Natasha de Vere - Plants Plenary

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Natasha de Vere - Plants Plenary

  1. 1. Barcode Wales / Codbar Cymru: A complete DNA Barcode Dataset of a Nation’s Native Flowering Plants: Creation, Applications and Public Engagement Natasha de Vere, Tim Rich, Col Ford, Sarah Trinder, Charlie Long, Chris Moore, Danielle Satterthwaite, Helena Davies, Joe Moughan, Addie Griffith, Laura Jones, Joel Allainguillaume, Mike Wilkinson, Tatiana Tatarinova, Hannah Garbett, Les Baillie, Jenny Hawkins
  2. 2. Barcode Wales: Cod Bar Cymru • DNA barcode the native flowering plants and conifers of Wales • Develop applications that utilise this research platform
  3. 3. Sample collection• 1143 native flowering plants and conifers• 455 genera, 95 families, 34 orders• 4272 individuals sampled, 3637 herbarium, 635 freshly collected• All specimens verified by taxonomic expert• Herbarium vouchers and full collection details for all samples
  4. 4. DNA extraction, amplification and sequencing• Qiagen kits, modified for herbarium material• rbcL: 5 primer combinations• matK 29 primer combinations• Macrogen Europe for Sanger sequencing
  5. 5. Sequence editing and multiple alignment• Sequencher 4.9. contig assembly and manual editing• rbcL alignment: MUSCLE• matK alignment: Transalign and Geneious Pro 5.4.4• Sequences BOLD and Genbank
  6. 6. The workforce: research training
  7. 7. Analysis• Interspecific and intraspecific divergence• Species discrimination: BLASTn Barcode Gap: min. interspecific p-distance > • Discrimination at than max. intraspecific different spatial (CBOL Plant Working scales, using species Group 2009) distribution records• Test discrimination using • Scripts written in GenBank data Python
  8. 8. Recoverability rbcL matK rbcL & matKNo. of spp. sequenced 1117 (98%) 1031 (90%) 1025 (90%)No. of spp. with > 1 individual 1041 (91%) 814 (71%) 808 (71%)sequencedMean no. of individuals per 3 2 2spp.Mode of individuals per spp. 3 3 3Range of individuals per spp. 1-9 1-8 1-8Total no. of individuals 3304 2419 2349sequenced In total 5,723 barcode sequences obtained for the 1143 species
  9. 9. Fresh vs Herbarium matK: Fresh = 5 primer combinations Herbarium = 29 primer combinations
  10. 10. Effect of herbarium specimen age Spearman Rank Correlation: rbcL rho = 0.993*** matK rho = 0.986***
  11. 11. Intra and interspecific divergence rbcL matKNo. of spp. showing intraspecific variation 66/1041 136/814 (6.3%) (16.7%)Mean intraspecific divergence: all individuals (SD) 0.0001 0.0003 (0.0005) (0.0009)Mean intraspecific divergence: theta(SD) 0.0001 0.0004 (0.0006) (0.0011)Mean coalescent depth (max. intraspecific) (SD) 0.0001 0.0004 (0.0006) (0.0012)Mean interspecific divergence (SD) 0.0063 0.0174 (0.0069) (0.0231)Using uncorrected p-distances
  12. 12. Relative discrimination 69 99 75 100 68 98 74 99 56 96 57 95808 species
  13. 13. Recoverability and discrimination 49 66 49 65 49 551143 species
  14. 14. Testing discriminationrbcL GenBank sequences Species % Genus % Family % Failed %Sequences correctly identified 57 93 99 1(n = 1346)Taxa correctly identified 58 94 100 0(n = 592)matK GenBank sequences Species % Genus % Family % Failed %Sequences correctly identified 67 95 99 1(n = 1380)Taxa correctly identified 72 96 99 1(n = 533) GenBank sequences queried against Barcode Wales database using BLASTn
  15. 15. rbcL discriminationScale n Mean discrimination % (SD)10x10 km 253 72 (4)2x2 km 1116 90 (9)Species lists generated for each square,discrimination assessed by presence of abarcode gap
  16. 16. matK discriminationScale n Mean discrimination % (SD)10x10 km 253 81 (3)2x2 km 1116 93 (7)Species lists generated for each square,discrimination assessed by presence of abarcode gap
  17. 17. rbcL & matK discriminationScale n Mean discrimination % (SD)10x10 km 253 82 (3)2x2 km 1116 93 (6)Species lists generated for each square,discrimination assessed by presence of abarcode gap
  18. 18. DNA barcoding and drug discovery• Collect wildflower honey from throughout UK• Test antibacterial properties of honey against MRSA and Clostridium difficile• DNA barcode honey• Identify plant derived phytochemicals• New drug discovery routes
  19. 19. Drug discovery – prelim results• 150 honey samples• Agar diffusion assay, plates with MRSA, activity present in some samples• Successfully amplified rbcL from honeyNext:• Identify cause of antimicrobial activity• Next gen sequencing of honey samples
  20. 20. DNA barcoding and phylogenetics Good matchML tree for rbcL, with APGIII.RAxML (GTR+CAT) 56% of species form1000 bootstraps, on the monophyletic groupsCIPRES supercomputer 44% with bootstrap supportcluster >70%
  21. 21. DNA barcoding and phylogenetic ecology ML tree for rbcL, threatened species traced using Mesquite
  22. 22. DNA barcoding and art-science
  23. 23. DNA barcoding and community engagement
  24. 24. DNA barcoding and community engagement
  25. 25. Thank you!• Funding from Welsh Government, National Botanic Garden of Wales, National Museum Wales, Countryside Council for Wales, Spirent Communications plc• Sponsorship from the people of Wales• www.gardenofwales.org. uk• Science at the Garden of Wales on facebook

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