Difficulties barcoding in the dark: the case of crustacean stygofauna from eastern Australia

2012 ◽  
Vol 26 (6) ◽  
pp. 583 ◽  
Author(s):  
Maria G. Asmyhr ◽  
Steven J. B. Cooper
Keyword(s):  
Dna Barcoding ◽  
Assessment Tool ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Universal Primers ◽  
Sequence Comparisons ◽  
Technical Problems ◽  
Groundwater Fauna ◽  
Taxonomic Descriptions ◽  
Eastern Australia

The eastern Australian aquifers remain mostly unexplored; however, recent surveys suggest that there could be substantial levels of subterranean biodiversity hidden in these aquifers. Groundwater fauna (stygofauna) is often characterised by short-range endemism. Furthermore, high levels of cryptic species, and lack of formal taxonomic descriptions and taxonomic expertise for many of the groups demand innovative approaches for assessing subterranean biodiversity. Here we evaluate the potential of using DNA barcoding as a rapid biodiversity assessment tool for the subterranean groundwater fauna of New South Wales, Australia. We experienced low amplification success using universal and more taxon-specific primers for PCR amplification of the barcoding gene (COI) in a range of crustacean stygofauna. Sequence comparisons of the most commonly used COI universal primers in selected crustacean taxa revealed high levels of variability. Our results suggest that successful amplification of the COI region from crustacean stygofauna is not straightforward using the standard ‘universal’ primers. We propose that the development of a multiprimer (taxon specific) and multigene approach for DNA barcode analyses, using next-generation sequencing methodologies, will help to overcome many of the technical problems reported here and provide a basis for using DNA barcoding for rapid biodiversity assessments of subterranean aquatic ecosystems.

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

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.

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

Forests ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 56 ◽  
Author(s):  
Feng Wu ◽  
Mei Li ◽  
Baowen Liao ◽  
Xin Shi ◽  
Yong Xu
Keyword(s):  
Dna Barcoding ◽  
Species Identification ◽  
Dna Barcode ◽  
Phylogenetic Reconstruction ◽  
Pcr Amplification ◽  
Guangdong Province ◽  
Nuclear Ribosomal Dna ◽  
Success Rates ◽  
Mangrove Species ◽  
Identification Rate

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.

scholarly journals Research on Phylogenetic Relationship of Lotus Populations Collected in Thua Thien Hue Province, Vietnam based on the Chloroplast Genome by DNA Barcode

2021 ◽  
Author(s):  
Dang Thanh Long ◽  
Hoang Thi Kim Hong ◽  
Le Ly Thuy Tram ◽  
Nguyen Thi Quynh Trang
Keyword(s):  
Chloroplast Genome ◽  
Dna Barcoding ◽  
Phylogenetic Analyses ◽  
Dna Barcode ◽  
Haplotype Diversity ◽  
Universal Primers ◽  
Nucleotide Position ◽  
Accurate Identification ◽  
Low Levels ◽  
Relationship Of

Background: The DNA barcoding is currently an effective and widely used tool that enables rapid and accurate identification of plant species. Methods: DNA barcoding of 9 chloroplast genes (rbcL, matK, trnH-psbA, accD-psaI, ndhA, psbE-petL, Rpl32-trnL, trnW-psaJ, trnSGCU-trnGGCC) were used to provide the theoretical basis for species identification, genetic diversity analysis of lotus population collected in Thua Thien Hue province, Vietnam. Universal primers were used and sequence products were analyzed using the MEGA X program. Result: The results showed that high levels of haplotype diversity (Hd), ranging from 0.618-0.869 and low levels of nucleotide diversity (Pi), ranging from 0.180 × 10-3-3.280 × 10-3 base on a total of nine gene regions of chloroplast genome. The neutrality tests show an excess of rare nucleotide position variations in individuals’ white lotus and derived haplotypes recent expansion. While the evolution of the individuals in the pink lotus may have to decrease. The phylogenetic analyses indicated that combined sequences were not insufficient to make a difference to the DNA barcoding in the individual’s lotus of the N. nucifera species this is in the study. The standardized and accurate barcode information of lotus is provided for researchers. It lays the foundation for the conservation, evaluation, innovative utilization and protection of Nelumbonaceae germplasm resources.

scholarly journals Real-time DNA barcoding in a remote rainforest using nanopore sequencing

2017 ◽  
Author(s):  
Aaron Pomerantz ◽  
Nicolás Peñafiel ◽  
Alejandro Arteaga ◽  
Lucas Bustamante ◽  
Frank Pichardo ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Barcoding ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Field Work ◽  
Sequence Information ◽  
Nanopore Sequencing ◽  
Consensus Sequences ◽  
Oxford Nanopore ◽  
Research Facilities

AbstractAdvancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet. Here we tested the feasibility for in situ molecular analyses of endemic fauna using a portable laboratory fitting within a single backpack, in one of the world’s most imperiled biodiversity hotspots: the Ecuadorian Chocó rainforest. We utilized portable equipment, including the MinION DNA sequencer (Oxford Nanopore Technologies) and miniPCR (miniPCR), to perform DNA extraction, PCR amplification and real-time DNA barcode sequencing of reptile specimens in the field. We demonstrate that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as we generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens. In addition, we generated sequence information at Universidad Tecnológica Indoamérica in Quito for the recently re-discovered Jambato toad Atelopus ignescens, which was thought to be extinct for 28 years, a rare species of blind snake Trilepida guayaquilensis, and two undescribed species of Dipsas snakes. In this study we establish how mobile laboratories and nanopore sequencing can help to accelerate species identification in remote areas (especially for species that are difficult to diagnose based on characters of external morphology), be applied to local research facilities in developing countries, and rapidly generate information for species that are rare, endangered and undescribed, which can potentially aid in conservation efforts.

scholarly journals Identification of Common Adulterants in Walnut Beverage Based on Plant DNA Barcode Technology

2018 ◽  
Author(s):  
Qing Han ◽  
Yan Zhang ◽  
Ying Shu ◽  
Jia Chen ◽  
Yalun Zhang ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Raw Materials ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Soybean Genome ◽  
Universal Primers ◽  
Universal Primer ◽  
Plant Dna ◽  
Calculation Results ◽  
Peanut Genome

AbstractWalnut beverage is a common vegetable protein drink that is rich in proteins and has a wide consumption market. In this study, a plant DNA barcode technology was used to establish a method to identify common adulterated ingredients (peanut and soybean) in walnut beverage. In this experiment, universal primers were designed, and PCR amplification was performed. The universal primers were screened by sequencing and comparing the amplified products. Results showed that the primers rbcL-4 and matK-4 amplified walnut, peanut, sesame, soybean, and hazelnut. Peanut genomic DNA and soybean genomic DNA were added to the genomic DNA of walnut in different proportions. Primer rbcL-4 can detect 10% peanut genome DNA, and primer matK-4 can detect 10% soybean genome DNA. Calculation results of the extraction rate revealed that primer rbcL-4 can detect 8.88% peanut raw materials and primer matK-4 can detect 2.30% soybean raw materials. The combination of these two primers can be used as a universal primer for the identification of adulterated components in walnut beverage. This experiment could serve as a theoretical reference for related research and detection.

scholarly journals Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Pathogenicity Test ◽  
Kashmir Valley ◽  
Fusarium Equiseti ◽  
Infected Root ◽  
Fusarium Sp ◽  
High Degree

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

scholarly journals Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chayapol Tungphatthong ◽  
Santhosh Kumar J. Urumarudappa ◽  
Supita Awachai ◽  
Thongchai Sooksawate ◽  
Suchada Sukrong
Keyword(s):  
High Resolution ◽  
Dna Barcoding ◽  
High Resolution Melting ◽  
Dna Barcode ◽  
Herbal Medicines ◽  
Efficient Tool ◽  
Hrm Analysis ◽  
Mitragyna Speciosa ◽  
Leaf Powder

AbstractMitragyna speciosa (Korth.) Havil. [MS], or “kratom” in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.

Research Paper DNA barcode identification of fish products from Guiyang markets in southwest China

2022 ◽  
Author(s):  
Qian Tang ◽  
Qi Luo ◽  
Qian Duan ◽  
Lei Deng ◽  
Renyi Zhang
Keyword(s):  
Dna Barcoding ◽  
Molecular Identification ◽  
Fish Consumption ◽  
Southwest China ◽  
Dna Barcode ◽  
Guizhou Province ◽  
Accurate Identification ◽  
Continuous Growth ◽  
Fish Products ◽  
Fresh Frozen

Nowadays, the global fish consumption continues to rise along with the continuous growth of the population, which has led to the dilemma of overfishing of fishery resources. Especially high-value fish that are overfished are often replaced by other fish. Therefore, the accurate identification of fish products in the market is a problem worthy of attention. In this study, full-DNA barcoding (FDB) and mini-DNA barcoding (MDB) used to detect the fraud of fish products in Guiyang, Guizhou province in China. The molecular identification results showed that 39 of the 191 samples were not consistent with the labels. The mislabelling of fish products for fresh, frozen, cooked and canned were 11.70%, 20.00%, 34.09% and 50.00%, respectively. The average kimura 2 parameter distances of MDB within species and genera were 0.27% and 5.41%, respectively; while average distances of FDB were 0.17% within species and 6.17% within genera. In this study, commercial fraud is noticeable, most of the high-priced fish were replaced of low-priced fish with a similar feature. Our study indicated that DNA barcoding is a valid tool for the identification of fish products and that it allows an idea of conservation and monitoring efforts, while confirming the MDB as a reliable tool for fish products.

scholarly journals PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

2007 ◽  
Vol 32 (5) ◽  
pp. 373-380 ◽  
Author(s):  
Jorge F. Pereira ◽  
Mariana D.C. Ignacchiti ◽  
Elza F. Araújo ◽  
Sérgio H. Brommonschenkel ◽  
Júlio C.M. Cascardo ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Pathogenic Fungus ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Sequence Comparisons ◽  
Copy Numbers ◽  
A Genome ◽  
Close Relationship ◽  
Pcr Products ◽  
Broom Disease

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

scholarly journals Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1042-1048 ◽  
Author(s):  
C. L. Trout ◽  
J. B. Ristaino ◽  
M. Madritch ◽  
T. Wangsomboondee
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Pcr Amplification ◽  
Accurate Method ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Direct Pcr ◽  
Field Samples ◽  
Pcr Products ◽  
Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

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