dna barcoding: a new prespective in taxonomy


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DNA Barcoding A New Perspective in Taxonomy:

DNA Barcoding A New Perspective in Taxonomy Pawan Saini 2010-11-153 Department of Plant Breeding and Genetics 5


Contents What is DNA barcoding ? Criteria of a DNA barcode sequence Barcoding regions (Animals & Plants) CBOL and major barcoding projects Case studies Applications Strengths and weaknesses Conclusion 6

DNA Barcoding:

DNA Barcoding DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome (Mitochondria and Chloroplast) 7 (Hebert et al ., 2003; Kumar and Jain, 2011)

DNA Barcode:

DNA Barcode DNA - four nitrogen bases Adenine = Thymine = Cytosine = Guanine = 8 (Hollingsworth, 2008) Green Red Blue Black

Origin of DNA Barcode:

Origin of DNA Barcode Ribosomal RNA (small subunit 16S RNA of prokaryotes and 18S RNA of eukaryotes) Cytochrome c oxidase 1 ( CO1 gene) 9

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10 Dr. Paul D. N. Hebert Founder of DNA Barcoding

Features of DNA Barcode Gene:

Features of DNA Barcode Gene Low intra-species variability Species level genetic variability and divergence Short sequence length Size of sequence : 600-700 bp Universality 11 (Kumar and Jain, 2011)

Barcoding Region:

Barcoding Region 12 (Kumar and Jain, 2011)

rRNA Genes:

rRNA Genes 13 Carl Woese (1990) Archaea , Bacteria & Eukaryota

Mitochondrial Gene Sequence:

Mitochondrial Gene Sequence 14 Small region of mitochondrial CO1 gene is used in animals for identification. (Hebert et al., 2003)

Identification of Plants :

Identification of Plants Use of the CO1 sequence “is not appropriate for most species of plants” Slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals Plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants (Kress et al ., 2005) 15

Chloroplast Genome Sequences:

Chloroplast Genome Sequences 16 CBOL-Plant Working Group: rbcL and matK standard two-locus barcode for plants (Ratnasingham and Hebert, 2007)

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17 How is DNA Barcoding Done?

Steps involved in DNA Barcoding:

Steps involved in DNA Barcoding 18

Steps involved in DNA Barcoding contd…:

Steps involved in DNA Barcoding contd… 19

DNA Barcoding Organizations:

DNA Barcoding Organizations 20

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CBOL’s Major Project (Source : http://www.barcoding.si.edu ) 21

CBOL-Initiated Projects:

CBOL-Initiated Projects Fish Barcode of Life (FISH-BOL) 30,000 marine/freshwater species by 2010 All Birds Barcoding Initiative (ABBI) 10,000 species by 2010 Tephritid Barcoding Initiative (TBI) 2,000 pest/beneficial species and relatives by 2010 Mosquitoes Barcoding Initiative (MBI) 3,300 species (completed -2009) 22

Barcode of Life Data System (BOLD):

Barcode of Life Data System (BOLD) Aids the collection, management, analysis, and the use of DNA barcodes Repository for the specimen and sequence records Aids the assembly of barcode data and maintains records 23

International Barcode of Life Project (iBOL):

International Barcode of Life Project (iBOL) Activated in October, 2010 Headquarter: Canada First phase: 2010-2015 Acquisition of DNA barcode records of 5 million specimen representing 500,000 species Release each barcode record in two phases Website: http://www.ibolproject.org 24

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25 iBOL’s Compaigns Formicidae Barcode of Life Bee Barcode of Life Initiative Trichoptera Barcode of Life Coral Reef Barcode of Life All Fungi Barcoding Shark Barcode of Liife Health BOL Lepidoptera Barcode of Life The Mammal Barcode of Life Marine Barcode of Life Polar Barcode of Life Sponge Barcoding Project

Canadian Center for DNA Barcoding (CCDB):

Canadian Center for DNA Barcoding (CCDB) Biodiversity Institute of Ontario (BIO) - University of Guelph Member of CBOL Laboratory protocols Maintain Barcode of Life Data System 26

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27 Why Barcoding Life?

Ten Reasons:

Ten Reasons Works with fragments Works for all stages of life Unmasks look-alikes 28

Ten Reasons contd…:

Ten Reasons contd… Reduce ambiguity Makes expertise go further Democratizes access 29 Known Species: >150,000 Flies, Mosquitoes, >250,000 Flowering Plants, >300,000 Beetles, >30,000 Crabs, Lobsters

Ten Reasons contd…:

Opens the way for an electronic handheld field guide, the Life Barcoder Sprouts new leaves on the tree of life Demonstrates value of collections Speeds writing the encyclopedia of life 30 Ten Reasons contd…

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31 Current Tech.

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Future Tech. 32

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33 Barcode Count

Kingdom of Life Barcoded:

Kingdom of Life Barcoded 34 15,64,478 11,19,458 95,595 http://www.boldsystems.org/taxbrowser_root.php?PHPSESSID=845ba099cb63fd89eeebc44e6f77e6f9

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35 Case Studies

CASE 1: Delimiting Cryptic Species :

CASE 1: Delimiting Cryptic Species Neotropical skipper Butterfly, Astraptes species 10 different species in North-Western Costa Rica 36 (Hebert et al ., 2004)

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Last- instar caterpillars of ten species in A. fulgerator complex 37


CO1 gene – 484 specimens from North-Western Costa Rica Ten species in one: Astraptes fulgerator 38 Conclusion (Hebert et al ., 2004)

CASE 2: DNA Barcodes for Phyllanthus species:

CASE 2: DNA Barcodes for Phyllanthus species Phyllanthus ( Euphorbiaceae ) medicinally important Tropical & subtropical regions In India : 41 species 13 Herbs, 3 Trees and 25 Shrubs 39

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Phyllanthus niruri Phyllanthus amarus Phyllanthus debilis Phyllanthus fraternus 40

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Phyllanthus sp.collections from India 41


P. niruri P. amarus Not present in India Conclusion 42

CASE 3: Identification of Dalbergia species from Western Ghats of India :

CASE 3: Identification of Dalbergia species from Western Ghats of India Leaf sample : 15 accessions 37 primer pairs specific Chloroplast genes - matK and rbcL genes 43 (Bhagwat et al ., 2010)


Conclusion 15 accessions categorized as 6 divergent species D. melanoxylon D. candenatensis D. rubiginosa D. latifolia D. volubilis D. paniculata 44

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45 Life Forms Gene Sequence & Specimens Result Reference Orchid matK 1600 sp. Identified Science Daily, Feb. 7, 2008 Indian Mosquitoes CO1 gene & 520 specimen 150 sp. Identified Pradeep Kumar et al ., 2008 Canadian Mosquitoes CO1 gene of 617 bp fragment 37 sp. Identified Hebert et al ., 2006 True Bug CO1 gene & 110 specimens 30 morphologically distinct bug sp. Gaikwad et al ., 2009 Butterflies & Skippers CO1 gene 100 sp. Identified Gaikwad et al ., 2009 Cockroach CO1 gene & 217 specimens Differ 4% from other cockroach sp. Tan & Cost, 2010 Amphibian CO1, 16S, 12S rRNA & Cyt b gene and 20 specimens CO1 & 16S RNA sequence useful Meenakshi et al. , 2010 Snakes CO1 & Dried Venom Pook and McEwing, 2005 Crustaceans Echinogammarus ischnus CO1 gene Adrians Radulovici , 2011 Endangered Whale Rhincodon typus CO1 gene, 16S rRNA & Cyt b genes NBFGR, Lucknow


Applications Identifying disease vector Identifying agricultural pests 46

Applications contd… :

47 Rhincodon typus Duikers, Spiral-horned antelope, Red river hogs, Old world monkeys, Alligators and Crocodiles (Tropical Africa and America) Protecting endangered sp. Applications contd…

Applications contd…:

Phyllanthus sp. Tracking adulteration in products Applications contd… 48

Applications contd…:

49 Monitoring water quality Applications contd…


Strengths Alternative taxonomic identification tool Identification of new species Works for all life stages Reveal cryptic, undescribed species 50

Weaknesses :

Weaknesses No universal DNA barcode gene Difficult to resolve recently diverged species Identifies inter-specific genetic variation only Single gene approach 51


Conclusion DNA barcoding has emerged and established itself as an important tool for species-identification and phylogenetic studies. Apart from this, it has proved useful in protecting endangered animals, identifying agricultural pests and disease vectors, tracking adulteration in products and sustaining environment. 52

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