Do Next Generation Sequencing approaches provide the answer for DNA barcoding of plants?

1. Do Next Generation Sequencing approaches provide the answer for DNA barcoding of plants? Hannah McPherson Marlien van der Merwe Paul Rymer Mark Edwards Maurizio Rossetto

2. Reproduced from Crisp et al. 2004 Landscape-levelstudies of the Australian flora • Species and population dynamics • Historical and current processes shaping distributions and assemblages of native trees • Using a range of molecular tools, life history traits and modelling

3. Reproduced from Crisp et al. 2004 Next generation sequencing • Exploring new molecular tools and approaches • NGS to assemble whole chloroplast genomes • Use of whole chloroplast as a barcode?

4. Reproduced from Crisp et al. 2004 Technical approach • Full genome shotgun sequencing • Solexa Illumina platform (7Gb/lane) • 8 labelled paired-end libraries multiplexed in one lane • Sub-sampled data from single lanes • No reference sequence

5. * Nightcap N * Sydney S Reproduced from Crisp et al. 2004 Sampling • 2 locations • 20 rainforest tree species • 4 individuals pooled from each species for each site

6. Reproduced from Crisp et al. 2004 reality check: sampling from rainforests • Collecting and identifying samples • Preserving leaf material • DNA extraction • 9/20 plants successfully sequenced from both North and South

7. Reproduced from Crisp et al. 2004 questions • Can we bioinformatically assemble chloroplast genomes from whole genomic shotgun sequencing without a reference? • What levels of variation do we find across a broad range of species/families? • Can we mine the data for non-chloroplast regions too? • Is whole/partial chloroplast genome sequencing a viable option for barcoding?

8. Angiosperm Phylogeny Atherospermataceae Monimiaceae Lauraceae Proteaceae Euphorbiaceae Urticaeae Malvaceae Sapindaceae, Meliaceae Pittosporaceae From Angiosperm Phylogeny Website http://www.mobot.org/MOBOT/Research/APweb/welcome.html Model organism tree

9. Brachychiton Malvaceae Gossypium, Theobroma Malvales

10. Laurales Calycanthaceae Calycanthus Atherospermataceae Doryphora Monimiaceae Wilkiea Lauraceae Cinnamomum From Angiosperm Phylogeny Website http://www.mobot.org/MOBOT/Research/APweb/welcome.html

11. assembling chloroplast genomes • Map trimmed reads to whole cp genome of closest relative available on Genbank (CLC) • Consensus of N & S • De Novo assembly (CLC and Velvet) • N & S separately • Local BLAST / cpDNA genome database • Assemble contigs to N & S reference (Geneious Pro)

12. Align with annotated

13. assembling chloroplast genomes

14. NC_008325 Daucus carota Pittosporum multiflorum Toona ciliata Synoum glandulosum NC_008334 Citrus sinensis Diploglottis cunninghamii Brachychiton acerifolius NC_008641 Gossypium barbadense Claoxylon australe NC_010433 Manihot esculenta NC_004993 Calycanthus floridus var. glaucus Cinnamomum oliveri Aligned with MAFFT RAXML tree from Cipres Sci Gateway ~40Kbp excluding gaps Wilkiea huegelii Doryphora sassafras

15. Reproduced from Crisp et al. 2004 quantifying variation • Map trimmed reads to newly constructed references (assembled contigs) • SNP detection (CLC) • SNP verification • exploring data • Sanger sequencing

16. SNP detection • Synoum glandulosum (~140Kbp) • SNPs between N and S • ~1 in 550bp • SNPs within N and S • N ~1 in 2800bp • S ~1 in 4500bp

17. SNP detection

18. Reproduced from Crisp et al. 2004 data mining • Chloroplast barcoding genes • Universal cpSSR markers • Other data BLAST • The question of coverage

19. choroplast barcoding loci Vijayan and Tsou 2010

20. Weising and Gardner 1999 universal cpSSR primers

21. Reproduced from Crisp et al. 2004 data mining • 26S coverage~35-300 • Rpb2 only returned when sequence available in same family or sister family coverage ~3-5 • Resistance genes – good return but coverage ~2-10 • Leafy – no returns

22. Reproduced from Crisp et al. 2004 data mining • Matches were good • Seem to be in more conserved bits • Single copy nuclear genes present but low coverage • Some difficulty retrieving regions depending on available data for BLAST

23. Reproduced from Crisp et al. 2004 viability for barcoding • Large portion of the chloroplast genome retrieved and easily assembled even without a reference • Potential for retrieving other regions with increased coverage/ carefully designed multiplexing

24. Reproduced from Crisp et al. 2004 to sum up the story so far • We can assemble large portions of chloroplast genomes from whole genomic shotgun sequencing even without a reference • Variation is low and varies from family to family • Single copy nuclear genes present but low coverage? • Is whole/partial chloroplast genome sequencing a viable option for barcoding?

25. Reproduced from Crisp et al. 2004 acknowledgements • Friends of the Botanic Gardens Trust • Southern Cross University – Robert Henry Nicole Rice Stirling Bowen • Evolutionary Ecology team at the Royal Botanic Gardens Sydney • Emma McIntosh Alexander Dohms Juelian Siow Ashlee Wakefield