植物物种的快速鉴定与iFlora: DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比, DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（Pedicularis L.）植物具对生（轮生）叶的种类70%以上分布在中国,近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81.40%和89.57%,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

植物物种的快速鉴定与iFlora：DNA条形码在马先蒿属中的应用

DNA条形码技术就是利用一段较短的标准DNA序列对物种进行快速鉴定。与基于植物外部形态特征的传统分类鉴定方法相比,DNA条形码具有高效、准确,且易于实现自动化和标准化的特点。马先蒿属（PedicularisL．）植物具对生（轮生）叶的种类70％以上分布在中国．近缘种间形态上非常相似,鉴定较为困难。研究选取马先蒿属具对生（轮生）叶类群43种164份样品,利用叶绿体基因（rbcL、matK、trnH-psbA）和核基因（ITS）条形码片段,采用建树法和距离法检验4个条形码对这些物种的鉴定效果。结果表明,ITS片段用于建树法和距离法的鉴别率分别为81．40％和89．57％,其鉴别率高于3个叶绿体基因片段和任一基因片段的组合条码。另外,利用ITS成功解决了一些疑难种的分类问题。DNA条形码在马先蒿属研究中的实用性为新一代植物志（iFlora）实现物种的快速和准确鉴定提供了有力支持,并能为分类学、生态学、进化生物学、居群遗传学和保护遗传学等分支学科的研究提供重要信息。     

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.     

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular‐based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA‐trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2‐parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA‐trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species‐level identification of the genus Ocimum.     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Barcoding Poplars (Populus L.) from Western China

Background

Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.

Methodology/Principal Findings

Here, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.

Conclusions/Significance

In this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).     

Phylogenomic relationships and species identification of the olive genus Olea (Oleaceae)

The olive genus Olea includes c. 30–40 taxa in three subgenera (Olea, Tetrapilus, and Paniculatae) within the family Oleaceae. Historically, the Olea genus was classified into four groups that were overall well supported by reconstructed phylogenies, despite incomplete sampling of subgenus Tetrapilus and poor resolution within clades. These analyses also showed that the genus was not monophyletic. Reliable identification of Olea species is important for both their conservation and utilization of this economically important genus. In this study, we used phylogenomic data from genome skimming to resolve relationships within Olea and to identify molecular markers for species identification. We assembled the complete plastomes, and nrDNA of 26 individuals representing 13 species using next-generation sequencing and added 18 publicly available accessions of Olea. We also developed nuclear SNPs using the genome skimming data to infer the phylogenetic relationships of Olea. Large-scale phylogenomic analyses of 138 samples of tribe Oleeae supported the polyphyly of Olea, with Olea caudatilimba and Olea subgenus Tetrapilus not sharing their most recent common ancestor with the main Olea clade (subgenus Paniculatae and subgenus Olea). The interspecific phylogenetic resolution was poor owing to a possible rapid radiation. By comparing with the plastome data, we identified the markers ycf1b and psbE-petL as the best Olea-specific chloroplast DNA barcodes. Compared with universal barcodes, specific DNA barcodes and super-barcode exhibited higher discriminatory power. Our results demonstrated the power of phylogenomics to improve phylogenetic relationships of intricate groups and provided new insights into barcodes that allow for accurate identification of Olea species.     

Plant DNA barcoding: from gene to genome

DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single‐locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole‐chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource‐effective and does not yet offer the speed of analysis provided by single‐locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super‐barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single‐locus barcodes and super‐barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L.(Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, marK, and trnH-psbA),were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%,respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.     

Promise and Challenge of DNA Barcoding in Venus Slipper (Paphiopedilum)

Orchidaceae are one of the largest families of flowering plants, with over 27,000 species described and all orchids are listed in CITES. Moreover, the seedlings of orchid species from the same genus are similar. The objective of DNA barcoding is rapid, accurate, and automated species identification, which may be used to identify illegally traded endangered species from vegetative specimens of Paphiopedilum (Venus slipper), a flagship group for plant conservation with high ornamental and commercial values. Here, we selected eight chloroplast barcodes and nrITS to evaluate their suitability in Venus slippers. The results indicate that all tested barcodes had no barcoding gap and the core plant barcodes showed low resolution for the identification of Venus slippers (18.86%). Of the single-locus barcodes, nrITS is the most efficient for the species identification of the genus (52.27%), whereas matK + atpF-atpH is the most efficient multi-locus combination (28.97%). Therefore, we recommend the combination of matK + atpF-atpH + ITS as a barcode for Venus slippers. Furthermore, there is an upper limit of resolution of the candidate barcodes, and only half of the taxa with multiple samples were identified successfully. The low efficiency of these candidate barcodes in Venus slippers may be caused by relatively recent speciation, the upper limit of the barcodes, and/or the sampling density. Although the discriminatory power is relatively low, DNA barcoding may be a promising tool to identify species involved in illegal trade, which has broad applications and is valuable for orchid conservation.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

DNA barcoding of endangered Indian Paphiopedilum species

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-β subunit (rpoB), RNA polymerase-β' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.     

Deciduous trees and the application of universal DNA barcodes: a case study on the circumpolar Fraxinus

The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32-trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA "universal" barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed.     

Testing species delimitation in sympatric species complexes: the case of an African tropical tree, Carapa spp. (Meliaceae)

Plant species delimitation within tropical ecosystems is often difficult because of the lack of diagnostic morphological characters that are clearly visible. The development of an integrated approach, which utilizes several different types of markers (both morphological and molecular), would be extremely useful in this context. Here we have addressed species delimitation of sympatric tropical tree species that belong to Carapa spp. (Meliaceae) in Central Africa. We adopted a population genetics approach, sampling numerous individuals from three locations where sympatric Carapa species are known to exist. Comparisons between morphological markers (the presence or absence of characters, leaf-shape traits) and molecular markers (chloroplast sequences, ribosomal internal transcribed spacer region (ITS) sequences, and nuclear microsatellites) demonstrated the following: (i) a strong correlation between morphological and nuclear markers; (ii) despite substantial polymorphism, the inability of chloroplast DNA to discriminate between species, suggesting that cytoplasmic markers represent ineffective DNA barcodes; (iii) lineage sorting effects when using ITS sequences; and (iv) a complex evolutionary history within the genus Carapa, which includes frequent inter-specific gene flow. Our results support the use of a population genetics approach, based on ultra-polymorphic markers, to address species delimitation within complex taxonomic groups.     

Conflicting phylogenies of Larix (Pinaceae) based on cytoplasmic and nuclear DNA

The phylogeny of Larix species was studied using chloroplast DNA RFLPs and nuclear ITS sequences and AFLPs. The study resolved the sister relationships between the Eurasian and Asian species and the monophyletic origin of each major geographic group. It also provides strong evidence for an early splitting of American Larix from other species in the genus. The discrepancy between phylogenies based on chloroplast DNA and nuclear DNA, previously observed by Qian et al. [Can. J. For. Res. 25 (1995) 1197], is confirmed. Various explanations for the incongruence between chloroplast DNA and nuclear DNA are discussed, and a tentative reconstruction of Eurasian Larix phylogeography is proposed.     

Exploring Genetic Divergence in a Species-Rich Insect Genus Using 2790 DNA Barcodes

DNA barcoding using a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) has proven to be successful for species-level identification in many animal groups. However, most studies have been focused on relatively small datasets or on large datasets of taxonomically high-ranked groups. We explore the quality of DNA barcodes to delimit species in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by using different analytical tools. The genus Tanytarsus is the most species-rich taxon of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species worldwide, some of which can be notoriously difficult to identify to species-level using morphology. Our dataset, based on sequences generated from own material and publicly available data in BOLD, consist of 2790 DNA barcodes with a fragment length of at least 500 base pairs. A neighbor joining tree of this dataset comprises 131 well separated clusters representing 121 morphological species of Tanytarsus: 77 named, 16 unnamed and 28 unidentified theoretical species. For our geographically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120–242 molecular operational taxonomic units (OTUs) depending on whether Objective Clustering, Automatic Barcode Gap Discovery (ABGD), Generalized Mixed Yule Coalescent model (GMYC), Poisson Tree Process (PTP), subjective evaluation of the neighbor joining tree or Barcode Index Numbers (BINs) are used. We suggest that a 4–5% threshold is appropriate to delineate species of Tanytarsus non-biting midges.     

阿蠓属一新种及一中国新记录种（昆虫纲：双翅目：蠓科）

本研究整理了2018年至2021年海南岛采集的阿蠓属Alluaudomyia昆虫标本,结合形态学特征和DNA条形码进行物种鉴定,首次记录了环纹阿蠓种团the annulata group在我国分布,鉴定发现了该种团内一新种拟环纹阿蠓Alluaudomyia pseudannulata Wu & Li sp. nov.,及一中国新记录种环足阿蠓Alluaudomyia annulipes Wirth & Delfinado, 1964。详细描述了两种阿蠓的形态特征,提供了鉴定特征图、海南岛物种分布图以及CO I条形码。     

DNA barcoding of Oryza: conventional,specific, and super barcodes

Key message

We applied the phylogenomics to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

Abstract

Rice (genus Oryza) is one of the most important crops in the world, supporting half of the world’s population. Breeding of high-yielding and quality cultivars relies on genetic resources from both cultivated and wild species, which are collected and maintained in seed banks. Unfortunately, numerous seeds are mislabeled due to taxonomic issues or misidentifications. Here, we applied the phylogenomics of 58 complete chloroplast genomes and two hypervariable nuclear genes to determine species identity in rice seeds. Twenty-one Oryza species were identified. Conspecific relationships were determined between O. glaberrima and O. barthii, O. glumipatula and O. longistaminata, O. grandiglumis and O. alta, O. meyeriana and O. granulata, O. minuta and O. malampuzhaensis, O. nivara and O. sativa subsp. indica, and O. sativa subsp. japonica and O. rufipogon. D and L genome types were not found and the H genome type was extinct. Importantly, we evaluated the performance of four conventional plant DNA barcodes (matK, rbcL, psbA-trnH, and ITS), six rice-specific chloroplast DNA barcodes (psaJ-rpl33, trnC-rpoB, rps16-trnQ, rpl22-rps19, trnK-matK, and ndhC-trnV), two rice-specific nuclear DNA barcodes (NP78 and R22), and a chloroplast genome super DNA barcode. The latter was the most reliable marker. The six rice-specific chloroplast barcodes revealed that 17% of the 53 seed accessions from rice seed banks or field collections were mislabeled. These results are expected to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

     

Species-Specific Identification from Incomplete Sampling: Applying DNA Barcodes to Monitoring Invasive Solanum Plants

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling–through this, DNA barcoding will greatly benefit the current fields of its application.