BIOFOR' 02

Sustainable Forestry, Wood Products & Biotechnology

Vitoria-Gasteiz, Espagne 11-1 4 novembre 2002

COMPTE-RENDU DE MISSION

Philippe Vigneron Jean-Marc Gion

Forêts Programme

Arbres et Plantations

BIOFOR'02- Sustainable Forestry, Wood Products & Biotechnology Vitoria-Gasteiz, Espagne

1 1-14 novembre 2002

Compte-rendu de mission Philippe Vigneron

Jean Marc Gion

Organisateurs : NEIKER - Instituto V asco de Investigaci6n y Desarrollo Agrario Gouvernement Autonome Basque Région Aquitaine

Jean Marc Gion et Philippe Vigneron ont participé à ce congrès et ont présenté une communication orale et un poster. Le CIRAD est par ailleurs co-auteur de la communication orale de Jacqueline Grima-Pettenati.

Vigneron Ph, Gion J-M & Verhaegen D, CIRAD Forêt. Montpellier (FRANCE) UR2PI­Congo: Genomic researches: new tools for genetic improvement of eucalypts in Congo. Voir

' )

annexe et diaporama fichier vig et gion03.pdf

J.M. Gion, M. Lalanne, C. Plomion , INRA: Wood proteomics in maritime pine. Voir

annexe et poster Pgion.pdf

Jacqueline Grima-Pettenati, CNRS : Functional genomics of wood formation in Eucalyptus: a Southem Europe project. Voir fichiers GPl.jpg et GP2.jpg

Les résumés (1 à 3 pages) des communications orales et des posters sont disponibles auprès de Ph. Vigneron ou J.M. Gion.

Les objectifs du congrès sont exposés ci-dessous (from the Symposium announcement)

The congress aims to address, in the broadest sense, possible applications of biotechnology for sustainable for estry and for improving quality of wood products. The importance of Biotechnology for forestry and forest products is growing rapidly. The recent scientific innovations in this field offer increasing possibilities of applications. These facts have motivated the Organizing Committee to organize this congress to share and disseminate novel achievements obtained in R&D projects and to identify gaps for future research. Furthermore this congress represents a platform where scientists and indus trial partners can meet, discuss industrial needs, problems and possible solutions and can establish collaborations. For this purpose, the congress will include an active participation of enterprises from the forest sector, of wood processing industries and biotech companies. Moreover, within the frame of this congress, facilities will be given to hold independent project meetings or workshops for interested groups. This option is particularly atractive for establsihing collaborations among participants in view of the approaching Framework VI

Programme of the EU.

Severa! members of the Management Committee COST action E28 "Genosilva: European Forest Genomics Network" of the European Commission participate in this event. Furthermore, the National Forest Genomics Network of Spain is under constitution and the

partners will make relevant contributions. In arder to underpin the importance of Genomics in Forest Research, extended results of the QoL R&D project "UHD map of Coniferous species (QLK5-1999-01159)" will be presented involving severa! conifer species. Within the congress Satellite Meetings will be held: - The Genetic Resources Group and the management committee of IEFC (European Institute of Cultivated Forests) will meet as part of the IMACFORD project - task B: Development of an integrated research network for the sustainable management and utilisation of fast growing forest resources in Europe.Furthermore, the working group: Management of Forest Habitats which belongs to Natura2000 will present and discuss results and experiences.

Trois types de réunions ont été tenues de façon concomitante . Les industriels et les producteurs basques et aquitains ont débattu des aspects transformations - commercialisation d'une part et des aspects certification d'autre part. Les chercheurs ont présenté et discuté leurs travaux de laboratoire. Il faut regretter que malgré les buts poursuivis, il n'y ait pas eu suffisamment de rencontres entre les différents acteurs : chercheurs, producteurs, transformateurs. Ce compte-rendu de mission rapporte l'état des recherches sur Eucalyptus tel qu'il a été présenté par les différents intervenants.

Les travaux sur Eucalyptus

Multiplication végétative

Les papiers présentés montrent l'importance croissante de l'embryogénèse somatique et des graines artificielles pour le déploiement industriel des conifères, principalement aux US, Canada et Nouvelle Zélande. Le système est au point et utilisé en routine. Rien n'a été présenté sur Eucalyptus ou autres feuillus.

L' Afocel a présenté un poster sur la cryoconservation d'espèces diverses, dont les eucalyptus: E. Dumas et al. « Cryopreservation: a powerful tool for the management of genetic resources for breeding and preservation purpose ».

Transformation génétique

De nombreux travaux prometteurs ont été publiés sur conifères, bouleau et peuplier mais l'Eucalyptus reste le parent pauvre. Les travaux sur la transformation génétique des eucalyptus sont relativement anciens mais se sont toujours heurtés à la phase clef qui est la régénération d'une plante entière après

l'obtention d'un cal cellulaire transformé. Visiblement, si on observe la transformation de cals cellulaires, l'écueil de la régénération est toujours présent.

Le papier présenté par Gallego (Université de Vigo, Espagne) sur la transformation de E. globulus n'est pas concluant : il présente une étude des différentes. conditions de transformation par infiltration sous vide avec Agrobacterium et obtient des cultures de feuilles ou de bourgeons exprimant le gène GUS (j'allais dire une fois de plus) mais reste très évasif sur la régéneration. Le poster « Efficient transformation systems for testing wood quality genes in hardwoods » de la compagnie privée ArborGen (South Carolina, USA) par MA Hinchee, Briggs T, Chang S, Frampton KA, Gause KC, Richardson P, Thomas RD, Wilde HD est un peu plus optimiste. ArborGen semble avoir fait des progrès significatifs dans le

domaine de la régénération de clones d'E. grandis, d'hybrid�s E.grandis x urophylla et d'E. camaldulensis après transformation avec des constructions génétiques destinées à modifier et amémliorer la qualité du bois pour l'industrie papetière. Ils ne donnent pas de détail sur l'identité des génes réprimés ou surexprimés. Le nombre de clones obtenus est encore faible (2 grandis x uro) et seuls 10 % des clones se régénèrent. Les premiers essais de terrain sont en cours aux US avec un total de 75 clones, essentiellement E. camaldulensis.

GENOMIQUE et BIODIVERSITE

Les travaux présentés lors de cette session et de la suivante (Industrial needs and applications) tournent autour de trois axes complémentaires de recherche : la cartographie génétique, la recherche de QTL et 1' analyse du transcriptome. Les travaux de cartographie sur les arbres forestiers utilisent de nombreux types de marqueurs (SSR, EST, gènes candidats, RAPD, AFLP . . . ) . De nombreuses cartes génétiques saturées sont maintenant publiées tant pour des conifères que des feuillus. Ces cartes génétiques servent au positionnement des QTL puis des gènes candidats. Deux problèmes majeurs se posent :

la taille réduite des populations de cartographie (familles de pleins ou demi frères) qui ne permet qu'une estimation très grossière des taux de recombinaison entre marqueur et donc une localisation très imprécise des marqueurs sur le génome, la faiblesse statistique des dispositifs au champ et la difficulté qu'il y a de distinguer les composantes génétiques et environnementales.

Ceci conduit généralement à une grande imprécision de la localisation des QTL et de l'intensité de leurs effets, au faible nombre de QTL détectés eu égard au caractère polygénique des propriétés mesurées, à la détection de faux QTL. . . Pour différentes raisons, dont la rapidité de croissance, l'importance des dispositifs de terrains, la faible taille du génome, 1' existence de familles de pleins frères présentant des effectifs importants, la grande diversité allélique . . . les travaux sur Eucalyptus sont maintenant parmi les plus avancés en la matière.

Les quatre partenaires du projet «Génomique fonctionnelle de la formation du bois d'Eucalyptus» ont fait le point sur l'état de la génomique Eucalyptus en Europe. Ce projet associe ENCE (Espagne), RAIZ (Portugal), CNRS (Toulouse, France) et le CIRAD. Il est présenté par Jacqueline Grima-Pettenati.

-JACQUELINE GRIMA-PETTENATI Functional genomics of wood formation in Eucalyptus: a Southem Europe project.

L'objectif du projet est d'identifier (CNRS) et de cartographier (ENCE, RAIZ et CIRAD) des gènes candidats (GC) et des QTL responsables des variations des propriétés du bois dans l'objectif de développer de la sélection assistée par marqueurs. Des GC « expressionels » (c'est à dire exprimés) ont été isolés en utilisant la technique d'hybridation soustractive (Suppression Soustractive Hybridization) entre feuilles et xylème. Une collection de 2700 EST (pour Expressed Sequence Tag) a été obtenue. La séquence des EST permet dans un certain nombre de cas de trouver, par comparaison à des banques de données, le gène en cause. Ce séquençage est en cours et certains gènes sont maintenant disponibles pour la cartographie (qui se fera à Montpellier sur la carte de la famille 14.144 x 9.2 1 ). Afin de cibler au mieux les gènes spécifiquement exprimés dans le xylème, l'étude se poursuit en comparant divers types de xylèmes : bois juvénile/bois adulte, bois tendu/bois normal. . .

Tous ce travail fait l'objet de la thèse d'Etienne Paux.

-CRISTINA MARQUES Forest Biotechnology "To Go" at RAIZ

RAIZ est un institut de recherche privé dont la fonction est d'aider à la compétitivité de la filière pâte et papier du Portugal. L'un des thèmes de recherche est la sélection de clones d'Eucalyptus globulus. RAIZ utilise les marqueurs pour le suivi de la diversité génétique de la population de clones et l'identification des variétés. Dans le cadre de notre partenariat, leur premier travail est de faire le tri dans les séquences exprimées dans le xylème. Il est en effet difficile et probablement inutile de cartographier les 2700 séquences. De nombreux gènes exprimés n'ont probablement rien à voir avec les propriétés du bois. En se basant sur la littérature existante, les bases de données génomiques, RAIZ a pour l'instant sélectionné 163 gènes (dont 80 prioritaires) sur la base de leurs fonctions physiologiques ou métaboliques supposées dans la formation du bois. Ce sont ces gènes qui seront cartographiés sur E. globulus et E. urophylla x grandis et dont on cherchera les colocalisations avec des QTL.

ROBERTO ASTORGA & GABRIEL TOV AL Industrial Applications of Biotechnology at ENCE

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La division forestière d'ENCE gère 150 000 ha, essentiellement des Eucalyptus, en Espagne et en Uruguay. Ils produisent environ 3 millions de m3 de bois par an. La division «Pâte »

produit environ 1 million de tonnes de pâte Kraft par an. Le programme d'amélioration travaille essentiellement sur E. globulus. Les marqueurs moléculaires (SSR) ont été utilisés pour l'analyse de la diversité génétique des populations de base, de celle de la population d'amélioration ainsi que pour le contrôle de l'identité des variétés déployées. Ils sont en train de développer une nouvelle banque de microsatellites (SSR) et commencent leur cartographie. Des QTL seront recherchés pour la résistance à la sécheresse, l'enracinement et la qualité du bois (avec le groupe des 4 partenaires). Nous sommes en discussion avec Roberto Astorga pour l'utilisation de leurs microsatellites.

PHILIPPE VIGNERON Genomic researches : new tools for genetic improvement of eucalyptus in Congo

Nous avons brossé un tableau des différents aspects de la recherche en génétique moléculaire sur Eucalyptus. Le résumé de la présentation orale est donné en annexe. La présentation diaporama est disponible.

La présentation orale a été axée sur : l'importance de disposer à la fois de nombreux marqueurs mais aussi, et c'est crucial, de familles de grande taille pour pouvoir construire des cartes génétiques à la fois saturées (génome bien couvert) et précises en terme de positionnement des marqueurs les uns par rapport aux autres ; la nécessité d'avoir une mesure fiable des caractères pour lesquels on recherche des QTL ; l'importance de disposer de généalogies complexes et diversifiées pour multiplier les chances de mise en évidence de QTL, de trouver du polymorphisme utile dans les populations d'amélioration, de tester la valeur des allèles dans des milieux différents ; l'importance des collaborations pour développer des travaux en génomique fonctionnelle (cf. notre collaboration ENCE-RAIZ-CNRS-CIRAD);

l'utilité d'une carte consensus des principales espèces du sous genre Symphyomyrtus, travail auquel le CIRAD, ENCE et RAIZ vont s'atteler en utilisant des marqueurs communs (SSR et EST).

Les deux dernières diapos ont servi à montrer en quoi le schéma de SRR du Congo constituait un outil privilégié pour ces recherches et leurs applications.

Réunion ENCE - RAIZ- CIRAD - CNRS

En marge du congrès, les quatre partenaires se sont réunis pour discuter des travaux en cours. Etienne Paux doit soutenir sa thèse en 2004. ll aura d'ici là constitué les banques d'EST associées aux divers types de xylèmes : juvénile vs adulte et cinétique de la mise en place du xylème tendu vs normal. Le protocole d'analyse de cette cinétique a été simplifié et limité à 3 dates.

Le travail bibliographique réalisé par RAIZ permet de faire un tri a priori des EST à cartographier. La construction de primers sera faite très prochainement en coordonant les efforts de RAIZ et du CIRAD afin d'éviter les doublons. Leur cartographie sera réalisée sur E. urophylla x grandis au cours de l' année 2003.

na été par ailleurs décidé de saisir toute opportunité de financement pour poursuivre�t étendre notre collaboration. Le projet EUCAGENE sera revu et augmenté (protéomique, banque BAC ?) puis soumis à fmancement dès que possible.

Programme et communications

Lundi 11 novembre

Session: MICROPROPAGATION and PHYSIOLOGY

ROBERTO RODRIGUEZ -University of Oviedo, Spain Keynote on Micropropagation and Physiological Aspects

JENNY AITKEN Somatic embryogenesis and organogenesis of conifers

JAN DICK Genetic bases of adventitous root formation.

ARMAND SEGUIN Molecular approaches for a better understanding of the defense response in forest tree species.

BORJA DIEGO Age- and phase-change-related gene expression in Pinus radiata D. Don.

MARiA BERD ASCO DNA methylation analysis useful for characterizing product quality: the example of Pinus radiata d, J?on in vitro

tissue culture.

CARMEN DIAZ-SALA Molecular and cellular approaches to the study of maturation-related decline of adventitious root formation

SONIA GONÇALVES Identification of genes differentially expressed during embryigenesis in maritime pine ( Pinus pinas ter).

CARLOS RAMIREZ Regeneration of Mexican pines by somatic embryogenesis

CONCEPCION A VILA Molecular biology of ammonium metabolism in earl y stages of pine development

Session: GENETIC TRANSFORMATION in forest species

CHRISTIAN WALTER- Forest Research Inst., New Zealand Keynote on Genetic Transformation in Forest species: Modem Biotechnology as a new and promising tool in

plantation forestry: Current status and future challenges

TREVOR FENNING Perspectives and Prospects for Forest Biotechnology in Europe

ZIV SHANI Plant growth modulation by Arabidopsis thaliana endo-1, 4-B-Glucanase ( cell ) in transgenic plants

Mardi 12 novembre

Session: GENETIC TRANSFORMATION in forest species (suite)

MATTHIAS FLADUNG Towards the sa fe use of transgenic trees

TUIJA ARONEN Transgenic silver birch ( Betula pendula) with lignin modifications

ODED SHOSEYOV Recombinant cellulose crosslinking proteins: novel fiber-moclification biomaterials.

PEDRO PABLO GALLEGO Genetic transformation of Eucalyptus globulus

MA TIHIAS FLADUNG Isolation of tree-specific genes and promoters by a transposon-tagging approach

KRYSTYNA KLIMASZEWSKA Trans gene integration patterns and expression levels in transgenic !ines of three spruce species

SUBHASH MINOCHA Genetic manipulation of polyamine metabolism in poplar

JEAN-FRANCOIS TRONTIN Current prospects and limits to genetic transformation ofPinus pinaster Ait. using somatic embryogenesis as

regeneration way

SUSANA TERESO Establishment of a genetic transformation system in maritime pine (Pinus pinaster Sol.)

CHRISTIAN WALTER Wood cells in the Petri dish: cell differentiation, secondary cell-wall formation and genetic transformation of

secondary cell wall forrning tissue of Pinus radiata d. Don.

Session GENOMICS and BIODIVERSITY

LADISLAW PAULE -Faculty of Forestry, Technical University, Zvolen, Slovakia Keynote: Genetic diversity and differentiation of principal tree species

ENRIQUE RITTER - NEIKER, Spain Keynote: Advances and challanges in Forest Genornics

ENRIQUE RITTER Construction and application of a multifunctional and saturated genetic map for coniferous species

TORSTEN MARKUSSEN QTL analysis for wood quality and growth traits in the P. pinaster reference population

ANA ARAGONES Construction of a genetic map and QTL analyses in Pinus radia ta

DANIEL PRAT Linkage mapping and QTL analysis for growth of young Pseudotsuga menziesii plants

VIRGINIE ACHERE Construction of linkage maps and application in Norway spruce (Picea abies)

TORSTEN MARKUSSEN Construction of Linkage maps and QTL analyses in Pinus silvestris

DANIEL PRAT Molecular dissection of growth and wood quality traits in !arch

GUILLAUME BESNARD Compared transmission of genomic blocks in Norway spruce [Picea abies (L.) Karst] progenies obtained under

two different environmental conditions.

FRANCISCO J. RUIZ CANTON

Transcriptone analysis of wood formation in maritime pine

JEAN-MARC FRIGERIO Seeking candidate genes for water deficit adaptation in maritime pine by transcript expression profiling

AINHOA LOPEZ Cbaracterization of selected Pinus pinea L. Trees

Mercredi 13 novembre

Session INDUSTRIAL NEEDS and APPLICATIONS

LUC HARVENGT- AFOCEL, France Keynote: Forest, Industry and Biotecbnology. Needs, difficulties and sorne solutions for the future.

PRAMOD GUPTA- WEYERHAEUSER, USA Keynote: Biotechnology of somatic embryogenesis, industrial application and implementation of this technology for reforstation

ENRIQUE TORIJA - BECKMANN Strategies for Automating Nucleic Acid Preparation

CARLAPETRARROLI-ABI High throughput marker analysis

GONZALO FIRPO - ABI New Trends in BIOINFORMATICS

JORDI NA V ARRO - AMERSHAM Latest developments in high throughput 2D Proteornics

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Les présentations des 4 partenaires du projet de développement de banque d'EST xylème d'eucalyptus

JACQUELINE GRIMA-PETTENA TI

Functional genornics of wood formation in Eucalyptus: a Southern Europe project.

CRISTINA MARQUES

Forest Biotechnology "To Go" at RAIZ

ROBERTO ASTORGA & GABRIEL TOY AL

lndustrial Applications of Biotechnology at ENCE

PHILIPPE VIGNERON

Genornic researches : new tools for genetic improvement of eucalyptus in Congo

WOLFGANG SCHUCH Commercial introduction of elite conifer germplasm in forest plantations using soma tic embryogenesis.

YILL-SUNG PARK Implementation of industrial clonai forestry using conifer soma tic embryogenesis in eastern Canada

ALAIN RIVAL Large scale micropropagation of tropical tree crops: oil palm as a case study

BEATRIZ CUENCA Cork oak phenotypic selection based on cork production and quality, biotecbnological propagation and

comercial application.

WORKSHOP: SCIENCE MEETS INDUSTRY

POSTER PRESENTATIONS

H-L. Pasonen, Pappinen A, Degefu Y, Timonen S, Brumos J Effects of a sugarbeet chitinase 4 gene on mycorrhiza formation in transgenic silver birches ( Betula pendula Roth) in vitro

S-K Seppanen, Pappinen A, Vahala J, Teeri T, von Weissenberg K Modification of lignin by an ti sense suppression of 4CL in forest trees.

z. Lorenzo, Soto A, Gil L Utilisation of nSSR for Quercus il ex x Quercus su ber hybrids identification.

O. Nicolaou, Parkes BD High-throughput gene tes ting in radiata pine.

BD Parkes, Sathyapala SK, Gough K, Urangia RM, Grunwell J, Dorey R A new age in forestry genetics: radiata pine somatic embryogenesis clones deployed in commercial plantings.

PA Balk, Verkerk-Bakker B, Konings MCJM, van der Geest AHM & van Wordragen MF Differentiai expression of dehydrin genes in relation to dorrnancy development and release in bud tissue of Pi nus sylvestris.

P. Alonso Rodriguez et al. Cytokinins and morphogenesis in P. pinea cotyledons.

S. Espinel et al. The P. radiata breeding programme in the Basque Country

D. Breton et al.

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Effect of cultural methods on proliferation and their after-effect on maturation of maritime pine (Pin us pinas ter) somalie embryos

E. Dumas et al. Cryopreservation: a powerful tool for the management of genetic resources for breeding and preservation purpose

R. Hasbun et al. Ageing and architecture determine specifie DNA methylation profiles in trees: Application to selected tree propagation.

C. Walter, T. Pearson Expression of a new gene in transgenic Pinus radiata in a field trial

S. Deroles, Walter C, Lill T & Javellana J Biolistic transformation of Pi nus radiata using a partie le inflow gun

J. Charity, L. Holland, L. Grace, C. Walter Expression of BAR, NPTTI and UIDA in transgenic Pinus radiata after Agrobacterium-mediated transformation. 1-F. Trontin et al. Bacterial strain, embryonal-suspensor masses (ESM) genotype and age affect transformation efficiency of Pi nus pinas ter Ait. via

Agrobacterium tumefaciens

S. Jeandroz et al. Mitochondrial DNA variation in french populations of Norway spruce (Picea abies [L.] Karst).

S. Espinel et al. Genetic origin of the oak reforestations in the Basque Country.

M Fladung, M Kaldorf, W Gieffers, B Ziegenhagen, S Kumar Field analysis of transgenic aspen

S Kumar, M Fladung Transgene integration in Populus.

H Hônicka, M Fladung Transformation ofPopulus with genes inducing male or female sterility

F Deutsch, J Kumlehn, M Fladung Induction of haploid poplar through microspore culture

A. Ramboer et al.

Identification and characterisation of differentially expressed genes involved in anoxia root of sessile and pedonculate oaks.

Ma.T.Martin et al. A modified ACM medium to maintain and in vitro collection ofPopulus tremula.

1. Creanga et al. Quercus biodiversity analyzed by means of Ieaf fractality

M. Goicoechea et al. Role of eucalyptus Myb factors in vascular tissues formation.

S. Marucci, Lucca F, Hopp E. et al. Use of Molecular mark ers in Eucalyptus dunnii and E. grandis breeding programmes from Argentin a.

N. S. Nehra et al Method for production and regeneration of transgenic loblolly pi ne clones from diverse elite families

Pablo Goikoetxea et al. Gene flow in Oaks

A. Ramboer et al. Development of microsatellite markers in maritime pine

A. Ramboer et al. Molecular methods for oak wood forensics

J.M. Gion, M. Lalanne, C. Plomion Wood proteomics in maritime pine

Celia Femandez et al. Somatic embryogenesis in mature Quercus robur trees

D. Lopez-Vela, Hem{mdez 1 et al. Induction of somatic embryogenesis in half-sib families of selected Quercus suber trees

K. Hoefig, Moyle R.L, Putterill J, Walter C. Tools to genetically engineer male sterility in Pinus radiata 1 expression analysis of four male cone promoters in Arabidopsis.

L.D. Phillips, Moody J., Geddes B., MacAskill U., Narayan R., Wagner A. Expression studies of Reporter and Selection transgenes in Biolistically transformed Pin us radiata

M. Valbuena, S. C. Gonzâlez-Martinez, Â.Soto, C. Collada, P. Goikoetxea, L. Gil Genetic relatedness and parentage analysis in a Quercus petraea and Quercus pyrenaica mixed oak stand in central Spain

MA Hinchee, Briggs T, Chang S, Frampton KA, Gause KC, Richardson P, Thomas RD, Wilde HD Efficient transformation systems for testing wood quality genes in hardwoods

HD Wilde, Foutz KR, Thomas RD, Zhao Y, Hinchee MA Transgenic eastern cotton wood (populus deltoïdes) with resistance to als-targeting herbicides

Kellison R, McCord S, Hinchee M A guiding organization for forest biotechnology.

C. Ramirez-S., Soltero-Q. R, Santacruz-R. ME, Baena-A. R, Âvila-S. JE, Gonzâlez-A. V, Pelayo-O. C. Production of embryogenic tissue in mature zygotic embryos ofPinus maximartinezii rzedowski.

Rafael A. Canas, Francisco M Canovas, Francisco R. Canton. Accumulation pattern of asparagine synthetase during the initial stages of pi ne development.

Herran A, Estioko L, Rodriguez MJB, Becker D, Lebrun P, Billote N, Baudouin L, Kullaya A, Bourdeix R, Konan JL, Barker JHA, Aldam C, Rohde W, Ritter E. Exploitation of high density DNA marker maps in coconut.

Volosyanchuk, RT, Smulders, M.J.M. Gene tic diversity in Ukrainian na tura! populations of black poplar.

Volosyanchuk R, Los S, Yatsyk R, Hayda Yu, Polupan A., Bohomolov V, Kuznyetsova T, Neyko 1, Gout R, Shvadchak 1. lnventory of genetic resources of broadleaved forest tree species in Ukraine

Billote N, Marseillac N, Risterucci A-M, Asmady, Herran A, Singh R, Amblard P, Durand-Gasselin T, Brottier P, Courtois B, Cheah SC, Rohde W, Ritter E Reference and SSR multi-parent linkage maps for molecular breeding in oil palm (Elaeis guineensis Jacq.).

Kellison R, McCord S, Hinchee M A guiding organization for forest Biotechnology.

P Garnier-Géré, Bedon F, Pot D, Austerlitz F, Léger P, Kremer A, Plomion C. DNA sequence polymorphism, linkage disequilibrium and haplotype structure in candidate genes of wood quality traits in maritime pine

(Pinus pinaster Ait.).

R Minocha, Minocha SC, Long S.

Monitoring of environ mental stress in forest trees using biochemical and physiological markers

Garaicoechea S et al. Characterisation of local olive germplasm in the Basque Country in nothem Spain.

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Annexe

GENOMIC RESEARCHES : NEW TOOLS FOR GENETIC I MPROVEMENT OF EUCAL YPTS IN CONGO.

Vigneron Ph, Gion J-M & Verhaegen D CIRAD Forêt. Montpellier (FRANCE)

UR2PI- Congo

Tree improvement is a long term and costly process which implies to address a lot of problems. The most difficult one is probably the precise assessment of the genetic value for a range of traits in a largely unpredictable environment. Growth, fibre yield and quality, wood physical and chemical properties, resistance to pests and diseases and cutting ability heavily rely to environmental conditions which disturb genetic prediction and gain. Development of molecular markers and functional genomic studies give new opportunities for a more direct and precise assessment of genotype values.

The Congolese plantations were established to supply pulp and paper industries. Due to severe environrnental constraints, pure Eucalyptus species are not sui table for industrial plantations in Congo. Nevertheless, good yield can be achieved using hybrids such as E. urophylla xE. grandis. A breeding programme for eucalypts hybrids is conducted using Reciprocal Recurrent Selection as· co.re of the breeding strategy (Vigneron, 1991). RRS is specially designed to irnprove hybrid varieties resulting from the combination of two unrelated populations (e.g. species) with complementary characteristics. Individuals from each pure parental population are crossed and performances of hybrid pro genies are used for back:ward parent's selection as weil as forward ortets selection. Several kinds of genotypes have to be assessed for a range of traits : families, parents through progeny and relatives, ortets and clones.

In order to improve selection accuracy as weil as genetic diversity management through generations, molecular genetic studies were developed. This paper reports progress and perspectives for marker assisted selection in CIRAD eucalypts breeding programmes.

Genome organisation and genetic diversity.

Linkage maps of individual E. urophylla and E. grandis trees have been constructed using RAPD markers (Verhaegen and Plomion, 1996), microsatellites (from Embrapa) and candidate genes (Gion et al., 2001) segregating in a pseudo-testcross configuration in a large F1 full sib family. The

maps cover respectively 1193 cM and 1021 cM in 13 and 11 linkage groups forE. urophylla andE. grandis parents. In addition, 16 FS families were used to develop genetic maps for a larger sample of parents. Interspecific synteny was partially demonstrated between E. urophylla and E. grandis. It should facilitate genetic studies at subgenera level.

Widespread Eucalyptus species exhibit a high level of genetic diversity. Optimal constitution of collection and breeding populations with appropria te weight of relevant provenances needs a better knowledge of level and structure of genetic variability. DNA markers with neutral or unknown adaptative significance are used to identify pattern of variation resulting from time of isolation, ma ting system, gene flow, genetic drift and population structure. A monitoring of microsatellites and RAPD markers is under progress for both E. urophylla and E. grandis natural populations. These markers are able to give a picture of the evolution of gene tic diversity across breeding cycles. As an example, gene flew within breeding seed orchard of E. grandis were assessed in Madagascar (Chaix, PhD thesis, in prep.). The study reveals a random-like mating system, as expected by breeders, but an unexpectedly high level of pollen pollution.

Beside neutra} diversity, breeders are interested in allelic and functional diversity of useful genes controlling traits of interest. Functional genomics researches presented herein after will help to

choose these genes.

Functional genomics

A global candidate gene investigation for eucalyptus is developed by CIR.AD as a part of a larger European initiative. This approach involves three major steps : (i) identification of expressional candidate gene (CG), (ii) study of colocalisation between CG and quantitative trait loci (QTL) for particular pedigrees (two parents of an inter specifie cross), and fmally (iii) analysis of CG effect in breeding populations.

The identification of expressional CG can be approached at mRNA or protein levels. We plan to develop a proteomic approach in order to identify differentially expressed genes for traits of interest. Proteomics presents high potential for studying genes and genomes. It takes into account post­transcriptional events such as alternative splicing, translation regulation and allows the analyse of post-translational modifications. The study of proteome will be realized using the two dimensional electrophoresis for proteins separation and quantification combined with mass spectrometry methods for protein characterization. These approach will be developed for wood formation and vegetative propagation related traits.

The analyse of genetic architecture of quantitative trait i.e. number, localization and.effect of QTL, is developed since 10 years. Using a large full sib family, a large number of QTL were found for

a range of traits (Verhaegen et al., 1997 ; Gion, 2001). Unfortunately, change in linkage disequilibrium between groups of genotypes, evolution of genetic control with age or season, genotype by environment interaction affect QTL stability and usefulness. The precise identification and isolation of the genes themselves becomes of major importance. CIR.AD Forêt start a functional candidate genes approach with the well known genes involved in lignification process. The observed colocalisations between few genes and severa} QTL for wood quality such as lignin content and Syringyl/Guaïacyl ratio, are consistent with results from transgenesis studies. Development of electronic databases (from Eucalyptus or others angiosperms such as Arabidopsis), differentiai display studies of Expressed Sequence Tags or proteins will provide thousands of potential candidate genes. According to the existing knowledge of their roles in biochemical or regula tory pathways, sorne genes will be localized on genetic maps, searching for QTL colocalisations. Validation of CG heavily relies to adequate phenotype assessment of a number of genotypes in various environments for a range of

traits such as wood physical or chemical properties. Adequate pedigrees, field trials and non destructive routine tests (NIR) are developed to support genomics studies.

The use of CG in MAS relies to the study of allelic variation in breeding population. Studies of single nucleotide polymorphisms (SNPs) of exonic regions of genes within breeding or natural populations enhance the possibility to find the most useful alleles for breeding. A first survey of SNPs is planned for few genes within the breeding population of E. urophylla andE. grandis. In a second step, the individuals displaying the different classes of alleles will be assessed for quality traits. SNPs as well as others markers will be used for MAS to i) set-up the core collection established on the basis of useful genetic variation, ii) choose parents for hybrid families, iii) select the ortets for next clonai tests.

Conclusion

Recent advances in molecular genetics allow a better knowledge of genes involved in complex quantitatifs characters : sequences, functions, expression level, localization . . . Going from lab to field and effective genetic and economie gain needs strong interconnection with traditional breeding and field trials. The current hybrid breeding strategy in Congo provides a lot of possibilities to do so : comprehensive collection of E. urophylla, E. grandis, E. pellita and others Symphyomyrtus species provenances, full-sib progeny tests for nearly two hundreds of parents in factorial mating designs

(more thau 800 families), multisite clonai tests (hundreds of clones), family and clone by spacing and fertilisation interaction trials, which allow an integrative approach of Eucalyptus genome functions and diversity.

Gion JM (2000). Genetic architecture of quantitative traits in eucalyptus: from anonymous markers to candidate genes. PhD Thesis of the Université Rennes 1.

Gion, Rech, Grima-Pettenati, Verhaegen, Plomion (2001). Mo/ecu/ar Breeding. 6: 441-449

Gion J-M, Boudet C., Grima-Pettenati J, Ham Pichavant F, Plomion C, Baillères H and Verhaegen D (2001). Proceedings of the IUFRO Conference Developing The Eucalypt of the future. Valdivia Chili 10 to 15 september 2001.

Verhaegen D and Plomion C (1996). Genome 39, 1051-1061.

Verhaegen D, Plomion, Gion J-M, Poitel M, Costa P, Kremer A (1997 a). Theoretical and Applied Genetics 95, 597-608.

Vigneron Ph (1991). In 'Proceedings ofillFRO congress: Intensive Forestry, the role of eucalypts' pp. 345-360. (Durba

J.-M. 6ION12, C. LALANNE2, H. DUMAZET2, C. PLOMION2 1. CIRAD Forêt. Programm� Arbr2S et Plantations TA 10/C, 34398 Mont�llier Cedex 5, Franc�.

2. INRA, Equi� de Génétique et Amélioration des Arbr2S Forestiers, BP45, F-33610 Cestas, France.

ln Differences in chemical, anatomical and physical characteristics of wood within a single tree could be explained by : (i) variations between normal and reaction wood, (ii) variations within annual ring, i.e. early versus late wood, and (iii)

variations due to juvenile wood with extremely variable properties ranging from the core to the bark particularly in the earl y years of cambium activity. This variability makes it possible to correlate gene/protein expression profiles with wood properties, and ultimately to identify candidate genes involved in the genetic control of wood quality and end-product properties. Recent improvements in realisation of two-dimensional gel electrophoresis (e.g.

immobilized pH gradient) and protein characterisation (mass spectrometry) allow considerable progress in proteome analysis. These advances, although not very used in plant genomics, afford opportunities to have an overlook of gene product expression and address physiological question at a leve! that is much relevant !han mRNA.

andmethads SIX tnes Il Wllll :

t/Oppo•ite wood

• On 14 years-old clones, sampling differentiating xylem were collected on

two years bended trees (opposite and compression sides).

TWo dlmenslonaluel etecuophoresls:

Compression wood

Protein

extraction

4/ Latewood

• On 14 years-old clones, samples of differentiating xylem were collected on April, 151 and September, 151.

l" dimension: l soelectric Focusing - Using a 4 to 7 pH gradient

- Using 24 cm t PG strip

• On 35 years-old clones, sampling differentiating xylem at the top Uuveline wood) and the bottom (mature wood) of the bote.

2•d dimension : SDS-PAGE (tSKD < Motecutar Weight < 90KD)

Constant polyacrylamide gel have becn used

Image AIIBIJS8S: Spot quantification analysis were carried out with Image Master 2D software (Amersham) after a colloïdal blue staining .

1/ A xvtem pepdde map for marldme plne:

- Characterization in progress for 300 xylem proteins using two mass

spectrometry methods (MALDI-TOF and MS/MS).

..

MW

(K.D) "

p chain tubulin a chain tubulin 2/tdentmcadon If dlfferendattv eqressellaratelns : W@[IJ� rn rn - Significant di fferentially expressed spots are represented in red (p<O.O 1 ).

-Non Significant differentially expressed spots are represented in blue. Mature

Wood JuveniJe Compression Oppos1te Early

Wood Wood Wood Wood La te

Wood

30% of analysed proteins were differentially expressed.

, :�_- ... -· ··

! . · · "' · l. . - .; . '

.. .

20% of analysed proteins were differentially expressed.

3/lnte ntllhl resutts ln IIUIIIIC lllta ase :

Differentially expressed proteins

11% of analysed proteins were differentially expressed.

Global scheme of PROTIC data base SPOTS RELATIONSHIP Modelisation of PROTIC data base

UPDATE QUERY PROJECf Set of data from

one analysis

SAMPLE ·Plants

. Culture conditions

- Sampling

GEL Mcthods ( 1 st dimension,

equilibration, 2nd dJmcnsion, staining)

. Samplc stock state L---------' · Extraction mctbod

ACQUISITION OF GEL PICfURE · Picturc

· Acquisition mclhods

· Spots detection

· Detection mcthods

·Calibration

·Calibration mcthods

,---��� ·Relation Iwo by two

·Management of spots group SPOTS -X, y -Arca

· Pic·hcight

1\'-----------' IDENTTFICATION · Management of identifications

· ldcnlification mcthods

(MS, WB, scqucncing ... )

· Identification rcsuhs

Genomic researches : New tools for genetic improvement

of eucalypts.

Philippe Vigneron Jean-Marc Gion

Daniel V er haegen Gilles Chaix

ktf UNITE DE RICHERO-IE SUR lA PRO!JUCTW!TE DES PLANTATIONS INDUSTRIEllES Bottt Postale 1291 POINTE-NOIRE République du Conso Tél. (242) 94 31 64 Fax (242} 94 47 95 t·INIII, URlo&'t:.!�•comf'

Biofor02 Vitoria Gasteiz

Recurrent Reciprocal Selection E. urophyl� � � . "" • � E. grandis

Provenance-p;?� test� ---- ......... Aovenance-progeny tests

.. � .. ..

��� Intra specifie crosses

�� ... ... ...

Hybrid progeny--_. tests lst clonai

tests --_. 2nd clonai tests

• ~ � Intra specifie crosses

.,.Jr ... ...

Hybrid progeny tests

............... lst clonai tests

............... 2nd clonai tests

• Genome organization and genetic diversity

• Gene expression and genetic architecture of quantitative traits

.

� ... ,

Genetic mapping

- �1111 silJ faLIIli11)1: ' x ' ...... Jill"""

E. urophylla E. grandis 400 Full Sibs

c:=) Two saLhlraLted genetic Illalps

- 4x4 �aLctoriaL1 Illalting:

� 14.128 ..._

i: 14.133 � � 14.136 � 14.144

E. grandis 9.37 9.40 9.41 9.42 "" "" "" "" "" "" "" "" "" "" "" "" "" "" "" ""

16 F.S progenies ;.._

q Eigth paLrtiaL1 genetic Illaps

M05_.527-(1) f A17_,700.(I)

15 5-

V20_ 337- (2) Z03_ 92!5+(3)

3te-K10l� Y11_.815+ (l5)

A09_.586+ (2)

63oM

21.7-

MOS .14!3+(1) 5•1

-x11.:.�2e. (B)

' .,._., .. (6) ,- /EMBRA20- (9ij "' '"

1<48cM

GROUP 1

808_2322- (<46)

K17_.591+ (47) At3_.1174-(48) N07_.ll1&-(49) fm_.tw tell

U13_.428-(50)

R15_.519-(51) 1 MOI_,tl411dof IU2....10f Jâl

GROUP3

<47cM

'"'"'

.....

137cM GROUPS

X01_1160+ (28) xœ_1178·!291

X02_ 1210• (30)

��:��:rJ\ M12_,4!!9-(33)

20.3-1 jEMBRA07+ (1og A20_.938+ (11) 114_1288+ (12)

�TH�!,3� K16_14c42+ (15)

Y13_.86i+ {18) N05_.95&- (17) ....- N11_1406- (11) Z08_1� (18) N07_.735- (19) Ntt_.197- (20) 111l.1102-t (21) r 000_, .. 02- (9) L05_.750+ (22) 9.2-

Mto_.Jn- (tOI

K10_1551+ (23) 1 A1i_.e94+ (11)

..... ......

GROUP2

Q14 .044+ (23) jEMBRA02:J24l]

R15_.671· (42) 8011_. 718+ (25)

908_1045+ (43) l14_.520-(2e)

A10_.1304+ (_..) R115_.625-t-(27)

.....

003_.390-(45)

57 cM

GROUP4

X07_.-(34)

� K12_.657- (38)

K16_1183+ (JV)

147cM GROUP6

"'"'

GROUP7

OS cM

GROUP9

128cM

GROUP Il

., ...

N05_1i20- (40)

Y05_1299+ (41)

N14_ 1588· (42)

A16_.759-(43)

jEMBRA1Û (44}] N12_.482+ (45)

)cm ... •.-. nrM •

noM

147cM

R13_.377• (71)

A09_.1S1� (72)

Xll2._11lN•(73)

114_.797+ (74)

801_.&715-{75)

X07_1533• (77) )Xfr ... u ... ,_,

003_.286'1•(711)

N13_1368-(711)

11.2-

GROUPS

jEMBRA13-(95� U18_ 1352- (SIIS)

123eM

� ... -1.8 -• �=�laàli�-_._

xœ_.742-(WI

K11S_.474-{100)

zoe_.IS5H (101)

.....

GROUP 10 122"'

LEGEND

Y05_ .... (51)

IAOI_.Ifmo 1WI U1.,,..ofc.ll

E. urophylla E. grandis )K'It_.-.OrM 1 ,,_, ... (1)

J011_5<9-(2)

jEMBRA13� (95ij &cL-(1oeij

R13_,m•C71)

SSR

GENES

RAPD

t ,,_, ...

l06_480-

Partial genetic maps for 4 E, urophylla genotypes

14.144 14.128

4.133 f ::::::::: :::::�

4f.1

��-...__.

-� Mll.IIOo lllf_toœ-- zn..tuo _,...- DJ

_..,.- za..� / ,..,._. li,_,. -:.lll..JC6.

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IOO----'�c���

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3t dll -t-- Ktt.l74· a cM Il CIM

GROUP 1

14.144 -f-ICtt.tUt•

..... YU _ _..

14.133 - Kil_..,., -1101_12QD.

......... �1(11_ .. 14.136 -- 1-Ktt_tllt+

-......... \f ... _....�f"'-' ... 'l-eM ""-'"· Xll.f46-LM.?a•

.128 ......... I .dol _/ M10_M)Oe- MIO.M-

t- MID_)oiGo / .dol

.... ....... --­_.,._SIOt __ ..... ... SIOt

- \ .......... ...

::--·\l--t= 17cM ttcM

GROUP2

Percentage of coverage � 50% 14.144

1---- 14.133 -xtt..tO»> 14.128 14.136 -f-xtt..tD»>

� ·�;.��

_.,._,... ..... -�<o ....... 1 ' n•_no. �IUl..IIO-

- J-wta..UOo­(MtU3Sol

r ,._un•

riOLIII»-

- . ....---rll:tt...IU­

__ ,/ ... . .... ,... -Mtt.

r-ICI1_t1 .. _..._.,.. 1- KM....7N· e ........._ -1- Kil_ nt. \:Il eloi , .. -f-.... -. f: ... _ ...

GROUPJ

14.144 14.133 � ..... -

..... -,14.128 -· .... -· 14.136 .. �.-�I "�· J . .......

··��·- •a.·---=:::·��1: __ , .... \

... �.

.._,.. _......,. .,._,

•111411:t'-m•�-•ut_tTt· '"-

.f-'l't._ .. .,,.._ ... -•n•_...,.._

_G_ROUP 4

14.136 �"':t.JIII IUJ..I

14.12 8

=:�= l=; =�·--r·�···- l \ 1 ·-.-.... ----- - - :: -n...

ftoil..lt..._- liiDI'_Ja. acM H11 _ _.. -----------

oiOaM --- -- BMJ:».

-.... llol..Jt ..

.. ,__

GROUPS .... X tl._. GROUPS

14.128 14.144

=-·-·

14 133 14.136

::::::-: .......... __ ,..__ r ........ -/ �-Ktq,.mo�__,.KÏÛJO.

- Y1V� .,,_ ... � -'

IOLlQI.o -)(ll_U\0. _---::--101__.

�·u..,.,-- "'-'\ / .. , .. !OoLnG. -tt•UMt 114..1--�too:M ..._,..

GROUP6

---.... , ... ...,

Vl\..740o

zœ_t ... 1,.01_1 .. 11-\.'--+

14.144 14.128 14.133 -1-

··�-- ---

""' +--- 14.136 � --t ........ _.._ "'---...:......-.. ._ .. \ , .. IUI..Ia• ........ lt4...•7'0-X1t_10CI). Sdol

Zl ..

GROUP7

lttt.. ...

.. ._ ..... ---,.,._ ...

lUI ..... •

14.144

I ... _.,.. .,.._.,. ....

GROUP9

14.133 -xta,.ao.

14.136 �XIIJIIOo

14.144 t,.._.,. ....... --

14.133 14.136 14.144

= · .. ·-;r ·u..-KII_IOOo - .. \l_t.t<ll)oo .,._ ... __ "'-"""\ 14.128

::'-t;�; -MilL*­""' ,.._ .... ....

"''--,.,. _ _� , ..

Partial genetic maps for 4 E .. grandis genotypes

9.40 9.37

..._ ....

HU.52So

�NU_t41�

9.37 ..... _ ...

9.41 -MIO-""·

GROUP2

9.41 ....... 9.40

� ._J'It(..lfl·

........ - - - l -:���--=---=-�·

--�.42 - 1 ,- ........ �'UT'" ltll.tll).

\l""-"'" ' _ ..._, ,,._ ....

. , .. .,.. ......_ ....... -.. ........ .Jl_

.. �.... ' -1-""

... ......... ... __

J

''"

_110-, ' \ -'"-��-- ..- MIU»"

... _ .... / ....__.

' , .. _

...

. \ ',, XtZ..•JOo• \ -LOI�----.. AOII.lC»>• \ t�r�:::-'-EOl_IJO.

GROUP3

Kt1l.nt•

LOI. ...

..... ,.

Percentage of coverage -- 50%

9.40 !

9.3?,,

__ lt(_IOC)o --f- 1\(.Jœo. 9.41 -t- ----- .... ,... lUI_� 7dlll

31''* +-Rtl_lfG. - .at.TJV..

GROUP4

9.37

9.42 f-.. ..... lfG..

9.40 IE0(_3100 9.41 9.42 ........ '1'1).. ...

.. � ...

ltl\...11 .. -.,.... ... __ - ., ......

!IOt....lll-). -Rtl_!-. - •tt.. .. ----11111::1111

t .. � ....

=\ .. \t.�jl;:;--1 .... -:;ROUP5

100 ..... ��'LI

.,._ ...

..... ,...

9.42 9.41 1 -........

(HI2...11�) (10t_7JI:>o) -Ntt...XJOo -l1l.l14+

-KIQ..)W• .... - ........ \

9.37

ltt...114+ 9.37

"'"' ""-"'"� ()tU_nJO) (101....A0-)

.......

ILCI.IIno.} K\2..._. ........ KIQ..�/JICIM

t- ltll.f10.

GROUP6

- ttl...0.--

.......

Mtl..-r·

""-�""

., __

.... ,.. (IDI_IIIG·)

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::!;--.. ·· ,._uJ -t..œ_.o+ ...

GROUPS

9.40 9.41 .,_.._,,,.. ___ ,s,..

GROUP9

9.40 .......... f-Ktl.'r»

91.42

.,_.,.. �

9.41!,_.,.."

91.3

�--XI\.IIU·- - _ -- - :-.::.o.\ Xll7.110o

'l'tl_... 9 41 - -ltti_IU<� ..._ ....

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li(_ W.---

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9.41 9.37 - ��;11,.11 ..

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M1.t_.ao­

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40 LOI_A'fOo

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Mœ.IIIIOo ....... ""' .... ,........ -,........ ! .... ".:: t-"'·"'" I'I:II _UOO ...

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...

....

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GROUP Il

14.133

Homologous linkage groups

4 E. urophylla genotypes

14.144 14.128 14.136

Linkage group 1

4 E. grandis genotypes 9.37 9.40 9.41

K12_879+ 1 ....., ...._ \ \1 <<

E04_370.\

-1- A09_1084+ \ A09_1084+

Linkage group 3

9.42

K12_879+

114_344-

Homologies with others Eucalyptus urophylla andE. grandis maps

Homologies with several markers

Homologies with only one marker

No homology

SSR Mark ers

EMBRA12

EMBRA16

EMBRAll

EMBRA5

EMBRA9

EMBRA15

EMBRA2

EMBRAlO

EMBRA19

EMBRA7

EMBRA13

EMBRA20

EMBRA8

Linkage group number

CIRAD Brondaniet a/.(1998) E. urophylla 1 E. grandis E. urophylla 1 E. grandis

r··············��;·····················;�;····························· .. ········��·�················-·····��·�······1

i i , ........................................................................................................................................................... ! ! GL4a GL4 - GLll i i 1 f······················'""''''''····················· .. ···························································· .. ·····································t

i i i GL6 - GLlO GLlO i � ........................................................................................................................................................... 1 ! GL9 - GL4 GL4 1 i j ! ............................................................................................................................................................. : .............................................................................. !

! GL2 - i � i i l i GLlO - i !. ........................................................................... 1

�, ,. "' .,, '• .,. ' •"'M:� '(!, ,'; ' '

• --1� !"'"- • .:� .. !.'. ·f_<�+.�:i· "<!; _<;i&.� [=��-=!J

Towards a consensus genetic map for Symphyomyrtus subgenus

• Mapping of new codominant markers

- Single Sequence Repeat markers

- Expressed Sequence Tags

• Development of Bacterial Artificial Chromosome

library for the future

Genetic diversity and gene flow

Eucalyptus grandis breeding population

•• 1 '" 'iil

�è!.l� U �• Individual 1 ,>J.

�·

�-·

�

1 . ...Jl �.�,;:f

001

.:::: lndividual 2

�""" tJ '"l ��

h�ll\1 �1 ..l" -:ill

r a-� =-141-1

,. '

: : :

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JJL . .L_jJ. )._ f.::: lndividua16 ' l!ill1 Œm.!LU __

. 11,...'�1

�!lw

. T� ' : Allelel t Allele 2 'f

Allele3

Genetic Diversity Structure

E. grandis breeding population based on several provenances

·'u , : ::'

16 Provenance 4 13

18

Provenance 3

16

ii!�:��-- El 19 Provenance 1

=::}New organization of breeding groups

Gene Flow

• Balanced contribution of the different genotypes

• Low lev el of self pollination: 3 %

• Random like mating system

High level ofundesirable 48% 1 > alien pollen

:::=} High quality seed production and acliievemênt of genetic

gain if pollen pollutiQt.t·iis �voided

• Genome organization and genetic diversity

• Gene expression and genetic architecture of quantitative traits

.

,._ ., •

Three steps in candidate gene approach

Expressional candidate gene (CG)

==> Transcriptomic studies (messenger RNA) ==> Proteomic studies (pro teins)

Functional candidate gene

==> Genetic mapping

==> QTL detection

==> PQL detection

V ali dation of allelic effects

==>Test gene effect in breeding popl:llations (SNPs ... )

Proteomic approach

Bidimensional Electrophoresis :

Separating polypeptides according to their isoelectric point

90KD

Separating polypeptides according to

their molecular weights (MW)

15KD <MW< 90KD

Proteins extraction

� 4 7

t

First dimension: Isoelectric focusi

Second dimension : SDS-PAGE

Protein Identification (Mass spectrometry)

Protein Quantification (Differentiai expression analysis)

1 A « pool » of candidate proteinslgenes 1

Why functional CG are necessary ?

QTL Unstability with

Age

Environment

Pedigrees 1 populations

.-

Seasons:

E. urophylla

E. grandis

QTL change with age

GL l -----------------1 ... GL3 a

GL8 ----------� .. �­GL3----------------

GL3 Il GU========-�·�==������������� GL5 GL9 GL9 Il

·'c

QTL detection for different genotypes

.. .., lol.a...>w·.

ICI.t..ao. TNI,_ID&o .... _J__ Nll.la.

lt .

. 37 • .._....

·r··· .. . _ ..... IlOt.. ,... • •

't11_110< ,,._

Nn_�«�&-

...._w.. � ... KID.�

-f- NIUG-GROUP2

Sl.41 ___/' 114.11'1·

ZCQ_710o _ ,.,.._117•

loi lU• 1 f"'"-l(!�f7Po -RIJ.._..,._

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*

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l.OUIOO e

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Functional CG

Genetic Mapping

- Lignification genes

- Expressed sequence tag ( expressional CG) ...

Quantitative trait Loci analysis

- Several wood properties

- Other quantitative traits

Genetic mapping on existing maps:

For E. urophylla and E. grandis

ForE. urophylla only

27.0 -PAL

EgTubAJ

CCR 94 cM 136 cM

GROUPE2 GROUPE6

2s.o -

29.s -

31.8 -

COMT2 COMTl EgHypar

153 cM GROUPE7

E. urophylla

4CL

1.9 -ca: �&AOMT

49.0 -29.o -

26.4 -

120 cM 125 cM

GROUPElO ,_ GROUPE 11

QTL Detection

• Strong effect of QTL

4 QTL explaining 41 % of lignin content variation

3 QTL explaining 33 % of S/G ratio variation

• Association between traits

Growth and wood quality

• Colocalisation genes - QTL

Joint segregation Genes 1 Quantitative Trait:

CCR and PAL 12

10

Q) 8

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E. urophylla 2

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GenEtic IMtance (dVI)

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==> Strong association QTL - gene

S/G ratio

Klason

• Evidence for 4 candidate genes

- CCR ::::} lignin content, S/G ratio - PAL ::::} S/G ratio

- 4CL ::::} wood density

- COMT::::} spliting index

::::} consistent with results from transgenesis

• No co localisation with CAD and ·CCoAOMT

Allelic effect within a family

Combined effect of CCR and PAL favorable alle les

S 1 G ratio variation in a full sib family of E. urophylla xE. grandis

n = 37 1

n = 33 n = 36

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PAL+ CCR+ CCR- PAL·

Overall mean : 3.65 Mean: 4,35

Gene variation effect within breeding population ?

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t t

SNPl SNP2

1 1 1 QTL Intqval

SNP3=QTN DNA sequence (cM)

Towards a Marker Assisted Selection

��� 111111 Survey of allele diversity in the breeding population

Provenance-progeny tests

Control of population

genetic diversity

~

��� Intra specifie crosses

1 1111

Hybrid progeny tests

Il Il Il Il

Test of cross­breeding value of

alleles

lst clonai

Il Il � tests �

Selection of best alleles Il combinations 2nd clonai tests

A considerable amount of field trials

• Provenance-progeny trials

55 000 individual E. urophylla, E. grandis and E. pellita trees

• Hybrid progeny trials (factorial mating designs)

- 200 parent trees in tests

- 800 full sib hybrid families (50 to 100 trees)

• Clonai tests (multisite)

300 E. urophylla x E. grandis clon�s in long term tests

(3 sites x 3 reps x 9 ramets)

Gene 1 allele effect through pedigrees

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Annexe Wood proteomics in maritime pine.

J.-M. Gion1, C. Lalanne2, H. Dumazet2, C. Plomion2

1. Programme Arbres et Plantations. CIRAD-Forêt TAlO/C, Campus de Baillarguet 34398 Montpellier Cedex 5, France. gion@cirad.fr 2. INRA, Equipe de Génétique et Amélioration des Arbres Forestiers. 69 route d'Arcachon. F-33612 CESTAS Cédex France

Differences in wood characteristics within a single tree are a common feature. These include: (i) variation within annual ring in temperate zones, i.e. early vs late wood, (ii) variation due to juvenile wood with extremely variable properties ranging from the core to the bark particularly in the early years of cambium activity, and (iii) variation between normal and reaction wood and more precisely between opposite wood vs compression wood. These six types of wood possess distinct chemical, anatomical and physical characteristics. This variability makes it possible to identify candidate genes involved in the genetic control of wood quality and end-product properties. Recent improvements in realisation of two­dimensional gel electrophoresis ( e.g. immobilized pH gradient) and protein characterisation (mass spectrometry) allow considerable progress in proteome analysis. We have studied the

proteome of differentiating xylem for the previous six types of wood. Proteins'hàve been separated according to their isoelectric point (lsolelectric Focusing) using a 4 to 7 pH gradient and according to their molecular weights (SDS-PAGE) on a constant polyacrylamide gel. Along the six xylem conditions, a means of 600 proteins, for each condition comparisons, have been detected after colloïdal blue staining. Around 200 spots have been eut of 2D gels for identification with mass spectrometry (MS/MS) analyses. Our fust objective is to obtain a peptide map for differentiating xylem in maritime pine (i.e. reference gel with all spots characterized) and integrate all this information in a public database "PROTIC DB" under development. The second goal of our approach is to determine putative candidate genes/proteins which differentiate the studied xylem conditions. After spot quantification and volume normalization, the analysis of expression levels for the detected spots reveal that more significant differentially expressed proteins (a = 1%) have been detected between the juvenile vs late wood (30 % of the detected spots) in comparison to early wood vs late wood (20%) and opposite wood vs juvenile wood (11 %).