DNA Bar-code to Distinguish the Species
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Using mtDNA to distinguish species
By Roya ShariatiANU College of Archaeology
and Anthropology Supervisor: Prof. Colin Groves
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Carl Linnaeus 250 years ago
Carl Linnaeus is best known for creating the system of classifying living organisms that became the international standard.
“You say tomato, I say Lycopersicon esculentum. You say potato, I saySolanum tuberosum. But Carl Linnaeus was the real plant buff.”
Often called the father of classification, Swedish naturalist Linnaeus established the familiar dual Latin names by which all creatures are now known.
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DNA Barcoding A DNA barcode is
a short gene sequence
taken from standardized
portions of the genome, used to identify
species
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An organism’s chromosome complement
is its karyotype
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Category Genes
NADH dehydrogenase(complex I)
MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6
Coenzyme Q - cytochrome c reductase/Cytochrome b(complex III)
MT-CYB
cytochrome c oxidase(complex IV)
MT-CO1, MT-CO2, MT-CO3
ATP synthase MT-ATP6, MT-ATP8
Amino Acid 3-Letter 1-Letter MT DNA
Alanine Ala A MT-TA
Arginine Arg R MT-TR
Asparagine Asn N MT-TN
Aspartic acid Asp D MT-TD
Cysteine Cys C MT-TC
Glutamic acid Glu E MT-TE
Glutamine Gln Q MT-TQ
Glycine Gly G MT-TG
Histidine His H MT-TH
Isoleucine Ile I MT-TI
Leucine Leu LMT-TL1, MT-TL2
Lysine Lys K MT-TK
Methionine Met M MT-TM
Phenylalanine Phe F MT-TF
Proline Pro P MT-TP
Serine Ser SMT-TS1, MT-TS2
Threonine Thr T MT-TT
Tryptophan Trp W MT-TW
Tyrosine Tyr Y MT-TY
Valine Val V MT-TV
Transport chain
rRNAMitochondrial rRNA is encoded by MT-RNR1 (12S) and MT-RNR2 (16S).
tRNAThe following genes encode tRNA:
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The Mitochondrial Genome
Cyt b
D-Loop
ND5
H-strand
ND4
ND4LND3
COIII
COICOIL-strand
ND6
COI
ND2
ND1
COII
Small ribosomal RNA Large
ribosomal RNA
ATPase subunit 8ATPase subunit
6
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C O M PA R I S O N O F M I T O C H O N D R I A L G E N E O R D E R S A M O N G T Y P I C A L
V E R T E B R AT E S , E . P E L E C A N O I D E S , A N D S . L AV E N B E R G I . F I V E B L O C K S O F
G E N E R E G I O N S ( A , T R N A G L U – T R N A G L N ; B , AT P 8 - N D 3 ; C , T R N A L E U ( C U N ) – N D 6 ;
D , T R N A I L E – T R N A L Y S ; A N D E , T R N A A R G – T R N A S E R ( A G Y ) G E N E R E G I O N S ) R E TA I N
T H E T Y P I C A L V E R T E B R AT E G E N E O R D E R W I T H T H E E X C E P T I O N O F
S E V E R A L T R N A G E N E S ( A R R O W H E A D S ) . T H E PA R T I A L M T D N A S E Q U E N C E S
F O R F O U R G E N E J U N C T I O N S ( V E R T I C A L B A R : N D 1 - AT P 8 , N D 3 - N D 5 , N D 6 -
N D 2 , A N D N D 4 - C Y T B R E G I O N S ) , T H E G E N E O R D E R O F W H I C H G R E AT LY
D I F F E R F R O M T H AT O F O T H E R T Y P I C A L V E R T E B R AT E S , W E R E
D E T E R M I N E D F O R F O U R A D D I T I O N A L E . P E L E C A N O I D E S I N D I V I D U A L S
( D ATA AVA I L A B L E F O R M D D B J / E M B L / G E N B A N K W I T H A C C E S S I O N
N U M B E R S A B 0 4 6 4 7 5 – A B 0 4 6 4 9 0 )
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A remarkably short DNA sequence can contain more than enough information to resolve 10 or even 100 million species. For example, a 600-nucleotide segment of a protein-coding gene contains 200 nucleotides that are in the third position within a codon. At these sites, substitutions are (usually) selectively neutral and mutations accumulate through random drift. Even if a group of organisms was completely biased to either adenosine or thymine (or alternatively, to either guanosine or cytosine) at third nucleotide positions there would still be 2200 , or 1060 , possible sequences based on third-position nucleotides alone. DNA sequence analysis of a uniform target gene to enable species identification has been termed DNA bar-coding, by analogy with the Uniform Product Code barcodes on manufactured goods.
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The Phylogenetic species concept
tokogenetic versus phylogenetic relationships
The phylogenetic species concept
Speciation and phylogenetic relationships
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Applied tool for identifying regulated species:Disease vectors, agricultural pests, invasiveEnvironmental indicators, protected species Using minimal samples, damaged specimens, gut
contents, droppings
Research tool for improving species-level taxonomy:Associating all life history stages, gendersTesting species boundaries, finding new variants
“Triage” tool for flagging potential new species:Undescribed and cryptic species
Uses of DNA Barcodes
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Using DNA Barcodes
Establish reference library of barcodes from identified voucher
specimens
If necessary, revise species limits
Then:
Identify unknowns by searching against reference sequences
Look for matches (mismatches) against ‘library on a chip’
Before long: Analyze relative abundance in multi-species
samples
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Reactions to Barcoding: 2004 From ecologists and other users:
“This is what we need! How soon can we get started?”
From traditional taxonomists:
“Species should be based on lots of characters,
not just barcodes”
From forward-looking taxonomists:
“Using molecular data as species diagnostics isn’t new, but standardization
and broad implementation are great!”
From barcoding practitioners:
“I had my doubts at the beginning, but it really works as a tool for
identification (96% accurate in a recent mollusc paper) and it is at least as good as
traditional approaches to discovering new species.”
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What DNA Barcoding is NOT
Barcoding is not DNA taxonomy; no single gene (or character)
is adequate
Barcoding is not Tree of Life; barcode clusters are not
phylogenetic trees
Barcoding is not just COI; standardizing on one region has
benefits and limits
Molecules in taxonomy is not new; but large-scale and
standardization are new
Barcoding can help to create a 21st century research
environment for taxonomy
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What DNA Barcoding is NOT
Barcoding is not DNA taxonomy; no single gene (or character)
is adequate
Barcoding is not Tree of Life; barcode clusters are not
phylogenetic trees
Barcoding is not just COI; standardizing on one region has
benefits and limits
Molecules in taxonomy is not new; but large-scale and
standardization are new
BUT…Barcoding can help to create a 21st century research
environment for taxonomy
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Barcode Sequence
Voucher Specime
n
Species Name
Specimen
Metadata
Literature(link to content
or citation)
BARCODE Data Standard
Indices - Catalog of Life - GBIF/ECAT
Nomenclators - Zoo Record - IPNI
NameBank
Publication links - New species
GeoreferenceHabitatCharacter setsImagesBehaviorOther genes Trace
filesOther Database
sPhylogeneticPop’n GeneticsEcological
Primers
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Barcoding projects have four components:1.The Specimen Collection: Desired
Specimen must be collected for which we
want to generate DNA Barcodes.
2.The Laboratory Analysis: Barcoding
protocols as described in the Materials and
Method Section can be followed to obtain
DNA Barcode sequences from these
collected specimens.
3. The Database: One of the most
important components of the Barcode
Initiative is the construction of a public
library of species identifiers which could be
used to assign unknown specimens to known
species
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Consortium for the Barcode of Life (CBOL)
First barcoding publications in 2002
Cold Spring Harbor planning workshops in 2003
Sloan Foundation grant, launch in May 2004
Secretariat opens at Smithsonian, September 2004
First international conference February 2005
Now an international affiliation of:
130+ Members Org’s, 40 countries, 6 continents
Natural history museums, biodiversity organizations
Users: e.g., government agencies
Private sector biotech companies, database providers
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IDS – Identification System
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DNA from identified voucher
Create BARCODE reference
record
ID unknownsRefine taxonomy of
group
DNA from identified adult
voucher
Create BARCODE reference records
Associate immatures with
names
ID unknowns
Refine taxonomy of
group
DNA from unidentified
immature specimen
Repository of provisional vouchers
Add names to vouchered immatures
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DIAGNOSIS OF NEW SPECIES . TAXONOMY IS RAPIDLY ABSORBING GENETICS INTO ITS PANOPLY
OF APPROACHES. BARCODING SHOULD BE A USEFUL ADDITION TO THE EXISTING TOOLS FOR SPECIES
IDENTIFICATION, BUT IT IS NOT INTENDED TO REPLACE THEM. IN MANY GROUPS, ALPHA
TAXONOMY REQUIRES DATA FROM MORPHOLOGY, BEHAVIOR, ECOLOGY, NATURAL HISTORY, AND
GEOGRAPHIC VARIATION. THESE DATA CAN ONLY BE ENHANCED BY COMPLEMENTARY INFORMATION
REGARDING DNA SEQUENCES
RESULT
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http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An%20external%20file%20that%20holds%20a%20picture%2C%20illustration%2C%20etc.%0AObject%20name%20is%20zpq0040609290003.jpg%20%5BObject%20name%20is%20zpq0040609290003.jpg%5D&p=PMC3&id=1348015_zpq0040609290003.jpg
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References
1. Fang SG, Wan QH, Fijihara N. 2002. Formalin removal from archival tissue by critical point drying. BioTechniques 33:604-611.
2. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994 3:294-299.
3. Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270:313-322.
4. Hebert PDN, Ratnasingham S, deWaard JR. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B
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