View
241
Download
4
Category
Tags:
Preview:
Citation preview
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.
Save NatureConserve the ecosystem
Recommended