First record of abnormal body coloration in a rockfish Sebastes koreanus(Scorpaenoidei:Sebastidae)from coastal water of China based on morphological characteristics and DNA barcoding

The first record of abnormal body coloration in Sebastes koreanus Kim and Lee,1994,from the Yellow Sea of China,was documented based on morphological characteristics and DNA barcoding.The two rockfish specimens were collected from the coastal waters of Qingdao,China,and the whole body and all fins of them were red.Of the two red-colored rockfish,there were tiny deep red spots on each fin,2 red radial stripes behind and below the eyes and 1 large deep red blotch on the opercula,while the similar stripe and spot patterns are also present in the S.koreanus specimens with normal body coloration.The countable characteristics of the two specimens are in the range of the morphometry of S.koreanus.To further clarify the species identity and taxonomic status of the two specimens,DNA barcode analysis was carried out.The genetic distance between the red-colored rockfish and S.koreanus was 0,and the minimum net genetic distances between the red-colored rockfish and other Sebastes species except for S.koreanus were 3.0%,which exceeds the threshold of species delimitation.The phylogenetic analysis showed that the DNA barcoding sequences of the two red-colored rockfish clustered with the S.koreanus sequences.The above results of DNA barcode analysis also support that the two red-colored rockfish could be identified as the species of S.koreanus.The mechanism of color variation in S.koreanus is desirable for further research and the species could be an ideal model to study the color-driven speciation of the rockfishes.

DNA Barcoding of Insects and Its Direct Application for Plant Protection

The introduction of invasive insect pests across national borders has become a major concern in crop production. Accordingly, national plant protection organizations are challenge to reinforce their monitoring strategies, which are hampered by the weight and size of inspection equipment, as well as the taxonomic extensiveness of interrupted species. Moreover, some insect pests that impede farmer productivity and profitability are difficult for researchers to address on time due to a lack of appropriate plant protection measures. Farmers’ reliance on synthetic pesticides and biocontrol agents has resulted in major economic and environmental ramifications. DNA barcoding is a novel technology that has the potential to improve Integrated Pest Management decision-making, which is dependent on the ability to correctly identify pest and beneficial organisms. This is due to some natural traits such as phenology or pesticide susceptibility browbeaten by IPM strategies to avert pest establishment. Specifically, Deoxyribonucleic acid (DNA) sequence information was applied effectively for the identification of some micro-organisms. This technology, DNA barcoding, allows for the identification of insect species by using short, standardized gene sequences. DNA barcoding is basically based on repeatable and accessible technique that allows for the mechanisation or automation of species discrimination. This technique bridges the taxonomic bio-security gap and meets the International Plant Protection Convention diagnostic standards for insect identification. This review therefore discusses DNA barcoding as a technique for insect pests’ identification and its potential application for crop protection.

Analysis of the Authenticity of Stone Medicinal Plants by DNA Barcoding

The genus Pyrrosia belongs to the family Polypodiaceae and are medium-sized epiphytic ferns,where the dried leaves of Pyrrosia lingua,Pyrrosia sheareri,Pyrrosia lanceolata,and Pyrrosia calvata are commonly used in medicinal practice.In this study,the authenticity of the collected medicinal plant samples of Shiwei was identified with the help of DNA barcoding technology using the internal transcribed spacer 2(ITS2)as the identifying sequence.The experimental samples were analyzed using the basic local alignment search tool(BLAST)and the authenticity of the samples was further verified with the results of similarity comparison.The results proved that the sequences of the experimentally collected samples of Pyrrosia lingua,Pyrrosia sheareri,Pyrrosia lanceolata,and Pyrrosia calvata had a similarity of more than 97%when compared with the corresponding sequences that were uploaded on the Internet.

DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.

DNA Barcoding and Mini-DNA Barcoding Reveal Mislabeling of Salmonids in Different Distribution Channels in the Qingdao Area

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.

PCR Amplification System of DNA Barcoding Genes ITS, ITS2 and rbcL from 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.

16S rRNA is a better choice than COI for DNA barcoding hydrozoans in the coastal waters of China

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.

DNA Barcoding,Fungal Diversity,and Authentication of Wild Gourmet Mushrooms

The application of molecular tools for the identification of strains,populations and species has become a common practice in a variety of applied and basic investigations in many parts of the world.However,standardization of such applications varies widely among organisms and scientific fields.In this mini-review,the author provides a brief introduction to one of the most prominent effort for species identification-the international barcode of life(iBOL) project,discusses the features of fungal diversity including the proposed fungal barcode DNA fragment-the intergenic spacer regions(ITS) of the nuclear ribosomal RNA gene clusters,and illustrates the potential promises and problems of using ITS for barcoding and for analyzing the phylogeographic pattern of the wild gourmet mushroom Tricholoma matsutake species complex.The analyses show that identification to species level is often insufficient for practical applications and that sequences from multiple genes in combination with critical morphological and physiological evaluations are needed to identify strains,populations and species.

Identification of 24 Species of Calyptratae Entering Ningbo Port Using DNA Barcoding Technique

[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.

DNA barcoding discriminates Pampus minor(Liu et al.,1998) from Pampus species

Although Pampus minor has been classified as a new species, it still remains controversial. Was used a DNA barcoding technique based on homologous sequence analysis of the16S and CO1 genes to clarify the confusion over the identification of this species. Among 12 individuals whose genetic distance was 0.002, two haplotypes were found. According to the 16S sequences, the genetic distances ranged from 0.121 to 0.133 between P. minor and other Pampus species. Although the same the genetic distance between the two P minor haplotypes was generated using CO1 sequences, the haplotype of Pm22-23, Pm28, and Pm32-33 was the same as that of Pci EF607462 and EF607466, while the haplotype of Pm24-27 and Pm29-31 was the same as that of Pci EF607461 and EF607463-65. In addition, the genetic distance ranged only from 0.002 to 0.005 between P minor and Pa EF607460 and EF607458. Apart from this, the interspecies genetic distances varied from 0.135 to 0.143 between P minor and other t＇ampus species according to the C01 sequences. Phylogenetic trees, using combined 16S and CO1 data, strongly support the viewpoint that all the P. minor individuals form one clade that is in a sister position to Pampus sp. individuals （EU357803, FJ434342-FJ434343, and FJ652423-FJ652427）.

Integrating a DNA barcoding project with an ecological survey:a case study on temperate intertidal polychaete communities in Qingdao,China

In this study,we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes.Using 16S rDNA as a complementary marker and combining morphological and ecological characterization,some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status.We obtained 22 haplotype gene sequences of 13 taxa,including 10 CO1 sequences and 12 16S rDNA sequences.Based on intra-and inter-specific distances,we built phylogenetic trees using the neighbor-joining method.Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes,but other genes,such as 16S rDNA,could be used as a complementary genetic marker.For more accurate species identification and effective testing of species hypothesis,DNA barcoding should be incorporated with morphological,ecological,biogeographical,and phylogenetic information.The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.

DNA Barcoding Assessment of Green Macroalgae in Coastal Zone Around Qingdao, China

An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao, China, during the period of April- December, 2011. Three markers were applied in molecular discrimination, including the plastid elongation factor tufA gene, the internal transcribed spacer(ITS) region of the ribosomal cistron and rubisco large subunit gene 3' regions(rbcL-3P). DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures in amplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification. The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and species discrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samples identified by tufA and rbcL as Ulva flexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confusion with discrimination of the complex of U. linza, U. procera and U. prolifera(as the LPP complex) still existed for the three DNA markers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. In distinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be identical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distributed in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae.

Identification of Kalidium species(Chenopodiaceae)by DNA barcoding

DNA barcoding is an increasingly prevalent molecular biological technology which uses a short and conserved DNA fragment to facilitate rapid and accurate species identification. Kalidium species are distributed in saline soil habitat throughout Southeast Europe and Northwest Asia, and used mainly as forage grass in China. The discrimination of Ka-lidium species was based only on morphology-based identification systems and limited to recognized species. Here, we tested four DNA candidate loci, one nuclear locus （ITS, internal transcribed spacer） and three plastid loci （rbcL9 matK and ycflb）, to select potential DNA barcodes for identifying different Kalidium species. Results showed that the best DNA barcode was ITS locus, which displayed the highest species discrimination rate （100%）, followed by matK （33.3%）,ycflb （16.7%）, and rbcL （16.7%）. Meanwhile, four loci clearly identified the variant species, Kalidium cuspidatum （Ung.-Stemb.） Gmb.var.A. J. Li,as a single species in Kalidium.

DNA barcoding of the family Sparidae along the coast of China and revelation of potential cryptic diversity in the IndoWest Pacific oceans based on COI and 16S rRNA genes

Sparids are of considerable economic importance in marine fishery and aquaculture in China, and the species diversity of this group is considered relatively high. However, the accurate species identification and delimitation of sparids in China remain unaddressed. In this study, we used mitochondrial cytochrome oxidase subunit ?(COI) and 16S ribosomal RNA(16S) genes to conduct DNA barcoding and species delimitation in eleven sparid species from the coastal waters of China. Based on Kimura-2 parameter genetic distances, the mean intraspecific/interspecific variation for COI and 16S were calculated as 0.004/0.152 and 0.002/0.072, respectively. All the conspecific individuals formed monophyletic clusters in neighbour-joining trees of both markers. An obvious barcoding gap was detected for each species, and a common genetic threshold of 1.3% sequence divergence was defined for species delimitation in both markers. Although the sequence variation of 16S was generally lower than that of COI, the results indicated that sparid species could be ef fectively and accurately identified and delimited by COI as well as 16S. Thus, we propose that the COI gene serve as the standard DNA barcode for sparids, and that the 16S gene could also be an ideal candidate barcode. Moreover, each of the six sparid species( Argyrops spinifer, Rhabdosargus sarba, Dentex hypselosomus, Acanthopagrus latus, Acanthopagrus australis and Acanthopagrus berda) showed high intraspecific divergence(>1.3% genetic threshold) with the remarkable geographic lineages in the Indo-West Pacific oceans, which supported that potential unrecognized cryptic species were in them. The potential cryptic diversity revealed here might be primarily attributed to the allopatric divergences caused by the long-term geographic isolation between the Indian and West Pacific oceans or between the opposite sides of the Indian Ocean. The results further suggest that a revision of taxonomic status of these species is required, followed by development of a biodiversity conservation strategy.

Identification and DNA Barcoding of a New Sillago Species in Beihai and Zhanjiang,China,with a Key to Related Species

A new Sillago species,Sillago parasihama sp.n.,is identified based on 127 specimens collected from the southern coast of China.We compared the morphological characters between Sillago parasihama and all other 11 Sillago species with two posterior extensions on the swim bladder.The new species is like S.sihama in the countable characters and color pattern,but is different from the latter by the distinct swim bladders.The swim bladder of S.parasihama is without lateral process.The posterior sub-extensions of anterolateral extensions are unique with some dendritic or sometimes stunted blind tubule,which are unilateral and outward,ex-tending along the abdominal,and are about one-third to half of the body of swim bladder in length.But the swim bladder of S.si-hama with 8-10 lateral processes,the posterior sub-extensions of anterolateral extensions are kinky,long and complicated,extend-ing along the abdominal wall below the peritoneum to the base of posterior extensions.S.parasihama can be distinguished from other species in this group by color pattern,meristic,and morphometric characters.Moreover,the results of genetic analysis using sequences of the mitochondrial cytochrome c oxidase subunit I(COI)fragment show significant interspecific-level genetic distances(0.159-0.231)between S.parasihama and 8 congeners in the group,which also support the validity of new species.We also provide a distribution map and a key of the related species.

DNA barcoding and molecular phylogeny of Dumasia(Fabaceae:Phaseoleae)reveals a cryptic lineage

Dumasia taxonomy and classification have long been problematic.Species within this genus have few morphological differences and plants without flowers or fruits are difficult to accurately identify.In this study,we evaluated the ability of six DNA barcoding sequences,one nuclear(ITS)and five chloroplast regions(trnH-psbA,matK,rbcL,trnL-trnF,psbB-psbF),to efficiently identify Dumasia species.Most single markers or their combinations identify obvious barcoding gaps between intraspecific and interspecific genetic variation.Most combined analyses including ITS showed good species resolution and identification efficiency.We therefore suggest that ITS alone or a combination of ITS with any cpDNA marker are most suitable for DNA barcoding of Dumasia.The phylogenetic analyses clearly indicated that Dumasia yunnanensis is not monophyletic and is separated as two independent branches,which may result from cryptic differentiation.Our results demonstrate that molecular data can deepen the comprehension of taxonomy of Dumasia and provide an efficient approach for identification of the species.

Identification of three kinds of Plumeria flowers by DNA barcoding and HPLC specific chromatogram

DNA barcoding and HPLC specific chromatogram were used to identify three kinds of Plumeria flowers respectively. DNAs extracted from the three Plumeria species were amplified by PCR with universal primers, and the psbA-trnH region was selected. All the amplified products were sequenced and the results were analyzed by MEGA 5.0. Chemometric methods including principal components analysis and hierarchical clustering analysis were conducted on the SAS 9.0 software to demonstrate the variability among samples. In conclusion, the psbA-trnH of all samples were successfully amplified from total DNA and sequenced. These three varieties of Plumeria can be differentiated by the psbA-trnH region and clustered into three groups respectively through building neighbor joining tree, which conformed to their germplasm origins. However, it was hard to distinguish them by HPLC specific chromatograms combined with chemometrics analysis. These indicated that DNA barcoding was a promising and reliable tool for the identification of three kinds of Plumeria flowers compared to HPLC specific chromatogram generally used. It could be treated as a powerful complementary method for traditional authentication, especially for those varieties which are difficult to be identified by conventional chromatography.

Applying DNA Barcoding to Identify Actinidia in the Northeast of China

Identification of kiwifruit germplasm materials is the basis of protecting and utilizing these resources.However,identifying Actinidia arguta varieties based on morphology is difficult,especially for non-specialists.In this study,we collected 180 specimens comprising 60 varieties of three species(A.arguta,A.kolomikta,and A.polygama)from the Northeast of China.The emphasis of our study was on the feasibility of identifying A.arguta varieties.Here,we used common analysis methods(genetic distance,phylogenetic analysis and the DNA barcoding gap)and SNPs analysis to evaluate the discriminatory power of different DNA barcoding markers.The results revealed that common methods were insufficient to identify A.arguta varieties but SNPs analysis based on DNA barcoden was the potential method for identifying A.arguta varieties.Besides,Our study agree that DNA barcoding could be used to analysis the evaluation genetic relationship of the Actinidia plants.

The Relationships Between Toxicity, Species and Populations in Nassarius Based on Toxin Detection and Multiple Gene Barcoding

Nassarius causes many poisonings in China and other Asian countries.The toxicity of Nassarius is related to species and populations.However,the molecular assignment of different toxic Nassarius samples is still unclear,which deteriorates the poisoning.Our study combined toxicity,toxin detection and multiple genes DNA barcoding to assign the various toxic Nassarius species and populations from China coast.The toxicity test indicated that different Nassarius species and different populations within one Nassarius species had different toxicity.Based on High Performance Liquid Chromatography,the toxin was confirmed to be TTX.The toxin content varied in different samples.The COI,16 S and ITS sequences revealed different toxic Nassarius species as monophyletic clades.All the samples tested have been assigned to species level.However,the different toxic populations within N.succinctus,N.variciferus and N.semiplicata did not form separated clades,and the toxic and nontoxic species did not form two clades separately.Our study constructed the relationship between toxicity and taxonomy assignment of different toxic Nassarius samples based on toxicity detection and multiple gene DNA barcoding,which provides the basis for controlling poisoning and studying the toxin-resistance difference in Mollusca.It is also indicated that the toxicity of Nassarius is really related with species at the genetic level,but not absolutely related with populations.

Species delimitation in the green algal genus Codium (Bryopsidales) from Korea using DNA barcoding

Codium, one of the largest marine green algal genera, is difficult to delimit species boundary accurately based on morphological identification only. DNA barcoding is a powerful tool for discriminating species of seaweeds. The plastid elongation factor TU （tufA） is considered as maker to perform DNA barcoding of green algal species than rbcL gene due to universality and rapid evolution rate. We conducted DNA barcoding application to Codium specimens from the Jeju Island, Korea to overcome the limit of morphological identification and to confirm the species diversity. As a result of applying tufA marker, we newly generated fifty-five tufA barcodes to resolve eight species. TufA marker exhibited 6.1%–21.8% interspecific divergences, wider than the gap of rbcL exon 1, 3.5%–11.5%. Molecular analysis of rbcL exon 1 sequences of Codium revealed eight distinct species like tufA analysis separated in five phylogenetic groups. DNA barcoding of the genus Codium using tufA marker is more helpful to overcome the limit of morphological identification, and this is more potential to reveal cryptic species and to resolve the relationships among subspecies than rbcL analysis alone. The complement of tufA barcoding and rbcL analyses including morphology for the genus Codium in the northwestern Pacific will give much more reliable achievement for discovering species diversity and resolving the phylogenetic relationships.