Dna Barcodes Methods And Protocols Pdf


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Four species of Paphiopedilum were sampled and barcoded. The combination of barcode regions revealed the lack of variation in rbcL and trnH-psbA but they are still useful for preliminary identification followed up by matK for accurate identification. Chapple, D.

DNA Barcodes pp Cite as. DNA barcoding, a new method for the quick identification of any species based on extracting a DNA sequence from a tiny tissue sample of any organism, is now being applied to taxa across the tree of life. As a research tool for taxonomists, DNA barcoding assists in identification by expanding the ability to diagnose species by including all life history stages of an organism.

DNA Barcodes

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers rbcL , matK , ITS2. Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada.

Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance MPD and mean nearest taxon distance MNTD were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity.

Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.

This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Complete taxonomic information, collection records, voucher images and sequences for 17, specimens are publically available through BOLD Ratnasingham and Hebert in the plants of Canada project Available as of January 4, ; doi: dx.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. DNA barcoding employs sequence variation in short, standardized gene regions as a tool to discriminate species [ 1 ].

The ideal DNA barcode region is reliably amplified and sequenced across large assemblages of taxa and provides a high level of species discrimination [ 2 ]. Due to the extremely low rates of nucleotide substitution in mitochondrial genes in most plant lineages [ 3 ], COI was not a candidate.

However, building on their intense use for phylogenetics and molecular systematics, two plastid gene regions were considered as DNA barcodes for vascular plants and the large subunit of RuBisCo rbcL in combination with an intron maturase matK were adopted as standards [ 4 ; 5 ].

Because these regions often fail to resolve congeners [ 6 ; 7 ; 8 ; 9 ; 10 ; 11 ], there has been a subsequent trend, building on earlier suggestions [ 12 ; 13 ; 14 ], to couple them with the nuclear-encoded ribosomal internal transcribed spacer, ITS2 [ 2 ; 15 ; 16 ]. A considerable number of studies have now examined the performance of different markers with respect to both their ease of amplification and their capacity to resolve plant species [ 9 ; 10 ; 15 ; 17 ; 18 ; 19 ; 7 ; 20 ; 21 ; 22 ; 23 ; 24 ; 25 ; 26 ].

The efficacy of these gene regions in discriminating species has been determined by tree-based phylogenetic or basic local alignment BLAST algorithms. It was suggested that the efficacy of DNA barcodes in delivering species-level identifications could be improved by developing local libraries [ 7 ; 27 ], and it was later demonstrated that this approach did indeed improve resolution [ 9 ; 23 ].

The effectiveness of such libraries depends upon complete sampling of local floras, accurate identification of the specimens that are analyzed, and quality of the resultant sequences [ 28 ]. Comparisons among past studies are difficult due to high variance in taxonomic scope 30— species , biogeographic focus e.

Arctic and temperate floras, tropical trees , the number of DNA barcode markers employed 2—8 chloroplast and nuclear , and the methodologies used for making taxonomic assignments. In fact, no prior study has involved a large-scale comparative analysis of the capacity of the standard barcode markers rbcL , matK , ITS2 to deliver a species-level identification for different biogeographic communities using a standard barcode library with the same methods. This study addresses this gap by employing a DNA barcode library for the vascular plants of Canada to determine the method that yields the best species resolution and the marker rbcL , matK , ITS2 with the highest performance.

As well, this study examines the efficacy of custom DNA barcode libraries for identification success, and compares phylogenetic diversity measures between sites and among species—rich families to determine factors affecting species resolution.

Complete taxonomic information, collection records, voucher images and sequences for 17, specimens are publically available through BOLD [ 30 ] in the plants of Canada project Available as of January 4, ; doi: dx. The number of species at each locale is in parentheses. For the purpose of further analyses, the 28 checklists were clustered into six biogeographic regions: Arctic, Atlantic, Boreal, Pacific, Prairies, and Woodland Table 1 representing 12 of the 15 terrestrial Canadian ecozones [ 32 ].

To ensure standardization of naming, all specimens and checklists used in this study followed the nomenclature accepted by VASCAN [ 31 ]. The number of plant species present at each site is indicated in parentheses. To reduce redundancy, identical sequences were clustered in UCLUST [ 33 ] and each cluster was parsed to its respective species one species could be represented by more than one cluster. We calculated two metrics for each barcode region, mean phylogenetic distance MPD and mean nearest taxon distance MNTD [ 38 ] to examine their potential as predictors of the capacity of each region to resolve species.

MPD is the average of the branch lengths or distances across all pairs of taxa in a phylogeny. It summarizes the overall phylogenetic diversity of a community and is influenced by the number of taxa in a tree [ 39 ]. By comparison, MNTD is an average of the distance between nearest neighbours so it describes the terminal phylogenetic structure. MNTD is the more appropriate measure of species resolution because it excludes internal nodes and instead calculates the mean distance between closely related species.

Because both measures are influenced by polytomies in a phylogeny [ 40 ], we only included one representative per species to avoid bias created by an unequal number of sequences per species.

The phylogenetic matrices for each barcode were calculated using the maximum likelihood tree, and were partitioned by family and genus. Regression analysis was used to determine if there was a correlation between each phylogenetic diversity metric and the number of sequences for a family. We also compared MNTD values for the three markers using a common set of genera to determine the strength of the correlation in their divergence values.

The percentage of congeners in the six large families with low MNTD Asteraceae, Brassicaceae, Cyperaceae, Poaceae, Rosaceae, and Salicaceae was evaluated for the datasets representing the three barcodes for the six biogeographic regions. Our custom sequence library for Canadian plants was parsed based on the species present at each locality and the taxonomic resolution provided by each barcode was then evaluated using BLAST searches and by mothur in Qiime [ 43 ]. For both methods, the species known for each park were compared with the parent library to ascertain if barcode records allowed their identification to a family, genus, or species level.

The resolution for species with multiple sequences was recorded as that where the taxonomic assignment for all individuals was consistent e. Mothur employs a distance matrix to assign a sequence or cluster to a species based on a parent library. For its use, identifications were predicted using a posterior probability cut-off of 0. We also report the true level of success of mothur by comparing the taxonomic identification assigned to a given sequence by mothur with its correct assignment.

The data for each park was then used to generate a mean level of taxonomic resolution for each family, genus, and species. Data was checked for normality prior to conducting a Kruskal-Wallis KW test or one-way ANOVA to test for significant differences in species resolution among the three markers. The parks were then subdivided into six biogeographic regions Arctic, Atlantic, Boreal, Pacific, Prairies, Woodland and the data was pooled for each region to estimate the mean level of taxonomic resolution for the floras that were examined.

After checking for normality, KW or one-way ANOVA was used to test for a significant difference in species resolution among the regions for a particular barcode marker. We also evaluated taxonomic resolution for the species with data for all three barcode genes to compare the mean of the parks and the performance of different markers using an identical set of taxa.

The performance of the barcodes for 25 families with the most species was then compared based on the BLAST results to identify groups where barcodes delivered low taxonomic resolution. All statistical tests were performed in R ver 3. After the removal of identical sequences within any one species, the read library was reduced to sequences for rbcL , sequences for matK , and sequences for ITS2.

This analysis showed that rbcL had considerably less sequence variation clusters; sequences than matK clusters; sequences while ITS2 was most diverse clusters; sequences. Both values were generally lower in the Arctic than in the other biogeographic regions for all three markers see Table C in S1 File for details , suggesting that species resolution should be most challenging in the north Table C in S1 File. These estimates of genetic diversity further predict that ITS2 will deliver the best taxonomic resolution followed by matK and rbcL.

When employing a posterior probability cut—off of 0. Analysis of the dataset consisting of species represented by all three barcodes generated similar results to the park data Table D in S1 File.

Since BLAST yielded the highest species resolution for each marker, these results were employed for the further analyses. For mothur, species resolution is reported for both a posterior probability cut-off 0. Level of taxonomic resolution provided by rbcL , matK or ITS2 for 25 families of vascular plant that are species-rich in Canada.

The three colours show the proportion of species identified to a family blue , genus orange or species green level. MPD and MNTD were first proposed as measures of phylogenetic diversity within a community [ 38 ], and have commonly been used to study community assembly [ 44 ; 45 ; 46 ].

MPD was previously used to compare substitution rates among plant families for three barcode regions rbcL , matK , ITS2 , and a positive correlation was reported between these rates and their capacity to discriminate species [ 26 ]. The present study extended this work by examining the utility of MPD and MNTD as predictors of species resolution for the same three gene regions.

Both MPD and MNTD indicated that ITS2 should deliver the best species resolution, an expected result given the higher rates of nucleotide substitution in nuclear than organellar genomes of plants [ 47 ; 48 ]. The prediction was supported when mothur was used to generate taxonomic assignments, but matK delivered the best species resolution with BLAST.

Interestingly, BLAST yielded higher species resolution than mothur for all three markers, a result which was maintained even when analysis was restricted to the species with sequence data for all three regions. Our results support the need for DNA barcoding to utilize phylogenetic methods that incorporate indels to maximize the resolving power of a given marker.

The genome compartment exposed to the highest intraspecific gene flow is generally the best suited for making species assignments because it reduces the likelihood that introgressed alleles will gain establishment and blur species diagnosis. Gene flow raises effective population size, reducing exposure to genetic drift, diminishing the chance of introgressed alleles gaining fixation in the gene pool, and increasing the probability that a particular gene will track species relationships [ 50 ].

Since the nuclear genome tends to experience greater dispersal and gene flow than the plastid genome, nuclear markers are generally more effective in species diagnosis than their plastid counterparts [ 50 ; 51 ].

Hence, the incorporation of a nuclear marker with the core plastid barcodes offers the advantage of compensating for situations where plastid markers fail to provide resolution. ITS2 did outperform its plastid counterparts in several species-rich families i.

Lamiaceae, Poaceae, Cyperaceae, and Saxifragaceae; Table 4 examined in this study. However, it was less effective in other families, likely reflecting incomplete lineage sorting stemming from its larger effective population sizes or, in rare cases, when plastid dispersal exceeds that of the nucleus [ 50 ; 51 ].

Despite these situations, the incorporation of ITS2 is preferable over additional plastid markers such as psbA-trnH because it occurs in all plant species which is not true for any plastid marker including rbcL and psbA-trnH and existing primers are nearly universal as opposed to those for matK. The observed differences in taxonomic resolution for the three barcodes are undoubtedly influenced by selection, species demography, hybridization, lineage sorting, and phylogeographic structure reviewed by [ 52 ].

The higher resolution of matK compared to rbcL reflects the different selective pressures acting on these genes. Because it encodes the large subunit of RuBisCo which has an essential role in photosynthesis, rbcL is under strong purifying selection in autotrophic plants [ 53 ] reducing its rate of evolution and constraining its utility for distinguishing closely related species. By contrast matK , an intron maturase involved in the splicing of group IIa introns, appears to be under relaxed purifying selection as evidenced by nearly equal substitution rates for all three coding positions [ 53 ; 54 ; 55 ; 56 ].

The relatively high rates of nucleotide substitution in matK compared to other plastid genes is useful for species delimitation, but a lack of conserved priming sites often undermines sequence recovery. Nuclear markers have a larger effective population size than plastid markers and tend to evolve more rapidly [ 47 ; 48 ]. The higher rates of nucleotide substitution and dispersal in plant nuclear genomes support their inclusion for plant DNA barcoding [ 51 ].

Additionally, the presence of multiple alleles for nuclear genes makes it possible to identify hybrids. Currently the only plant nuclear locus that meets barcoding criteria is ITS2 see above and its inclusion adds depth to barcode reference libraries by tracking a different genomic compartment.

The present analysis shows that MPD and MNTD are strong predictors of barcode resolution, identifying families and genera where taxonomic resolution is low. They were particularly useful in revealing genera with low resolution in families where divergences are high. For example, the Fabaceae has a relatively high MPD for both rbcL and matK but low species resolution, reflecting its inclusion of several genera e.

The latter genera explain the lower than average species resolution in this family for all markers, but this outcome was especially surprising for Lupinus because it was previously observed to have high genetic diversity in North America and low genetic diversity in the Andes due to a recent adaptive radiation [ 57 ].

Further research is needed to determine if a similar radiation occurred in North America. MNTD is a better predictor of species resolution than MPD because it quantifies the distance between pairs of closely related species and it is also less influenced by polytomies than MPD or PD [ 40 ].

As such, it is a better estimator of the efficacy of a given DNA barcode. The low correlation between MNTD values for the three barcode regions in different genera implies they are evolving independently S1 Fig. As a consequence, the use of multiple barcode markers consistently improves taxonomic resolution because a particular marker can compensate for the deficits in resolution of its counterparts [ 7 ; 9 ; 10 ; 15 ; 17 ; 18 ; 19 ; 20 ; 21 ; 22 ; 23 ; 24 ; 25 ; 26 ].

This complementarity supports the use of specific barcodes that optimize species resolution for different groups [ 58 ; 59 ]. The patterns of variation in the phylogenetic matrices MPD, MNTD agree with the earlier conclusion [ 15 ; 16 ] that ITS2 has higher discriminatory power than matK or rbcL when specimens are analyzed against a local reference library. For example, Burgess et al.

Analysis of the same community with the barcode library for all Canadian plants lowered species resolution i.

DNA barcodes: methods and protocols

A DNA barcode in its simplest definition is one or more short gene sequences taken from a standardized portion of the genome that is used to identify species through reference to DNA sequence libraries or databases. These methods include the latest information on techniques for generating, applying, and analyzing DNA barcodes across the Tree of Life including animals, fungi, protists, algae, and plants. Thorough and intuitive, DNA Barcodes: Methods and Protocols aids scientists in continuing to study methods from wet-lab protocols, statistical, and ecological analyses along with guides to future, large-scale collections campaigns. Skip to main content Skip to table of contents. Advertisement Hide. This service is more advanced with JavaScript available.


DNA barcode Identification Taxonomy Discovery Ecology Evolution. Download protocol PDF. Springer Nature is developing a new tool to find.


DNA Barcodes: Methods and Protocols

Imagine getting bitten by a spider, but being unable to tell what kind of spider it was poisonous or not?! To help organize our understanding of the diversity of species in the living world, Carl Linneaus invented a system for naming and classifying organisms in We still use this system today, and call it taxonomy. In Linnaean taxonomy, all the different kinds of living organisms can be organized practically into groupings with shared characteristics, where every species can be given a unique name.

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers rbcL , matK , ITS2. Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance MPD and mean nearest taxon distance MNTD were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is highest.

DNA barcoding, a new method for the quick identification of any species based on extracting a DNA sequence from a tiny tissue sample of any organism, is now being applied to taxa across the tree of life. As a research tool for taxonomists, DNA barcoding assists in identification by expanding the ability to diagnose species by including all life history stages of an organism. As a biodiversity discovery tool, DNA barcoding helps to flag species that are potentially new to science. As a biological tool, DNA barcoding is being used to address fundamental ecological and evolutionary questions, such as how species in plant communities are assembled.

Paul D.

DNA Barcoding

DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that, by comparison with a reference library of such DNA sections also called " sequences " , an individual sequence can be used to uniquely identify an organism to species, in the same way that a supermarket scanner uses the familiar black stripes of the UPC barcode to identify an item in its stock against its reference database. Different gene regions are used to identify the different organismal groups using barcoding. The 16S rRNA gene for example is widely used in identification of prokaryotes, whereas the 18S rRNA gene is mostly used for detecting microbial eukaryotes.

Sara M. Handy and Jonathan R. Deeds U. Natalia V.

DNA barcoding

Methods and Protocols

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Introduction. Front Matter. Pages PDF · DNA Barcodes: Methods and Protocols. W. John Kress, David L. Erickson. Pages PDF.

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