Transcript benefits of dna barcoding

Some
major
episodes in
the history
of life.
Trends in Ecology
& Evolution
Feb. 2003. Vol. 18, Iss. 2
3 of these 5 are the most
downloaded papers in TREE
1. Taxonomy: renaissance or
Tower of Babel?
Jim Mallet et al
4. A plea for DNA taxonomy
Tautz et al
5. The encyclopedia of life
Edward Wilson
DNA barcoding
a new diagnostic tool for rapid species recognition identification,
and discovery
New Scientist, 26 June, 2004
James Hanken, Museum of Comparative Zoology, Harvard University, USA
BARCODING LIFE
Barcoding is a standardized approach to identifying plants and animals by
minimal sequences of DNA, called DNA barcodes.
DNA Barcode: A short DNA sequence, from a uniform locality on the
genome, used for identifying species.
Mark Stoeckle, The Rockefeller University; Paul E. Waggoner, Connecticut Agricultural Experiment Station; Jesse
H. Ausubel, Alfred P. Sloan Foundation
By harnessing advances in electronics and genetics, barcoding will
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help many people quickly and cheaply recognize known species and retrieve
information about them
speed discovery of the millions of species yet to be named
provide vital new tools for appreciating and managing the Earth’s immense
and changing biodiversity.
Standardization
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accelerate construction of a comprehensive, consistent reference library of
DNA sequences
speedy development of economical technologies for species identification.
The goal is that anyone, anywhere, anytime be able to identify quickly and
accurately the species of a specimen whatever its condition.
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Results so far suggest that a mitochondrial gene will enable identification of
most animal species.
For plants, mitochondrial genes do not differ sufficiently to distinguish among
closely related species. Promising approaches to standardize plant
identification use one or possibly more barcode regions are under
development.
An Internal ID System for All Animals
The Mitochondrial Genome
DNA
D-Loop
Small ribosomal RNA
Cytochrome b
ND1
ND6
Typical Animal Cell
COI
ND5
mtDNA
L-strand
H-strand
ND4
ND4L
ND3
Mitochondrion
COII
COIII
ATPase subunit 8
ATPase subunit 6
ND2
Why barcode animals with mitochondrial DNA?
Mitochondria, energy-producing organelles in plant and animal cells, have
their own genome. Twenty years of research have established the utility of
mitochondrial DNA sequences in differentiating among closely-related
animal species.
Four properties make mitochondrial genomes especially suitable for
identifying species
* Greater differences among species
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Copy number. There are 100-10,000 more copies of
mitochondrial than nuclear DNA per cell, making recovery,
especially from small or partially degraded samples, easier
and cheaper.
Relatively few differences within species in most cases. Small intraspecific
and large interspecific differences signal distinct genetic boundaries
between most species, enabling precise identification with a barcode.
Introns, which are non-coding regions interspersed between coding
regions of a gene, are absent from mitochondrial DNA of most animal
species, making amplification straightforward. Nuclear genes are often
interrupted by introns, making amplification difficult or unpredictable.
How Barcoding is Done
From specimen to sequence to species
C
NO
DI
3I
I
Collecting
Voucher Specimen
DNA extraction
CO1 gene
N
N
D
1D
2
DNA sequencing
Trace file
Database of Barcode
Records
What are the main limits to
barcoding encountered so far?
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Groups with little sequence diversity
Resolution of recently diverged
species
Hybrids
Nuclear pseudogenes
What do barcode differences among and within animal
species studied so far suggest?
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Barcodes identify most animal species unambiguously.
Approximately 2-5% of recognized species have shared or overlapping
barcodes with closely-related species. Many of the species with
overlapping barcodes hybridize regularly.
In all groups studied so far, distinct barcode clusters with biologic covariation suggest cryptic species.
Barcoding
North
American birds
highlights
probable
cryptic species
Barcodes affirm the unity
of the species Homo sapiens.
Comparisons show we differ
from one another by only 1 or 2
nucleotides out of 648, while
we differ from chimpanzees at
60 locations and gorillas at 70
locations.
Can barcodes aid
understanding history
of animal and plant
species?
What isn’t DNA barcoding?
• It is not intended to, in any way, supplant
or invalidate existing taxonomic practice.
• It is not DNA-taxonomy; it does not
equate species identity, formally or
informally, with a particular DNA
sequence.
• It is not intended to duplicate or compete
with efforts to resolve deep phylogeny,
e.g., Assembling the Tree of Life (ATOL).
“the role of any molecular diagnostic is to aid research, not to serve as an
end in itself. Barcoding … is independent of questions as to whether
individual taxa are species, what species are (or should be), and where
they fit in a unified tree of life…. Barcoding is not an end in itself, but
will boost the rate of discovery. The unique contribution of DNA
barcoding to … taxonomy and systematics is a compressed timeline for
the exploration and analysis of biodiversity.”
Barcoding must adhere to standards
for specimen and data management
Sequence data
Voucher specimens
and electronic
databases
Digital images
Strengths
• Offers alternative taxonomic identification
tool for situations in which morphology is
inconclusive.
• Focus on one or a small number of genes
provides greater efficiency of effort.
• Cost of DNA sequencing is dropping rapidly
due to technical advances.
• Potential capacity for high throughput and
processing large numbers of samples.
• Once reference database is established,
can be applied by non-specialist.
Weaknesses
• Assumes intraspecific variation is negligible, or at
least lower than interspecific values.
• No single gene will work for all taxa (e.g., COI is
not appropriate for vascular plants, or even for
some animals).
• Single-gene approach is less precise than using
multiple genes; may introduce unacceptable error.
• Some of the most attractive aspects rely on future
technology, e.g., handheld sequencer
Simple & Ambitious!
Advocates
ID all species
Discover new
species
Speed up ID’s
Revitalize biological collections
Opposition
Won’t work
Destroy traditional
systematics
Service industry
Pseudo taxonomy
A global science project
Making Every Species Count
► 5 years
► 5M specimens
► 500K species
Official launch of iBOL – CN Tower, Toronto, September 25, 2010
iBOL launches with 1M records, 100K species
iBOL structure: participating nations
Central Nodes ($25M)
Regional Nodes ($5M)
National Nodes ($1M)
ICI is an alliance of researchers and biodiversity organisations in 21 nations.
All nations active in specimen assembly, curation and data analysis.
Sequencing and informatics support by regional and central nodes.
Central Nodes
Regional Nodes
Developing Nodes
Collection and
Databasing
Curation and
Identification
Sequencing
Mirrored
Databases
Data Analysis
and Access
Theme 1:
DNA Barcode Library
WG1.1 Vertebrates
WG1.2 Land Plants
WG1.3 Fungi
WG1.4 Animal Parasites, Pathogens & Vectors
WG1.5 Agricultural & Forestry Pests & Parasitoids
WG1.6 Pollinators
WG1.7 Freshwater Bio-Surveillance
WG1.8 Marine Bio-Surveillance
WG1.9 Terrestrial Bio-Surveillance
WG1.10 Polar Life
iBOL WG 1.5
• Bringing genomics to the fight against plant pests
and invasive species
• Assembling a DNA barcode reference library of
pests and their parasitoids
• 2015 target: 25,000 of the most important pest
species
BENEFITS OF DNA BARCODING
• DNA barcoding can speed up identification of new species.
• DNA barcodes can be linked to readily observable
morphological characters.
• DNA barcoding can provide an avenue to encourage new
participants into taxonomy.
• Applied taxonomic research areas will benefit from barcoding.
• Food adulteration
Barcode of Life Community
Networks, Projects, Organizations
• Promote barcoding
as a global standard
• Build participation
• Working Groups
• BARCODE standard
• International
Conferences
• Increase production
of public BARCODE
records
CBOL Member Organizations: 2009
• 200+ Member organizations, 50 countries
• 35+ Member organizations from 20+ developing countries
GenBank, EMBL, and DDBJ
Global, Open Access to Barcode Data
NBII, 25 February 2009
http://www.insdc.org/
BARCODE Records in INSDC
Specimen
Metadata
Geo reference
Habitat
Character sets
Images
Behavior
Other genes
Other Databases
Phylogenetic
Pop’n
Genetics
Ecological
Voucher
Specimen
Barcode
Sequence
Trace
files
Primers
Literature
(link to content or
citation)
Species
Name
Indices
- Catalogue of
Life
- GBIF/ECAT
Nomenclators
- Zoo Record
- IPNI
- NameBank
Publication
Databases links
-- New
speciessp.
Provisional
Linkout from GenBank to BOLD
NBII, 25 February 2009
Linkout from GenBank to Taxonomy
NBII, 25 February 2009
Link from GenBank to Museums
NBII, 25 February 2009
Current Norm: High throughput
Large labs, hundreds of samples per day
Large capacity PCR and
sequencing reactions
ABI 3100 capillary
automated sequencer
Emerging Norm: Table-top Labs
Faster, more portable: Hundreds of samples per hour
Integrated DNA
microchips
Table-top microfluidic
systems
Producing Barcode Data: 201?
Barcode data anywhere, instantly
• Data in seconds to
minutes
• Pennies per sample
• Link to reference
database
• A taxonomic GPS
• Usable by nonspecialists
Adoption by Regulators
• Food and Drug Administration
– Reference barcodes for commercial fish
• NOAA/NMFS
– $100K for Gulf of Maine pilot project
– FISH-BOL workshop with agencies, Taipei, Sept 2007
• Federal Aviation Administration – $500K for birds
• Environmental Protection Agency
– $250K pilot test, water quality bioassessment
• FAO International Plant Protection Commission
– Proposal for Diagnostic Protocols for fruit flies
• CITES, National Agencies, Conservation NGOs
– International Steering Committee, identifying pilot projects
Establishing a DNA Barcode
for Land plants
Santiago Madriñán Restrepo
Universidad de los Andes, Bogotá, Colombia
[email protected]
COI or cox1 in Plants
• Low sequence divergence
• Other mitochondrial genes
– Exhibit incorporation of foreign genes
– Frequent transfer of some genes to the nuclear
genome
Barcode representation of DNA fingerprints of Indian CASHEW varieties (Archak et al 2003
Project Partners
Part n er Org ani z ations
Scient ists
Ro yal Bota n ic Gar d ens, Ke w, UK
Ro b yn Cowan
Mark Cha s e
Natural His t or y Mu seum (Lon d on) , UK
Mark Car ine
To rte lla, Pty ch o mn iaceae, As p le n ium ,
Natural His t or y Mu seum , De n mark
Git te Pet e rs e n
Ho rdeu m , Scale s ia, Cr o cus
New Yo rk Bota n ica l Garde n , USA
Kenn e th Cam e ron
Elapho g lo s sum , C u pres s us, Lab ordia
Ro yal Bota n ic Gar d en Edin b u rgh, UK
Pete r Hol lin gs wo rt h
Pod o carpu s , Arau caria, As t er e lla,
Anastr o ph yllu m
So u th Afr ican Na tio nal Biodi ver s it y
Ins t itu te , (Cape T o wn), S o ut h Africa
Feroza h Co n rad
Encephala rt os, M imet e s
Unive rsidad d e los Andes,
Tar g et gr o ups
Con o s t ylis , Pin us, Equis e tu m ,
Dact ylo rhiza macu lata/incarn ata
com p lex
Co lombia
Santi a go Madr iñá n
Lauraceae
Ins t itu to de Bi o lo g ia UNAM, Me x ico
Gerar d o A. Salaza r
Agave
Unive rsida d e Esta d ual de Feira de
Santana, Bras il
Cássio v a n d e n Ber g
Laelia, Ca t tle ya
Unive rs ity of Cape To wn, So u th Afric a
Ter ry Hed d er s on
Anastr o ph yllu m - Barbi lop h ozia,
Bryu m
Impe rial Co lle ge , UK (& RBG Ke w)
Tim o th y Barrac lo u gh
(Data anal ysis)
Plant Barcode Proposals
Molecules and their useful rangesin phylogenetic relationships
Species
Genera
Family
Order Class Divisions
Spacers
[ITS]
mt DNA
Nu rDNA
; more sufficient statistically significant results
; sufficient statistically significant results
3/28/2016
45 1991
Taylor, et al.,
Other Regions
• Internal transcribed spacer regions of nuclear
ribosomal DNA (ITS)
– often highly variable in angiosperms at the generic and
species level
– divergent copies are often present within single
individuals
• Non-coding plastid regions
– Highly length variable
• rbcL (
– Not variable enough at species level for many plant
groups
Plastid DNA
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Monomorphic
High copy number
Highly diagnostic
Regions and Primers
Gene
matK
rpoC 1
rpoB
accD
YCF5
ndhJ
Prime r
2.1
2.1a
5
3.2
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
Dire ction
f
f
r
r
f
f
r
r
f
f
r
r
f
f
r
r
f
f
r
r
f
f
r
r
Seque n ce 5' - 3'
CCTATCCATCTGGAAATCTTAG
ATCCATCTGGAAATCTTAGTTC
GTTCTAGCACAAGAAAGTCG
CTTCCTCTGTAAAGAATTC
GTGGATACACTTCTTGATAATGG
GGCAAAGAGGGAAGATTTCG
TGAGAAAACATAAGTAAACGGGC
CCATAAGCATATCTTGAGTTGG
AAGTGCATTGTTGGAACTGG
ATGCAACGTCAAGCAGTTCC
CCGTATGTGAAAAGAAGTATA
GATCCCAGCATCACAATTCC
AGTATGGGATCCGTAGTAGG
GGRGCACGTATGCAAGAAGG
TTTAAAGGATTACGTGGTAC
TCTTTTACCCGCAAATGCAAT
GGATTATTAGTCACTCGTTGG
ACTTTAGAGCATATATTAACTC
ACTTACGTGCATCATTAACCA
CCCAATACCATCATACTTAC
CATAGATCTTTGGGCTTYGA
TTGGGCTTCGATTACCAAGG
ATAATCCTTACGTAAGGGCC
TCAATGAGCATCTTGTATTTC
Sister taxa: Cattleya and Sophronitis
“Corsage orchids”
Cássio van den Berg
Universidade Estadual de Feira de Santana, Brasil
Cattleya: 43 spp. in 2 subgenera
Unifoliate species = 18 species, allopatric species “complex”
Bifoliate species = 25 well-defined species, 6 species pairs
Sophronitis: 63 spp. in 3 subgenera (as “sections”)
Sect. Cattleyodes+Hadrolaelia – 17 well-defined species
Sect. Parviflorae – 40 spp. messy complex, genetic data indicate ca. 15 spp.
Sect. Sophronitis – 6 allopatric closely related species
C.labiata
C. aclandiae
S. perrinii
S. sp. nov.
% species discriminated
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ITS: 90.5%
psbA-trnH: 60%
matK: 33.3%
ndhJ: 37.1%
rpoB: 9.9%
rpoC1:9.9%
accD: 6.05 %
Nuclear non-coding
Plastid non-coding
Plastid coding
• accD, rpoB, rpoC1: variation too low for use as a single barcode
• matK and ndhF: more variable but with great variation of rate among
subgenera
• Non-coding regions (ITS and psbA-trnH spacer) performed better, but
required great manual effort for indel alignment
Lauraceae
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Big family
Largely unstudied
VERY difficult to id.
Economically important
accD
Lauraceae
accD
matK
ndhJ
rpoB
rpoC
1
Genera
17
17
16
17
17
Species
42
40
36
42
43
Specimens
47
58
42
48
49
Sp. w/ >1
specimen
5
11
6
6
6
Actinodaphne glabra K-8202
Actinodaphne pruinosa K-8203
Aiouea dubia AN-417
Beilschmiedia pendula BCI-065681
Beilschmiedia pendula BCI-170421
Beilschmiedia pendula BCI-257596
Beilschmiedia tawa K-5519
Caryodaphnopsis cogolloy JAUM-s.n.
Cinnamomum camphora K-6469
Cinnamomum dictyoneuron K-8201
Cinnamomum obtusifolium K-8303
Cinnamomum triplinerve BCI-206711
Cinnamomum triplinerve BCI-240764
Cinnamomum triplinerve SM-Ama-005
Cinnamomum zeylanicum K-8306
Cryptocarya triplinervis K-5522
Dodecadenia grandiflora K-5520
Endlicheria sp. 1 SM-Ama-006
Endlicheria sp. 2 SM-Ama-003
Lauraceae sp. 1 A-1535
Lauraceae sp. 2 NN-BCI?
Laurus azorica K-21989
Laurus nobilis SM-s.n.
Lindera benzoin K-16947
Litsea cubeba K-15475
Nectandra cissiflora BCI-216314
Nectandra cissiflora BCI-233922
Nectandra cissiflora BCI-244145
Nectandra cissiflora BCI-269005
Nectandra cuspidata FC-1579
Nectandra cuspidata Gamboa-s.n.
Nectandra 'fuzzy' BCI-036152
Nectandra 'fuzzy' BCI-105750
Nectandra lineata BCI-065446
Nectandra lineata BCI-220065
Nectandra purpurea BCI-151022
Nectandra purpurea BCI-277412
Nectandra purpurea BCI-415163
Nectandra sp. 1 AN-410
Nectandra sp. 1 AN-411
Neolitsea aciculata K-17739
Ocotea callophyla SM-s.n.
Ocotea cernua BCI-206437
Ocotea cernua BCI-215988
Ocotea cernua BCI-412951
Ocotea floribunda HD-1166
Ocotea guianensis A-818
Ocotea oblonga BCI-309078
Ocotea oblonga BCI-403510
Ocotea puberula BCI-146684
Ocotea puberula BCI-272219
Ocotea puberula BCI-716317
Ocotea sp. 1 HD-1167
Ocotea whitei BCI-008086
Ocotea whitei BCI-306277
Persea americana SM-s.n.
Persea caerulea Sánchez-4911
Persea rimosa K-8204
Rhodostemonodaphne frontinoensis Brant-1387
Rhodostemonodaphne kunthiana HD-1175
Rhodostemonodaphne kunthiana Madriñán-717
Rhodostemonodaphne penduliflora SM-s.n.
Sassafras albidum K-16948
matK
ndhJ
rpoB
rpoC1
matK
974 bp
ndhJ
428 bp
Cryptocarya triplinervis K-5522
Laurus nobilis SM-s.n.
A comparative study of different DNA barcoding
markers for the identification of
some members of Lamiacaea
Fabrizio De Mattia, Ilaria Bruni, Andrea Galimberti, Francesca Cattaneo, Maurizio Casiraghi, Massimo Labra
Università degli Studi di Milano Bicocca, ZooPlantLab, Dipartimento di Biotecnologie e Bioscienze, Piazza
della Scienza 2, 20126 Milano, Italy
Food Research International 44 (2011) 693–702
The objective is to evaluate the efficacy of a DNA barcoding approach as a tool for the recognition of
commercial kitchen spices belonging to the Lamiaceae family that are usually sold as enhancers of food
flavor. A total of 64 spices samples, encompassing six different genera (i.e. Mentha, Ocimum, Origanum,
Salvia, Thymus and Rosmarinus) were processed with a classical DNA barcoding approach by amplifying
and sequencing four candidate barcode regions (rpoB, rbcL, matK and trnH-psbA) with universal primers.
Results suggest that the non-coding trnH-psbA intergenic spacer is the most suitable marker for molecular
spices identification followed by matK, with interspecific genetic distance values ranging between about 0%
to 7% and 0% to 5%, respectively. Both markers were almost invariably able to distinguish spices species
from closest taxa with the exclusion of samples belonging to the genus Oregano. Moreover, in a context of
food traceability the two markers are useful to identify commercial processed spice species (sold as dried
plant material). We also evaluated the potential benefits of a multilocus barcode approach over a single
marker and although the most suitable combination was the matK+trhH-psbA, the observed genetic
distances values were very similar to the discriminatory performance of the trnH-psbA. Finally, this
preliminary work provide clear evidences that the efficacy of a DNA barcoding approach to the recognition
of commercial spices is biased by the occurrence of taxonomic criticisms as well as traces of hybridization
events within the family amiaceae. For this reason, to better define a more practical and standardized DNA
barcoding tool for spices traceability, the building of a dedicated aromatic plants database in which all
species and cultivars are described (both morphologically and molecularly) is strongly required.
Fig. 1. Neighbor-joining reconstructions
obtained with MEGA 4.0 for three out of
the four molecular datasets produced in this
study. Each tree encompasses all the
samples analysed for the six taxonomical
group considered: a) trnH-psbA, b) matK, c)
rbcL. Bootstrap values lower than 70% not
showed. Details on samples, species,
cultivar, provenance and accession numbers
for each marker can be retrieved from Table
1. Each taxonomic group has been shown on
the tree with squared brackets
.
Overall Results
• Standardized universal primers
• Different levels of variation in different
groups at different taxonomic levels
• Variable ID success with a single region
• Score on basis of
– Amplification success
– Sequence variation
Non-COI regions for other taxa
• Land plants:
– Chloroplast matK and rbcL approved Nov 09
– Non-coding plastid and nuclear regions being
explored
www.kew.org/barcoding
What barcode providers want
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High PCR and sequencing success rates
Bigger window into older, compromised samples
Better software integration to eliminate bottlenecks
Smaller labs/developing countries:
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Lower equipment and maintenance costs
Simplification for techs with less training
Install anywhere without lab renovations
Willing to accept slower throughput
What barcode users want
• Answers to specific questions:
– Is this thing on this list of species or not?
– Is this thing a member of this genus/family?
– Which of the species on this list is this thing?
– What species is this thing?
• Production-scale capabilities:
– Hundreds to thousands of installations
– Lower but constant throughput
– Rapid turnaround
– The right price-point and limited life cycle costs
What barcode users would do with the
reference libraries
• Inspection stations at every port and international airport for:
– Agricultural pest control
– Illegal trade in endangered species
– Violations of trade quotas
• Regular Federal and State water quality surveys
• Federal, State and local food inspection
• Public health monitoring and diagnoses
IISR
Barcoding of insect pests of Spices (IIHR,
IISR initiative (TK Jacob) - COI
Barcoding for checking adulterants in traded
spices (Sasikumar B) – ITS, rbcL, Mat K
etc