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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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slide1
Standard land plant barcoding requires a multi loci approach?

Robyn Cowan

Sujeevan Ratnasingham

Peter Gasson

slide2
Mitochondrial DNA in land plants:
  • 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

Partners

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
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
slide6
So what are the characteristics of a good barcode?
  • 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**

slide7
What sort of marker should we use?
  • 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) ?
slide8
Advantages of plastid DNA (hence its use in phylogenetics)
  • Monomorphic (separation of different copies not required in hybrids)
  • High copy number (can even be amplified from highly degraded DNA)
  • Potentially highly diagnostic (in spite of its reputation to the contrary)

However, will not detect hybrids, introgression, paralogy

slide9
Coding or non-coding?

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)

criterion for locus selection
Criterion for locus selection
  • Species level sequence divergence
  • Appropriate length (200-800bp)
  • Presence of conserved primer target sites
  • At least 200bp exon sequence
our strategy
Our Strategy
  • 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
standard pcr recipe
Standard PCR Recipe
  • 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
slide16
Trial regions

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

slide17
Trial groups

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

slide23
Trial regions

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

slide24
Agavaceae X 22 sp.

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

slide26
Users of DNA Barcoding:

‘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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