Development of DNA Barcode and Species-Specific Markers forHelopeltis antoniiSignoret andPachypeltis maesarum(Kirkaldy) (Heteroptera: Miridae), Pests of Cashew in India

Scutellaria L. (family Lamiaceae) includes approximately 470 species found in most parts of the world and is commonly known as skullcaps. Scutellaria L. is a medicinal herb used as a folk remedy in Korea and East Asia, but it is difficult to identify and classify various subspecies by morphological methods. Since Scutellaria L. has not been studied genetically, to expand the knowledge of species in the genus Scutellaria L., de novo whole-genome assembly was performed in Scutellaria indica var. tsusimensis (H. Hara) Ohwi using the Illumina sequencing platform. We aimed to develop a molecular method that could be used to classify S.indica var. tsusimensis (H. Hara) Ohwi, S. indica L. and three other Scutellaria L. species. The assembly results for S.indica var. tsusimensis (H. Hara) Ohwi revealed a genome size of 318,741,328 bp and a scaffold N50 of 78,430. The assembly contained 92.08% of the conserved BUSCO core gene set and was estimated to cover 94.65% of the genome. The obtained genes were compared with previously registered Scutellaria nucleotide sequences and similar regions using the NCBI BLAST service, and a total of 279 similar nucleotide sequences were detected. By selecting the 279 similar nucleotide sequences and nine chloroplast DNA barcode genes, primers were prepared so that the size of the PCR product was 100 to 1000 bp. As a result, a species-specific primer set capable of distinguishing five species of Scutellaria L. was developed.

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Character-based DNA barcoding for authentication and conservation of IUCN Red listed threatened species of genus Decalepis (Apocynaceae)

Scientific Reports ◽

10.1038/s41598-017-14887-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 8

Author(s):

Priyanka Mishra ◽

Amit Kumar ◽

Gokul Sivaraman ◽

Ashutosh K. Shukla ◽

Ravikumar Kaliamoorthy ◽

...

Keyword(s):

Dna Barcode ◽

Market Demand ◽

Coding Region ◽

Hemidesmus Indicus ◽

Decalepis Hamiltonii ◽

Tuberous Roots ◽

Wild Harvesting ◽

Species Specific ◽

Database Platform ◽

Direct Use

Abstract The steno-endemic species of genus Decalepis are highly threatened by destructive wild harvesting. The medicinally important fleshy tuberous roots of Decalepis hamiltonii are traded as substitute, to meet the international market demand of Hemidesmus indicus. In addition, the tuberous roots of all three species of Decalepis possess similar exudates and texture, which challenges the ability of conventional techniques alone to perform accurate species authentication. This study was undertaken to generate DNA barcodes that could be utilized in monitoring and curtailing the illegal trade of these endangered species. The DNA barcode reference library was developed in BOLD database platform for candidate barcodes rbcL, matK, psbA-trnH, ITS and ITS2. The average intra-specific variations (0–0.27%) were less than the distance to nearest neighbour (0.4–11.67%) with matK and ITS. Anchoring the coding region rbcL in multigene tiered approach, the combination rbcL + matK + ITS yielded 100% species resolution, using the least number of loci combinations either with PAUP or BLOG methods to support a character-based approach. Species-specific SNP position (230 bp) in the matK region that is characteristic of D. hamiltonii could be used to design specific assays, enhancing its applicability for direct use in CITES enforcement for distinguishing it from H. indicus.

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Trace element concentrations in seaweeds of the Arabian Gulf identified by morphology and DNA barcodes

Botanica Marina ◽

10.1515/bot-2021-0027 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Hanan Al-Adilah ◽

Dhia Al-Bader ◽

Mohammed Elkotb ◽

Ioanna Kosma ◽

Puja Kumari ◽

...

Keyword(s):

Trace Element ◽

Inductively Coupled Plasma ◽

Arabian Gulf ◽

Dna Barcode ◽

Principal Component ◽

Extreme Environment ◽

Inductively Coupled ◽

The World ◽

Food And Feed ◽

Species Specific

Abstract Even though seaweeds have been considered a nutrient-rich dietary source of minerals in other parts of the world, there is little knowledge about trace element accumulation in seaweeds of the Arabian Gulf. The Arabian Gulf is of particular interest due to being an extreme environment, as it features some of the highest temperatures and salinities observed in any marine waters in the world. This study determined the minerals contents using inductively-coupled plasma-mass spectrometry (ICP-MS) in 10 of the most common seaweeds of this region (Iyengaria stellata, Padina boergesenii, Chondria sp., Feldmannia indica, Codium papillatum, Sargassum aquifolium, Ulva chaugulii, Ulva tepida and Ulva sp.) supported by morphological and molecular (DNA barcode)-based identification. The finding of U. chaugulii reported here is a new record both for Kuwait and the Arabian Gulf. Most of the seaweeds were rich in essential minerals including Ca, Mg, Na, K, Fe and Zn and their contents were higher than those of other mineral-rich foods. Principal component analysis revealed species-specific distributions of minerals in seaweeds. U. tepida and I. stellata were found to be exceptionally rich in most of the macro- and trace elements along with low As and Se, and thus can be utilized for food and feed applications.

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New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes

Insects ◽

10.3390/insects10120447 ◽

2019 ◽

Vol 10 (12) ◽

pp. 447 ◽

Cited By ~ 1

Author(s):

Linyu Zheng ◽

Yue Zhang ◽

Wenzhao Yang ◽

Yiying Zeng ◽

Fan Jiang ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Dna Barcode ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Specific Primers ◽

Oxidase Subunit ◽

Preimaginal Stages ◽

Specific Fragment ◽

Species Specific

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

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Application of eDNA method in the detection of Cordulegaster (Insecta: Odonata) species

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65041 ◽

2021 ◽

Vol 4 ◽

Author(s):

Judit Fekete ◽

Dominik Buchner ◽

Florian Leese ◽

Judit Padisák ◽

Gábor Várbíró

Keyword(s):

Pilot Study ◽

Dna Barcode ◽

Illumina Miseq ◽

Iucn Red List ◽

Universal Primers ◽

Specific Primers ◽

Tissue Samples ◽

Difficult Time ◽

Species Specific ◽

Dragonfly Species

The aim of this pilot study was to investigate the potential of eDNA techniques to detect the presence of the two dragonfly species Cordulegaster heros and Cordulegaster bidentata. Both species are classified as “near threatened” according to the IUCN Red List and are strictly protected in several countries. Monitoring these species with traditional sampling methods is often difficult, time-consuming and invasive. In this pilot study, we first collected tissue samples from C. heros and C. bidentata to sequence the traditional DNA-barcode gene fragment COI. We then collected further dragonfly COI sequences from BOLD to design species-specific primers. This, however, was impossible given the enormous variability of COI. Therefore, we refrained from species-specific eDNA assays and followed eDNA metabarcoding protocol using universal (BF2/BF2) and a newly designed dragonfly specific primer. For the evaluation of the method, we took water samples from places where Cordulegaster specimens are known to occur. After the extraction, we used two sequential PCR steps for obtaining the desired amplicon (two-step PCR) using universal primers in the first step, and group (dragonfly) specific primers or universal primers. Amplicons were sequenced on an Illumina MiSeq platform and then analysed the data with the JAMP pipeline. With the newly designed primers and we could effectively detect the targeted dragonfly species from tissue samples, and also from filtered environmental samples. The detection of the species with the traditional method is time consuming and involves the destruction of the specimens. In comparison, with the eDNA method we could easily detect these near threatherned odonates and other dragonfly species in a non-invasive way.

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Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid Identification of Dendrobium officinale

10.20944/preprints201810.0404.v1 ◽

2018 ◽

Author(s):

Lu Yang ◽

Hua Zhou ◽

Huili Lai ◽

Fei Fu ◽

Wenru Wu

Keyword(s):

Color Change ◽

Dna Barcode ◽

Its Region ◽

Rapid Identification ◽

Isothermal Amplification ◽

Dendrobium Officinale ◽

Lamp Method ◽

Loop Mediated Isothermal Amplification ◽

Specificity And Sensitivity ◽

Species Specific

Background: Dendrobium officinale is not only an ornamental plant, but also a valuable medicinal herb that is both effective and widely used in traditional Chinese medicine. However, distinguishing D. officinale from other Dendrobium species is usually a difficult task that need much time and complex technologies due to their very similar external morphologies. The aim of this study is to develop a fast, even on-spot approach to identify D. officinale. Methods: We used DNA barcode-based loop-mediated isothermal amplification (LAMP) method with species-specific LAMP primers targeting the internal transcribed spacer (ITS) region of the rDNA of D. officinale. LAMP reaction time and temperature were optimized and the specificity and sensitivity of LAMP species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of D. officinale and allowed for rapid amplification (within 40 min) of the ITS region under a constant and mild temperature range of 65 °C without using thermocyclers. Besides, by using SYBR® Green I dye as the color developing agent, the color change was easily observed with naked eye. Reaction mixture containing DNA of D. officinale changed from orange to green, while the other Dendrobium species and the negative control retained original orange color. The specificity of this LAMP-based method was confirmed by testing 17 samples of D. officinale and 32 adulterant samples from other Dendrobium species. Conclusions: This LAMP-based rapid identification method does not require expensive equipment or specialized techniques and can be used in field surveys for accurate and fast on site identification.

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A Rapid Colorimetric Assay for On-Site Authentication of Cephalopod Species

Biosensors ◽

10.3390/bios10120190 ◽

2020 ◽

Vol 10 (12) ◽

pp. 190

Author(s):

Giuseppina Tatulli ◽

Paola Cecere ◽

Davide Maggioni ◽

Andrea Galimberti ◽

Pier Paolo Pompa

Keyword(s):

Dna Barcoding ◽

Genomic Dna ◽

Large Scale ◽

Colorimetric Assay ◽

Dna Barcode ◽

Mitochondrial Coi ◽

Cephalopod Species ◽

Accurate Design ◽

Relevant Group ◽

Species Specific

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5’ end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.

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Does GenBank provide a reliable DNA barcode reference to identify small alien oysters invading the Mediterranean Sea?

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315414001027 ◽

2014 ◽

Vol 95 (1) ◽

pp. 111-122 ◽

Cited By ~ 12

Author(s):

F. Crocetta ◽

P. Mariottini ◽

D. Salvi ◽

M. Oliverio

Keyword(s):

Mediterranean Sea ◽

Native Species ◽

Dna Barcode ◽

Genetic Data ◽

Molecular Data ◽

The Public ◽

Variable Species ◽

The Mediterranean ◽

Species Specific

The Mediterranean Sea is currently under siege by a conspicuous alien pressure, and, within some families (e.g. the Ostreidae), the number of native species seems to be remarkably outnumbered by that of the alien ones. We wanted to test the reliability of the molecular data currently available on the small alien oysters recently invading the Mediterranean Sea. Samples from Greece and Turkey, encompassing the known species-specific morphological variation, were sequenced for the markers with the widest taxonomic coverage in the group of small oysters (i.e. the 16S rDNA and the COI). The sequences obtained have been compared with those available in GenBank, and a possible identification at the species level has been finally tested in a DNA-barcoding fashion. The present results clearly demonstrated that our samples belong to a single, morphologically highly variable species. Their 16S sequences were closely related to a sequence registered under the name Dendostrea folium, with a genetic distance which does not warrant conspecificity. Additionally, a remarkable number of sequences retrieved from the GenBank (of both genes) did not form a monophyletic group according to the published classification of the vouchers, suggesting—at least in part—an origin from specimens not properly identified. Both genes seem promising for use as DNA-barcode, although the COI will probably prove more effective. Therefore, we urge the availability of a baseline of oyster pedigreed DNA barcode sequences in the public databases, to allow the use of such genetic data to reliably monitor bio-invasions in the Mediterranean Sea.

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Developing an efficient DNA barcoding system to differentiate between Lilium species

BMC Plant Biology ◽

10.1186/s12870-021-03229-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yixin Liu ◽

Mingfang Zhang ◽

Xuqing Chen ◽

Xi Chen ◽

Yue Hu ◽

...

Keyword(s):

Dna Barcoding ◽

Dna Barcode ◽

Identification System ◽

Representative Species ◽

Lilium Species ◽

Edible Species ◽

Medicinal Properties ◽

Species Specific ◽

Genomic Regions

Abstract Background Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. Results To develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). Conclusions A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

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