scientifc, USA) and a netic trees based on the trnH-psbA and trnL-trnF regions NanoDrop 2000 Spectrophotometer (Nanodrop Tech- using 22 Iranian Fritillaria species and ten Turkey Fri- nologies, Wilmington, DE, USA), respectively. tillaria species respectively, and the phylogenetic trees showed that it is impossible to distinguish these Fritil- Genome sequencing, assembly and annotation laria species. Rønsted et al. [20] presented the same Te shotgun libraries (450 bp) were constructed using result based on matK, rpl16, trnK, and ITS sequences for ~ 2 μg of total DNA according to the manufacturer’s Fritillaria. Terefore, these fndings demonstrate that the instructions (Illumina Inc., San Diego, CA). A total Wu et al. Chin Med (2021) 16:52 Page 3 of 11 of 11 cp genomes from seven Fritillaria species were sequences were aligned using the MAFFT program [40], sequenced using Illumina HiSeq X platform (Illumina, and then adjusted manually in Bioedit. Phylogenetic trees San Diego, CA, USA), and we obtained > 2 Gb data for were constructed by the maximum likelihood (ML) and each sample.