Barcode UK: A complete DNA barcoding resource for the flowering plants and conifers of the United Kingdom

Jones, Laura; Twyford, Alex D.; Ford, Col R.; Rich, Tim C. G.; Davies, Helena; Forrest, Laura L.; Hart, Michelle; McHaffie, Heather; Mr, Brown; Hollingsworth, Peter

doi:10.1111/1755-0998.13388

Cited by 51 publications

(57 citation statements)

References 51 publications

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“…The past has shown that ITS-based studies have struggled with amplification success (Braukmann et al 2017;Gill et al 2019;Jones et al 2021). However, this work eliminates the most pressing issues, namely the lack of PCR optimisations and the lack of a comprehensive primer evaluation.…”

Section: Discussionmentioning

confidence: 99%

“…Aside from marker choice, primer choice has repeatedly been identified as one of the key factors to facilitate accurate recovery of taxa in a sample (Krehenwinkel et al 2017;Elbrecht et al 2019;Hajibabaei et al 2019;Kelly et al 2019;Piñol et al 2019;Li et al 2020). However, despite the availability of multiple ITS primer sets (White et al 1990;Gu et al 2013;Cheng et al 2016;Moorhouse-Gann et al 2018;Tremblay et al 2019), we have identified persistent problems with the amplification success of ITS in multiple recent large-scale barcoding studies (Braukmann et al 2017;Gill et al 2019;Jones et al 2021). The lack of a side-by-side evaluation of current ITS primers makes it impossible to identify and solve the underlying issues of low ITS amplification success rates, compared to other barcode markers.…”

Section: Introductionmentioning

confidence: 99%

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Internal transcribed spacer primer evaluation for vascular plant metabarcoding

Kolter¹,

Gemeinholzer²

2021

MBMG

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The unprecedented ongoing biodiversity decline necessitates scalable means of monitoring in order to fully understand the underlying causes. DNA metabarcoding has the potential to provide a powerful tool for accurate and rapid biodiversity monitoring. Unfortunately, in many cases, a lack of universal standards undermines the widespread application of metabarcoding. One of the most important considerations in metabarcoding of plants, aside from selecting a potent barcode marker, is primer choice. Our study evaluates published ITS primers in silico and in vitro, through mock communities and presents newly designed primers. We were able to show that a large proportion of previously available ITS primers have unfavourable attributes. Our combined results support the recommendation of the introduced primers ITS-3p62plF1 and ITS-4unR1 as the best current universal plant specific ITS2 primer combination. We also found that PCR optimisation, such as the addition of 5% DMSO, is essential to obtain meaningful results in ITS2 metabarcoding. Finally, we conclude that continuous quality assurance is indispensable for reliable metabarcoding results.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Internal transcribed spacer primer evaluation for vascular plant metabarcoding

Kolter¹,

Gemeinholzer²

2021

MBMG

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“…Compiling a barcode library specific to the study would also be advantageous. While work by (Jones et al, 2021) has resulted in increasing the number of ITS2 sequences within the DNA barcode database by 1105 species, the utilisation of a custom reference database may allow all future research to be conducted at the species level.…”

Section: Discussionmentioning

confidence: 99%

Using multi-marker DNA metabarcoding to reveal the diet of a scarce woodland bird

Stenhouse

Bellamy

Kirby

et al. 2021

Preprint

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Understanding the role diet plays in the structure of food webs is vital, and dietary knowledge is key for conservation management success. There is limited knowledge of the diets of woodland bird species, due largely to difficulties in accurately identifying plant and invertebrate taxa being consumed. Here, we show the effectiveness of multi-marker faecal metabarcoding to provide the most in-depth dietary analysis of a generalist passerine, the Hawfinch (Coccothraustes coccothraustes, Linnaeus), to date. Faecal samples were obtained from 2016-2019 from Hawfinch populations prior to and during the breeding season throughout the UK. DNA was extracted from 263 samples and amplified using Internal Transcribed Spacer 2 (ITS2) and cytochrome C oxidase subunit I (COI) barcodes. Using high-throughput sequencing (HTS), we identified 49 and 97 ITS2 and COI zero radius operational taxonomic units (zOTUs) respectively which equated to reputed dietary items. The herbivorous element of Hawfinch diet was dominated by naturally occurring taxa such as beech (Fagus sylvatica, Linnaeus), hornbeam (Carpinus betulus, Linnaeus) and oak (Quercus sp., Linnaeus). The most taxon rich and commonly recorded invertebrate taxon identified was Lepidoptera. We found Hawfinch diet varied spatially, as well as between sexes. Hawfinch showed broad dietary plasticity and utilised multiple resources within their foraging environments. Our study shows the potential of multi-marker DNA metabarcoding to reveal subtle dietary differences, but also highlights the challenges of studying omnivorous species using metabarcoding methods.

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“…The barcode gene region(s) must be suitable for differentiating the taxa in the study system to the taxonomic level required and have primers universal enough to amplify all taxa. Recent research has shown a combination of rbcL and ITS2 to work better than other combinations for detecting the highest number of species at the lowest level of taxonomic discrimination (Jones, Twyford, et al, 2021). Several studies have worked towards developing more universal primers for ITS2 (e.g., Kolter & Gemeinholzer, 2021;Moorhouse-Gann et al, 2018) and primers to amplify shorter regions to account for pollen degradation in historical samples (Simanonok et al, 2021).…”

Section: Monitoring Impacts On Human Healthmentioning

confidence: 99%

“…Compared to morphology-based identifications, DNA-based identifications of pollen provide several advantages: there are more people with the required laboratory and bioinformatics expertise; greater taxonomic resolution can be obtained (Lau et al, 2019); there is potential for higher throughput (Sickel et al, 2015); and it is possible to identify the entire pollen composition of a sample (e.g., pollen load carried by an individual pollinator), rather than being limited to a subsample that is tractable by microscopic identification. DNA metabarcoding still has some challenges, mainly in respect to assigning abundances (Bell et al, 2019) and species resolution, which depends on the gene region used for metabarcoding and the quality and completeness of the relevant reference database (Jones, Twyford, et al, 2021). In most cases, however, pollen DNA metabarcoding resolves taxonomy equally well or better than traditional morphological based methods (Keller et al, 2015;Leontidou et al, 2018;Macgregor et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Pollen DNA metabarcoding and related methods in global change ecology: prospects, challenges, and progress

Bell

Turo

Lowe

et al. 2022

Preprint

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Anthropogenic activities are leading to changes in the environment at global scales, and understanding these changes requires rapid, high-throughput methods of assessment. Pollen DNA metabarcoding and related methods provide advantages in throughput and efficiency over traditional methods, such as microscopic identification of pollen and visual observation of plant-pollinator interactions. Pollen DNA metabarcoding is currently being applied to assessments of plant-pollinator interactions and their responses to land-use change such as increased agricultural intensity and urbanisation, surveillance of ecosystem change, and monitoring of spatiotemporal distribution of allergenic pollen. In combination with historical specimens, pollen DNA metabarcoding can compare contemporary and past ecosystems. Current technical challenges with pollen DNA metabarcoding include the need to understand the relationship between sequence read and species abundance, develop methods for determining confidence limits for detection and taxonomic classification, increase method standardisation, and improve of gaps in reference databases. Future research expanding the method to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens could open further avenues for research. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for assessing these changes and providing timely management recommendations to preserve biodiversity and the evolutionary and ecological processes that support it.

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Barcode UK: A complete DNA barcoding resource for the flowering plants and conifers of the United Kingdom

Cited by 51 publications

References 51 publications

Internal transcribed spacer primer evaluation for vascular plant metabarcoding

Internal transcribed spacer primer evaluation for vascular plant metabarcoding

Using multi-marker DNA metabarcoding to reveal the diet of a scarce woodland bird

Pollen DNA metabarcoding and related methods in global change ecology: prospects, challenges, and progress

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