A rapid method of non-destructive DNA extraction from individual springtails (Collembola)

Aoyama, Hiroaki; Saitoh, Seikoh; Fujii, Saori; Nagahama, Hideki; Shinzato, Naoya; Kaneko, Nobuhiro; Nakamori, Taizo

doi:10.1007/s13355-015-0340-0

Cited by 13 publications

(6 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A total of 99 and 134 unique sequences of mtCOI and mt16S were found, respectively. Of these, mtCOI sequences from nine species were already published as part of the study by Aoyama et al (2015). We found that the 454 sequencer frequently yielded multiple sequence types from a single specimen (76 and 103 specimens out of 190 total specimens sequenced for mtCOI and mt16S genes, respectively), which were considered to be unlikely as PCR or sequencing errors because they were found in both directions of reads and from multiple conspecific specimens whose PCR reactions were constructed independently.…”

Section: Collection Of Reference Sequencesmentioning

confidence: 97%

“…To construct a reference database linking metabarcoding sequences to morphological species, mtCOI and mt16S gene sequences were collected as follows: total DNA was extracted from collembolan specimens by a nondestructive method (Aoyama et al 2015). After DNA extraction, the specimens were mounted on glass slides and identified to the species level based on morphological traits observed under a microscope according to the literature (Suma 2009;Tanaka 2010;Niijima and Hasegawa 2011;Hasegawa and Niijima 2012;Ichisawa 2012;Itoh et al 2012;Hasegawa and Tanaka 2013;Furuno et al 2014;Nakamori et al 2014).…”

Section: Collection Of Reference Sequencesmentioning

confidence: 99%

See 1 more Smart Citation

A quantitative protocol for DNA metabarcoding of springtails (Collembola)

Saitoh

Aoyama

Fujii

et al. 2016

Genome

Self Cite

View full text Add to dashboard Cite

Abstract:We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.Key words: metabarcoding, Collembola, 16S, COX1, quantification. Résumé :Les auteurs ont mis au point un protocole pour le métacodage à barres de l'ADN chez les collemboles, un ordre important parmi les micro-arthropodes du sol. Des amorces PCR dégénérées ont été conçues pour les gènes codant pour la sous-unité I de la cytochrome c oxydase mitochondriale (mtCOI) et pour l'ARN ribosomique 16S mitochondrial (mt16S) sur la base des génomes mitochondriaux déjà séquencés chez les collemboles. La meilleure paire d'amorces a été choisie sur la base de sa capacité à amplifier chaque gène, sans égard à l'espèce.

show abstract

Section: Collection Of Reference Sequencesmentioning

confidence: 97%

Section: Collection Of Reference Sequencesmentioning

confidence: 99%

A quantitative protocol for DNA metabarcoding of springtails (Collembola)

Saitoh

Aoyama

Fujii

et al. 2016

Genome

Self Cite

View full text Add to dashboard Cite

show abstract

“…Longer heating steps are present in all approaches with digestion steps for non-destructive DNA isolation [ 11 , 15 , 18 , 19 , 21 , 22 , 35 , 36 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. Longer or repetitive heating steps are also used in direct PCR methods [ 13 , 37 , 41 ], the HotSHOT method [ 20 , 41 ], the Chelex method [ 35 , 43 ], the TE-boiling method [ 50 ], and the TNES method [ 51 ] for arthropods.…”

Section: Discussionmentioning

confidence: 99%

A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of Schizophora

Stein

Wagner

Bräsicke

et al. 2022

Insects

View full text Add to dashboard Cite

While the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new—and the modification of existing—non-destructive approaches with the avoidance of further DNA template cleaning.

show abstract

“…Partial regions of the mitochondrial cytochrome c oxidase subunit 1 (CO1; 658 bp) and 16S ribosomal RNA (16S; 445 bp) genes were selected as primary and supporting DNA barcoding markers, respectively. The supporting marker previously used for Japanese Collembola (Aoyama et al, 2015;Saitoh et al, 2016;Babenko & Nakamori, 2017;Potapov et al 2017;Nakamori et al, 2020;Ohira et al, under review) was included in this study for cases for which that the primary marker was not available. To study a specimen both morphologically and molecularly, DNA was extracted as described in Aoyama et al (2015), with complete specimens being boiled in buffer.…”

Section: Methods Of Molecular Analysismentioning

confidence: 99%

“…The supporting marker previously used for Japanese Collembola (Aoyama et al, 2015;Saitoh et al, 2016;Babenko & Nakamori, 2017;Potapov et al 2017;Nakamori et al, 2020;Ohira et al, under review) was included in this study for cases for which that the primary marker was not available. To study a specimen both morphologically and molecularly, DNA was extracted as described in Aoyama et al (2015), with complete specimens being boiled in buffer. The DNA barcode sequence fragments were amplified using PCR and the DNA sequences of the fragments were determined by a direct Sanger sequencing.…”

Section: Methods Of Molecular Analysismentioning

confidence: 99%

The genus Ceratrimeria Bӧrner, 1906 (Collembola: Neanuridae, Pseudachorutinae) in the fauna of Japan

Babenko¹,

Nakamori²,

Ohira³

et al. 2021

Far East. entomol.

View full text Add to dashboard Cite

Two Japanese species, Ceratrimeria takaoensis (Kinoshita, 1916) and C. yasumatsui (Uchida, 1940), were redescribed based on fresh material, including that from the type localities and using modern morphological criteria. The main diagnostic characters of the genus Ceratrimeria are also discussed. In addition, nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 and 16S ribosomal RNA genes of specimens obtained from the type localities of both congeners are analyzed allowing for their species statuses to be confirmed. Barcoded specimens are deposited in the National Museum of Nature and Science, Tsukuba, Japan and the DNA sequence data are available in the International Nucleotide Sequence Databases.

show abstract

A rapid method of non-destructive DNA extraction from individual springtails (Collembola)

Cited by 13 publications

References 21 publications

A quantitative protocol for DNA metabarcoding of springtails (Collembola)

A quantitative protocol for DNA metabarcoding of springtails (Collembola)

A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of Schizophora

The genus Ceratrimeria Bӧrner, 1906 (Collembola: Neanuridae, Pseudachorutinae) in the fauna of Japan

Contact Info

Product

Resources

About