DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Jugl...DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Juglandaceae to evaluate an assembly-free reads accurate identification(AFRAID)method of species identification,a novel approach for precise species identification in plants.Specifically,we determined(1)the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae,(2)the minimum size of chloroplast dataset for species discrimination,and(3)minimum amount of next generation sequencing(NGS)data required for species identification.We found that species identification rates were highest when whole chloroplast genomes were used,followed by taxon-specific DNA barcodes,and then universal DNA barcodes.Species identification of 100%was achieved when chloroplast genome sequence coverage reached 20%and the original sequencing data reached 500,000 reads.AFRAID accurately identified species for all samples tested after 500,000 clean reads,with far less computing time than common approaches.These results provide a new approach to accurately identify species,overcoming limitations of traditional DNA barcodes.Our method,which uses next generation sequencing to generate partial chloroplast genomes,reveals that DNA barcode regions are not necessarily fixed,accelerating the process of species identification.展开更多
The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term o...The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term overfishing,the ichthyoplankton structure has been dramatically altered.Understanding the species composition and distribution of fish eggs and larvae is one of the most essential tasks to accurately regulate fishery resources and formulate effective management policies;however,little is known about the ichthyoplankton in this region.In this study,an integrated strategy of morphology identification(MI)and mitochondrial COI DNA barcoding was used to identify species of fish eggs and larvae collected from the ECSCZA.MI revealed 15 fish egg species belonging to 12 families and 12 fish larva species belonging to 12 families;in contrast,DNA barcoding altogether identified 30 species,including 18 fish egg species and 13 fish larva species.One species was shared between the egg and larva samples.Our study offers useful tools and critical scientific information for further understanding the diversity,distribution,and conservation management of various ichthyoplankton species in the marine environment.展开更多
[Objective] A study on the classification of 24 species of Calyptratae entering Ningbo port using DNA barcoding technique was carried out.[Method] The CO I genes of the 24 species of Calyptratae were first sequenced.B...[Objective] A study on the classification of 24 species of Calyptratae entering Ningbo port using DNA barcoding technique was carried out.[Method] The CO I genes of the 24 species of Calyptratae were first sequenced.Based on the comparison and analysis of the obtained sequences,the phylogenetic tree was constructed using MEGA6.0.[Result] The cluster analysis showed the classification of the 24 species of Calyptratae was consistent with the morphological classification at the family and genus levels.However,the cluster analysis could not fully distinguish the closely-related species.[Discussion] The DNA barcoding technique cannot be singly used for classifying and identifying Calyptratae.It should be combined with morphological classification methods,and can be treated as a beneficial supplement for morphological classification methods.展开更多
[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA barcoding genes ITS, ITS2 and rbcL were amplified from seven Xanthium ...[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA barcoding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species under the same PCR condition： PCR amplification was started with initial denaturation at 95 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA barcoding genes ITS, ITS2 and rbcL were all amplified successfully. [Conclusion] This study indicates that PCR amplification conditions for DNA barcoding genes ITS,ITS2 and rbcL in plants may be consistent.展开更多
The aim is to select a universal DNA barcode for identifying all poisonous medicinal plants in Chinese pharmacopoeia and their poisonous related species or adulterants. We chose 4 commonly used regions as candidate DN...The aim is to select a universal DNA barcode for identifying all poisonous medicinal plants in Chinese pharmacopoeia and their poisonous related species or adulterants. We chose 4 commonly used regions as candidate DNA barcodes(ITS2, psb A-trn H,matK and rbc L) and compared their identification efficiency in 106 species from 27 families and 65 genera totally. Data analysis was performed including the information of sequence alignment, inter/intra-specific genetic distance and data distribution, identification efficiency and the situation of Neighbor-Joining(NJ) phylogenetic trees. We found ITS2 sequence region had high variation, stable genetic distance and identification efficiency relatively. The topological structure of NJ phylogenetic tree showed monophyletic. Our findings show that ITS2 can be applied as a universal barcode for identifying poisonous medicinal plants in Chinese pharmacopoeia and their poisonous related species or adulterants.展开更多
Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and ident...Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psb A-trn H, rbc L, mat K, trnL(UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL(UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%——20% of the processed samples, while the amplification rates of the trnL(UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL(UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.展开更多
Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for specie...Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for species identification, however, the choice between mitochondrial cytochrome c oxidase subunit I（COI） and large subunit ribosomal RNA gene（16S） as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances（K2P） documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvious ＂barcoding gap＂ was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Considering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.展开更多
Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identif...Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identification techniques often fail to distinguish between them accurately, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Herba Anoectochili and its adulterants accurately, this study identified 41 Herba Anoectochili and its adulterant samples based on the ITS2 sequence. Sequence characteristics, Basic Local Alignment Search Tool(BLAST) application, genetic distance, construction of phylogenetic tree, secondary structure prediction, and other methods showed the ITS2 sequence to accurately identify Herba Anoectochili from its adulterants. Furthermore, in this study, we designed a specific primer, based on the ITS2 sequence, and established a real-time PCR detection system for the rapid, sensitive, and specific identification of the original plant of Herba Anoectochili. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Herba Anoectochili.展开更多
There is an increasing demand for salmonid authentication due to the globalization of the salmonid trade.DNA barcoding and mini-DNA barcoding are widely used for identifying fish species based on a fragment of the mit...There is an increasing demand for salmonid authentication due to the globalization of the salmonid trade.DNA barcoding and mini-DNA barcoding are widely used for identifying fish species based on a fragment of the mitochondrial cytochrome c oxidase subunit I(COI)sequence.In this study,rainbow trout(Oncorhynchus mykiss),steelhead trout(O.mykiss),and Atlantic salmon(Salmo salar)collected from two salmonid aquaculture bases in China were authenticated by DNA barcoding(about 650 bp)and mini-DNA barcoding(127 bp)to evaluate the accuracy of the two methods in the identification of different salmonid species.The results revealed that both methods could effectively distinguish O.mykiss and S.salar with 100%accuracy.However,the two methods failed to separate rainbow trout(O.mykiss)and steelhead trout(O.mykiss),which are the same species but cultured in different water environments.Moreover,salmonid samples from three main distribution channels in the Qingdao area(traditional supermarkets,online supermarkets,and sushi bars)were identified by the two methods.Substitution of S.salar with O.mykiss was discovered,and the 27.78%overall substitution rate of salmonids in the Qingdao area was higher than those in other regions reported in previous studies.In addition,the mislabeling rates of salmonids from traditional supermarkets,online supermarkets,and sushi bars were compared in this study.The mislabeling rate was significantly greater in sushi bars(50%)than in the other two channels(16.67%),suggesting that stronger monitoring and enforcement measures are necessary for the aquatic food catering industry.展开更多
基金supported by the funds from Natural Science Foundation of Hebei Province(C2022402017).
文摘DNA barcoding has been extensively used for species identification.However,species identification of mixed samples or degraded DNA is limited by current DNA barcoding methods.In this study,we use plant species in Juglandaceae to evaluate an assembly-free reads accurate identification(AFRAID)method of species identification,a novel approach for precise species identification in plants.Specifically,we determined(1)the accuracy of DNA barcoding approaches in delimiting species in Juglandaceae,(2)the minimum size of chloroplast dataset for species discrimination,and(3)minimum amount of next generation sequencing(NGS)data required for species identification.We found that species identification rates were highest when whole chloroplast genomes were used,followed by taxon-specific DNA barcodes,and then universal DNA barcodes.Species identification of 100%was achieved when chloroplast genome sequence coverage reached 20%and the original sequencing data reached 500,000 reads.AFRAID accurately identified species for all samples tested after 500,000 clean reads,with far less computing time than common approaches.These results provide a new approach to accurately identify species,overcoming limitations of traditional DNA barcodes.Our method,which uses next generation sequencing to generate partial chloroplast genomes,reveals that DNA barcode regions are not necessarily fixed,accelerating the process of species identification.
文摘The East China Sea(ECS)off the Coast of Zhoushan Archipelago,Zhejiang(ECS-CZA)is home to abundant fishery resources and an important spawning,feeding,and nursing ground for a variety of fish species.Due to long-term overfishing,the ichthyoplankton structure has been dramatically altered.Understanding the species composition and distribution of fish eggs and larvae is one of the most essential tasks to accurately regulate fishery resources and formulate effective management policies;however,little is known about the ichthyoplankton in this region.In this study,an integrated strategy of morphology identification(MI)and mitochondrial COI DNA barcoding was used to identify species of fish eggs and larvae collected from the ECSCZA.MI revealed 15 fish egg species belonging to 12 families and 12 fish larva species belonging to 12 families;in contrast,DNA barcoding altogether identified 30 species,including 18 fish egg species and 13 fish larva species.One species was shared between the egg and larva samples.Our study offers useful tools and critical scientific information for further understanding the diversity,distribution,and conservation management of various ichthyoplankton species in the marine environment.
文摘[Objective] A study on the classification of 24 species of Calyptratae entering Ningbo port using DNA barcoding technique was carried out.[Method] The CO I genes of the 24 species of Calyptratae were first sequenced.Based on the comparison and analysis of the obtained sequences,the phylogenetic tree was constructed using MEGA6.0.[Result] The cluster analysis showed the classification of the 24 species of Calyptratae was consistent with the morphological classification at the family and genus levels.However,the cluster analysis could not fully distinguish the closely-related species.[Discussion] The DNA barcoding technique cannot be singly used for classifying and identifying Calyptratae.It should be combined with morphological classification methods,and can be treated as a beneficial supplement for morphological classification methods.
基金Supported by Science and Technology Project of Jiangsu Entry-Exit Inspection and Quarantine Bureau(2012KJ54)~~
文摘[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA barcoding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species under the same PCR condition： PCR amplification was started with initial denaturation at 95 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA barcoding genes ITS, ITS2 and rbcL were all amplified successfully. [Conclusion] This study indicates that PCR amplification conditions for DNA barcoding genes ITS,ITS2 and rbcL in plants may be consistent.
基金supported by National Standardization Research Project(Nos.ZYBZH-Y-TJ-43,ZYBZH-C-JS-37,ZYBZH-C-JS-29,and ZYBZH-Y-JIN-34)Independent project of Institute of Chinese Materia Medica,Academy of Chinese Medical Sciences(No.ZXKT17021)+1 种基金Key Research Project of China Academy of Chinese Medical Sciences of the 13th Five-Year Plan(No.ZZ10-007)National key research and development plan(No.2017YFB1002303)
文摘The aim is to select a universal DNA barcode for identifying all poisonous medicinal plants in Chinese pharmacopoeia and their poisonous related species or adulterants. We chose 4 commonly used regions as candidate DNA barcodes(ITS2, psb A-trn H,matK and rbc L) and compared their identification efficiency in 106 species from 27 families and 65 genera totally. Data analysis was performed including the information of sequence alignment, inter/intra-specific genetic distance and data distribution, identification efficiency and the situation of Neighbor-Joining(NJ) phylogenetic trees. We found ITS2 sequence region had high variation, stable genetic distance and identification efficiency relatively. The topological structure of NJ phylogenetic tree showed monophyletic. Our findings show that ITS2 can be applied as a universal barcode for identifying poisonous medicinal plants in Chinese pharmacopoeia and their poisonous related species or adulterants.
基金supported by the Major Scientific and Technological Special Project for“Significant New Drugs Creation(No.2014ZX09304307)the Key Projects in he National Science and Technology Pillar Program(No.2011BAI07B08)
文摘Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psb A-trn H, rbc L, mat K, trnL(UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL(UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%——20% of the processed samples, while the amplification rates of the trnL(UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL(UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.
基金The National Natural Science Foundation of China under contract No.41006078the Fundamental Research Funds for the Central Universities under contract No.2010121037+1 种基金the Public Science and Technology Research Funds Projects of Ocean under contract Nos 201005012-3 and 201005015-5the Natural Science Foundation of Fujian Province of China under contract No.2011J05116
文摘Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for species identification, however, the choice between mitochondrial cytochrome c oxidase subunit I（COI） and large subunit ribosomal RNA gene（16S） as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances（K2P） documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvious ＂barcoding gap＂ was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Considering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.
基金supported by the National Key Research and Development Program "Chinese Medicine Modernization Research" Key Project(No.2017YFC1702100)Wuhan University of Technology Professional Degree Graduate Team Guidance Project(No.201701)Wuhan University of Technology Graduate Public Experimental Course Construction Project(No.201906)
文摘Herba Anoectochili is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the similar morphological characteristics of Herba Anoectochili and its adulterants, traditional identification techniques often fail to distinguish between them accurately, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Herba Anoectochili and its adulterants accurately, this study identified 41 Herba Anoectochili and its adulterant samples based on the ITS2 sequence. Sequence characteristics, Basic Local Alignment Search Tool(BLAST) application, genetic distance, construction of phylogenetic tree, secondary structure prediction, and other methods showed the ITS2 sequence to accurately identify Herba Anoectochili from its adulterants. Furthermore, in this study, we designed a specific primer, based on the ITS2 sequence, and established a real-time PCR detection system for the rapid, sensitive, and specific identification of the original plant of Herba Anoectochili. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Herba Anoectochili.
基金the National Key Research and Development Program of China(No.2019YFD0901000)the Natural Science Foundation of Shandong Pro-vince,China(No.ZR2020MC194).
文摘There is an increasing demand for salmonid authentication due to the globalization of the salmonid trade.DNA barcoding and mini-DNA barcoding are widely used for identifying fish species based on a fragment of the mitochondrial cytochrome c oxidase subunit I(COI)sequence.In this study,rainbow trout(Oncorhynchus mykiss),steelhead trout(O.mykiss),and Atlantic salmon(Salmo salar)collected from two salmonid aquaculture bases in China were authenticated by DNA barcoding(about 650 bp)and mini-DNA barcoding(127 bp)to evaluate the accuracy of the two methods in the identification of different salmonid species.The results revealed that both methods could effectively distinguish O.mykiss and S.salar with 100%accuracy.However,the two methods failed to separate rainbow trout(O.mykiss)and steelhead trout(O.mykiss),which are the same species but cultured in different water environments.Moreover,salmonid samples from three main distribution channels in the Qingdao area(traditional supermarkets,online supermarkets,and sushi bars)were identified by the two methods.Substitution of S.salar with O.mykiss was discovered,and the 27.78%overall substitution rate of salmonids in the Qingdao area was higher than those in other regions reported in previous studies.In addition,the mislabeling rates of salmonids from traditional supermarkets,online supermarkets,and sushi bars were compared in this study.The mislabeling rate was significantly greater in sushi bars(50%)than in the other two channels(16.67%),suggesting that stronger monitoring and enforcement measures are necessary for the aquatic food catering industry.
