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Hannah McPherson - Plants Plenary


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Hannah McPherson - Plants Plenary

  1. 1. Do Next Generation Sequencingapproaches provide the answer for DNA barcoding of plants?Hannah McPherson Marlien van der MerwePaul Rymer Mark Edwards Maurizio Rossetto
  2. 2. Landscape-level studies 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 Reproduced from Crisp et al. 2004
  3. 3. Next generation sequencing Exploring new molecular tools and approaches NGS to assemble whole chloroplast genomes Use of whole chloroplast as a barcode? Reproduced from Crisp et al. 2004
  4. 4. 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 Reproduced from Crisp et al. 2004
  5. 5. Sampling  2 locations Nightcap N *  20 rainforest tree species  4 individualsSydney S * pooled from each species for each site Reproduced from Crisp et al. 2004
  6. 6. reality check: sampling from rainforests Collecting and identifying samples Preserving leaf material DNA extraction 9/20 plants successfully sequenced from both North and South Reproduced from Crisp et al. 2004
  7. 7. 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? Reproduced from Crisp et al. 2004
  8. 8. Angiosperm Phylogeny Model organism tree Atherospermataceae Monimiaceae Lauraceae Proteaceae Euphorbiaceae Urticaeae Malvaceae Sapindaceae, Meliaceae PittosporaceaeFrom Angiosperm Phylogeny Website
  9. 9. Malvales Brachychiton Malvaceae Gossypium, Theobroma
  10. 10. Laurales Calycanthaceae Calycanthus Atherospermataceae Doryphora Monimiaceae Wilkiea Lauraceae Cinnamomum
  11. 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. 12. Align with annotated
  13. 13. assembling chloroplast genomes17000015000013000011000090000 Toona Doryphora Claoxylon Brachychiton Synoum Pittosporum Cinnamomum Diploglottis Wilkiea Length/closest cpDNA ref Length mapped cpDNA Length assembled contigs
  14. 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 Wilkiea huegelii Aligned with MAFFT RAXML tree fromDoryphora sassafras Cipres Sci Gateway ~40Kbp excluding gaps
  15. 15. quantifying variation Map trimmed reads to newly constructed references (assembled contigs) SNP detection (CLC) SNP verification • exploring data • Sanger sequencing Reproduced from Crisp et al. 2004
  16. 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 4500bpreferencereferenceSynoum NSynoum N SSynoum S
  17. 17. SNP detection
  18. 18. data mining Chloroplast barcoding genes Universal cpSSR markers Other data BLAST The question of coverage Reproduced from Crisp et al. 2004
  19. 19. Citrus Toona Wilkiea Daucus Synoum Claoxylon Doryphora Gossypium Diploglottis Pittosporum Calycanthus Brachychiton Cinnamomum rbcL a-f F rbcL a-r R rbcL 1F rbcL 724R accD 1 F accD 2 F accD 3 R accD 4 R matK 2.1 F matK 2.1a F matK X F matK 3.2 R matK 5 R 390 F 1326 R matK_1F matK_1R matK_2F matK_2R rpoB 1 F rpoB 2 F rpoB 3 R rpoB 4 R rpoC1 1 F rpoC1 2 F rpoC1 3 R rpoC1 4 R ycf5 1 F ycf5 2 F ycf5 3 R ycf5 4 R ndhJ 1 F ndhJ 2 F ndhJ 3 R ndhJ 4 R trnH2 F psbAF R trn H (GUG) F psb A R choroplast barcoding loci atpF F atpH R psbK R psbI R trnL-c F trnL-d R trnL-e F trnL-f R trnL-g FVijayan and Tsou 2010 trnL-h R
  20. 20. universal cpSSR primers ccmp10R ccmp10F ccmp1R ccmp2R ccmp3R ccmp4R ccmp5R ccmp6R ccmp7R ccmp8R ccmp9R ccmp1F ccmp2F ccmp3F ccmp4F ccmp5F ccmp6F ccmp7F ccmp8F ccmp9FBrachychitonCinnamomumClaoxylonDiploglottisDoryphoraPittosporumSynoumToonaWilkieaDaucusGossypiumCalycanthusCitrus ` Weising and Gardner 1999
  21. 21. 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 Reproduced from Crisp et al. 2004
  22. 22. 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 Reproduced from Crisp et al. 2004
  23. 23. 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 Reproduced from Crisp et al. 2004
  24. 24. 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? Reproduced from Crisp et al. 2004
  25. 25. 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 Reproduced from Crisp et al. 2004