PLANT DNA BARCODING: HISTORY CASE STUDIES FUTURE PERSPECTIVES MARIA KUZMINA UNIVERSITY OF GUELPH, CANADA
Building the DNA barcode library for the flora of Canada using herbarium specimens
Encouraging start ... • COI is a successful barcode for animals but fails in plants for several reasons: • intron presence is variable across plants • exceptionally low rates of evolution COI
rbcL 2009 Chloroplast DNA markers Ideal barcode should be: • Universal primers • Bidirectional • Maximum discrimination among species Resources: • 190 species of the land plants • 7 plastid DNA regions Decision: • rbcL ( 61% species discrimination) psbA - trnH matK • matK ( 69% species discrimination)
Top opolog ological cor ical corresp espon onde denc nce e of of th the e DN DNA A ba barco code de ph phyloge ylogeny ny an and d th the Ang e Angiospe iosperm P m Phylog hylogen eny y Gr Grou oup p (AP (APG) G) The central role of the plastid gene rbc L in our overall understanding of the evolution of the angiosperms (Soltis et al., 2005) Angiosperm phylogeny website (Kress et al., 2009) (Stevens, 2001 onward)
Adding a nuclear marker ... • ITS was originally proposed but Nuclear Ribosomal DNA rejected • ITS2 was proposed later • variable short region • can be easily amplified across a diverse sample of plants • used previously to discriminate ITS1 ITS2 species 5.8S 26S 18S • good length for NGS (Chen et al, 2010)
Comparing “Apples with Oranges and Kiwi” Publicati Geographi No. Reported species resolution (%) on c area of species rbc L mat K ITS2 rbc L+ ma rbc L+ITS All 3 t K 2 A.Fazekas North 92 48 56 et al, 2008 America K.Burgess Koffler 436 80 89 93 et al, 2011 Scientific Reserve (KSR), Ontario M.Kuzmin Churchill, 312 54 63 69 a Manitoba et al, 2012 D.Percy North Incomplete lineage sorting OR plastid capture with 71 et al, 2014 America selective sweep M.Zarrei North Polyploidy and hybridization 83 et al, 2015 America T.Elliott Mont Focusing on quality control of collected material and data 582 et al, 2015 St.Hilaire, Quebec
What it boils down too ... Gene Pros Cons Easily amplified Poor taxonomic resolution rbcL (550 bp) Good length for NGS matK Good taxonomic Often difficult to amplify (800 bp) resolution Too long for most NGS platforms ITS2 Good taxonomic Paralogous copies (350 bp) resolution Not easy to align across a diverse Good length for NGS set of taxa
Plant DNA Barcode Library for All Canada Resource s Agriculture and Agri-Food Canada ( DAO ) Canadian Museum of Nature, National Herbarium of Canada ( CAN ) McGill University Herbarium, Macdonald Campus ( MTMG ) Ontario Agriculture College Herbarium ( OAC ) Private Herbarium of Bruce Bennett, White Horse, Yukon ( BABY ) Royal Ontario Museum, Green Herbarium ( TRT ) 6 5K The Manitoba Museum ( MMMN ) 5 4 Universite de Montreal, L'Herbier Marie-Victorin ( MT ) 3 University of Alberta Herbarium, ( ALTA ) 2 University of British Columbia Herbarium ( UBC ) 1 0 University of Manitoba Herbarium ( WIN ) 2008 2009 2010 2011 2012 2013 2014 2015 2016
Sampling ~18,000 specimens
Sequencing
Sequencing
Taxonomic Bias
Taxonomic Bias
Taxonomic Bias
Taxonomic Bias
species resolution (%) species resolution (%) 0 10 20 30 40 50 60 70 80 90 100 reserves Plant checklists from 28 national parks and rbcL rbcL matK matK Gene ITS2 ITS 2 BLAST species resolution (%) 0 10 20 30 40 50 60 70 80 90 100 rbcL rbcL matK matK Gene ITS2 ITS 2 mothu r Library resolution
species resolution species resolution (%) 20 30 40 50 60 70 80 90 (%) Plant checklists from 28 national parks and reserves combined in 6 biogeographic regions Arctic Arctic Boreal Boreal Pacific Pacific rbcL Region Prairie Prairie Woodland Woodland Atlantic Atlantic species resolution (%) 20 30 40 50 60 70 80 90 Arctic Arctic Boreal Boreal Pacific Pacific matK Region Prairie Prairie Woodland Woodland Atlantic Atlantic species resolution (%) 20 30 40 50 60 70 80 90 Arctic Arctic Library resolution Boreal Boreal Pacific Pacific ITS2 Region Prairie Prairie Woodland Woodland Atlantic Atlantic
The he DN DNA A ba barco code de ref efer eren ence ce li libr brar ary y for or mosse mosses: s: rb rbcL and and tr trnL-F f F for or 77 775 5 spe species of cies of Can Canad adian ian Br Bryop ophyt hyta Maria Kuzmina, Jennifer Doubt, Catherine La Farge, Juan Carlos Villarreal & Paul Hebert Canadian Museum of Nature Center for Biodiversity Genomics
Step one: sampling, imaging, databasing
Source location of the specimens inc include luded in d in th the DN e DNA A ba barco code de ref efer eren ence ce li libr brar ary y for or Can Canad adian mosse ian mosses ~ 2000 specimens 775 species ~ 3 records per species
Number of moss specimens analyzed by province
The he most ofte most often use n used d ph phyloge ylogene netic tic mar marker ers f s for or mosses mosses (Stech & Quandt, 2010) trn L-F rbc L ITS
Relationship between specimen age and sequence recovery Overall sequencing success: Time Specimens Species rbc L 84% 94% trn L-F 85% 98%
The Maximum Lik he Maximum Likelihood best elihood best rbc rbc L tr tree ee UNUSUAL: Monophyletic Many orders are Polyphyletic polyphyletic! 1665 specimens
Boot Bootst strap p consensus consensus rbc rbc L tr tree ee SURPRISINGLY: rbc L poorly supports beta taxonomy Bootstrap >80% but good at resolving genera! 1665 specimens
Species resolution with rbcL and trnL-F for species-rich orders of mosses
Ree eexam xamina ination tion of of tax taxono onomy ( my (red b ed bar ars) p ) provok oked ed by by rbc rbc L results esults
Acknowledgement s • Anuar Rodrigues • Stephanie deWaard • Jesse Sills • Sean Graham • Aaron Fazekas • Bruce Bennett • Timothy Dickinson • Jeffrey Saarela • Paul Catling • Steven Newmaster • Diana Percy • Erin Fenneman • Aurelien Lauron-Moreau • Bruce Ford • Lynn Gillespie • Bruce Ford • Jeannette Whitton • Lynn Gillespie • Linda Jennings • Erin Fenneman • Deborah Metsger • Aurelien Lauron-Moreau • Connor Warne • Bruce Ford • Allison Brown • Lynn Gillespie • Elizabeth Sears • Ragupathy Subramanyam • Jeremy De Waard
Reference Library for Targeted SNP-based Identification of Cibotium barometz Using NGS Natalia Ivanova Maria Kuzmina Evgeny Zakharov
Cibotium barometz Plant growing in the Botanischer Garten München - The golden brown hairs at the base of the frond Nymphenburg, Munich, Germany Photograph by: Mokkie Photograph by: Daderot, Public domain Creative Commons Attribution-Share Alike 4.0 http://tropical.theferns.info/image.php?id=Cibotium+barometz
Medicinal Use Anti- inflammatory Anti- Antioxidant rheumatic Anti- Antibacterial osteoporotic Styptic Tonic
Cibotium Phylogeny Geiger JMO, Korall P, Ranker TA, Kleist AC, Nelson CL (2013) Molecular Phylogenetic Relationships of Cibotium and Origin of the Hawaiian Endemics. Am Fern J, 103: 141 – 152, doi:10.1640/0002-8444-103.3.141
Cibotium barometz ID rps4 (ribosomal protein S4) – 94 bp atpA (ATP synthase alpha chain) – 86 bp trnG-trnR intergenic spacer – 95 and 102 bp rps4-trnS intergenic spacer – 84 bp and 87 bp atpB-rbcL intergenic spacer – 79 and 110 bp
Cibotium Reference Library – BOLD
Cibotium Reference Library – BOLD Average age of UBC Cibotium herbarium material – 58 years
Cibotium Reference Library – BOLD
SNP Summary rps4- rps4 atpA trnS 87-200 trnG- rps4- trnG- atpB- rtnR trnS rtnR rbcL 565-705 163-300 681-830 Shared C. barometz/cumingii SNPs Confirmed C. barometz SNPs Signature SNPs
Summary Assembled reference library with voucher specimens Confirmed available GenBank data for C.barometz and C.cumingii Increased coverage for the regions of interest Resulting reference library can be used for regulatory purposes
Thank you!
Genome2-ID unbiased and rapid species identification using NGS data David L. Erickson
Genome2-ID: Format reference database DECOMPOSE EACH SEQUECE INTO LIST OF OVERLAPPING “WORDS” A A T C G A T C G G A T C T A G A T C T C G A T A T A A A T C G A E. purpurea_NC1234 A T C G A T E. purpurea_NC1234 T C G A T C E. purpurea_NC1234 C G A T C G E. purpurea_NC1234 G A T C G G E. purpurea_NC1234 Example: plant chloroplast A T C G G A E. purpurea_NC1234 T C G G A T E. purpurea_NC1234 genomes as our reference . . . . ~ 150,000 bases in size G A T A T A E. purpurea_NC1234 DNA4 Technologies LLC
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