Standard land plant barcoding requires a multi loci approach?
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Standard land plant barcoding requires a multi loci approach?. Robyn Cowan. Sujeevan Ratnasingham. Peter Gasson. Mitochondrial DNA in land plants: undergoes rearrangements transfer of genes to nucleus incorporation of foreign genes substitution rates are VERY slow

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Standard land plant barcoding requires a multi loci approach?

Robyn Cowan

Sujeevan Ratnasingham

Peter Gasson


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  • Mitochondrial DNA in land plants: approach?

  • undergoes rearrangements

  • transfer of genes to nucleus

  • incorporation of foreign genes

  • substitution rates are VERY slow

  • (with a few notable exceptions e.g. Plantago, Cho & al.)


Partners l.jpg

Partners approach?

Instituto de Biologia UNAM,Mexico – Gerardo Salazar

Imperial College, UK - Timothy Barraclough

Natural History Museum, Denmark - Gitte Petersen

Natural History Museum (London), UK - Mark Carine

New York Botanical Garden, USA - Kenneth Cameron

Royal Botanic Garden Edinburgh, UK - Peter Hollingsworth

Royal Botanic Gardens, Kew, UK - Mark Chase

South African National Biodiversity Institute - Ferozah Conrad

University of Cape Town, South Africa - Terry Hedderson

U. Estadual de Feira de Santana, Brazil - Cássio van den Berg

Universidad de los Andes - Santiago Madriñán

U. of Wales Aberystwyth UK (previously University of Reading, UK) - Mike Wilkinson

Alfred P. Sloan Foundation

Gordon and Betty Moore Foundation


To develop a universal approach to barcoding of all landplants l.jpg
To develop a universal approach to barcoding of all landplants

  • Phase 1: primer development (protein motifs); complete genome sequences; problems: ferns; 46 pairs of sister taxa from mosses, liverworts, hornworts, lycopods, ferns/fern allies, gymnosperms, angiosperms – percent PCR success & percent polymorphisms

  • Phase 2: in depth trials of six markers identified in phase I on a range of well sampled taxa from across land plants


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  • So what are the characteristics of a good barcode? landplants

  • High inter-specific, low intra-specific sequence divergence

  • Universal amplification/sequencing with standard primers

  • Technically simple to sequence

  • Short enough to sequence in one reaction

  • Easily alignable (few insertions/deletions)

  • Readily recoverable from museum or herbarium samples and other degraded samples

**Universal + Variable**


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  • What sort of marker should we use? landplants

  • Mitochondrial DNA

  • Plastid

  • Ribosomal DNA (ITS)

  • Low-copy nuclear DNA (protein coding)

  • Length variable ?

  • Single loci

  • Multiple loci (one genomic compartment) ?

  • Multiple loci (two genomic compartments) ?


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However, will not detect hybrids, introgression, paralogy


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Coding or non-coding? landplants

Non-coding regions:

sometimes more variable

microsatellites difficult to sequence through

numerous indels-impossible to align, length variable

cannot translate to check for pseudoproteins and to aid aligment

sometimes contain rearrangements and coding insertions

(character based identification)



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Criterion for locus selection landplants

  • Species level sequence divergence

  • Appropriate length (200-800bp)

  • Presence of conserved primer target sites

  • At least 200bp exon sequence


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Our Strategy landplants

  • Identify suitable loci on the basis of in silico screens using Nicotiana cp sequence

  • Design universal primers (sets of 4 primers/locus) using amino acid and nucleic acid sequence data

  • Perform initial screen for universality (1 primer pair)

  • Screen for sequence variation using diverse species pairs

  • Improve universality (e.g. use all primer combinations)

  • Use statistical modelling approaches to identify optimal primer sets


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Standard PCR Recipe landplants

  • NH4 x1

  • Mg2+ 1.5mM

  • dNTPs 0.2mM

  • FW test primer 1M

  • RE test primer 1M

  • Taq DNA polymerase 2 units

  • BSA 0.1mg/ml

  • Template 40ng

  • Water to 20l




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Trial regions landplants

Selected seven genes that represent the different levels of universality and variability. Blue= high, green = medium, yellow= low.


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Trial groups landplants

Asterella Anastrophyllum-Barbilophozia Tortella Bryum Triquetrella Homalothecim Tortella Elaphoglossum Asplenium Equisetum Cupressus Pinus Araucaria Labordia Conostylis Dactylorhiza maculata/incarnata Mimetes Inga Hordeum Scalesia Crocus Laelia Cattleya Mormodes Deiregyne Lauraceae


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Summary landplants


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Trial regions landplants

Selected seven genes that represent the different levels of universality and variability. Blue= high, green = medium, yellow= low.


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Agavaceae X 22 sp. landplants

Crocus X 9 sp.

Aulosepalum X 8 sp.(?all)

Cattleya X 30sp.(2 clades approx 43 sp.)

Dactylorhiza 15 sp. (species complex)

Sophrinitis 27 sp. (approx. 37 sp.)

Scalesia X 4 (species complex)

Conostylis X 42 (?all)

Equisetum X 14

Pinus X 66

Hordeum X 10

Lauraceae



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Users of DNA Barcoding: landplants

‘The Traffic Light approach’

Green - non-problematic taxa (current markers appropriate, silver standard)

Orange - need for gold standard

(polyploidy, introgression, paralogy)

Red - barcoding needs investigation, species complex, etc


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