scholarly journals Development of novel PCR primer sets for DNA metabarcoding of aquatic insects, and the discovery of some cryptic species

2021 ◽  
Author(s):  
Masaki Takenaka ◽  
Koki Yano ◽  
Tomoya Suzuki ◽  
Koji Tojo
Keyword(s):  
Cryptic Species ◽  
Dna Barcoding ◽  
Environmental Science ◽  
Pcr Primers ◽  
12S Rrna ◽  
Universal Primer ◽  
Data Registration ◽  
Dna Database ◽  
Dna Metabarcoding ◽  
Primer Sets

DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification by using standardized genetic region(s). It can be a powerful tool in various fields of biology such as for revealing the existence of cryptic species and/or rare species and in environmental science such as when monitoring river biota. Biodiversity reduction in recent times has become one of the most serious environmental issues on a worldwide scale. DNA barcoding techniques require the development of sets of universal PCR primers for DNA metabarcoding. We tried to develop universal primer sets for the DNA barcoding of all insect groups. In this study, we succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region. A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. Such resulting database enhancements will serve as a powerful tool for increasingly accurate assessment of biodiversity and genetic diversity.

scholarly journals Ichthyoplankton metabarcoding as a tool for studying fish reproductive dynamics

2021 ◽  
Vol 4 ◽  
Author(s):  
Daniel Teixeira ◽  
Heron Hilário ◽  
Gustavo Rosa ◽  
Guilherme Santos ◽  
Gilmar Santos ◽  
...  
Keyword(s):  
Fish Assemblages ◽  
Fish Larvae ◽  
Reference Sequence ◽  
12S Rrna ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Fish Eggs ◽  
Migration Routes ◽  
Dna Metabarcoding ◽  
Primer Sets

The study of ichthyoplankton composition, abundance and distribution is paramount to understand the reproductive dynamics of local fish assemblages. The analysis of these parameters allows the identification of spawning sites, nursery areas and migration routes. However, due to the lack of characters in early life stages, the morphological identification of ichthyoplankton is often impractical and many studies identify only fish larvae. Additionally, its accuracy shows great variation between taxonomists and laboratories according to their experience and specialty. DNA barcoding emerged as an alternative to provide assertive identification of fish eggs and larvae, but it becomes too expensive and laborious when the study demands the processing of huge amounts of organisms. DNA metabarcoding can overcome these limitations as a rapid, cost-effective, broad and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. Here, we present the identification of a sample containing 68 fish eggs and another containing 293 fish larvae from a single site in the São Francisco River Basin, Eastern Brazil, through DNA metabarcoding. We used a low-cost saline DNA extraction followed by PCR amplification with three primer sets targeting the 12S rRNA gene: MiFish (~170bp), Teleo_1 (~60bp), and NeoFish (~190bp). The latter was recently developed by our research group specifically for the identification of Neotropical fishes. All the amplified samples were sequenced in a single multiplexed Illumina MiniSeq run. We performed the filtering steps and assigned Amplicon Sequence Variants (ASVs) using a DADA2/Phyloseq based pipeline and a custom 12S reference sequence database including 101 species and 70 genera from the Jequitinhonha and São Francisco basins. The species Cyphocharax gilbert, Leporinus taeniatus, Megaleporinus elongatus, Prochilodus argenteus, P. costatus and Psalidodon fasciatus were detected by all three primer sets in the larva pool, while Pterygoplichthys etentaculatus was detected solely by NeoFish (Fig. 1). Within the egg pool, all three markers detected the species Characidium zebra, Curimatella lepidura, M. elongatus, Pimelodus fur and P. costatus, but Brycon orthotaenia was detected only by NeoFish, P. maculatus only by Teleo, and P. pohli by MiFish and Teleo (Fig. 1). The consistency in species detection among all three markers underpins the credibility of this method to accurately describe the sample composition. Considering that most of species were exclusive to the larvae or egg pool, our experiment highlights the importance of including the identification of fish eggs in reproduction studies, as it can provide additional information about which species are spawning in an area. Furthermore, the application of DNA metabarcoding to the study of ichthyoplankton can help decision makers create more informed guidelines for conservation of economically and ecologically important fish species.

scholarly journals Pengujian Dua Pasang Primer Universal Untuk Amplifikasi Gen Cytochrome Oxidase I Copepoda

Jurnal MIPA ◽  
2015 ◽  
Vol 4 (1) ◽  
pp. 93
Author(s):  
Thalita C. P. Sumampow
Keyword(s):  
Cytochrome Oxidase ◽  
Cryptic Species ◽  
Dna Barcoding ◽  
Cytochrome Oxidase I ◽  
Coi Gene ◽  
Universal Primer ◽  
Cytochrome Oxidase I Gene ◽  
I Gene

Copepoda merupakan zooplankton kaya manfaat dengan diversitas yang sangat tinggi dan terdiri dari banyak spesies kriptik. Identifikasi cepat, akurat, dan hemat dapat dilakukan dengan menggunakan teknik DNA Barcoding. Kesuksesan teknik tersebut sangat dipengaruhi oleh penggunaan primer yang tepat. Tujuan penelitian ini adalah untuk menguji kemampuan dua pasang primer universal, yakni LCO1490-HCO2198 dan FF2d-FR1d, mengamplifikasi gen COI Copepoda. Dalam penelitian ini, pasangan primer LCO1490-HCO2198 tidak berhasil mengamplifikasi gen target. Sekuens-sekuens hasil amplifikasi menggunakan pasangan primer FF2d-FR1d diidentifikasi melalui BLAST. Hasil yang diperoleh menunjukan bahwa sekuens-sekuens tersebut memiliki persentase kemiripan sebesar 92% dengan bakteri Pandoraea pnomenusa. Melalui hasil yang didapatkan disimpulkan bahwa kedua pasangan primer universal LCO1490-HCO2198 dan FF2d-FR1d tidak cukup spesifik untuk amplifikasi gen cytochrome oxidase I Copepoda.Copepoda is a very beneficial and highly diverse zooplankton with many cryptic species. A fast, reliable, and affordable identification can be done through DNA Barcoding. The success of this technique is affected by the usage of correct primers. The aim of this research was to test the ability of two universal primer pairs, which were LCO1490-HCO2198 and FF2d-FR1d, amplifying COI gene of Copepoda. In this research, LCO1490-HCO2198 primer pairs weren’t able to amplify COI gene of Copepoda. Sequences which were successfully amplified using FF2d-FR1d primer pairs were identified through BLAST. The result shows that the sequences are 92% similar to bacteria named Pandoraea pnomenusa. It can be concluded that both primer pairs are not specific enough to amplify cytochrome oxidase I gene of Copepoda.

scholarly journals Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

2021 ◽  
Author(s):  
Masayuki K. Sakata ◽  
Mone U. Kawata ◽  
Atsushi Kurabayashi ◽  
Takaki Kurita ◽  
Masatoshi Nakamura ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Environmental Dna ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Rrna Gene ◽  
Biodiversity Monitoring ◽  
Natural Ecosystems ◽  
Universal Primer ◽  
Primer Sets

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

scholarly journals Benchmarking DNA barcodes: an assessment using available primate sequences

Genome ◽  
2006 ◽  
Vol 49 (7) ◽  
pp. 851-854 ◽  
Author(s):  
Mehrdad Hajibabaei ◽  
Gregory AC Singer ◽  
Donal A Hickey
Keyword(s):  
New Species ◽  
Cryptic Species ◽  
Dna Barcoding ◽  
Species Identification ◽  
Dna Sequences ◽  
Phylogenetic Relationships ◽  
Primate Species ◽  
Dna Barcodes ◽  
Global Biodiversity ◽  
Efficient Sequence

DNA barcoding has been recently promoted as a method for both assigning specimens to known species and for discovering new and cryptic species. Here we test both the potential and the limitations of DNA barcodes by analysing a group of well-studied organisms—the primates. Our results show that DNA barcodes provide enough information to efficiently identify and delineate primate species, but that they cannot reliably uncover many of the deeper phylogenetic relationships. Our conclusion is that these short DNA sequences do not contain enough information to build reliable molecular phylogenies or define new species, but that they can provide efficient sequence tags for assigning unknown specimens to known species. As such, DNA barcoding provides enormous potential for use in global biodiversity studies.Key words: DNA barcoding, species identification, primate, biodiversity.

scholarly journals Reassessment of the mitochondrial 12S-rRNA gene for DNA barcoding of museum specimens of shelled marine gastropods from Japan

2021 ◽  
Vol 322 ◽  
pp. 01028
Author(s):  
Nao Fukunaga ◽  
Moe Shimizu ◽  
Shinnosuke Teruya ◽  
Nazifa Naziha Razali ◽  
Satoko Nakashima ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
12S Rrna ◽  
Rrna Gene ◽  
Museum Specimens ◽  
Biodiversity Monitoring ◽  
Marine Gastropods ◽  
12S Rrna Gene ◽  
Reference Databases ◽  
Mitochondrial 12S Rrna ◽  
Comprehensive Reference

DNA barcoding is an effective and powerful tool for taxonomic identification and thus very useful for biodiversity monitoring. This study investigated the usefulness of the mitochondrial 12S-rRNA gene for the DNA barcoding of shelled marine gastropods. To do so, we determined partial 12S-rRNA sequences of 75 vouchered museum specimens from 69 species of shelled gastropods from Japan. The specimens have been identified morphologically, and natural history data catalog. Sequence analyses through BLAST searches, maximum likelihood phylogenetic analysis, and species delimitation analysis suggested that the 12S-rRNA gene is helpful for barcoding shelled marine gastropods. They thus could be helpful to complement barcoding studies using other markers such as COI. The analyses successfully confirmed all samples’ identity at higher taxonomy (subfamily and above), but much less so at the species level. Our result thus also underlines the lingering problem of DNA barcoding: The lack of comprehensive reference databases of sequences. However, since we provided sequences of properly curated, vouchered museum specimens in this study, our result reported here has thus also helped to give taxonomically reliable reference sequences for biodiversity monitoring and identifications of shelled gastropods which include many important fisheries species.

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