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Dna barcoding


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Dna barcoding

  1. 1. DNA Barcoding
  2. 2. Linnaeus classification vs. DNA barcoding DNA Barcodes: DNA segments that map well on taxonomy DNA Barcodes - challenge the nature of Linnaean taxonomy
  3. 3. Introduction  DNA barcoding is a technology using gene sequences to differentiate species, similar to the way retail stores rely on short, standardized barcodes to differentiate the hundreds of thousands of items they sell
  4. 4. Overview  What is DNA barcoding?  Criteria of DNA barcode sequence  Barcoding regions (Animals and Plants)  Major barcoding projects  Applications
  5. 5. Simple & Ambitious! Advocates Opposition ID all species Discover new species Speed up ID’s Revitalize biological collections Won’t work Destroy traditional systematics Service industry Pseudo taxonomy
  6. 6. What???  DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. (Hebert et al. 2003, Kumar and Jain, 2011)  Short DNA sequences are called as DNA barcodes- ranging from 400- 800 bp.
  7. 7. The birth of DNA barcoding  The use of DNA sequences for species identification has a long history (e.g. Nanney, 1982; Bartlett and Davidson, 1991; see also Sperling, 2003; Will and Rubinoff, 2004; Will et al., 2005; Cameron et al., 2006; Meier, 2008).  But it received significant attention only after it was formally proposed as ‘‘DNA barcoding’’ in 2003. (Shiyang et al., 2012)
  8. 8. The birth of DNA barcoding  University of Guelph (2003), the DNA barcoding initiative has gathered momentum, gained extensive international participation in the form of the International Barcode of Life Project (iBOL), and captured attention of the scientific community, government agencies, and the general public.  Dr. Paul D.N. Hebert- Father of DNA barcoding
  9. 9. Significant limitations – traditional approach  Phenotypic plasticity and genetic variability in the characters employed for species recognition can lead to incorrect identifications.  Overlooks morphologically cryptic taxa, which are common in many groups (Knowlton, 1993; Jarman and Elliott 2000).  Morphological keys are often effective only for a particular life stage or gender, many individuals cannot be identified.  Although modern interactive versions represent a major advance, the use of keys often demands such a high level of expertise that misdiagnoses are common (Hebert et al., 2003)
  10. 10. Successful DNA barcode a) Short enough to be quickly sequenced, b) Easily identified in all species of organisms. c) Variable enough to provide a unique sequence for each species d) Size of sequence : 600 – 700 bp e) Universality (Kress, 2006)
  11. 11. Which gene fragments? Prokaryote s • rRNA genes used for species identification Animals, Birds, Fishes • Small regions of the mitochondrial COI gene Plants • rbcL gene sequence and matK gene sequence of chloroplast Fungi • ITS region (Kumar and Jain, 2011)
  12. 12. Sequencing can be done by a single organelle region  Approximately ~650-bp sequences - C-terminal fragment of the mitochondrial gene named cytochrome oxidase subunit I (COI) has been proposed as universal marker for species level identification in several animal group.  Barcodes (mtDNA) have become one of the most contentious and animated issues in the application of genetic information to global biodiversity assessment and species identification. (Saccone et al. 1999). DNA barcoding for animals
  13. 13. DNA Barcoding in microbes  1.5 million species of fungi exist, but 10% are formally described  Variously used barcode, 400- to 600-bp region of the nuclear large ribosomal subunit , the internal transcribed spacer (ITS) gene sequences.  Partial elongation factor 1- (EF-1) sequences focused on species of Penicillium subgenus Penicillium (Trichocomaceae, Eurotiales, Eurotiomycetes, Ascomycota) (Kress et al., 2005)
  14. 14. Technical problems with defining species using mtDNA  Use of COI sequence is not appropriate for most species of plants.  Slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals (Kress et al., 2005, Hollingworth, 2008) Recombination Not enough variability to discriminate species Variable enough
  15. 15. DNA barcode for plants  Compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK– psbI spacer, and trnH–psbA spacer)  Assessments of recoverability, sequence quality, and levels of species discrimination, they recommended the 2-locus combination of rbcL+matK as the plant barcode. (CBOL Plant Working Group, 2009)
  16. 16.  The studies on Cucumis sp for the application of DNA barcode shows the possibility of discrimination at species level not the varietal level using the rbcL+matK gene barcode.
  17. 17.  The barcode clearly differentiated the species C. sativus and C. melo which will help for the future application in cucumis taxonomy and phylogeny studies
  18. 18. Proposed Plant barcoding regions - Second International Barcode of Life conference (Hollingsworth, 2008)
  19. 19. Characteristics of different markers in plant barcoding (Hollingsworth et al., 2011)
  20. 20.  Biological invasions – major threats to global biodiversity.  So they have distinguished invasive and non invasive species by using 242 samples belonging to 26 species from 10 genera (Onagraceae, Haloragaceae, Hydrocharitaceae and Cabombaceae ) of aquatic plants and assessed using the chloroplast loci trnH-psbA, matK and rbcL (Ghahramanzadeh et al., 2013)
  21. 21. Proportion of individuals successfully amplified and sequenced from two plastid loci P.A - percentage amplification; P.S.O - percentage sequences obtained; L - consensus sequence length; V.S - number of variable sites.  matK locus could not be amplified or sequenced reliably
  22. 22.  Success rates of universal primers for amplification of matK and rbcL loci in 26 different plant species (covering 14 families) from Saudi Arabia were tested.
  23. 23. Plant Specimens used
  24. 24. Contd..,  Success rate in PCR was higher for rbcL (88%) compared with matK (69%).  Universal primers of both matK and rbcL failed (primer mismatch at the annealing site) to amplify the DNA from 3 plant species - Asteraceae (Anthemis deserti, Pulicaria undulate, and Sonchus oleraceus).
  25. 25.  Samples of Phyllanthus used in raw drug trade were obtained from 25 shops in southern India.  Authenticated species were identified by using morphological keys
  26. 26. Contd..,  Validated by developing species specific DNA barcode using the chloroplast DNA region psbA-trnH, matK, trnE-trnF  matK and trnE-trnH failed to amplify for Phyllanthus sp  Six different species were identified by psbA-trnH
  27. 27. DNA Barcoding process
  28. 28. Why Barcoding? 1. Works with fragments 2. Works for all stages of life 3. Unmasks look-alikes
  29. 29. Contd.., 4. Reduce ambiguity 5. Makes expertise to go further 6. Opens the way for an electronic handheld field guide, the Life Barcoder 7. Demonstrates the value of collection 8. Speeds writing the Encyclopedia of Life (Savolainen et al., 2005)
  30. 30. Composition of the DNA Barcode Library by 2014 (iBOL Consortium, 2013)
  31. 31. Consortia  iBOL - International Barcode of Life.  CBOL – Consortium for the Barcode of Life  ECBOL - European Consortium for the Barcode of Life Databases  BOLD – Barcode of Life Database  INSDC - International Nucleotide Sequence Database Collaboration. Labs  CCBD – Canadian centre for DNA Barcoding
  32. 32. IBOL – International Barcode of Life  Largest biodiversity genomics initiative  From 25 nations Scientists are working together to construct DNA barcode reference library  First phase of operations (2010-2015) - barcode five million specimens representing 500,000 species.
  33. 33. CBOL – Consortium for the Barcode of Life  International initiative (2004)  To develop DNA barcoding as a global standard for the identification of biological species  200 Member Organizations from 50 countries ECBOL - European Consortium for the Barcode of Life  Established as part of the research infrastructure efforts of EDIT, the European Distributed Institute of Taxonomy.
  34. 34. The Barcode of Life Database (BOLD)  At the University of Guelph, public workbench for barcoding projects.  Researchers can assemble, test, and analyze their data records Sequence statistics Barcode clusters for animals: 280,889 Barcode Sequences :2,145,877 Formally described species Animals : 132,785 Plants :42,770 Fungi & Other Life: 2,502
  35. 35. International Nucleotide Sequence Database Collaboration  GenBank, EMBL, and DDBJ which comprise the International Nucleotide Sequence Database Collaboration.  Permanent public repository for barcode data records.
  36. 36. CCBD – Canadian centre for DNA Barcoding  Largest 'barcode factory', generating hundreds of thousands of data records per year  Training barcode researchers from around the world. Networks  iBOL's partners consist of national, regional and central nodes, each of which have network of projects, institutions and labs.  The first national barcode network - Canada, followed by others in the Netherlands, Mexico, Australia, and other countries.
  37. 37. Bioinformatics – major role  In case of non-availability of sequences, sequencing has to be done in vitro for which a recently developed software ecoPrimers can be helpful.  Further,basic sequence statistics computation and phylogenetic analysis can be performed by MEGA and PHYLIP/PAUP tools (Bhargava et al., 2013)
  38. 38. Ecoprimers  To identify new barcode markers in particular metabarcode markers and their associated PCR primers  Scans a large database of whole genomes to find such markers without a prior knowledge and selects highly conserved primers  Further, eco-Primers tests an amplified region for its discriminatory power between the different taxa.  ecoPrimers selects the primer pairs and evaluates their quality by optimizing two indices, Bc and Bs  Bc estimates the amplification range of a primer pair and Bs evaluates the discrimination capacity of the amplified marker.
  39. 39. Softwares available for barcoding of plants.
  40. 40. Databases available for extracting sequence and structure information of molecular markers
  41. 41. 8,000 described species • Mosquito Barcoding Initiative - MBI plans to barcode at least five specimens from 80 percent of the 3,200 known mosquito species • Bee-BOL, the Bee Barcode of Life Initiative - for all 20,000 bee species • Shark Barcode of Life project aims to barcode the 1,000 marine and 100 freshwater shark species.
  42. 42. Total specimen recorded (iBOL consortium, 2013)
  43. 43. Species Identification really matters!  Cataloguing hidden diversity  Basic research in taxonomy  Improving environmental monitoring  Controlling Agricultural Pests  Identifying Disease Vectors  Environmental Sustainability : Sustaining Natural Resources  Protecting Endangered Species  Taxonomists, ecologists, conservationists, foresters, agriculturalists, forensic scientists, customs and quarantine officer
  44. 44. Under construction
  45. 45. Conclusion  Cytochrome oxidase subunit I (COI) - universal marker for species level identification in several animal group.  In spite of failure in certain cases, the currently used barcode sequences are rbcL and matK  It will be highly useful for plant barcoding until the discovery of more efficient and robust primers for a broader coverage of plant species.
  46. 46.  Thus, there is a need for protocol development to enhance the amplification strategies for enhanced success in barcoding of plant species. Biological barcoding is likely to provide a new frame work for cataloging, categorizing and monitoring the planet’s biodiversity. In the space of few years DNA barcoding has moved from fantasy to reality.
  47. 47. Earth is home to an estimated 10 million species of plants and animals Human brain can learn to identify a few thousand species, a small fraction of life’s diversity
  48. 48. 26 - 31 October 2013 Fifth International Barcode of Life Conference. Kunming, Yunnan, China Upcoming Event
  49. 49. Discussion
  50. 50. Thank you :) Presented by: R. Veera Ranjani