Making Barcoding work in Plants

1. Making Barcoding work in Plants David Erickson Department of Botany Smithsonian Institution ericksond@si.edu

2. Advertisement Methods in Molecular Biology DNA BARCODES: METHODS AND PROTOCOLS Humana Press • Out in Spring 2012 David Erickson - Plant Barcoding

3. Processes • DNA extraction • Archival • PCR • Sequencing • Analysis David Erickson - Plant Barcoding

4. Processes • DNA extraction • Disruption • LESS tissue is better: 0.05g dry = 2 hole punches • We use either 96 well Polypropylene plate (=$5 each) with 5mm stainless ball bearing – grind with tissue-lyser • Costar brand plates with 8 strip caps from Qiagen; beads from Qiagen • Single tube with lysing matrix ($2/sample) – forensic samples • http://www.qbiogene.com/fastprep/lysing_tubes.shtml • Extraction • GUSCN+Silica membrane • Ivanova, Fazekas, Hebert (2008) Plant Mol Biol Reporter • (DOI 10.1007/s11105-008-0029-4) • Archival • PCR • Sequencing • Analysis David Erickson - Plant Barcoding

5. Processes • DNA extraction • DNA Archival • pH matters….8.0 or higher is better using Tris-HCl • DNA is an acid so it will change the pH of the solution as you mix the Tris with your DNA • check it if you want it to last? • Make a working stock and an Archival stock • After 5-6 rounds of freeze-thaw, DNA starts to fail • PCR • Sequencing • Analysis David Erickson - Plant Barcoding

6. Processes • DNA extraction • DNA Archival • PCR • Dilution is the solution to Pollution • Dilute DNA 10x-100x before PCR • When PCR fails, we re-dilute 10x and try again • Taq does not matter much • Exception in repeat regions (=poly A), • then use phusion taq (New England Biolabs) • (Fazekas et al. 2010. Biotechniques 48:277-285) • Use 30 cycles – • PCR propagates error – sequence reactions don’t • Sequencing • Analysis David Erickson - Plant Barcoding

7. Processes • DNA extraction • DNA Archival • PCR • Sequencing • Lots of cycles for coding genes help (40+ cycles are good) • Lots of cycles are less good for non-coding DNA (psbA-trnH) • Use ExoSap mixture for purification of PCR (0.3ul ExoSap + 10ul PCR) • Typical Seq reaction in 10ul: 0.5ul BD, 1.75ul 5X Seq buffer, 5ul PCR, 0.02ul 100uM primer, 2.73 H2O • Analysis David Erickson - Plant Barcoding

8. Processes • DNA extraction • DNA Archival • PCR • Sequencing • Analysis • Its Complicated… • Ecological Forensics • Community phylogenetics • Meta-Genomics/Barcoding David Erickson - Plant Barcoding

9. Diet Analysis

10. Community Phylogenetics

11. Barcodes vs. Phylomatic Community Phylogenetics Barcode Phylogeny: Phylogenetically clustered = Low Plateau and Slope Habitats Phylogenetically Over-dispersed = High Plateau, Mixed and Young Habitats = Phylogenetically Random = Stream and Swamp Habitats Phylomatic Phylogeny: Phylogenetically clustered = High Plateau, Low Plateau and Young Habitats Phylogenetically Over-dispersed = Swamp and Slope Habitats Phylogenetically Random = Stream and Mixed Habitats 50-ha Forest Dynamics Plot on BCI, Panama (281 species): Community Phylogeny Kress et al. 2009

12. Diet Analysis