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Standard land plant barcoding requires a multi loci approach?

Standard land plant barcoding requires a multi loci approach?. Robyn Cowan. Sujeevan Ratnasingham. Peter Gasson. Mitochondrial DNA in land plants: undergoes rearrangements transfer of genes to nucleus incorporation of foreign genes substitution rates are VERY slow

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Standard land plant barcoding requires a multi loci approach?

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  1. Standard land plant barcoding requires a multi loci approach? Robyn Cowan Sujeevan Ratnasingham Peter Gasson

  2. Mitochondrial DNA in land plants: • undergoes rearrangements • transfer of genes to nucleus • incorporation of foreign genes • substitution rates are VERY slow • (with a few notable exceptions e.g. Plantago, Cho & al.)

  3. Partners Instituto de Biologia UNAM,Mexico – Gerardo Salazar Imperial College, UK - Timothy Barraclough Natural History Museum, Denmark - Gitte Petersen Natural History Museum (London), UK - Mark Carine New York Botanical Garden, USA - Kenneth Cameron Royal Botanic Garden Edinburgh, UK - Peter Hollingsworth Royal Botanic Gardens, Kew, UK - Mark Chase South African National Biodiversity Institute - Ferozah Conrad University of Cape Town, South Africa - Terry Hedderson U. Estadual de Feira de Santana, Brazil - Cássio van den Berg Universidad de los Andes - Santiago Madriñán U. of Wales Aberystwyth UK (previously University of Reading, UK) - Mike Wilkinson Alfred P. Sloan Foundation Gordon and Betty Moore Foundation

  4. To develop a universal approach to barcoding of all landplants • Phase 1: primer development (protein motifs); complete genome sequences; problems: ferns; 46 pairs of sister taxa from mosses, liverworts, hornworts, lycopods, ferns/fern allies, gymnosperms, angiosperms – percent PCR success & percent polymorphisms • Phase 2: in depth trials of six markers identified in phase I on a range of well sampled taxa from across land plants

  5. So what are the characteristics of a good barcode? • High inter-specific, low intra-specific sequence divergence • Universal amplification/sequencing with standard primers • Technically simple to sequence • Short enough to sequence in one reaction • Easily alignable (few insertions/deletions) • Readily recoverable from museum or herbarium samples and other degraded samples **Universal + Variable**

  6. What sort of marker should we use? • Mitochondrial DNA • Plastid • Ribosomal DNA (ITS) • Low-copy nuclear DNA (protein coding) • Length variable ? • Single loci • Multiple loci (one genomic compartment) ? • Multiple loci (two genomic compartments) ?

  7. Advantages of plastid DNA (hence its use in phylogenetics) • Monomorphic (separation of different copies not required in hybrids) • High copy number (can even be amplified from highly degraded DNA) • Potentially highly diagnostic (in spite of its reputation to the contrary) However, will not detect hybrids, introgression, paralogy

  8. Coding or non-coding? Non-coding regions: sometimes more variable microsatellites difficult to sequence through numerous indels-impossible to align, length variable cannot translate to check for pseudoproteins and to aid aligment sometimes contain rearrangements and coding insertions (character based identification)

  9. trnH-psbA spacer region

  10. Criterion for locus selection • Species level sequence divergence • Appropriate length (200-800bp) • Presence of conserved primer target sites • At least 200bp exon sequence

  11. Our Strategy • Identify suitable loci on the basis of in silico screens using Nicotiana cp sequence • Design universal primers (sets of 4 primers/locus) using amino acid and nucleic acid sequence data • Perform initial screen for universality (1 primer pair) • Screen for sequence variation using diverse species pairs • Improve universality (e.g. use all primer combinations) • Use statistical modelling approaches to identify optimal primer sets

  12. Standard PCR Recipe • NH4 x1 • Mg2+ 1.5mM • dNTPs 0.2mM • FW test primer 1M • RE test primer 1M • Taq DNA polymerase 2 units • BSA 0.1mg/ml • Template 40ng • Water to 20l

  13. Results of First PCR

  14. Number of Variable Sites

  15. Trial regions Selected seven genes that represent the different levels of universality and variability. Blue= high, green = medium, yellow= low.

  16. Trial groups Asterella Anastrophyllum-Barbilophozia Tortella Bryum Triquetrella Homalothecim Tortella Elaphoglossum Asplenium Equisetum Cupressus Pinus Araucaria Labordia Conostylis Dactylorhiza maculata/incarnata Mimetes Inga Hordeum Scalesia Crocus Laelia Cattleya Mormodes Deiregyne Lauraceae

  17. Summary

  18. Trial regions Selected seven genes that represent the different levels of universality and variability. Blue= high, green = medium, yellow= low.

  19. Agavaceae X 22 sp. Crocus X 9 sp. Aulosepalum X 8 sp.(?all) Cattleya X 30sp.(2 clades approx 43 sp.) Dactylorhiza 15 sp. (species complex) Sophrinitis 27 sp. (approx. 37 sp.) Scalesia X 4 (species complex) Conostylis X 42 (?all) Equisetum X 14 Pinus X 66 Hordeum X 10 Lauraceae

  20. Samples with unique ‘barcode’

  21. Users of DNA Barcoding: ‘The Traffic Light approach’ Green - non-problematic taxa (current markers appropriate, silver standard) Orange - need for gold standard (polyploidy, introgression, paralogy) Red - barcoding needs investigation, species complex, etc

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