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Do Next Generation Sequencingapproaches provide the answer for DNA barcoding of plants?Hannah McPherson Marlien van der Me...
Landscape-level studies of the      Australian flora                   Species and population                    dynamics...
Next generation sequencing Exploring new molecular tools and  approaches NGS to assemble whole chloroplast  genomes Use...
Technical approach Full genome shotgun sequencing Solexa Illumina platform (7Gb/lane)     •    8 labelled paired-end lib...
Sampling                     2 locations   Nightcap N                *    20 rainforest tree                      specie...
reality check: sampling from           rainforests Collecting and identifying samples Preserving leaf material DNA extr...
questions   Can we bioinformatically assemble chloroplast    genomes from whole genomic shotgun    sequencing without a r...
Angiosperm Phylogeny    Model organism tree                                   Atherospermataceae                          ...
Malvales                       Brachychiton           Malvaceae                       Gossypium,                       The...
Laurales           Calycanthaceae Calycanthus           Atherospermataceae Doryphora           Monimiaceae Wilkiea        ...
assembling chloroplast genomes   Map trimmed reads to whole cp genome of    closest relative available on Genbank (CLC)  ...
Align with annotated
assembling chloroplast genomes17000015000013000011000090000                                                               ...
NC_008325 Daucus carota                  Pittosporum multiflorum                      Toona ciliata                      S...
quantifying variation Map trimmed reads to newly constructed  references (assembled contigs) SNP detection (CLC) SNP ve...
SNP detection    Synoum glandulosum (~140Kbp)             •   SNPs between N and S                  •    ~1 in 550bp     ...
SNP detection
data mining Chloroplast barcoding genes Universal cpSSR markers Other data BLAST The question of coverage             ...
Citrus                                                                              Toona                                 ...
universal cpSSR primers                                                                                                   ...
data mining 26S coverage ~35-300 Rpb2 only returned when sequence  available in same family or sister family  coverage ~...
data mining Matches were good Seem to be in more conserved bits Single copy nuclear genes present but  low coverage So...
viability for barcoding Large portion of the chloroplast genome  retrieved and easily assembled even  without a reference...
to sum up the story so far   We can assemble large portions of chloroplast    genomes from whole genomic shotgun    seque...
acknowledgements Friends of the Botanic Gardens Trust Southern Cross University – Robert  Henry Nicole Rice Stirling Bow...
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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 Websitehttp://www.mobot.org/MOBOT/Research/APweb/welcome.html
  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

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