DNA BarcodingIn Animals

CONTENTS

• HISTORY• INTRODUCTION

• BARCODE OF LIFE COMMUNITY

• HOW BARCODING IS DONE

• WHY BARCODING IS DONE

• APPLICATIONS

• DNA BARCODING IN PAKISTAN

• BARCODE DATA ANALYSIS

• CONCLUSION

I. Taxonomic Identification• Morphological characters• Breeding• Biology• Host plants etc.

II. Molecular Identification RNA/DNA nucleotide data Protein analysis

Biological Unit Identification

Molecular Identification

Use of DNA/RNA and proteins sequence data for individual or species identification by

• Sequence comparisons • Constructing phylogenetic trees• Other available tools

Why Molecular Identification?

• Clear genetic basis• Deformed/broken samples are OK• DNA from fossilized specimens can be used• Identification of immature stages, or concealed

stages possible• Quick and unbiased• Reliable

Deoxyribonucleic Acid

• DNA is:

• The hereditary material in humans and almost all other organisms.

• Same in nearly every cell of body• Mostly located in the cell nucleus

(nuclear DNA)• Also found in small amount in the

mitochondria (mitochondrial DNA).

ghr.nlm.nih.gov

Barcodes:• Uniform Product Code• Consists of hidden language

made up of series vertical bars lines of varying width

• Used in identification by optical or laser scanner

The system of showing information in the form of barcodes

Barcoding

en.wikipedia.org/wiki/Barcode

DNA Barcoding• DNA barcoding is a standardized approach to identifying

plants and animals by minimal sequences of DNA, called DNA barcodes.

• A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species.

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

Marker

• The standard region used to generate DNA barcode

• Different in different species

• In animals it is COI or cox1 (cytochrome C oxidase 1) present in mitochondrial gene

www.dnabarcodes.org

An Internal ID System The Mitochondrial Genome

The Chosen Gene

Why COI Gene?

• Standard region • Lack insertions or deletions• Ease of isolation• Greater differences among

species• High copy number (100-10,000 )• Relatively few differences within

species• Absence of introns• Range of mutational rates in

different regions of the molecule

Standard Barcode region for Prokaryote

• SSU • lSU

Nuclear DNA - rRNA• Easily available• High copy number• High degree of

variation• Find and Amplify

• Inter Transcribed spacer

• Ribosomal genes code for rRNA• Spacer regions are transcribed but then removed• Region has restriction site polymorphism between

species

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

www.springer.com/us/book/9781617795909

History Of DNA Barcoding

• Carl Woese gave this concept first time.

• He used rRNA and molecular markers like rDNA and mtDNA to discover Archea i.e. prokaryotes and then for drawing evolutionary tree.

• In 2003, Paul Hebert, researcher at the University of Guelph in Ontario, Canada, proposed “DNA barcoding” as a way to identify species. Paul Hebert

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

Our Planet: Home to approximately 10-100 million species

Our Brain:Able to recall and recognize perhaps 1000 species

Why DNA Barcoding?

Ten Reasons For Identifying Species By DNA Barcodes

1. Works with fragments2. Works for all stages of life3. Unmasks look-alikes4. Reduces ambiguity5. Makes expertise go further6. Democratizes access7. Opens the way for an electronic handheld field guide, the Life

Barcode8. Sprouts new leaves on the tree of life9. Demonstrates value of collections10. Speeds writing the encyclopaedia of life

• Promote barcoding as

a global standard• Build participation• Working Groups• BARCODE standard• International Conferences• Increase production of public BARCODE records

Networks, Projects, Organizations

Barcode of Life Community

http://www.barcodeoflife.org/content/about/barcoding-landscape

There are four components of

barcoding projects:

1. The Specimen Collection 

2. The Laboratory Analysis 

3. The Data Base

4. The Data Analysis

Components Of Barcoding Projects

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

Method

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

DNA Barcoding Lab Scenes

Barcode Analysis

• Nucleotide identities/matches

• Distance analysis• Barcoding gap• Cluster analysis

Methods

Applications Of DNA Barcoding

Identification Of All Life Stages

www.springer.com/us/book/9781617795909

Identification Of Fragments Or Products Of Organisms

• Illegally traded bushmeat, sharkfins, skins

?

Food Safety and Accuracy

For Example

Disease Vectors

Culex pipiensWEST NILE VIRUS

Culicens incidensNON-VECTOR

Egg raft

?

?

Dengue transmission cycle West Nile Virus transmission cycle

Differentiating Cryptic Species

• Astraptes fulgerator, skipper butterfly.

• Wide-ranging; southern U.S. to northern Argentina.

• In northwestern Costa Rica, comprises complex of 10 sympatric species that are distinct in DNA sequence (COI), larval coloration, food plants, and subtle morphological traits.

Sympatric larvae of Astraptes fulgerator

Food plant: Celtis iguanaea

Food plant: Trigonia (2 species); larvae will starve if reared on plants used by other larval types.

Sisterspecies vs congeners

Panthera leo (lejon)

Panthera tigris (tiger)Motacilla flava (gulärla) Motacilla alba (sädesärla)Carabus nitens (guldlöpare) Carabus coriaceus (läderlöpare)

Sisterspecies vs congeners

Sylvia minula (ökenärtsångare)

Sylvia curucca (ärtsångare)

Sisterspecies vs congeners

Pipistrellus pipistrellus (Pipistrell)

Pipistrellus pygmaeus (dvärgfladdermus)

Identification of stomach contents, trace ecological food-chains

Other Applications

• Customs control• Invasive species control• Police • Agriculture• Forestry• Conservation• Education • Estimating species diversity• Resolving commercial disputes• Etc

www.springer.com/us/book/9781617795909 www.ijergs.org

iBOL nodes

Canadian Centre for DNA Barcoding

NIBGE

Funded by:

HEC ; iBOL

Pakistan Form Barcode Focal Point

Barcoding Species From Pakistan

http://ibol.org/pakistan-to-form-barcode-focal-point/

• About 2,000 arthropod species have been barcoded under the NIBGE-BIO collaborative project "sequencing DNA barcodes of economically important insect species of Pakistan.“

• DNA barcoding is also being done on spiders, fishes and mosquitos by different universities and researchers to identify their species.

http://www.dawn.com/news/1203605

Illegal Wildlife Trade

• Pakistan is using DNA barcoding to check illegal wild life trade.

CASE STUDIES:

• Last year, over 200 black pond turtles destined for Bangkok, were confiscated at the Karachi airport.

• Another consignment of turtles of the same species was caught at the Chinese border which was then repatriated to Pakistan.

• The DNA testing method was first tried in March 2015 when customs officials confiscated a shipment of nearly 2,000 kilograms of freshwater turtle meat worth $60 million.

• The confiscated consignment was of turtles native to the Indus River and listed in the (CITES), that restricts trade to ensure its survival.

• As many as 4,000 turtles were killed and were being shipped out of Pakistan.

Continued…

Strength• Offers alternative taxonomic identification tool for situations

in which morphology is inconclusive. • DNA barcoding can speed up identification of new species • Potential capacity for high throughput and processing large

numbers of samples• Identify specimens – a global identification system• Discover new species – aid and speed up the discovery of the

remaining biodiversityFocus on one or a small number of genes provides greater efficiency of effort.

• Cost of DNA sequencing is dropping rapidly due to technical advances.

• Once reference database is established, can be applied by non-specialist.

Thank you!