Post on 25-May-2015
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
Barcode Wales: Cod Bar Cymru
• DNA barcode the native flowering plants and conifers of Wales
• Develop applications that utilise this research platform
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
DNA extraction, amplification and sequencing
• Qiagen kits, modified for herbarium material
• rbcL: 5 primer combinations
• matK 29 primer combinations
• Macrogen Europe for Sanger sequencing
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
The workforce: research training
Analysis
• Interspecific and intraspecific divergence
• Species discrimination: BLASTn
Barcode Gap: min. interspecific p-distance > than max. intraspecific (CBOL Plant Working Group 2009)
• Test discrimination using GenBank data
• Discrimination at different spatial scales, using species distribution records
• Scripts written in Python
Recoverability
rbcL matK rbcL & matK
No. of spp. sequenced 1117 (98%) 1031 (90%) 1025 (90%)
No. of spp. with > 1 individual sequenced
1041 (91%) 814 (71%) 808 (71%)
Mean no. of individuals per spp.
3 2 2
Mode of individuals per spp. 3 3 3
Range of individuals per spp. 1 - 9 1 - 8 1 - 8
Total no. of individuals sequenced
3304 2419 2349
In total 5,723 barcode sequences obtained for the 1143 species
Fresh vs Herbarium
matK: Fresh = 5 primer combinations Herbarium = 29 primer combinations
Effect of herbarium specimen age
Spearman Rank Correlation: rbcL rho = 0.993*** matK rho = 0.986***
Intra and interspecific divergence
rbcL matK
No. of spp. showing intraspecific variation 66/1041 (6.3%)
136/814 (16.7%)
Mean intraspecific divergence: all individuals (SD) 0.0001 (0.0005)
0.0003 (0.0009)
Mean intraspecific divergence: theta(SD)
0.0001 (0.0006)
0.0004 (0.0011)
Mean coalescent depth (max. intraspecific) (SD) 0.0001 (0.0006)
0.0004 (0.0012)
Mean interspecific divergence (SD) 0.0063 (0.0069)
0.0174 (0.0231)
Using uncorrected p-distances
Relative discrimination
808 species
69
75
57
56
74
68
99
100
98
99
96
95
Recoverability and discrimination
1143 species
49
66
49
65
49
55
Testing discrimination
rbcL GenBank sequences Species % Genus % Family % Failed %
Sequences correctly identified (n = 1346)
57 93 99 1
Taxa correctly identified (n = 592)
58 94 100 0
matK GenBank sequences Species % Genus % Family % Failed %
Sequences correctly identified (n = 1380)
67 95 99 1
Taxa correctly identified (n = 533)
72 96 99 1
GenBank sequences queried against Barcode Wales database using BLASTn
rbcL discrimination
Scale 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 a barcode gap
matK discrimination
Scale 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 a barcode gap
rbcL & matK discrimination
Scale 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 a barcode gap
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
Drug discovery – prelim results
• 150 honey samples
• Agar diffusion assay,
plates with MRSA,
activity present in
some samples
• Successfully amplified
rbcL from honey
Next:
• Identify cause of antimicrobial activity
• Next gen sequencing of honey samples
DNA barcoding and phylogenetics
ML tree for rbcL, RAxML (GTR+CAT) 1000 bootstraps, on the CIPRES supercomputer cluster
Good match with APGIII.
56% of species form monophyletic groups
44% with bootstrap support >70%
DNA barcoding and phylogenetic ecology
ML tree for rbcL, threatened species traced using Mesquite
DNA barcoding and art-science
DNA barcoding and community engagement
DNA barcoding and community engagement
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