DNA Barcodes: Methods and Protocols: W. John Kress and David L. Erickson
DNA Barcodes: Methods and Protocols: W. John Kress and David L. Erickson
DNA Barcodes: Methods and Protocols: W. John Kress and David L. Erickson
1. What Is DNA
Barcoding?
The taxonomic impediment that exists today for many systematists,
field ecologists, and evolutionary biologists, i.e., determining the
correct identification for any plant or animal sample in a rapid,
repeatable, and reliable fashion, is a reality we all must accept (1).
This taxonomic problem was a major reason for the development of
a new method for the quick identification of any species based on
extracting a DNA sequence from a tiny tissue sample of any organism. Appropriately called DNA barcoding, referring to the
UPC labels one finds on commercial products, DNA barcodes
consist of a standardized short sequence of DNA between 400
and 800 bp long that, in theory, can be easily isolated and characterized for all species on the planet (2, 3). By harnessing advances
W. John Kress and David L. Erickson (eds.), DNA Barcodes: Methods and Protocols, Methods in Molecular Biology, vol. 858,
DOI 10.1007/978-1-61779-591-6_1, Springer Science+Business Media, LLC 2012
3. DNA Barcoding
Methods in Brief
The process of DNA barcoding entails two basic steps: (1) building
the barcode library of known species and (2) matching, or assigning
the barcode sequence of the unknown sample against the barcode
library for identification. The first step requires taxonomic expertise in selecting one or preferably several individuals per species to
serve as reference samples in the barcode library. All taxonomists
should generate DNA barcodes for the taxa in their monographs
or at the least they should deposit verified DNA samples with their
associated voucher specimens in core DNA barcode institutions.
Tissue samples that yield high-quality DNA extractions in some
cases can be obtained from specimens already housed in museum
collections and herbaria. However, in most cases new tissues will
be taken directly from live specimens in the field before they are
prepared, labeled, and stored as voucher specimens in museum collections. These vouchers then serve as the permanent record that
connects the DNA barcode to a particular species of plant, fungus,
or animal.
Once the reference barcode library is complete for the organisms under study, whether they comprise a geographic region, a
taxonomic group, or a target assemblage (e.g., medicinal plants,
timber trees, etc.), then the DNA barcodes generated for the
4. A Short History
of DNA Barcoding
To be practical as a DNA barcode, a gene region must satisfy three
criteria: (1) contain significant species-level genetic variability and
divergence, (2) possess conserved flanking sites for developing
universal PCR primers for the widest taxonomic application, and
(3) be of appropriate sequence length so as to facilitate current
capabilities of DNA extraction and sequencing. A fourth criterion for a successful DNA barcode relates to sequence quality and
has been proposed by some as an important consideration (see
CBOL Plant Working Group 2009). A short DNA sequence of
600 bp in the mitochondrial gene for cytochrome c oxidase subunit 1 (CO1; 2) generally fits these criteria and was accepted early
on as a practical, standardized species-level barcode for many
animals (see http://www.barcoding.si.edu/). The inability of CO1
to work as a barcode in plants and fungi (6, 8) required that botanists find a more appropriate marker. A number of candidate gene
regions were immediately suggested as possible barcodes for plants
(e.g. refs. 6, 7, 9, 10, 20, 21), but until 2009 none were universally
accepted by the plant taxonomic community. This lack of consensus was in most part due to the limitations inherent in a plastid
marker (i.e., low sequence variability) relative to CO1. In 2008,
The Consortium for the Barcode of Life Plant Working Group
convened a lengthy discussion on selecting an appropriate plant
barcode and eventually published a paper (11) in which the largest
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