Evaluation and application of DNA barcoding for the species identification of Dicranostigma
AbstractDicranostigma Hook. f. & Thomson (Papaveraceae, Chelidonieae) is an oligotypic genus consisting of 3 species. Species delimitation and identification of species in this genus have been problematic because of high levels of morphological plasticity. Dicranostigma leptopodum is a traditional medicinal plant in China, which has the efficacy of antimicrobial, antivirus, antitumor, anti-hemolytic and improving immunity, thus correct identification of those three morphologically similar species is important to ensure the safety of clinical application. Therefore, it is needed to develop new effective DNA barcodes through genome skimming technology for accurate identification of medicinal materials. In order to identify the species and clarify the species boundary of Dicranostigma, we collected 22 populations covering the distribution area of this genus in China and three individuals were randomly selected from each population. In total, 66 individuals were sequenced using genome skimming technology, and the complete plastome sequences and nuclear ribosome DNA regions (ITS, ETS) were acquired. Several barcoding candidates were compared regarding their identification efficacy, including three universal DNA barcodes (matK, rbcL, psbA-trnH), nrITS and ETS, super barcodes (the complete plastome sequences, combined sequences of coding region, combined sequences of noncoding region), and specific DNA barcodes (cemA-ycf4, rpl16 intron, rps2, petA-psbJ, petN-trnC). Based on variety of sequences, intraspecific and interspecific Kimura 2-Parameter (K2P) distances, DNA barcoding gaps and capability of authentication at specific and populational levels, we have further evaluated the suitability of each barcode in the barcoding of plants of Dicranostigma, and the major findings are as follows: (1) In this paper, nine DNA barcoding markers were screened out from chloroplast genome and nrDNA region, which could identify all species of Dicranostigma, i.e. matK, matK+rbcL, complete chloroplast genome, combined sequences of coding region, combined sequences of noncoding region, cemA-ycf4, rpl16 intron, rps2, petA-psbJ, petN-trnC. Within 9 DNA barcoding markers, the barcoding gap of super barcodes, rpl16 intron and rps2 was obvious. (2) Among the candidate DNA barcodes, super barcode has the highest discrimination rate at specific level and populational level; except for petN-trnC, all the other specific barcodes could identify 3 species of Dicranostigma, but the discrimination rate was low at the populational level; within universal DNA barcodes, only matK could identify all species of Dicranostigma, but barcoding gap was unobvious; and nrDNA barcodes could not identify all species of Dicranostigma. (3) Phylogenetic analyses based on super barcodes and several DNA barcoding markers (matK, cemA-ycf4, rpl16 intron, rps2, petA-psbJ) shows that each species of Dicranostigma form a seperate clade and well spported. Dicranostigma lactucoides is the basal clade within the genus
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