DNA Bar-code to Distinguish the Species

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  • 1. Using mtDNA to distinguish species
    By RoyaShariati
    ANU College of Archaeology and Anthropology
    Supervisor: Prof. Colin Groves
  • 2. Carl Linnaeus 250 years ago
    Carl Linnaeus is best known for creating the system of classifying living organisms that became the international standard.
    “You say tomato, I say Lycopersiconesculentum. You say potato, I saySolanumtuberosum. But Carl Linnaeus was the real plant buff.”
    Often called the father of classification, Swedish naturalist Linnaeus established the familiar dual Latin names by which all creatures are now known.
  • 3. DNA Barcoding
    A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species
  • 4. An organism’s chromosome complement is its karyotype
  • 5.
  • 6. tRNA
    The following genes encode tRNA:
    Transport chain
    rRNA
    Mitochondrial rRNA is encoded by MT-RNR1 (12S) and MT-RNR2 (16S).
  • 7. D-Loop
    Small ribosomal RNA
    Large
    ribosomal RNA
    Cyt b
    ND1
    ND6
    COI
    COI
    ND5
    L-strand
    ND2
    H-strand
    ND4
    COI
    ND4L
    COII
    ND3
    ATPase subunit 8
    COIII
    ATPase subunit 6
    The Mitochondrial Genome
  • 8.
  • 9. Comparison of mitochondrial gene orders among typical vertebrates, E. pelecanoides, and S. lavenbergi. Five blocks of gene regions (A, tRNAGlu–tRNAGln; B, ATP8-ND3; C, tRNALeu(CUN)–ND6; D, tRNAIle–tRNALys; and E, tRNAArg–tRNASer(AGY)gene regions) retain the typical vertebrate gene order with the exception of several tRNA genes (arrowheads). The partial mtDNA sequences for four gene junctions (vertical bar: ND1-ATP8, ND3-ND5, ND6-ND2, and ND4-cyt b regions), the gene order of which greatly differ from that of other typical vertebrates, were determined for four additional E. pelecanoides individuals (data available form DDBJ/EMBL/GenBank with accession numbers AB046475–AB046490)
  • 10. Aremarkably short DNA sequence can contain more than enough information to resolve 10 or even 100 million species. For example, a 600-nucleotide segment of a protein-coding gene contains 200 nucleotides that are in the third position within a codon. At these sites, substitutions are (usually) selectively neutral and mutations accumulate through random drift. Even if a group of organisms was completely biased to either adenosine or thymine (or alternatively, to either guanosine or cytosine) at third nucleotide positions there would still be 2200 , or 1060 , possible sequences based on third-position nucleotides alone. DNA sequence analysis of a uniform target gene to enable species identification has been termed DNA bar-coding, by analogy with the Uniform Product Code barcodes on manufactured goods.
  • 11. The Phylogenetic species concept
    tokogenetic versus phylogenetic relationships
    The phylogenetic species concept
    Speciation and phylogenetic relationships
  • 12. Applied tool for identifying regulated species:
    Disease vectors, agricultural pests, invasive
    Environmental indicators, protected species
    Using minimal samples, damaged specimens, gut contents, droppings
    Research tool for improving species-level taxonomy:
    Associating all life history stages, genders
    Testing species boundaries, finding new variants
    “Triage” tool for flagging potential new species:
    Undescribed and cryptic species
    Uses of DNA Barcodes
  • 13. 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
  • 14. Reactions to Barcoding: 2004
    From ecologists and other users:“This is what we need! How soon can we get started?”
    From traditional taxonomists:“Species should be based on lots of characters, not just barcodes”
    From forward-looking taxonomists:“Using molecular data as species diagnostics isn’t new, but standardization and broad implementation are great!”
    From barcoding practitioners:“I had my doubts at the beginning, but it really works as a tool for identification (96% accurate in a recent mollusc paper) and it is at least as good as traditional approaches to discovering new species.”
  • 15. What DNA Barcoding is NOT
    Barcoding is not DNA taxonomy; no single gene (or character) is adequate
    Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees
    Barcoding is not just COI; standardizing on one region has benefits and limits
    Molecules in taxonomy is not new; but large-scale and standardization are new
    Barcoding can help to create a 21st century research environment for taxonomy
  • 16. What DNA Barcoding is NOT
    Barcoding is not DNA taxonomy; no single gene (or character) is adequate
    Barcoding is not Tree of Life; barcode clusters are not phylogenetic trees
    Barcoding is not just COI; standardizing on one region has benefits and limits
    Molecules in taxonomy is not new; but large-scale and standardization are new
    BUT…Barcoding can help to create a 21st century research environment for taxonomy
  • 17. BARCODE Data Standard
    Voucher Specimen
    Species Name
    Specimen Metadata
    Indices - Catalog of Life - GBIF/ECAT
    Nomenclators - Zoo Record - IPNI
    NameBank
    Publication links - New species
    GeoreferenceHabitatCharacter setsImagesBehaviorOther genes
    BarcodeSequence
    Trace files
    Primers
    Other Databases
    Literature(link to content or citation)
    PhylogeneticPop’n GeneticsEcological
  • 18. Barcoding projects have four components:
    1.The Specimen Collection: Desired Specimen must be collected for which we want to generate DNA Barcodes.
    2.The Laboratory Analysis: Barcoding protocols as described in the Materials and Method Section can be followed to obtain DNA Barcode sequences from these collected specimens.
    3. The Database: One of the most important components of the Barcode Initiative is the construction of a public library of species identifiers which could be used to assign unknown specimens to known species
  • 19. Consortium for the Barcode of Life (CBOL)
    First barcoding publications in 2002
    Cold Spring Harbor planning workshops in 2003
    Sloan Foundation grant, launch in May 2004
    Secretariat opens at Smithsonian, September 2004
    First international conference February 2005
    Now an international affiliation of:
    130+ Members Org’s, 40 countries, 6 continents
    Natural history museums, biodiversity organizations
    Users: e.g., government agencies
    Private sector biotech companies, database providers
  • 20.
  • 21. IDS – Identification System
  • 22. DNA from identified voucher
    Create BARCODE reference record
    ID unknowns
    Refine taxonomy of group
    DNA from unidentified immature specimen
    Create BARCODE reference records
    Associate immatures with names
    DNA from identified adult voucher
    Repository of provisional vouchers
    ID unknowns
    Add names to vouchered immatures
    Refine taxonomy of group
  • 23. Diagnosis of new species. Taxonomy is rapidly absorbing genetics into its panoply of approaches. Barcoding should be a useful addition to the existing tools for species identification, but it is not intended to replace them. In many groups, alpha taxonomy requires data from morphology, behavior, ecology, natural history, and geographic variation. These data can only be enhanced by complementary information regarding DNA sequences
    RESULT
  • 24.
  • 25. http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An%20external%20file%20that%20holds%20a%20picture%2C%20illustration%2C%20etc.%0AObject%20name%20is%20zpq0040609290003.jpg%20%5BObject%20name%20is%20zpq0040609290003.jpg%5D&p=PMC3&id=1348015_zpq0040609290003.jpg
  • 26.  References 
    1. Fang SG, Wan QH, Fijihara N. 2002. Formalin removal from archival tissue by critical point drying. BioTechniques 33:604-611.
    2. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994 3:294-299.
    3. Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London, Series B 270:313-322.
    4. Hebert PDN, Ratnasingham S, deWaard JR. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London, Series B