Three newly recorded species of Acerentomata(Hexapoda:Protura) from China,with analysis of DNA barcodes

Three newly recorded species in the order Acerentomata in Protura from China are described:Filientomon duodecimsetosum Nakamura,2004,Verrucoentomon anatoli Shrubovych & Bernard,2012 and Verrucoentomon louisanne Shrubovych & Bernard,2012.The important morphological characters of Chinese specimens are described in detail.An updated key to Chinese Verrucoentomon species is provided.In addition,their DNA barcodes are sequenced and analyzed.

Crane flies(Diptera: Tipuloidea: Tipulidae) from Dayaoshan National Nature Reserve, China and analysis of DNA barcodes, with description of one new species in the genus Indotipula

This paper reports six species of crane flies from Dayaoshan National Nature Reserve, Guangxi Zhuang Autonomous Region, China, including one new species, Indotipula jinxiuensis sp. nov. The males of Pselliophora guangxiensis Yang Yang, 1988 and Holorusia basiflava Yang Yang, 1993 and female of Pselliophora xanthopimplina Enderlein, 1921 are redescribed and illustrated with new morphological characters. The females of P. guangxiensis and H. basiflava are described and illustrated for the first time. A key for separating known species of Indotipula Edwards, 1931 from China is provided. DNA barcodes of all species in this study are provided and analyzed.

DNA Barcodes in Fig Cultivars (Ficus carica L.) Using ITS Regions of Ribosomal DNA, the psbA-trnH Spacer and the matK Coding Sequence

Molecular markers provide a useful method for genotype characterization and allow a high precision determination of the genetic relationship between cultivars and varieties. A system based on DNA sequences—which is known as DNA barcoding—will choose one or several standard loci which can be sequenced and compared to differentiate between species. In this research, the ITS, matK, and trnH-psbA sequences were evaluated for the molecular identification of seven F. carica genotypes, generating complete sequences for the first two loci, but unable to produce bidirectional sequences by using the trnH-psbA sequence. The ITS sequence presented the highest variation rates, while the phylogeny constructed with the matK sequence obtained the highest percentage of solved monophyletic groups. Through Pearson’s correlation analysis, it was possible to determine the existence of a significant correlation between the ITS region and psbA-trnH, and the matK and psbA-trnH sequences, but not between ITS and matK. The phylogenies constructed with the ITS + matK barcodes and ITS + matK + psbA-trnH presented the highest percentage for resolution. However, considering the cost efficiency and the facilitated recovery by using PCR, the matK + ITS combination is recommended.

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.

Applications of Three DNA Barcodes in Assorting Intertidal Red Macroalgal Flora in Qingdao, China

This study is part of the endeavor to construct a comprehensive DNA barcoding database for common seaweeds in China. Identifications of red seaweeds, which have simple morphology and anatomy, are sometimes difficult solely depending on morphological characteristics. In recent years, DNA barcode technique has become a more and more effective tool to help solve some of the taxonomic difficulties. Some DNA markers such as COI（cytochrome oxidase subunit I） are proposed as standardized DNA barcodes for all seaweed species. In this study, COI, UPA （universal plastid amplicon, domain V of 23S rRNA）, and ITS （nuclear internal transcribed spacer） were employed to analyze common species of intertidal red seaweeds in Qingdao （119.3°-121°E, 35.35°-37.09°N）. The applicability of using one or a few combined barcodes to identify red seaweed species was tested. The results indicated that COI is a sensitive marker at species level. However, not all the tested species gave PCR amplification products due to lack of the universal primers. The second barcode UPA had effective universal primers but needed to be tested for the effectiveness of resolving closely related species. More than one ITS sequence types were found in some species in this investigation, which might lead to confusion in further analysis. Therefore ITS sequence is not recommended as a universal barcode for seaweeds identification.

Chloroplast gene matK holds the barcodes for identification of Momordica(Cucurbitaceae) species from Indian subcontinent

DNA barcoding is a supplementary tool in plant systematics,extensively used to resolve the species level controversies.This paper details the identification of DNA barcodes for seven species of Momordica,using the chloroplast gene mat K.Since the species M.cymbalaria has been confused as a member of the genus Luffa,26 accessions of Momordica belonging to seven Indian species and two accessions of Luffa acutangula were included in this study.Analysis of mat K sequences has yielded distinct barcodes in M.charantia var.charantia,M.subangulata subsp.renigera,M.cochinchinensis,M.balsamina,M.cymbalaria and also in Luffa acutangula.Evolutionary status of each species was reflected as nucleotide polymorphisms in each sequence.The wild species M.dioica and M.sahyadrica have yielded one barcode but failed to get differentiated.Further,this study provides conclusive proof that M.cymbalaria is a member of Momordica genus.The phylogram generated was successful to distinguish the monoecious species of this genus,M.charantia,M.balsamina and M.cymbalaria,from the dioecious species M.dioica,M.sahyadrica,M.subangulata subsp.renigera and M.cochinchinensis.Thus,mat K locus,by accumulating the evolutionary sequence variations,is proven efficient to differentiate the Momordica species and to reveal their relatedness.

Emergence of Plastidial Intergenic Spacers as Suitable DNA Barcodes for Arid Medicinal Plant <i>Rhazya stricta</i>

The desert plant Rhazya stricta has anticancer and antimicrobial properties, and is widely used in indigenous medicines of Saudi Arabia. However, the therapeutic benefits rely on an accurate identification of this species. The authenticity of R. stricta and other medicinal plants and herbs procured from local markets can be questionable due to a lack of clear phenotypic traits. DNA barcoding is an emerging technology for rapid and accurate species identification. In this study, six candidate chloroplastid barcodes were investigated for the authentication of R. stricta. We compared the DNA sequences from fifty locally collected and five market samples of R. stricta with database sequences of R. stricta and seven closely related species. We found that the coding regions matK, rbcL, rpoB, and rpoC1 were highly similar among the taxa. By contrast, the intergenic spacers psbK-psbI and atpF-atpH were variable loci distinct for the medicinal plant R. stricta. psbK-psbI clearly discriminated R. stricta samples as an efficient single locus marker, whereas a two-locus marker combination comprising psbK-psbI + atpF-atpH was also promising according to results from the Basic Local Alignment Search Tool and a maximum likelihood gene tree generated using PHyML. Two-dimensional DNA barcodes (i.e., QR codes) for the psbK-psbI and psbK-psbI + atpF-atpH regions were created for the validation of fresh or dried R. stricta samples.

Evaluating the Discriminatory Power of DNA Barcodes in Panicoideae, Poaceae

DNA barcoding is a powerful technique for species identification with little morphological knowledge, by using short sections of DNA from a specific region of the genome. Two core barcode markers, rbcL and matK, and a supplementary nuclear ribosomal internal transcribed spacer region were used to examine the effectiveness of the markers for Poaceae barcoding using 133 individuals of 36 taxa across 23 genera of Korean Panicoideae. We also aimed to establish a DNA barcode database for the major weeds of Korean Panicoideae. All three markers revealed a good level of amplification and sequencing success. As a single DNA marker, the ITS region achieved the highest species resolution, followed by matK. Resolving power was increased when nrlTS was incorporated into the core barcode markers. The best resolving power was obtained with a combination of matK ＋ ITS with 89.7%, followed by rbcL ＋ matK ＋ ITS with 89.3%. Thus, rbcL may be not necessary as a DNA barcode for Panicoideae species identification, when considering cost and effectiveness. Instead, a combination of matK ＋ ITS is proposed as the most suitable DNA barcode for the species identification of Panicoideae, Poaceae. We conclude that DNA barcoding using a combination of matK ＋ ITS could be one of powerful techniques for the identification of Poaceae species, The barcode sequences were deposited to the National Center for Biotechnology Information （NCBI） database for public use.

Generating barcodes for nanopore sequencing data with PRO

DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequencing makes it difficult to identify barcodes accurately,which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing run.Here,we present a comprehensive study of the generation of barcodes and develop a tool,PRO,that can be used for selecting optimal barcode sets and demultiplexing.We formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically NP-complete.For practical applications,we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing accuracy.Specifically,the maximum size of the barcode kits designed by PRO is 2,292,which keeps the length of barcodes the same as that of the official ones used by Oxford Nanopore Technologies(ONT).We validated the performance of PRO on a simulated nanopore dataset with high error rates.The demultiplexing accuracy of PRO reached 98.29%for a barcode kit of size 2,922,4.31%higher than that of Guppy,the official demultiplexing tool.When the size of the barcode kit generated by PRO is the same as the official size provided by ONT,both tools show superior and comparable demultiplexing accuracy.

Dual-stimuli responsive photonic barcodes based on perovskite quantum dots encapsulated in whispering-gallery-mode microspheres

Micro/nanoscale photonic barcodes hold great potential for broad applications in items tracking,mul-tiplexed bioassays and anti-counterfeiting.The ever-increasing demand in advanced anti-counterfeiting applications calls for micro/nanoscale barcodes with accurate recognition,large encoding capacity and high security level.Here,we proposed a strategy to construct the dual-stimuli responsive photonic barcodes based on the perovskite quantum dots(PQDs)doped polymer whispering-gallery-mode(WGM)microcavities via swelling-deswelling method.Benefiting from the well-defined spherical microcavities,the photoluminescence(PL)spectra of as-prepared composites exhibit a series of sharp peaks characteristics resulting from the effective WGM modulation,which constitutes the fingerprint of a specific resonator and thus allows a definition of photonic barcodes.On this basis,we achieved responsive photonic barcodes based on the volatile polar-solvent-controlled luminescence in the mi-crospheres benefitting from the space-confined microcavities and the ionic feature of the PQDs.More-over,the light-controlled photonic barcodes have further been acquired through reversibly regulating the inactivation and activation of the energy transfer(ET)process between the PQDs and photochromic dyes.The well-established protocols of PQDs@WGM enable the development of distinct responsive barcodes with multi-responsive features,which will pave an avenue to new types of flexible WGM-based components for optical data recording and security labels.

Dynamic photonic barcodes for molecular detection based on cavity-enhanced energy transfer

Optical barcodes have demonstrated a great potential in multiplexed bioassays and cell tracking for their distinctive spectral fingerprints.The vast majority of optical barcodes were designed to identify a specific target by fluorescence emission spectra,without being able to characterize dynamic changes in response to analytes through time.To overcome these limitations,the concept of the bioresponsive dynamic photonic barcode was proposed by exploiting interfacial energy transfer between a microdroplet cavity and binding molecules.Whispering-gallery modes resulting from cavity-enhanced energy transfer were therefore converted into photonic barcodes to identify binding activities,in which more than trillions of distinctive barcodes could be generated by a single droplet.Dynamic spectral barcoding was achieved by a significant improvement in terms of signal-to-noise ratio upon binding to target molecules.Theoretical studies and experiments were conducted to elucidate the effect of different cavity sizes and analyte concentrations.Timeresolved fluorescence lifetime was implemented to investigate the role of radiative and non-radiative energy transfer.Finally,microdroplet photonic barcodes were employed in biodetection to exhibit great potential in fulfilling biomedical applications.

Assessment of candidate plant DNA barcodes using the Rutaceae family

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention.Here,seven regions (psbA-trnH,matK,ycf5,rpoC1,rbcL,ITS2,and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae.To evaluate each barcode’s utility for species authentication,PCR amplification efficiency,genetic divergence,and barcoding gaps were assessed.We found that the ITS2 region exhibited the highest inter-specific divergence,and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests.The ITS2 locus had the highest identification efficiency among all tested regions.In a previous study,we found that ITS2 was able to discriminate a wide range of plant taxa,and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore,we propose that ITS2 is a promising candidate barcode for plant species identification.

Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes

To ensure the safety of medications,it is vital to accurately authenticate species of the Apocynaceae family,which is rich in poisonous medicinal plants.We identified Apocynaceae species by using nuclear internal transcribed spacer 2(ITS2)and psb Atrn H based on experimental data.The identification ability of ITS2 and psb A-trn H was assessed using specific genetic divergence,BLAST1,and neighbor-joining trees.For DNA barcoding,ITS2 and psb A-trn H regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified.The PCR amplification for ITS2 and psb A-trn H sequences was 100%.The sequencing success rates for ITS2 and psb A-trn H sequences were 81%and 61%,respectively.Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psb A-trn H region were downloaded from Gen Bank.Moreover,the analysis showed that the interspecific divergence of Apocynaceae species was greater than its intra-specific variations.The results indicated that,using the BLAST1 method,ITS2 showed a high identification efficiency of 97%and 100%of the samples at the species and genus levels,respectively,via BLAST1,and psb A-trn H successfully identified 95%and 100%of the samples at the species and genus levels,respectively.The barcode combination of ITS2/psb A-trn H successfully identified 98%and 100%of samples at the species and genus levels,respectively.Subsequently,the neighbor joining tree method also showed that barcode ITS2 and psb A-trn H could distinguish among the species within the Apocynaceae family.ITS2 is a core barcode and psb A-trn H is a supplementary barcode for identifying species in the Apocynaceae family.These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.

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).

Microfluidic generation of barcodes with in situ synthesized perovskite quantum dot encapsulation

Perovskite quantum dots(PQDs)are new class of optoelectronic materials,which have been widely studied for their extraordinary physical properties.Attempts to develop these materials are tending to make their fabrication much controllable and extend their values in different areas.Here,we present a novel strategy for one-step in situ synthesis of PQD-encapsulated barcode particles with the assistance of microfluidic technique.By changing the halide ratio in perovskite precursor solutions that emulsified in microfluidic devices,a series of PQDs with different colors have been successfully fabricated,which made them ideal materials as barcodes.Because of the stable encapsulation of ethyleneglycol dimethacrylate(EGDMA)resin,the PQD-encapsulated barcode particles were with no cytotoxicity and could be anti-quenched.It was demonstrated for the first time that the PQD-encapsutated barcode particles by microfluidics were valuable for multiplex biomolecular encoding and assays.These features indicate that the PQD-encapsutated barcode particles by microfluidics are ideal for many practical applications and have a broad prospect in biomedical field.

Deciphering mycorrhizal fungi in cultivated Phalaenopsis microbiome with next-generation sequencing of multiple barcodes

Identifying the species composition of a microbial ecosystem is often hampered by difficulties in culturing the organisms and in the low sequencing depth of traditional DNA barcoding.Metagenomic analysis,a huge-scale nucleotide-sequence-based tool,can overcome such difficulties.In this study,Sanger sequencing of 500 nrITS clones uncovered 29 taxa of 19 fungal genera,whereas metagenomics with next-generation sequencing identified 512 operational taxonomic units(OTUs)for ITS1/2 and 364 for ITS3/4.Nevertheless,high throughput sequencing of PCR amplicons of ITS1/2,ITS3/4,nrLSU-LR,nrLSU-U,mtLSU,and mtATP6,all with at least 1,300×coverage and about 21 million reads in total,yielded a very diverse fungal composition.The fact that 74%of the OTUs were exclusively uncovered with single barcodes indicated that each marker provided its own insights into the fungal flora.To deal with the high heterogeneity in the data and to integrate the information on species composition across barcodes,a rank-scoring strategy was developed.Accordingly,205 genera among 64 orders of fungi were identified in healthy Phalaenopsis roots.Of the barcodes utilized,ITS1/2,ITS3/4,and nrLSU-U were the most competent in uncovering the fungal diversity.These barcodes,though detecting different compositions likely due to primer preference,provided complementary and comprehensive power in deciphering the microbial diversity,especially in revealing rare species.

One-step and wash-free multiplexed immunoassay platform based on bioinspired photonic barcodes

Multiplex,rapid and accurate virus quantification plays a great value in biomedical detection.Here,a novel one step,wash-free immunoassay platform based bioinspired PhC barcodes for multiplexed virus quantification was explored.PhC barcodes were decorated with PDA by self-polymerization of DA,thus this nanocomposite hybridized PhC barcodes facilitated the adsorption of FITC labelled antibodies and quenched itself photolumines-cent,allowing a fast responsive composite platform.In the presence of target analyte,the FITC-labelled detection antibody was released from the surface of PDA decorated microcarrier to specifically bind to the target ana-lyte,thus recovered the photoluminescence.In addition,the PhC microcarrier was enabled to carry out various color barcode for different targets detection though tuning internal periodic structures.Based on these excellent performances of the nanocomposite barcode,this method can not only capture H1N1,H5N1,SARS-CoV-2 si-multaneously with rapid,accuracy but also accomplish multiplex quantification detection with high-sensitivity.Furthermore,our developed platform was also achieved with high-sensitivity and high-specificity through the verification of clinical samples,thus laying out a new avenue for multiplex virus detection in clinical diagnosis.

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.

Morphological and molecular description of a new species of sandfly,Sergentomyia(Neophlebotomus)ashwanii sp.nov.(Diptera:Psychodidae)from Western Ghats,India

Objective:To report a new species of sandfly,Sergentomyia(Neophlebotomus)ashwanii sp.nov.(Diptera:Psychodidae)from Western Ghats,India.Methods:A systematic sandfly survey was conducted in the Thrissur and Kollam districts of Kerala,India using mechanical aspirators,light and sticky traps,both indoor and outdoor habitats,for a period of one year.Deoxyribonucleic acid barcoding of samples was performed targeting mitochondrial cytochrome oxidase I(COI)gene and sequence generated was subjected to phylogenetic analysis.Results:Sergentomyia(Neophlebotomus)ashwanii,a new sandfly species is recorded and described in this communication.A single row of 10-12 pointed teeth in the cibarium with 4-6 small denticles or fore-teeth are the key characteristics that is distinctive from other members of the subgenus Neophlebotomus.Mitochondrial COI barcode followed by phylogenetic analysis of the nucleotide sequence confirms that specimens of the species belong to the same taxonomic group while the genetic distance(14.2%)with the congeners established it to be a different species.Conclusions:The Western Ghats'being an important biodiversity hotspot and has dearth of systematic entomological surveys on sandflies.The current study tried to fill the void and also report a new sandfly species.