But it won't replace the traditional methods of identifying and classifying species by their appearance.only group that proved tricky was jellyfish and sea anemones, which seem to evolve too slowly for DNA differences between species to be a badge of identity.
Problems4 coding region genes and 3 non coding spacershave pooled data across laboratories including sequence data from 907 samples, representing445 angiosperm, 38 gymnosperm, and 67 cryptogam species
International scholarly Research Network
Molecular Ecology Resources (2013) 13, 21–31
Excluding outgroups Fuchsia and Circaea.
Notwithstanding their failure in certain cases, the currently used primers for rbcL and matK are useful for plant barcoding until the discovery of more efficient and robust primers for a broader coverage of plant species. Thus, there is a need for protocol development to enhance the amplification strategies including the development of new primers or primer cocktails for enhanced success in barcoding of plant species of different regions.
matK, trnE-trnF and psbA-trnH
1. Barcoding can identify a species from bits and pieces. 2. Barcoding can identify a species in its many forms, from eggs and seed, through larvae and seedlings, to adults 3. Barcoding can distinguish among species that look alike, and enabling a more accurate view of biodiversity.
4. A library of digital barcodes will provide an unambiguous reference that will facilitate identifying species invading and retreating across the globe5. scientists can equip themselves with barcoding to speed up the identification of known organisms and facilitate rapid recognition of new species.6. Barcoding links biological identification to advancing frontiers in DNA sequencing, miniaturization in electronics, and computerized information storage. Integrating those links will lead to portable desktop devices and ultimately to hand-held barcoders7. Compiling the library of barcodes begins with the multimillions of specimens in museums, herbaria, zoos and gardens, and other biological repositories. This collections will strengthen their ongoing efforts to preserve Earth's biodiversity. 8. Helps to create an on-line encyclopedia of life on Earth, with a web page for every species of plant and animal.
The International Barcode of Life project (iBOL) is the largest biodiversity genomics initiative ever undertaken. Hundreds of biodiversity scientists, genomics specialists, technologists and ethicists from 25 nations are working together to construct a richly parameterized DNA barcode reference library that will be the foundation for a DNA-based identification system for all multi-cellular life. In the first phase of operations (2010-2015), iBOL collaborators will barcode five million specimens representing 500,000 species. During construction of the barcode library, iBOL participants will also be building the infrastructure needed to use it in real-world situations such as conservation, ecosystem monitoring, forensics and control of agricultural pests and invasive species.
The Consortium for the Barcode of Life (CBOL) is an international initiative devoted to developing DNA barcoding as a global standard for the identification of biological species. Established in 2004 through support from the Alfred P. Sloan Foundation, CBOL promotes barcoding through Working Groups, networks, workshops, conferences, outreach, and training. CBOL has 200 Member Organizations from 50 countries and operates from a Secretariat Office located in the Smithsonian Institution's National Museum of Natural History in Washington, DC.
The specimen records created with the DNA Barcoding Assistant can be stored and converted into an Excel spreadsheet which can be send out via the standard email app. This is a huge time saver and it minimizes the chance of error creeping in while copying data from field book to spreadsheet to database.
Data in seconds to minutesPennies per sampleLink to reference databaseA taxonomic GPSUsable by non-specialists
Linnaeus classification vs. DNA barcoding
DNA Barcodes: DNA segments that
map well on taxonomy
DNA Barcodes - challenge the nature
of Linnaean taxonomy
DNA barcoding is a technology using gene sequences to differentiate
species, similar to the way retail stores rely on short, standardized
barcodes to differentiate the hundreds of thousands of items they sell
What is DNA barcoding?
Criteria of DNA barcode sequence
Barcoding regions (Animals and Plants)
Major barcoding projects
Simple & Ambitious!
ID all species
Speed up ID’s
Revitalize biological collections
DNA barcoding is a technique in which species identification is
performed by using DNA sequences from a small fragment of the
genome, with the aim of contributing to a wide range of ecological and
conservation studies in which traditional taxonomic identification is not
(Hebert et al. 2003, Kumar and Jain, 2011)
Short DNA sequences are called as DNA barcodes- ranging from 400-
The birth of DNA barcoding
The use of DNA sequences for species identiﬁcation has a long history
(e.g. Nanney, 1982; Bartlett and Davidson, 1991; see also Sperling,
2003; Will and Rubinoﬀ, 2004; Will et al., 2005; Cameron et al., 2006;
But it received signiﬁcant attention only after it was formally proposed
as ‘‘DNA barcoding’’ in 2003.
(Shiyang et al., 2012)
The birth of DNA barcoding
University of Guelph (2003), the DNA
barcoding initiative has gathered momentum,
gained extensive international participation in
the form of the International Barcode of Life
Project (iBOL), and captured attention of the
scientific community, government agencies,
and the general public.
Dr. Paul D.N. Hebert- Father of DNA
Significant limitations – traditional approach
Phenotypic plasticity and genetic variability in the characters employed
for species recognition can lead to incorrect identifications.
Overlooks morphologically cryptic taxa, which are common in many
groups (Knowlton, 1993; Jarman and Elliott 2000).
Morphological keys are often effective only for a particular life stage
or gender, many individuals cannot be identified.
Although modern interactive versions represent a major advance, the
use of keys often demands such a high level of expertise that
misdiagnoses are common
(Hebert et al., 2003)
Successful DNA barcode
a) Short enough to be quickly sequenced,
b) Easily identified in all species of organisms.
c) Variable enough to provide a unique sequence for each species
d) Size of sequence : 600 – 700 bp
Which gene fragments?
• rRNA genes used for species identification
• Small regions of the mitochondrial COI gene
• rbcL gene sequence and matK gene sequence of
• ITS region
(Kumar and Jain, 2011)
Sequencing can be done by a single organelle region
Approximately ~650-bp sequences - C-terminal
fragment of the mitochondrial gene named
cytochrome oxidase subunit I (COI) has been
proposed as universal marker for species level
identification in several animal group.
Barcodes (mtDNA) have become one of the most
contentious and animated issues in the application
of genetic information to global biodiversity
assessment and species identification.
(Saccone et al. 1999).
DNA barcoding for animals
DNA Barcoding in microbes
1.5 million species of fungi exist, but 10% are formally described
Variously used barcode, 400- to 600-bp region of the nuclear large
ribosomal subunit , the internal transcribed spacer (ITS) gene
Partial elongation factor 1- (EF-1) sequences focused on species of
Penicillium subgenus Penicillium (Trichocomaceae, Eurotiales,
(Kress et al., 2005)
Technical problems with defining species using
Use of COI sequence is not appropriate for most species of plants.
Slower rate of cytochrome c oxidase 1 gene evolution in higher plants
than in animals
(Kress et al., 2005, Hollingworth, 2008)
Not enough variability to discriminate species
DNA barcode for plants
Compared the performance of 7 leading candidate plastid DNA regions
(atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–
psbI spacer, and trnH–psbA spacer)
Assessments of recoverability, sequence quality, and levels of species
discrimination, they recommended the 2-locus combination of
rbcL+matK as the plant barcode.
(CBOL Plant Working Group, 2009)
The studies on Cucumis sp for the application of DNA barcode shows
the possibility of discrimination at species level not the varietal level
using the rbcL+matK gene barcode.
The barcode clearly differentiated the species C. sativus and C. melo
which will help for the future application in cucumis taxonomy and
Proposed Plant barcoding regions - Second
International Barcode of Life conference
Characteristics of different markers in plant
(Hollingsworth et al., 2011)
Biological invasions – major threats to global biodiversity.
So they have distinguished invasive and non invasive species by using
242 samples belonging to 26 species from 10 genera (Onagraceae,
Haloragaceae, Hydrocharitaceae and Cabombaceae ) of aquatic plants
and assessed using the chloroplast loci trnH-psbA, matK and rbcL
(Ghahramanzadeh et al., 2013)
Proportion of individuals successfully
ampliﬁed and sequenced from two plastid loci
P.A - percentage ampliﬁcation; P.S.O - percentage sequences obtained;
L - consensus sequence length; V.S - number of variable sites.
matK locus could not be ampliﬁed or sequenced reliably
Success rates of universal primers for amplification of matK and rbcL
loci in 26 different plant species (covering 14 families) from Saudi
Arabia were tested.
Success rate in PCR was higher for rbcL (88%) compared with matK
Universal primers of both matK and rbcL failed (primer mismatch at the
annealing site) to amplify the DNA from 3 plant species - Asteraceae
(Anthemis deserti, Pulicaria undulate, and Sonchus oleraceus).
Samples of Phyllanthus used in raw drug trade were obtained from 25
shops in southern India.
Authenticated species were identified by using morphological keys
Validated by developing species speciﬁc DNA barcode using the
chloroplast DNA region psbA-trnH, matK, trnE-trnF
matK and trnE-trnH failed to amplify for Phyllanthus sp
Six different species were identified by psbA-trnH
1. Works with fragments
2. Works for all stages of life
3. Unmasks look-alikes
4. Reduce ambiguity
5. Makes expertise to go further
6. Opens the way for an electronic handheld field guide, the Life Barcoder
7. Demonstrates the value of collection
8. Speeds writing the Encyclopedia of Life
(Savolainen et al., 2005)
Composition of the DNA Barcode Library by 2014
(iBOL Consortium, 2013)
iBOL - International Barcode of Life.
CBOL – Consortium for the Barcode of Life
ECBOL - European Consortium for the Barcode of Life
BOLD – Barcode of Life Database
INSDC - International Nucleotide Sequence Database
CCBD – Canadian centre for DNA Barcoding
IBOL – International Barcode of Life
Largest biodiversity genomics initiative
From 25 nations Scientists are working together to construct DNA
barcode reference library
First phase of operations (2010-2015) - barcode five million specimens
representing 500,000 species.
CBOL – Consortium for the Barcode of Life
International initiative (2004)
To develop DNA barcoding as a global standard for the identification of
200 Member Organizations from 50 countries
ECBOL - European Consortium for the Barcode of
Established as part of the research infrastructure efforts of EDIT,
the European Distributed Institute of Taxonomy.
The Barcode of Life Database (BOLD)
At the University of Guelph, public workbench for barcoding projects.
Researchers can assemble, test, and analyze their data records
Barcode clusters for animals: 280,889
Barcode Sequences :2,145,877
Formally described species
Animals : 132,785
Fungi & Other Life: 2,502
International Nucleotide Sequence Database
GenBank, EMBL, and DDBJ which comprise the International Nucleotide
Sequence Database Collaboration.
Permanent public repository for barcode data records.
CCBD – Canadian centre for DNA Barcoding
Largest 'barcode factory', generating hundreds of thousands of data
records per year
Training barcode researchers from around the world.
iBOL's partners consist of national, regional and central nodes, each of
which have network of projects, institutions and labs.
The first national barcode network - Canada, followed by others in the
Netherlands, Mexico, Australia, and other countries.
Bioinformatics – major role
In case of non-availability of sequences, sequencing has to be done in
vitro for which a recently developed software ecoPrimers can be
Further,basic sequence statistics computation and phylogenetic
analysis can be performed by MEGA and PHYLIP/PAUP tools
(Bhargava et al., 2013)
To identify new barcode markers in particular metabarcode markers
and their associated PCR primers
Scans a large database of whole genomes to ﬁnd such markers without
a prior knowledge and selects highly conserved primers
Further, eco-Primers tests an ampliﬁed region for its discriminatory
power between the different taxa.
ecoPrimers selects the primer pairs and evaluates their quality by
optimizing two indices, Bc and Bs
Bc estimates the ampliﬁcation range of a primer pair and Bs evaluates
the discrimination capacity of the ampliﬁed marker.
Databases available for extracting sequence and
structure information of molecular markers
8,000 described species
• Mosquito Barcoding Initiative - MBI plans to barcode at
least five specimens from 80 percent of the 3,200
known mosquito species
• Bee-BOL, the Bee Barcode of Life Initiative - for all
20,000 bee species
• Shark Barcode of Life project aims to barcode the 1,000
marine and 100 freshwater shark species.
Total specimen recorded
(iBOL consortium, 2013)
Species Identification really matters!
Cataloguing hidden diversity
Basic research in taxonomy
Improving environmental monitoring
Controlling Agricultural Pests
Identifying Disease Vectors
Environmental Sustainability : Sustaining Natural Resources
Protecting Endangered Species
Taxonomists, ecologists, conservationists, foresters, agriculturalists,
forensic scientists, customs and quarantine officer
Cytochrome oxidase subunit I (COI) - universal marker for species level
identification in several animal group.
In spite of failure in certain cases, the currently used barcode
sequences are rbcL and matK
It will be highly useful for plant barcoding until the discovery of more
efficient and robust primers for a broader coverage of plant species.
Thus, there is a need for protocol development to enhance the
amplification strategies for enhanced success in barcoding of plant
Biological barcoding is likely to provide a new frame work for
cataloging, categorizing and monitoring the planet’s biodiversity.
In the space of few years DNA barcoding has
moved from fantasy to reality.
Earth is home to an estimated 10
million species of plants and animals
Human brain can learn to identify a few
thousand species, a small fraction of
26 - 31 October 2013 Fifth International Barcode of Life
Kunming, Yunnan, China