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Comparison of the abilities of universal, super, and specific DNA barcodes to discriminate among the original species of Fritillariae cirrhosae bulbus and its adulterants.

ABSTRACT: Fritillariae cirrhosae bulbus is a famous type of traditional Chinese medicine used for cough relief and eliminating phlegm. The medicine originates from dried bulbs of five species and one variety of Fritillaria. Recently, immature bulbs from other congeneric species, such as F. ussuriensis, have been sold as adulterants of Fritillariae cirrhosae bulbus in medicine markets owing to the high price and limited availability of the genuine medicine. However, it is difficult to accurately identify the bulbs from different original species of Fritillariae cirrhosae bulbus and its adulterants based on traditional methods, although such medicines have different prices and treatment efficacies. The present study adopted DNA barcoding to identify these different species and compared the discriminatory power of super, universal, and specific barcodes in Fritillaria. The results revealed that the super-barcode had strong discriminatory power (87.5%). Among universal barcodes, matK provided the best species resolution (87.5%), followed by ITS (62.5%), rbcL (62.5%), and trnH-psbA (25%). The combination of these four universal barcodes provided the highest discriminatory power (87.5%), which was equivalent to that of the super-barcode. Two plastid genes, ycf1 and psbM-psbD, had much better discriminatory power (both 87.5%) than did other plastid barcodes, and were suggested as potential specific barcodes for identifying Fritillaria species. Phylogenetic analyses indicated that F. cirrhosa was not a "good" species that was composed of multiple lineages, which might have affected the evaluation of the discriminatory ability. This study revealed that the complete plastid genome, as super barcode, was an efficient and reliable tool for identifying the original species of Fritillariae cirrhosae bulbus and its adulterants.

SUBMITTER: Chen Q

PROVIDER: S-EPMC7018091 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fritillariae Bulbus is a precious Chinese herbal medicine that is grown at high elevation and used to relieve coughs, remove phlegm, and nourish the lungs. Historically, Fritillariae Bulbus has been divided into two odourless crude drugs: Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus. However, now the Chinese Pharmacopoeia has described five Fritillariae Bulbus-the new additions include Fritillariae Pallidiflorae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Hupehensis Bulbus. Because the morphology of dried Fritillariae Bulbus is similar, it is difficult to accurately identify the different types of Fritillariae Bulbus. In the current study, we develop a method combining DNA barcoding and high-performance liquid chromatography (HPLC) to help distinguish Fritillariae Cirrhosae Bulbus from other Fritillariae Bulbus and guarantee species traceability of the five types of Fritillariae Bulbus. We report on the validation of an integrated analysis method for plant species identification using DNA barcoding that is based on genetic distance, identification efficiency, inter- and intra-specific variation, calculated nearest distance, neighbour-joining tree and barcoding gap. Our results show that the DNA barcoding data successfully identified the five Fritillariae Bulbus by internal transcribed spacer region (ITS) and ITS2, with the ability to distinguish the species origin of these Fritillariae Bulbus. ITS2 can serve as a potentially useful DNA barcode for the Fritillaria species. Additionally, the effective chemical constituents are identified by HPLC combined with a chemical identification method to classify Fritillaria. The HPLC fingerprint data and HCA (hierarchical clustering analysis) show that Fritillariae Cirrhosae Bulbus is clearly different from Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus, but there is no difference between Fritillariae Cirrhosae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Pallidiflorae Bulbus. These results show that DNA barcoding and HPLC fingerprinting can discriminate between the five Fritillariae Bulbus types and trace species to identify related species that are genetically similar.

Project description:A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF-atpH, and psbK-psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85-100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK-psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69-71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.

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Comparison of the abilities of universal, super, and specific DNA barcodes to discriminate among the original species of Fritillariae cirrhosae bulbus and its adulterants.

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