Prospects and Problems for Identification of Poisonous Plants in China using DNA Barcodes

Objective Poisonous plants are a deadly threat to public health in China. The traditional clinical diagnosis of the toxic plants is inefficient, fallible, and dependent upon experts. In this study, we tested the performance of DNA barcodes for identification of the most threatening poisonous plants in China. Methods Seventy-four accessions of 27 toxic plant species in 22 genera and 17 families were sampled and three DNA barcodes （motK, rbcL, and ITS） were amplified, sequenced and tested. Three methods, Blast, pairwise global alignment （PWG） distance, and Tree-Building were tested for discrimination power. Results The primer universality of all the three markers was high. Except in the case of ITS for Hemerocollis minor, the three barcodes were successfully generated from all the selected species. Among the three methods applied, Blast showed the lowest discrimination rate, whereas PWG Distance and Tree-Building methods were equally effective. The ITS barcode showed highest discrimination rates using the PWG Distance and Tree-Building methods. When the barcodes were combined, discrimination rates were increased for the Blast method.

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.

Prospects for discriminating Zingiberaceae species in India using DNA barcodes

We evaluated nine plastid （matK, rbcL, rpoCl, rpoB, rp136-rpsS, ndhJ, trnL-F, tmrnH-psbA, accD） and two nuclear （ITS and ITS2） barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India. Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction （PCR） amplicons in all the accessions tested. However, only 35 （58%） and 4o accessions （66~） yielded ITS and ITS2 sequences, respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species （75%） into monophyletic groups and five species into two para- phyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species （6o%）, and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence ofnatural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of iTS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling.

Identification of Dian Ji Xue Teng(Kadsura interior) with DNA barcodes

Objective: To identify Kadsura interior(Dian Ji Xue Teng, Schisandraceae) by using DNA barcoding.Methods: We analyzed five DNA barcodes(ITS, ITS2, psb A-trn H, mat K and rbc L) using DNA barcoding in terms of distance-based,tree-based and character-based identification to distinguish Kadsura interior and its adulterants.Results: In distance-based and tree-based identification, K. interior could be distinguished easily from the species of Schisandra and K. coccinea.In character-based identification, there are two single nucleotide polymorphisms(SNPs) in ITS and one SNP in psb A-trn H which can be used to distinguish K. interior from K. heteroclita and K. longipedunculata.Conclusion: The results indicate that DNA barcoding can be used to identify K. interior. ITS and psb A-trnH sequence can be the most ideal DNA barcode for discriminating K. interior and its adulterants by the combination analysis of distance-based, tree-based and character-based identification(SNPs).

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia

Fargesia,the largest genus within the temperate bamboo tribe Arundinarieae,has more than 90 species mainly distributed in the mountains of Southwest China.The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals,including the giant panda.However,species-level identification of Fargesia is difficult.Moreover,the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes(rbcL,matK,and ITS) in bamboos.With progress in the sequencing technologies,complete plastid genomes(plastomes) and nuclear ribosomal DNA(nrDNA)sequences have been proposed as organelle barcodes for species identification;however,these have not been tested in bamboos.We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes.Our analysis indicates that complete plastomes have substantially higher discriminatory power(28.6%) than standard barcodes(5.7%),whereas nrDNA sequences show a moderate improvement(65.4%) compared to ITS(47.2%).We also found that nuclear markers performed better than plastid markers,and ITS alone had higher discriminatory power than complete plastomes.The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia.However,neither of these sequences were able to discriminate all the sampled species,and therefore,more nuclear markers need to be identified.

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.

Pharmacognostical s tudies o n Spermacoce p usilla Wallich

Background:Spermacoce pusilla Wallich(S.pusilla)is widely used in Guangdong province of China.It has valuable medicinal value in treating trauma,fractures,carbuncle,swelling,and poisonous snake bites.Method:The present study was carried out to provide a scientific basis for the identification and authenticity of S.pusilla with the help of pharmacognostical parameters,which has not been done before.In this study,a series of related identification information such as source,character,microscopic observation,physical and chemical reaction,and molecular markers were employed to establish accurate,comprehensive pharmacognostic identification information of S.pusilla.Results:The study provided some basic data regarding the genuine crude drug.The identification efficiency of ITS sequences obtained in this study is high,the psbA-trnH sequence was obtained from S.pusilla and sequenced for the first time in this study.Conclusion:In this study,traditional pharmacognosy identification and molecular marker identification of S.pusilla in the Guangdong region were carried out,and a systematic and comprehensive pharmacognosy identification information system was established.

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 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.

DNA barcoding of Antarctic marine zooplankton for species identification and recognition

Polar zooplankton are particularly sensitive to climate change, and have been used as rapid-responders to indicate climate-induced changes in the fragile Antarctic ecosystem. DNA barcoding provides an alternative approach for rapid zooplankton species identification. Ninety-four specimens belonging to 32 Antarctic zooplankton species were barcoded to construct a compre- hensive reference library. An 830 to 1 050 base-pair region of the mitochondrial cytochrome c oxidase subunit I （mtCOI） gene was obtained as DNA barcodes. The intraspecific variation of the gene ranged from 0 to 2.6% （p-distance）, with an average of 0.67% （SD=0.67%）. The distance between species within the same genera ranged from 0.1% （Calanus） to 29.3%, with an average of 15.3% （SD=8.4%）. The morphological and genetic similarities between Calanus propinquus and C. simillimus raise new questions about the taxonomic status of C. simillimus. With the exception of the two Calanus species, the intraspecific genetic divergence was much smaller than the interspecific divergence among congeneric species, confirming the existence of a barcode gap for Ant- arctic zooplankton. In addition, species other than Calanus sp. formed a monophyletic group. Therefore, we have confirmed DNA barcoding as an accurate and efficient approach for zooplankton identification in the Antarctic area （except for Hydromedusa, Tu- nicata, and other gelatinous zooplankton）. Indicator vector analysis further confirmed this conclusion. The new primer sets issued here may facilitate the study of Antarctic marine zooplankton species composition by environmental metagenetic analysis.

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.

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.

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 Barcode Screening and Preliminary Study on Phylogeny of Sea Snake Based on Genes COI and cytb

[Objectives]The most common gene fragment used in animal DNA barcode technology is COI,but it is not necessarily suitable for all species.This study was conducted to screen genes suitable for the DNA barcode of sea snakes.[Methods]All COI and cytb gene sequences on GenBank were searched and downloaded.After the comparison with Mega software,clustering trees of MrBayes system were established.[Results]Interspecies distances were greater than intraspecies distances for the two genes.The topological structures of their molecular hierarchical clustering trees were clear,and the support rates were high.[Conclusions]Therefore,it is concluded that not the DNA barcode of each species must be gene COI.Cytb is more suitable in terms of the mitochondrial gene of sea snakes.

Dietary analysis of the House Swift(Apus nipalensis)in Hong Kong using prey DNA in faecal samples

Background:To understand the dietary composition of the highly aerial swift(Apodidae),ecologists conventionally depend on the morphological identification of prey items from food boluses or stomach contents,but these techniques are often invasive,require expertise in identification,and often cannot produce accurate identifications at the species level.Methods:DNA barcoding was used to analyse the dietary composition of House Swifts(Apus nipalensis)in Hong Kong,China.Faecal samples from five different colonial nest sites were collected between 2019 and 2020.We used universal primers to amplify a region of the cytochrome C oxidase gene from prey DNA in the faecal samples for identification purposes.Results:Ten different orders and 44 families from three different classes of Arthropoda were identified in the collected faecal samples.Hymenoptera,Hemiptera and Diptera were the most prevalent groups of prey found in the samples.Differences in the dietary composition of House Swifts during the breeding(April to September)and nonbreeding(October to March)season were also found.Hymenoptera,particularly ants(Formicidae),were predominant in the diet during the breeding season,whereas Diptera and Hemiptera were predominant during the non-breeding season.Conclusion:The prey groups identified in this study were similar to those identified in a previous study of the diet of House Swift,which also suggests a possible role of House Swifts in reducing the numbers of local insect pests.This study demonstrates the usefulness of applying molecular tools for the dietary analysis of aerial feeders.Conserving local forested areas may be crucial for the maintenance of House Swift population.

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.

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.

Molecular Identification of Dried Shellfish Products Sold on the Market Using DNA Barcoding

The dried shellfish products with rich nutrients and low-calorie content are favorite food in China,especially in coastal areas.However,the species of dried shellfish products in the market are usually unknown,as the taxonomic features were removed during the production process.This study described the application of DNA barcoding technique to the identification of 100 dried shellfish(scallop,squid,octopus and cuttlefish)products in markets.Samples were authenticated by comparing mitochondrial cytochrome oxidase subunit I(COI)gene and 16S ribosomal RNA(16S rRNA)gene sequences with public reference taxonomic databases.The results showed that all the 100 products can be identified at species level.Sixty four scallop adductor products were processed using the bay scallop,Argopecten irradians,and one was from Portuguese oyster,Crassostrea angulata.All the 27 squid,2 cuttlefish and 6 octopus products were produced by the Jumbo flying squid,Dosidicus gigas.The neighbour-joining tree is in agreement with the results of DNA barcoding analysis.The 64 scallop samples formed one A.irradians cluster,leaving Sca65 clustered with the reference oyster sequence C.angulata(MH997922).All the 35 cephalopod(squid,octopus and cuttlefish)samples formed a D.gigas cluster.This investigation revealed a low variety of dried shellfish products sold on the market,and highlighted the high rate of mislabeling and species substitution.Our present work provides a practical method for tracing and authenticating shellfish products.