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

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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