DNA Barcoding Analysis and Phylogenetic Relation of Mangroves in Guangdong Province, China

Mangroves are distributed in the transition zone between sea and land, mostly in tropical and subtropical areas. They provide important ecosystem services and are therefore economically valuable. DNA barcoding is a useful tool for species identification and phylogenetic reconstruction. To evaluate the effectiveness of DNA barcoding in identifying mangrove species, we sampled 135 individuals representing 23 species, 22 genera, and 17 families from Zhanjiang, Shenzhen, Huizhou, and Shantou in the Guangdong province, China. We tested the universality of four DNA barcodes, namely rbcL, matK, trnH-psbA, and the internal transcribed spacer of nuclear ribosomal DNA (ITS), and examined their efficacy for species identification and the phylogenetic reconstruction of mangroves. The success rates for PCR amplification of rbcL, matK, trnH-psbA, and ITS were 100%, 80.29% ± 8.48%, 99.38% ± 1.25%, and 97.18% ± 3.25%, respectively, and the rates of DNA sequencing were 100%, 75.04% ± 6.26%, 94.57% ± 5.06%, and 83.35% ± 4.05%, respectively. These results suggest that both rbcL and trnH–psbA are universal in mangrove species from the Guangdong province. The highest success rate for species identification was 84.48% ± 12.09% with trnH-psbA, followed by rbcL (82.16% ± 9.68%), ITS (66.48% ± 5.97%), and matK (65.09% ± 6.00%), which increased to 91.25% ± 9.78% with the addition of rbcL. Additionally, the identification rate of mangroves was not significantly different between rbcL + trnH-psbA and other random fragment combinations. In conclusion, rbcL and trnH-psbA were the most suitable DNA barcode fragments for species identification in mangrove plants. When the phylogenetic relationships were constructed with random fragment combinations, the optimal evolutionary tree with high supporting values (86.33% ± 4.16%) was established using the combination of matK + rbcL + trnH-psbA + ITS in mangroves. In total, the 476 newly acquired sequences in this study lay the foundation for a DNA barcode database of mangroves.

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DNA Barcoding Mushroom Spawn Using EF-1α Barcodes: A Case Study in Oyster Mushrooms (Pleurotus)

Frontiers in Microbiology ◽

10.3389/fmicb.2021.624347 ◽

2021 ◽

Vol 12 ◽

Author(s):

Peng Zhao ◽

Sen-Peng Ji ◽

Xian-Hao Cheng ◽

Tolgor Bau ◽

Hong-Xin Dong ◽

...

Keyword(s):

Species Identification ◽

Dna Barcode ◽

Elongation Factor ◽

Pcr Amplification ◽

Interspecific Variation ◽

28S Rdna ◽

Oyster Mushroom ◽

Success Rates ◽

Mushroom Species ◽

Oyster Mushrooms

Oyster mushrooms (genus Pleurotus) are widespread and comprise the most commonly cultivated edible mushrooms in the world. Species identification of oyster mushroom spawn based on cultural, morphological, and cultivated characteristics is time consuming and can be extraordinarily difficult, which has impeded mushroom breeding and caused economic loss for mushroom growers. To explore a precise and concise approach for species identification, the nuclear ribosomal internal transcribed spacer (ITS), 28S rDNA, and the widely used protein-coding marker translation elongation factor 1α (EF-1α) gene were evaluated as candidate DNA barcode markers to investigate their feasibility in identifying 13 oyster mushroom species. A total of 160 sequences of the candidate loci were analyzed. Intra- and interspecific divergences and the ease of nucleotide sequence acquisition were the criteria used to evaluate the candidate genes. EF-1α showed the best intra- and interspecific variation among the candidate markers and discriminated 84.6% of the species tested, only being unable to distinguish two closely related species Pleurotus citrinopileatus and Pleurotus cornucopiae. Furthermore, EF-1α was more likely to be acquired than ITS or 28S rDNA, with an 84% success rate of PCR amplification and sequencing. For ITS and 28S rDNA, the intraspecific differences of several species were distinctly larger than the interspecific differences, and the species identification efficiency of the two candidate markers was worse (61.5 and 46.2%, respectively). In addition, these markers had some sequencing problems, with 55 and 76% success rates of sequencing, respectively. Hence, we propose EF-1α as a possible DNA barcode marker for oyster mushroom spawn.

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Phylogenetic Analysis of Black Peper (Piper Spp.) Population Collected in Different Locations of Vietnam Based on the ITSU1-4 Gene Region

10.21203/rs.3.rs-844711/v1 ◽

2021 ◽

Author(s):

Sonexay Rasphone ◽

Long Thanh Dang ◽

Hoan Nguyen ◽

Ngoc Quang Nguyen ◽

Oanh Thi Duong ◽

...

Keyword(s):

Dna Barcoding ◽

Ribosomal Dna ◽

Maximum Likelihood Method ◽

Dna Barcode ◽

Population Expansion ◽

Nuclear Ribosomal Dna ◽

Likelihood Method ◽

Gene Region ◽

Universal Primers ◽

Intraspecific Distance

Abstract Background: The internal transcribed spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses, and it has been recommended as a core plant DNA barcode. To compare and find out the analysis genetic diversity difference some pepper individuals collected in different localities in Vietnam when using the ITS of nuclear ribosomal DNA. The ITS gene region from the nuclear genomes were tested for their suitability as DNA barcoding regions of thirty-nine pepper individuals. Universal primers were used, and sequenced products were analyzed using the Maximum Likelihood method and Tamura-Nei model in the MEGA X program.Results: We did not observe high variability in intraspecific distance within the ITSu1-4 gene region between individuals, ranged from 0.000 to 0.155 (mean = 0.033). The size of the gene region has fluctuated from 667 to 685 bp between different individuals with the percentage (G + C) contained in the ITSu1-4 gene region was ranged from 54.776% to 60.805%, mean = 60.174%. The values of Fu’s Fs, D, Fu and Li’s D* and F* were negative as well (Fs = -0.209, D = -1.824; P < 0.05, D* = -1.205; not significant, P > 0.10 and F* = -1.699; not significant, 0.10 > P > 0.05), indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred. The value Strobeck’s S the obtained between individuals in a population is high (S = 0.684). The results of evolutionary relationships of taxa obtained 3 groups with the highest value of Fst is shown in the pairs of groups II and III (Fst = 0.151), and the lowest is in groups II and I (Fst = 0.015). All of the new sequences have been deposited in GeneBank under the following accession numbers MZ636718 to MZ636756.Conclusions: This database is an important resource for researchers working on Species of pepper in Vietnam and also provides a tool to create ITSu1-4 databases for any given taxonomy.

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Molecular identification of species of Physalis (Solanaceae) using a candidate DNA barcode: the chloroplast psbA–trnH intergenic region

Genome ◽

10.1139/gen-2017-0115 ◽

2018 ◽

Vol 61 (1) ◽

pp. 15-20 ◽

Cited By ~ 3

Author(s):

Shangguo Feng ◽

Kaili Jiao ◽

Yujia Zhu ◽

Hongfen Wang ◽

Mengying Jiang ◽

...

Keyword(s):

Intergenic Region ◽

Morphological Traits ◽

Dna Barcode ◽

Pcr Amplification ◽

Genetic Distances ◽

Success Rates ◽

Medicinal Species ◽

Distance Methods ◽

Identification Of Species ◽

Related Plant

Physalis L., an important genus of the family Solanaceae, includes many commercially important edible and medicinal species. Traditionally, species identification is based on morphological traits; however, the highly similar morphological traits among species of Physalis make this approach difficult. In this study, we evaluated the feasibility of using a popular DNA barcode, the chloroplast psbA–trnH intergenic region, in the identification of species of Physalis. Thirty-six psbA–trnH regions of species of Physalis and of the closely related plant Nicandra physalodes were analyzed. The success rates of PCR amplification and sequencing of the psbA–trnH region were 100%. MEGA V6.0 was utilized to align the psbA–trnH sequences and to compute genetic distances. The results show an apparent barcoding gap between intra- and interspecific variations. Results of both BLAST1 and nearest-distance methods prove that the psbA–trnH regions can be used to identify all species examined in the present study. In addition, phylogenetic analysis using psbA–trnH data revealed a distinct boundary between species. It also confirmed the relationship between species of Physalis and closely related species, as established by previous studies. In conclusion, the psbA–trnH intergenic region can be used as an efficient DNA barcode for the identification of species of Physalis.

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Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

Genome ◽

10.1139/gen-2015-0205 ◽

2016 ◽

Vol 59 (12) ◽

pp. 1150-1156 ◽

Cited By ~ 4

Author(s):

Sundar Poovitha ◽

Nithaniyal Stalin ◽

Raju Balaji ◽

Madasamy Parani

Keyword(s):

Dna Barcoding ◽

Species Identification ◽

Dna Barcode ◽

Species Differentiation ◽

Molecular Method ◽

Peninsular India ◽

Plant Materials ◽

Medicinal Value ◽

Formed Part ◽

Suitable Marker

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes rbcLa and matK together with the suggested additional regions trnH-psbA and ITS2. No intraspecies divergence was observed among the accessions studied. Interspecies divergence was 0%–9.6% with individual markers, which increased to 0%–12.5% and 0.8%–20.3% when using two- and three-marker combinations, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when the matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.

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MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples

Genes ◽

10.3390/genes11040445 ◽

2020 ◽

Vol 11 (4) ◽

pp. 445 ◽

Cited By ~ 5

Author(s):

Adeline Seah ◽

Marisa C.W. Lim ◽

Denise McAloose ◽

Stefan Prost ◽

Tracie A. Seimon

Keyword(s):

Dna Barcoding ◽

Species Identification ◽

Wildlife Conservation ◽

Sequence Similarity ◽

Dna Barcode ◽

Bioinformatics Pipeline ◽

Tissue Samples ◽

Consensus Sequences ◽

Sequencing Errors ◽

Fresh Frozen

The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples. We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols. For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29%–100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies. We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification.

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Land plants and DNA barcodes: short-term and long-term goals

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2005.1720 ◽

2005 ◽

Vol 360 (1462) ◽

pp. 1889-1895 ◽

Cited By ~ 316

Author(s):

Mark W Chase ◽

Nicolas Salamin ◽

Mike Wilkinson ◽

James M Dunwell ◽

Rao Prasad Kesanakurthi ◽

...

Keyword(s):

Dna Barcoding ◽

Dna Barcode ◽

Plastid Dna ◽

Land Plant ◽

Land Plants ◽

Nuclear Ribosomal Dna ◽

Short Term ◽

Species Limits ◽

Variable Regions

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL , trnL-F , etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH ) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the ‘genetic gaps’ that are useful in assessing species limits.

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Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding

Genome ◽

10.1139/gen-2015-0225 ◽

2017 ◽

Vol 60 (2) ◽

pp. 139-146 ◽

Cited By ~ 7

Author(s):

Stalin Nithaniyal ◽

Sophie Lorraine Vassou ◽

Sundar Poovitha ◽

Balaji Raju ◽

Madasamy Parani

Keyword(s):

Medicinal Plants ◽

Medicinal Plant ◽

Alternative Medicine ◽

Dna Barcoding ◽

Species Identification ◽

Dna Barcode ◽

Regulatory Agencies ◽

Vernacular Names ◽

Identification Of Species ◽

Whole Plants

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

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Short Communication: Phylogenetic analysis and molecular identification of Canar (Smilax spp.) in Java, Indonesia Based on DNA Barcoding Analysis

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d190202 ◽

2018 ◽

Vol 19 (2) ◽

pp. 364-368

Author(s):

LULUT DWI SULISTYANINGSIH ◽

MARLINA ARDIYANI ◽

ABINAWANTO ABINAWANTO ◽

ANDI SALAMAH

Keyword(s):

Phylogenetic Analysis ◽

Dna Barcoding ◽

Molecular Identification ◽

Short Communication ◽

Pharmacological Study ◽

Dna Barcode ◽

Phylogenetic Reconstruction ◽

Steroidal Saponin ◽

Morphological Variations ◽

Molecular Approaches

Sulistyaningsih LD, Abinawanto, Ardiyani M, Salamah A. 2018. Short Communication: Phylogenetic analysis and molecular identification of Canar (Smilax spp.) in Java, Indonesia Based on DNA Barcoding Analysis. Biodiversitas 19: 364-368. Smilax spp. (Smilacaceae) has long been used as medicinal herbs especially in East Asia and North America as they were known to be rich in steroidal saponin. Pharmacological study has been carried out in Indonesia. This genus is widespread in Indonesia and fairly abundant in Java and has been known either as edible fruit or medicinal plants. Characteristics of Smilax as a dioecious plant with high morphological variations make it thorny in species identification. Various molecular approaches have been devised to overcome identification problems such as DNA barcoding. This study, therefore was conducted to analyze the DNA barcoding application for phylogenetic and identification of Smilax in Java. A total of 31 samples were used in this study including 19 accession numbers from NCBI GeneBank. The genus Ripogonum was used as the out-group in phylogenetic reconstruction. Samples were successfully extracted by CTAB method with some modifications. rbcL region was used as the DNA barcode showed sufficient variation and conserved flanks. Two unidentified specimens have high similarity with S. leucophyla and lies in the same clade. The phylogenetic tree constructed by Maximum Likelihood analysis. The result showed that the monophyletic of Smilacaceae consisted of four clades. The genus Heterosmilax nested with Smilax though with low bootstraps value. It supports the monogeneric status of Smilacaceae.

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DNA barcoding British Euphrasia reveals deeply divergent polyploids but lack of species-level resolution

10.1101/164681 ◽

2017 ◽

Author(s):

Xumei Wang ◽

Galina Gussarova ◽

Markus Ruhsam ◽

Natasha de Vere ◽

Chris Metherell ◽

...

Keyword(s):

Genetic Variation ◽

Dna Barcoding ◽

Species Identification ◽

Dna Barcode ◽

Species Level ◽

Ecological Processes ◽

Evolutionary Patterns ◽

Ploidy Levels ◽

Self Fertilization ◽

Phylogenetic Framework

ABSTRACTBackground and aimsDNA barcoding is emerging as a useful tool not only for species identification but for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode datasets can provide insights into the mechanisms underlying the generation of species diversity. Here, we use DNA barcoding to study evolutionary processes in taxonomically complex British Euphrasia, a group with multiple ploidy levels, frequent self- fertilization, young species divergence and widespread hybridisation.MethodsWe sequenced the core plant barcoding loci, supplemented with additional nuclear and plastid loci, in representatives of all 19 British Euphrasia species. We analyse these data in a population genetic and phylogenetic framework. We then date the divergence of haplotypes in a global Euphrasia dataset using a time-calibrated Bayesian approach implemented in BEAST.Key resultsNo Euphrasia species has a consistent diagnostic haplotype. Instead, haplotypes are either widespread across species, or are population specific. Nuclear genetic variation is strongly partitioned by ploidy levels, with diploid and tetraploid British Euphrasia possessing deeply divergent ITS haplotypes (DXY = 5.1%), with haplotype divergence corresponding to the late Miocene. In contrast, plastid data show no clear division by ploidy, and instead reveal weakly supported geographic patterns.ConclusionsUsing standard DNA barcoding loci for species identification in Euphrasia will be unsuccessful. However, these loci provide key insights into the maintenance of genetic variation, with divergence of diploids and tetraploids suggesting that ploidy differences act as a barrier to gene exchange in British Euphrasia, with rampant hybridisation within ploidy levels. The scarcity of shared diploid-tetraploid ITS haplotypes supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, DNA barcoding can reveal insightful evolutionary patterns in taxonomically complex genera.

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Testing three proposed DNA barcodes for the wood identification of Dalbergia odorifera T. Chen and Dalbergia tonkinensis Prain

Holzforschung ◽

10.1515/hf-2014-0234 ◽

2016 ◽

Vol 70 (2) ◽

pp. 127-136 ◽

Cited By ~ 17

Author(s):

Min Yu ◽

Kai Liu ◽

Liang Zhou ◽

Lei Zhao ◽

Shengquan Liu

Keyword(s):

Economic Value ◽

Dna Barcode ◽

Pcr Amplification ◽

First Grade ◽

Wood Identification ◽

Dna Barcodes ◽

Success Rates ◽

Interspecific Differences ◽

Dalbergia Odorifera ◽

Dalbergia Tonkinensis

Abstract Dalbergia odorifera T. Chen is a first-grade state protected plant in China. However, it is difficult to distinguish it from the closely related species Dalbergia tonkinensis Prain, which is less important in economic value, by wood anatomical features. In this study, three potential DNA barcode sequences, namely rpoC1, trnH-psbA and internal transcribed spacer (ITS), were used to differentiate wood of D. odorifera from D. tonkinensis. The average quantities of DNA extracts from twigs, sapwood and heartwood were 16.3, 11.5 and 6.0 ng mg-1, respectively. The success rates for polymerase chain reaction (PCR) amplification for three loci, namely ITS, trnH-psbA and rpoC1, were 62.5, 100 and 81.25%, respectively. The success rate for bidirectional sequencing of amplified products was 100% for all the three loci. The identification power of the three proposed DNA barcodes has been calculated by the BLAST, tree-based method and the TAXONDNA method. The interspecific differences of the trnH-psbA region were greater than intraspecific variations. Moreover, the identification power of trnH-psbA was higher than that of ITS and rpoC1 regions at the species level. Finally, the trnH-psbA region is proposed as a DNA barcode for wood identification between D. odorifera and D. tonkinensis.

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