DNA Barcoding

Kandhan. S, M. Tech (Biotechnology) PSG College of Technology

Barcodes

• Consists of hidden language made up of series vertical bars lines of varying width

• Used in identification by optical or laser scanner

http://www.barcodesinc.com/generator/index.php

Aztec code

Cronto Sign

Digital matrix

EZ code

Nexcode

High capacity color code

Data matrix

Maxi code

PDF 417

SPARQ Code

Qode

QR Code

Shot code

What is this ?

DNA barcoding is a standardized approach

to identifying plants and animals by minimal

sequences of DNA, called DNA barcodes.

DNA barcode - short gene sequences taken from a standardized portion of the genome that is used to identify species

DNA Barcoding

How it all started in 2003

Propose a CO1-based (~650bp of the 5’ end)global identification system of animals, and show the success (96.4-100%) of assigningtest specimens to the correct phyla, order and species(Lepidoptera from Guelph) through a CO1-profile.

98% of congeneric species in 11 animal phyla showed>2% sequence divergence in CO1

Banbury Center, Cold Spring HarborMarch 2003, September 2003

Proc Royal Soc London B 2003

http:www.barcoding.Si.edu

BIG challenge: 1.9M species

1 square = 10,000 species Other plants

Collection andDatabasing

Central Nodes

Developing Nodes

Regional Nodes

Curation andIdentification

Sequencing MirroredDatabases

Data Analysisand Access

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.

CBOL Member Organizations: 2009

• 200+ Member organizations, 50 countries

• 35+ Member organizations from 20+ developing countries

WHERE I’M

Nucleus

Standard DNA barcode for animals

Animal Cell

Mitochondrion

DNA

mtDNA

D-Loop

ND5

H-strand

ND4

ND4L

ND3COIII

L-strand

ND6

ND2

ND1

COII

Small ribosomal RNA

ATPase subunit 8

ATPase subunit 6

Cytochrome b

COICOI

The Mitochondrial Genome

5’ cytochrome c oxidase subunit I distinguishes 95% species

(648 bp)

15,000 Base pair

Herbert et al,2003

Why COI ?

standard region

lack insertions or deletions

Protein closely-related species.

Greater differences among species

Copy number. (100-10,000 )

Relatively few differences within species

Absence of IntronsHerbert et al,2003

Barcode regions of plant

Nuclear DNAITS Plastid DNA loci

DiscriminationUniversalityRobustness

Plant Cell

Mat Krbc LtrnH-psbAatpF-Fpsb k1rpo C1rpo Brpo C2ndh Jtrn Lycf 5acc D

100,000 Base pair

• DiscriminationBarcoding regions must be different for each species. Ideally you are looking for a single DNA locus which differs in each species.

• UniversalitySince barcoding protocols (typically) amplify a region of DNA by PCR, you need primers that will amplify consistently.

• RobustnessSince barcoding protocols (typically) amplify a region of DNA by PCR, also need to select a locus that amplifies reliably, and sequences well.

% species discriminated

• 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

Based on recommendations by a barcoding consortium (Consortium forthe Barcode of Life, plant working group) the chloroplast genes rbcL andmatK universal plant barcodes.

– rbcL – chloroplast ribulose-1,5-bisphosphate carboxylate

– matK – chloroplast maturase K

Ratnasingham and Herbert, 2007

Why not COI Sequence divergentIncorporation of forgein genesFrequent transfer of some gene to Nucler gene0

Then plastidShortEasily alienableEasily recoverable from even herbarium sample Maternal interitence

mat K

rbc L

Comparison of Plant Barcode region

Standard Barcode region for Prokaryote

SSU lSU

Nuclear DNA - rRNA

Easily availableHigh copy numberHigh degree of variationFind and Amplify

Inter Transcribed spacer

Ribosomal genes code for rRNA

Spacer regions are transcribed but then removed

Region has restriction site polymorphism between species

Kress et al,2007 Chase et al ,2005 Conrad L. schock at al , 2012

Why Barcoding?

1)Works with fragments

2) Works for all stages of life

3)Unmasks look-alikes

4) Reduce ambiguity

5) Expertise to go further

6)Democratize access

7)Opens the way for an electronic handheld field guide, the life barcoder

8)Sprouts new leaves on the tree of life

9) Demonstrates the value of collection

10) Speed writing the life of encylcopedia(http://eol.org/)

How the DNA Barcoding done

Step Involved in it

Sample collection & recording

http://www.barcodeoflife.org/content/about/what-dna-barcoding

Sample collection

Biogeography classification

Expert Taxonomist

•Museum•Botanical garden

• Herbarium preparation

Wet lab Dry lab

DNA extraction, amplification & Sequencing

Amplification

Sequencing

Doyle and Doyle ,1998

Sanger, F. & Coulson, AR (1975)

Mullis et al ,1985

Sequence Align

UPLOAD IN BOLD AND OTHER DATABASE

CONVERT TO BARCODE

http://biorad-ads.com/DNABarcodeWeb/Bio-rad barcode generator

Program behind DNA Barcode generator

• Luca &Howell

• Python 2.5 to 2.6

• shell window

Hollingworth,2008

Current Norm: High throughputLarge labs, hundreds of samples per day

ABI 3100 capillary automated sequencer

Large capacity PCR and sequencing reactions

Emerging Norm: Table-top LabsFaster, more portable: Hundreds of samples per hour

Integrated DNA microchips Table-top microfluidic systems

Future in 20??

• Data in seconds to minutes

• Pennies per sample

• Link to reference database

• A taxonomic GPS

• Usable by non-specialists

Advantage Of DNA barcoding

• Protection of Endangered Species ( Conservation)• Tracking adulterations• Identifying Agricultural pest• Water quality testing• Identification of all life stages, eggs, larvae, nymphs, pupa, adults• Identification of fragments or products of organisms• Identification of stomach contents, trace ecological food-chains• Food control• Customs control• Invasive species control• Disease vector control• Police • Agriculture• Forestry• Education• Etc

Strength VS Weakness

• Alternative taxonomic Identification tool

• Identification of new species

• Work for all life stages

• Reveal undescribed species

• No universal DNA barcode region

• Difficult to resolve recently diverged species

• Identifies Inter-specific genetic variation only

• Single approach

Conclusion

DNA barcoding has emerged and established itself as a important tool for species-identification and phylogenetics studies

it has proved useful in protecting Endangered species, identifying agricultural pests and disease vectors, tracking adulteration in products and sustaining environment

Case studies

Hebert et al,2007

R.Sriama and Uma Shaanker,

Bha

Case studies

CONSERVE OUR ECOSYSTEM

This is where we stand today!

Why are u waiting for

Come out and play with DNA Bar-codingto conserve the environment

References• Smith, A., D.H. Janzen and P.D.N. Hebert. 2006. DNA barcodes reveal cryptic host-spceificity within the presumed

polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae). Proc. Natl. Acad. Sci. USA 103: 3657-3662.

• Hajibabaei, M., D.H. Janzen, J.M. Burns, W. Hallwachs and P.D.N. Hebert. 2006. DNA barcodes distinguish species of tropical Lepidoptera. Proc. Nat. Acad. Sci. USA: 103: 968-971.

• Ward, R.D., T.S. Zemlak, B.H. Innes, P.R. Last and P.D.N. Hebert. 2005. DNA barcoding Australia 's fish species. Phil. Trans. R. Soc. Lond. 360: 1847-1857.

• Hebert, P.D.N. and T.R. Gregory. 2005. The promise of DNA barcoding for taxonomy. System. Biol. 54: 852-859.

• Barrett, R.D.H. and P.D.N. Hebert. 2005. Identifying spiders through DNA barcodes. Can. J. Zool. 83: 481-491.

• Lambert, D.M., A. Baker, L. Huynen, O. Haddrath, P.D.N. Hebert and C.D. Millar. 2005. Is a large-scale DNA-based inventory of ancient life possible? J. Heredity: 96: 1-6.

• Hebert, P.D.N., M.Y. Stoeckle, T.S. Zemlak and C.M. Francis. 2004. Identification of birds through DNA barcodes. PLoS Biology 2: 1657-1663.

• Hebert, P.D.N., E.H. Penton, J. Burns, D.J. Janzen and W. Hallwachs. 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly, Astraptes fulgerator . Proc. Natl. Acad. Sci. USA: 101: 14812-14817.

• Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. deWaard. 2003. Biological identifications through DNA barcodes. Proc. Roy. Soc. Lond. Ser. B: 270: 313-321.

• Hebert, P.D.N., J.D.S. Witt and S.J. Adamowicz. 2003. Phylogeographic patterning in Daphnia ambigua: regional divergence and intercontinental cohesion. Limnol. Oceanograph. 48: 261-268.

• Witt, J.D.S., D.W. Blinn and P.D.N. Hebert. 2003. The recent evolutionary origin of the phenotypically novel amphipod, Hyalella montezuma offers an ecological explanation for morphological stasis in a closely allied species complex. Mol. Ecol. 12: 405-413.

• Derry, A.M., P.D.N. Hebert and E.E. Prepas. 2003. Evolution of rotifers in saline and subsaline lakes: a molecular phylogenetic approach. Limnol. Oceanograph. 48: 675-685.

• Gregory, T.R. and P.D.N. Hebert. 2002. Genome-size estimates for some oligochaete annelids. Can. J. Zool. 80: 1485-1489.

• Sutton, R.A. and P.D.N. Hebert. 2002. Patterns of sequence divergence in daphniid hemoglobin genes. J. Mol. Evol. 55: 375-385.

• Adamowicz, S.J., T.R. Gregory, M.C. Marinone and P.D.N. Hebert. 2002. New insights into the distribution of polyploid Daphnia : the Holarctic revisited and Argentina explored. Mol. Ecol.: 11: 1209-1217.

• Hardie, D.C., T.R. Gregory and P.D.N. Hebert. 2002. From pixels to picograms: a beginner’s guide to genome quantification by Feulgen image analysis densitometry. J. Histochem. and Cytochem. 50: 735-749.

• Hebert, P.D.N., E.A. Remigio, J.K. Colbourne, D.J. Taylor and C.C. Wilson. 2002. Accelerated molecular evolution in halophilic crustaceans. Evolution 56: 909-926.

• Cristescu, M.E.A. and P.D.N. Hebert. 2002. Phylogeny and adaptive radiation in the Onychopoda(Crustacea: Cladocera): evidence from multiple gene sequences. J. Evol. Biol. 15: 838-849.

• Cywinska, A. and P.D.N. Hebert. 2002. Origins of clonal diversity in the hypervariable asexual ostracodCypridopsis vidua. J. Evol. Biol. 15: 134-145.

• Hebert, P.D.N. and M.E.A. Cristescu. 2002. Genetic perspectives on invasions: the case of the Cladocera. Can. J. Fish. Aquat. Sci. 59: 1229-1234.

• Remigio, E.A., D.A.W. Lepitzki, J.S. Lee and P.D.N. Hebert. 2001. Molecular systematic relationships and evidence for a recent origin of the thermal spring endemic snails Physella johnsoni and Physella wrighti(Pulmorata: Physidae). Can. J. Zool. 79: 1941-1950.

• Remigio, E.A., P.D.N. Hebert and A. Savage. 2001. Phylogenetic relationships and remarkable radiation in Parartemia (Crustacea: Anostraca), the endemic brine shrimp of Australia: evidence from mitochondrial DNA sequences. Biol. J. Linn. Soc. 74: 59-71.

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