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Tracing forest reproductive material by its DNA: forest forensics
Berthold Heinze (BFW) & Work Package 7 partners:ALTERRA – J. Buiteveld; ASP – Barbara Fussi; IBBR-CNR -
G.G. Vendramin; INRA Bordeaux – Erwan Guichoux & Rémy Petit; TI – Céline Blanc-Jolivet & Mirko Leisebach
European Regulations for Forest Reproductive Material (seeds and plants)
• Council Directive 1999/105/EC of 22 December 1999 on the marketing of forest reproductive material
• Commission Regulation (EC) No 1597/2002 of 6 September 2002 … detailed rules for … the format of national lists of the basic material of forest reproductive material
• Commission Regulation (EC) No 1598/2002 of 6 September 2002 … detailed rules for … provision of mutual administrative assistance by official bodies
• et cetera
• assurance of quality, including intrinsic quality = genetics• based on paper documentation
genetic quality is important …
• correct species (e.g. oaks, pines)• adaptation to specific regions• adaptation to specific site conditions• adaptation to elevation
• reaction towards climatic and edaphic factors is in the genes!
what if …
• cheating …?• cheaper material e.g.
– from other countries– from more easily accessible stands– from more abundant sources– from older harvests
• seed often „looks“ the same• plant may look „a bit“ different, but not always
• genes are different … can they be identified?
there are no „geographic genes“, but …
• trees often carry a „reminder“ of glacial refugia• trees from one region / provenance / … are often more closely related to
each other
• seeds / plants form a single, defined source stand can sometimes be matched back to the parental trees
example 1: wider geographyorigin of Pinus pinaster material
Erwan Guichoux, Adline Delcamp, Christophe Boury & Rémy Petit, INRA Bordeaux
(based on work done by GG Vendramin, S. Gonzalez Martinez, C. PlomionCNR+INIA+INRA)
Ex : Traceability in Pinus pinaster
French Atlantic populations
Spanish Atlantic populations
Corsica
Central Spain populations
Southern Spain
Morocco
260 SNPs
A plantation with Central Spain origin!
12 SSRs
Objective: to develop a simple SNP‐based test to distinguish main breeding zones and native populations (collaboration INRA‐INIA‐CNR)
work done by GG Vendramin, S. Gonzalez Martinez, C. Plomion(CNR+INIA+INRA), Jaramillo‐Correa et al. 2015
Ex : Traceability in Pinus pinaster
Application for certification of Landes maritime pine seedlotsErwan Guichoux, Adline Delcamp, Annie Raffin, Laurent Bouffier, Marc Kleinhentz, Camille Lepoittevin, Sophie Gerber
K=2
K=8
Pop Origin
1 Morocco
2 Spain (Atlant.)
3 Landes
4 Portugal
5 Spain (Center)
6 Spain (East)
7 Spain (South)
8 CorsicaBased on 66 SNPs
Test in real conditions on 8 ONF plantations (~25 individuals/plantation)
Portugal Spain (Atlantic coast)
Landes
ONF
Landes origin validated!
Ex : Traceability in Pinus pinaster
exampe 2: material from a specific Larix decidua seed orchard?
• Berthold Heinze, Renate Slunsky, Michael Mengl• BFW, Austria
P3 – Lammerau – Hamet -Wienerwaldlärche
good quality of real „P3“ material
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indirect / relatedness:STRUCTURE software; genetic distances
• calculation of a „distance“ to the orchard on a population (seed lot) basis– simple Euclidian Distance based on STRUCTURE output:– Q matrix (share of each present population in the assumed original
populations)
threshold ?
German provenances ≠ P3
authenticated seed samples from P3
example 3: regional geographyorigin of Abies alba material
Application for certification of the origin of plantations
Francesca Bagnoli, Celeste Labriola, Andrea Piotti, Ilaria Spanu, Giovanni G. Vendramin
CNR, IBBR, Firenze, Italy
Postolache et al. 2014
24 natural populations genotyped with 16 nSSRs multiplexed
Ex : Traceability in Abies albaApplication for certification of the origin of plantationsFrancesca Bagnoli, Celeste Labriola, Andrea Piotti, Ilaria Spanu, Giovanni G. Vendramin
24 natural populations
10 planted populations
GeneClass2
Structure
PLANTATION
Genotyping with 16 nSSRs (K=4)
PcoA (24 natural populations and 10 plantations)
The plantations are separeted in one group, genetically closer toSouthern Apennine populations (yellow dots) and the other one toNorthern Apennine populations (blue dots). The plantation FOS is inbetween.
Ex : Traceability in Abies alba
example 4:species identification Tilia sp.
• Barbara Fussi • ASP Teisendorf
• Samples from a plant lot sent by a tree consultant• Species of delivered plants were questioned
Tilia sp.
• DNA extracted from cambium• Genetic fingerprint using 11 nuclear microsatellites established• Comparison with reference material led to identification of the sample as
Tilia cordata
‐40,000
‐35,000
‐30,000
‐25,000
‐20,000
‐15,000
‐10,000
‐5,000
0,000‐45,000 ‐40,000 ‐35,000 ‐30,000 ‐25,000 ‐20,000 ‐15,000 ‐10,000 ‐5,000 0,000
Pop 2
Pop 1
S_Linde
W_lindeSample in question was assigned to Tilia cordata
Tilia platyphyllos
Tilia cordata
example 5:identification of the number of harvested trees in a mixed seed lot of Douglas fir
• Barbara Fussi • ASP Teisendorf
Douglas fir
• Using the seed coat, the harvested tree (mother tree) can be identified• DNA extracted from seed coat of 74 seeds• Genetic fingerprint using 8 nuclear microsatellites• 9 unique trees were ientified
DNA from seed coatis identical with themother tree
conclusions
• DNA markers are useful in forest trees• Europe-wide patterns of variation can be obtained from e.g. chloroplast
DNA• more fine-scale variation is possible with highly variable microsatellites• or with single-nucleotide polymorphisms (SNPs)
– which are faster to produce• parentage analysis is possible for many species
– for matching seeds and plants to their biological parents
Got an identification problem? We can likely help you!visit our session: „Your problem, our solution“ to discuss it Wednesday afternoon (WP7) – cancelled …
..but here are some examples:
• introduce work done by participating institutes
• wanted to discuss problems that the audience has in identification of forestreproductive material
Species Speciesidentificat
ion
Cloneidentificat
ion
Provenance
identification(ZüF)
Population
genetics
Parentage
analysis
Varietyidentificati
on
Abies alba x x
Fagus sylvatica x x x
Picea abies x x x
Pseudotsuga menziesii x x x x
Larix decidua x x
Acer pseudoplatanus, A. platanoides
x
Quercus robur, Q. petraea, Q. rubra
x x x x
Taxus baccata, Alnusglutinosa, A. viridis, A. incana, Sorbustorminalis
x x
Populus sp., Malus sylvestris, Tilia sp., Betula sp., Pinus cembra
x
Prunus avium x x
Fraxinus excelsior, F. ornus x x x
Pinus sylvestris, P. nigra x x x
Carpinus betulus x x
Aesculus hippocastanum x
e.g. roots growing into the neighbor’s garden
local court case: alleged theft of a wild cherry log (worth ~ 500 € )
• could wood samples from the log be matched to the remaining rootstock with DNA?
cherry results
• log and rootstock material show identical patterns• other cherries are highly variable• 1 : 14 000 000 000 000 probability for erroneous identity
– for identity by chance, but not by descent– if all trees are unrelated
• 1 : 52 000 probability if trees are related– full sibs with identical ‚mother‘ and ‚father‘
• the log in question was apparently from the rootstock that is still there …
Species Multiplex Applications New
Fagus sylvatica 9 Population genetics, parentage… +
Larix decidua 10 Traceability
Pinus pinaster 2 Pedigrees
Pinus pinaster 2 Traceability +
Pinus sylvestris 1 Population genetics
Prunus avium 2 Parentage analysis
Quercus robur & Q. petraea 12 Traceability, mapping, species id
Quercus ilex 2 Population genetics, parentage…
Quercus alba 8 mapping +
Quercus spp (Americ white) 2 Species diagnose +
Robinia pseudoacacia 4 Population genetics, clonal reprod +
Symphonia 4 Population genetics +
Khaya 4 Traceability +
Swietenia 4 Traceability +
Armillaria sp. 2 Population genetics +
Platycarya sp. 4 Species diagnose +
SNP multiplexes for Sequenom
• Rapid progresses–creation of new SNP multiplex panels easy + extreme reproducibility
• Develop specialised multiplexes for specific purposes: geography & autochthony (high Fst), species id (high intersp Fst), parentage (tri-allelic)…
• Feasible to develop all purposes multiplexes for all relevant European tree species
Trees4Future Training Session in March-April 2016"Development of SNP markers using ddRAD-seq and
validation using Sequenom technology"6 selected projects
Conclusions