Search

International audience; AbstractKey messageA moderate genetic diversity, the absence of a significant genetic differentiation between wild and cultivated stands and a highly admixed genetic structure of sweet chestnut with two main clusters were observed in France using two different data sets with 10 and 18 microsatellites.ContextRenewed interest in European chestnut in France is focused on finding locally adapted populations partially resistant to ink disease and identifying local landraces.AimsWe genotyped trees to assess (i) the genetic diversity of wild and cultivated chestnut across most of its range in France, (ii) their genetic structure, notably in relation with the sampled regions, and (iii) to a lesser extent the relations between French chestnuts with 10 cultivated chestnuts from the Northwest of Spain that were previously classified in the Iberian or Italian groups.MethodsA total of 693 trees in 16 sampling regions in France were genotyped at 24 SSRs and 1401 trees in 17 sampling regions at 13 SSRs.ResultsGenetic diversity was moderate in most sampling regions, with redundancy between them. No significant differentiation was found between wild and cultivated chestnut. A genetic structure analysis with no a priori information found a low yet significant structure and identified two clusters at 18 SSRs. One cluster gathers trees from south-east France and Corsica (RPP1), and another cluster gathers trees from all other sampled regions (RPP2). A substructure was detected at 10 SSRs suggesting differentiation within both RPP1 and RPP2. RPP1 was split between south-east France and Corsica. RPP2 was split between northwest France, Aveyron, Pyrenees, and a last cluster gathering individuals from several other regions.ConclusionThe genetic structure within and between our sampling regions is likely the result of natural events (recolonization after the last glaciation) and human activities (migration and exchanges). These advances on our knowledge of chestnut genetic diversity and structure will benefit conservation and help our local partners’ valorization efforts.

KEY MESSAGE: High genetic diversity, absence of a significant genetic differentiation between wild and cultivated stands and highly admixed genetic structure of sweet chestnut were observed in France using two different datasets with 10 and 18 microsatellites. Results strongly suggest that the French gene pool is at the intersection between the Italian and Spanish gene pools. Context: Renewed interest in European chestnut in France is focused on finding locally adapted populations partially resistant to ink disease and identifying local landraces. Aims: We genotyped trees to assess (i) the genetic diversity of wild and cultivated chestnut across most of its range in France, (ii) their genetic structure, notably in relation with the sampled regions, and (iii) relations with its neighbors in Spain and Italy. Methods: A total of 693 trees in 16 sampling regions in France were genotyped at 24 SSRs, and 1,401 trees in 17 sampling regions at 13 SSRs. Results: Genetic diversity was high in most sampling regions, with redundancy between them. No significant differentiation was found between wild and cultivated chestnut. A genetic structure analysis with no a priori information found a low, yet significant structure, and identified two clusters. One cluster gathers trees from south-east France and Corsica (RPP1) and another cluster gathers trees from all other sampled regions (RPP2). A substructure was detected at 13 SSRs suggesting differentiation within both RPP1 and RPP2. RPP1 was split between south-east France and Corsica. RPP2 was split between north-west France, Aveyron, Pyrenees and a last cluster gathering individuals from several other regions. Conclusion: The genetic structure within and between our sampling regions is likely the result of natural events (recolonization after the last glaciation) and human activities (migration and exchanges). Notably, we provide evidence for a common origin of most French and Iberian chestnut trees, except those from south-east France that were associated with the Italian gene pool. This advances our knowledge of chestnut genetic diversity and structure, will benefit conservation and help our local partners9 valorization efforts.

Biogenesis of the secondary cell wall in trees involves the massive biosynthesis of the phenylalanine-derived polymer lignin. Arogenate dehydratase (ADT) catalyzes the last, and rate-limiting, step of the main pathway for phenylalanine biosynthesis. In this study, we found that transcript levels for several members of the large ADT gene family, including ADT-A and ADT-D, were enhanced in compression wood of maritime pine, a xylem tissue enriched in lignin. Transcriptomic analysis of maritime pine silenced for PpMYB8 revealed that this gene plays a critical role in coordinating the deposition of lignin with the biosynthesis of phenylalanine. Specifically, it was found that ADT-A and ADT-D were strongly down-regulated in PpMYB8-silenced plants and that they were transcriptionally regulated through direct interaction of this transcription factor with regulatory elements present in their promoters. Another transcription factor, PpHY5, exhibited an expression profile opposite to that of PpMYB8 and also interacted with specific regulatory elements of ADT-A and ADT-D genes, suggesting that it is involved in transcriptional regulation of phenylalanine biosynthesis. Taken together, our results reveal that PpMYB8 and PpHY5 are involved in the control of phenylalanine formation and its metabolic channeling for lignin biosynthesis and deposition during wood formation in maritime pine., Functional genomics unravels the role of two arogenate dehydratases in maritime pine during wood formation, and provides new insights into how lignin biosynthesis and deposition are regulated in conifers.

Although eucalypts are the most planted hardwood trees worldwide, the majority of them are frost sensitive. The recent creation of frost-tolerant hybrids such as Eucalyptus gundal plants (E. gunnii × E. dalrympleana hybrids), now enables the development of industrial plantations in northern countries. Our objective was to evaluate the impact of cold on the wood structure and composition of these hybrids, and on the biosynthetic and regulatory processes controlling their secondary cell-wall (SCW) formation. We used an integrated approach combining histology, biochemical characterization and transcriptomic profiling as well as gene co-expression analyses to investigate xylem tissues from Eucalyptus hybrids exposed to cold conditions. Chilling temperatures triggered the deposition of thicker and more lignified xylem cell walls as well as regulation at the transcriptional level of SCW genes. Most genes involved in lignin biosynthesis, except those specifically dedicated to syringyl unit biosynthesis, were up-regulated. The construction of a co-expression network enabled the identification of both known and potential new SCW transcription factors, induced by cold stress. These regulators at the crossroads between cold signalling and SCW formation are promising candidates for functional studies since they may contribute to the tolerance of E. gunnii × E. dalrympleana hybrids to cold.

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

Key message Molecular markers were used for paternity recovery in a maritime pine (Pinus pinasterAit.) polycross trial, facilitating forward selection. Different breeding strategies for seed orchard establishment were evaluated by comparing genetic gains and diversity. This work opens up new perspectives in maritime pine breeding.[br/] Context Polycross mating designs are widely used in forest tree breeding to evaluate parental breeding values for backward selection. Alternatively, polycross progeny trials may be used to select the best trees on the basis of individual breeding values and molecular pedigree analysis.[br/] Aims This study aimed to test such a forward selection strategy for the maritime pine breeding program.[br/] Methods In a maritime pine polycross trial, progeny with higher breeding values for growth and stem straightness was first preselected with or without relatedness constraints. After paternity recovery, the preselected trees were ranked on the basis of their breeding values, estimated from the recovered full pedigree. Finally, the best candidates were selected with three different strategies (forward, backward, mixed) and three levels of coancestry constraints to establish a virtual clonal seed orchard.[br/] Results Complete pedigrees were successfully recovered for most of the preselected trees. There was no major difference in expected genetic gains between the two preselection strategies which differed for relatedness constraints. Genetic gains were slightly higher for forward selection than for classical backward selection.[br/] Conclusion This seminal study opens up new perspectives for using forward selection within the French maritime pine breeding program. [br/]

Fungal degradation of wood is mainly restricted to basidiomycetes, these organisms having developed complex oxidative and hydrolytic enzymatic systems. Besides these systems, wood-decaying fungi possess intracellular networks allowing them to deal with the myriad of potential toxic compounds resulting at least in part from wood degradation but also more generally from recalcitrant organic matter degradation. The members of the detoxification pathways constitute the xenome. Generally, they belong to multigenic families such as the cytochrome P450 monooxygenases and the glutathione transferases. Taking advantage of the recent release of numerous genomes of basidiomycetes, we show here that these multigenic families are extended and functionally related in wood-decaying fungi. Furthermore, we postulate that these rapidly evolving multigenic families could reflect the adaptation of these fungi to the diversity of their substrate and provide keys to understand their ecology. This is of particular importance for white biotechnology, this xenome being a putative target for improving degradation properties of these fungi in biomass valorization purposes.

Annotation of the Eucalyptus grandis genome showed a large amplification of the dehydration-responsive element binding 1/C-repeat binding factor (DREB1/CBF) group without recent DREB2 gene duplication compared with other plant species. The present annotation of the CBF and DREB2 genes from a draft of the Eucalyptus gunnii genome sequence reveals at least one additional CBF copy in the E. gunnii genome compared with E. grandis, suggesting that this group is still evolving, unlike the DREB2 group. This study aims to investigate the redundancy/neo- or sub-functionalization of the duplicates and the relative involvement of the two groups in abiotic stress responses in both E. grandis and E. gunnii (lower growth but higher cold resistance). A comprehensive transcriptional analysis using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was performed on leaves, stems and roots from the two Eucalyptus species after cold, heat or drought treatment. A large CBF cluster accounted for most of the cold response in all the organs, whereas heat and drought responses mainly involved a small CBF cluster and the DREB2 genes. In addition, CBF putative target genes, known to be involved in plant tolerance and development, were found to be cold-regulated. The higher transcript amounts of both the CBF and target genes in the cold tolerant E. gunnii contrasted with the higher CBF induction rates in the fast growing E. grandis. Altogether, the present results, in agreement with previous data about Eucalyptus transgenic lines over-expressing CBF, suggest that these factors, which promote both stress protection and growth limitation, participate in the trade-off between growth and resistance in this woody species.

chapitre en open access; Maritime pine is a major species in Europe, especially in France, Portugal and Spain. This species has long been subjected to advanced forestry and genetic breeding since the early sixties in France. However there are strong limitations to improvement progress, i.e. quite long generation time, high genetic load as well as high genetic redundancy within breeding population. Sudden and drastic socio-economic and environmental changes in recent years would need a significant paradigm shift in current breeding technology to deliver suitable tested tree varieties in plantation forestry, i.e. multi-varietal forestry targeting a wide range of end-products through various silvicultural regimes. Field comparison of vegetative propagules is a key towards individual selection and efficient capture of the best genetic stocks. Highly efficient clonal propagation technology is also required for scaling up production of improved varieties. Somatic embryogenesis is considered as the key technology to fulfil such requirements in maritime pine. The species is characterized by increased recalcitrance to vegetative propagation through conventional cuttings as trees are reaching their adult vegetative or reproductive phase. Somatic embryogenesis initiation from mature trees is still challenging in conifers. Therefore the approach developed in Maritime pine is the postponed propagation of tested trees combining somatic embryogenesis initiation from immature zygotic embryos and stable cryopreservation of juvenile embryogenic tissue. This review describes the most recent achievements and challenges towards efficient somatic embryogenesis as a key technology for multi-varietal forestry in Maritime pine.

International audience; Polycross mating systems are widely used in forest tree breeding for genetic testing. Backward selection based on polycross testing assumes equal male reproductive success and true half-sib progeny. The main objectives of this study were, firstly, to investigate the departure from these assumptions in a maritime pine polycross trial and, secondly, to evaluate the consequences for heritability and breeding values estimations. A total of 984 offspring from 98 half-sib families was genotyped with single nucleotide polymorphism markers to recover the full pedigree. Paternity was assigned successfully for 89 % of the offspring at a 99 % confidence level. We thus concluded there was an 11 % pollen contamination rate, assuming contamination when no genotype from the polymix composition could be identified as a father. The paternal contribution to the offspring varied among the males, but the departure from half-sib assumption was moderate since the average genetic correlation within the family was 0.26. Heritability and breeding values for girth at breast height and stem sweep were estimated using individual-tree mixed models with either partial or full pedigree information. The results highlighted a minor bias in heritability estimation due to unknown paternity, as well as a high correlation for estimated breeding values between the partial and full pedigree models, suggesting that the genetic merit of the parental generation for backward selection was adequately predicted using the partial pedigree model. Finally, pedigree recovery was also discussed in a perspective of forward selection.

Somatic embryogenesis is affected by highly variable maturation yields in Pinus pinaster. Origins of this variability were investigated by testing effects of spatial and temporal division of initial embryogenic tissue into independently cultivated pieces. One embryogenic cell-line was proliferated to obtain six embryonal-suspensor masses (ESM) treated as six sub-lines within a single dish. After proliferation to reach 2, 4 and 8 dishes (12, 24, 48 ESM), the 48 ESM (six sub-lines) were maintained by weekly subculture (34 weeks). ESM observations and maturation tests were regularly performed during the amplification and maintenance periods. Relations between maturation yields as well as cotylfedonary embryo length and immature embryo morphology were analyzed. ESM yielded variable results, independently from culture spatial (dish) and temporal (sub-line) subdivision. Maturation yields and length of regenerated embryos globally decreased as a function of subculture number. Concomitantly, morphological degradation of immature embryos occurred, indicating a global loss of embryogenic ability. Maturation variability probably results from immature embryos heterogeneity, which could be increased by manipulations during subculture. This is the first time that this evolution along time in culture is precisely described in conifers somatic embryos.

International audience; Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Summary Embryonal-suspensor mass (ESM) lines characterized by a spiky morphotype (i.e., developed early embryos escaping from the ESM periphery) were recently shown to produce the best maturation yields in Maritime pine. How to select or preserve such a valuable morphotype during ESM maintenance (prior to maturation treatment) is still unknown. Several maintenance procedures were tested; 2400 ESM from 10 lines were subcultured each 7 or 14 d on maltose- or sucrosecontaining medium without plant growth regulator (PGR) or supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine or a low concentration of abscisic acid. Multiple components analyses based on growth rate, macromorphology data, and micromorphology data, collected for 6 mo., allowed for the association of the spiky morphotype with additional traits (defining the phenotype I) such as whitish aspect, high vigor, high growth rate, and complex cellular organization (resulting from high early embryogenic ability). Since a gradual increase in growth rate as well as a decrease in cellular organization were concomitantly observed during the 6 mo. experiment, we concluded that ESM growth and early somatic embryo development were disconnected. In some lines, the progressive loss of early embryogenic ability (aging process) could be decreased using maltose-based and PGR-free medium. For most lines, the aging effect was minimized using a weekly subculture. An improved procedure for ESM maintenance prior to the maturation step is thus proposed.

International audience; Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene ($\beta$-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.; Transformation génétique du pin maritime (Pinus pinaster Ait.). En appliquant nos protocoles d'embryogenèse somatique développés pour Pinus pinaster, des tissus et plantes transgéniques ont été obtenus après bombardement avec des microparticules (biolistique) ou coculture de masses embryonnaires (ESM) avec Agrobacterium tumefaciens. Les expériences de transformation ont été conduites à l'aide du gène de sélection hpt (résistance à l'hygromycine B) et du gène rapporteur gus (activité $\beta$-glucuronidase). L'hygromycine a permis de sélectionner efficacement les cellules transformées par ces deux méthodes en 4 à 19 semaines. Le nombre moyen de lignées résistantes à l'hygromycine exprimant le gène gus obtenu par gramme d'ESM varie de 7,0 à 8,5 (biolistique) ou de 0 à 67,3 (Agrobacterium). Les embryons matures obtenus à partir de certaines de ces lignées ont pu être convertis en plantules élevées en serre. Seulement 12 mois sont nécessaires de la transformation des ESM jusqu'à l'acclimatation des plantes. La nature transgénique des ESM, embryons somatiques et plantes, a été confirmée à l'aide de tests histochimiques " GUS " et de méthodes moléculaires.

International audience

International audience; Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.

Article fédérateur entre toutes les équipes Européennes travaillant sur le pin maritime. Assemblage du transcriptome des données venant de France (INRA Orléans + INRA Pierroton) + Espagne + Portugal = projet Sustainpine; International audience; Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

Two maturation media with high and low concentration of gellan gum were used to evaluate the maturation performances of four maritime pine ESM (embryonal-suspensor mass) lines. The maturation performance is influenced by sampling modalities; the outer part of the ESM yielded more cotyledonary embryos than the inner part or the whole colony. ESM lines showing several stage 1 embryos at the periphery (spiky) were more productive than those for which stage 1 embryos were rarely visible (smooth). This latter group develop preferably stage 3 embryos on the maturation medium containing high concentration of gellan gum. Biomass production is higher on a medium containing low concentration of gellan gum. However, sampling modalities did not affect the biomass production, and no relation was found between the biomass production and the maturation performance of each line. Stage 3 embryos developed on the medium with low concentration of gellan gum (0.45%, w/v) were shorter than those developed on medium with 0.9% of gellan gum. These short embryos were not able to germinate whereas about 48% of germination was reached with the longest embryos. The ability to develop primary root is dependent on the genotype while epicotyl elongation was observed among all lines.

The 5′-flanking region of a B (small) subunit gene of ADP-glucose pyrophosphorylase (EC 2.2.7.27) – called the agpB1 promoter – was cloned from the potato cultivar Desiree and its activity studied in transgenic potato (Solanumtuberosum L.) and tomato (Lycopersiconesculentum Mill.) plants using the gusA reporter gene. In potato, high β-glucuronidase (GUS) activity was found in the storage tissues of developing tubers, but gusA expression was also detected in phloem-associated parenchymas in stolons, stems and petioles, as well as in the starch sheath adjacent to the vascular tissues in leaf veins, stems and petioles. In leaves, promoter activity was limited to stomatal guard cells and to the starch sheath of the major veins, with no detectable activity in the mesophyll. No expression was observed in roots. β-Glucuronidase activity was finally detected in pollen grains and in ovary placental tissues. The potato promoter::gusA construct was introduced in transgenic tomatoes and was shown to be highly regulated during fruit development, with a tight parallelism between GUS activity, the extractable ADP-glucose pyrophosphorylase activity and fruit starch content. In conclusion, the agpB1 promoter appears to be specific to sink tissues, contrasting with the sAGP gene recently described by P.A. Nakata and T.W. Okita (1996 Mol Gen Genet 250: 581–592) which is transcribed in both sink and source tissues. Furthermore, and in contrast to sAGP, agpB1 transcription is stimulated by sucrose.

International audience

This work has been conducted under Research Infrastructure Concerted Action “Treebreedex” (http://treebreedex.eu, contract RICA-CT-2006-026076).; Vegetative propagation of forest trees offers advantages to both tree breeders and the forest industry. This review will describe benefits, type of vegetative propagation, and its integration into breeding programmes. Of all of the different methods for vegetative propagation, only rooted cuttings and somatic embryogenesis (and the combined use of both) offer any practical methods for large-scale commercial use. However, it is very difficult to fully appreciate the overall level of activity of the research and application of somatic embryogenesis of forest trees. Publications and reports only highlight a small fraction of the ongoing work. To this end, a survey was conducted across Europe (under EU Research Infrastructure Concerted Action “Treebreedex”) to document the species involved, the state-of-the-art of somatic embryogenesis, its stage of development and its application in tree improvement programmes and to commercial forestry. The results of this survey are presented and discussed. In addition, this review presents the challenges (biological, economic, public acceptance and regulatory) and their relationships to European forestry. Finally, a strategy to promote the use of this technology is proposed.

The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including beta-etherase that specifically cleaves the beta-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular beta-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. (c) 2012 Elsevier B.V. All rights reserved.

FCBA et l'INRA ont engagé des recherches il y a maintenant près de 20 ans pour développer une méthode de multiplication végétative performante du pin maritime. Ses applications dans le programme d'amélioration génétique seraient multiples, depuis la gestion durable des ressources génétiques jusqu'à la sélection plus efficace des meilleures variétés et leur déploiement facilité dans les plantations. Suite aux développements pionniers chez l'épicéa auxquels FCBA a contribué (années 80, Afocel) et comme chez la plupart des autres conifères, c'est le processus d'embryogenèse somatique à partir de graines immatures couplé à la cryoconservation des embryons somatiques obtenus qui offre actuellement les meilleures perspectives pratiques. Des progrès importants ont été obtenus pour la maîtrise de cette technologie, particulièrement depuis la mise en place en 2004 (et continue depuis) d'une collaboration spécifique sur ce thème entre les équipes " Biotechnologie & Sylviculture Avancée " de FCBA et "Amélioration, Génétique et Physiologie Forestières " de l'INRA. Nous faisons ici un bilan des avancées majeures à différentes étapes de l'embryogenèse somatique jusqu'à la mise en place d'essais au champ en cours d'évaluation. Nous portons également un regard sur les verrous techniques et les contraintes socio-économiques qu'il faudrait lever pour amener cette technologie prometteuse à franchir les portes de l'application pratique.

Reverse genetics, defined as ectopic candidate gene expression or silencing, has become an indispensable tool for functional dissection of traits of interest in forest trees. In maritime pine as in other conifers, long generation time and long life span, high genetic loads as well as high genetic redundancy are major obstacles to perform standard genetic approaches including association genetics. Validating marker associations with specific properties before transferring into breeding selection models is still challenging. Stable Agrobacterium-mediated genetic transformation of maritime pine was first reported by Trontin et al. (2002) and developed in both France (FCBA, INRA) and Portugal (IBET) with sufficient refinement (reviewed in Trontin et al. 2007) to envisage practical application in reverse genetics as an attractive alternative to association studies. As a result, the technology has been recently implemented in French (GenoQB, 2006-2009) and transnational (Sustainpine, 2010-2013) or European initiatives (ProCoGen 2012-2016). Somatic embryogenesis (SE) was revealed as a key tissue culture system for achieving genetic transformation, easy cryopreservation of transgenic tissue and efficient transgenic plant regeneration in maritime pine. Much consideration is given in France, Portugal and Spain to apply SE as a clonal propagation system for maritime pine improvement and deployment strategies in multivarietal forestry (MVF, Park YS, reviewed in Klimaszewska et al. 2007). By using this technology it is expected that we will obtain greater genetic gain, high flexibility in deployment of tested embryogenic varieties and easy balancing of genetic gain and diversity in plantations. Both SE and Agrobacterium-mediated transformation methods developed at FCBA, INRA or IBET are being successfully transferred to different partners through running the Sustainpine project (http://www.scbi.uma.es/sustainpine/).

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. Anew fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional beta-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix alpha 4' in the C-terminal domain, of the pre-sumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.

International audience; Association mapping is a recommended method to dissect the genetic basis of naturally occurring trait variation in non-model tree species with outcrossing mating systems and large population sizes. We report here the results of the first association-mapping study in maritime pine (Pinus pinaster Ait.), a conifer species of economical importance for timber and pulp production in south-western Europe. Two association samples were examined: 160 plus trees belonging to the first generation breeding population (G0, resulting from mass selection for overall good growth and form in the forest of South West of France) and 162 trees from the second generation breeding population (G1, resulting from biparental crosses between G0 trees). These samples were (1) genotyped for 184 in vitro SNPs discovered in 40 candidate genes for plant cell wall formation or drought stress resistance and 200 in silico SNPs detected in 146 contigs from the maritime pine EST database and (2) phenotyped for growth, stem straightness and wood chemistry traits in progeny or clonal experimental designs (from 768 to 5,080 phenotypes depending on the trait). First, SNP data were used to test for putative stratification in the breeding population. Then, two different approaches using pedigree records to account for inbreeding were used to test for associations. Despite the a priori low power of the designs, we identified two mutations that were significantly associated, one with variation in growth (in a HD-Zip III transcription factor) and the other with variation in wood cellulose content (in a fasciclin-like arabinogalactan protein).

International audience; *Introduction : Tree breeding is giving an increasing attention to wood properties in order to better fit the requirements of the saw, board, pulp and paper industries. In particular, it has been reported that lignin and cellulose content display moderate to high heritabilities making them prime candidates for genetic improvement of wood chemistry. Moreover, these traits have been shown to be negatively correlated at both phenotypic and genetic levels. However, they have generally been evaluated against a narrow genetic background, and little is known about their correlations with mandatory selection criteria such as growth and straightness. *Materials and methods : In this study, we first investigated the performance of near-infrared (NIR) spectroscopy combined with a non-destructive sampling method to assess chemical properties of wood in maritime pine. We afterwards estimated genetic parameters of growth, stem form and wood chemistry traits across a large genetic background in a progeny trial and clonally replicated progenies. *Results : Our results showed that removal of extractives prior to NIR spectra acquisition is highly recommended for achieving high accuracy in NIRS-PLSR prediction for wood chemistry traits in maritime pine. We further observed moderate heritabilities (0.15–0.55) for the studied traits. Wood chemistry traits were genetically inter-correlated (e.g., negatively between lignin and cellulose), whereas correlations with growth were not significant, indicating that growth and chemical properties could be improved independently.

BackgroundThere is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size ( approximately 23.8 Gb/C).Methodology/principal findingsA 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).Conclusions/significanceThis study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.

International audience; Methods for genetic transformation of maritime pine embryonal masses (EM) using Agrobacterium tumefaciens (At) have been independently established at 2 laboratories (FCBA and INRA) in France. The two procedures appeared roughly similar but technical differences were noticed during the decontamination and washing of co-cultivated EM, the subsequent reactivation culture step and the selection phase of transgenic lines. One major difference was the basal formulations of media used for At-mediated transformation development at FCBA (mDCR, modified Gupta and Durzan medium) and INRA (mLV, modified Litvay medium). Joined experiments were carried out in order to get insights on factors affecting the transformation rate based on a comparison of both procedures (one model genotype, C58pMP90 At strain, modified pCambia1301 binary vector harbouring the uidA-intron, hpt and bar genes). Our ultimate goal is to foster the transgenic approach as an efficient tool for establishing direct linkages between genes and phenotypes. We showed that the efficiency of the selected transformation procedures is tightly correlated with the basal medium formulation. Using mLV medium, currently recognized in both laboratories as a superior mineral base for maritime pine somatic embryogenesis, transformation yields were higher using the INRA procedure following either hygromycin or phosphinothricin selection. In contrast full advantage of the FCBA procedure could only be raised by using mDCR basal medium instead of mLV. In conjunction with strong phosphinothricin selection (1 mg l-1) the mDCR-based FCBA procedure was revealed as the best option to transform one model genotype of maritime pine (89.6 ± 18.4 phosphinothricin-resistant lines per g EM).

Summary • Here, embryo-specific patterns of glutamine synthetase (GS) genes were studied for the first time using pine somatic and zygotic embryogenesis as model systems. • GS1a expression was absent in zygotic embryos whereas it was detected in the cotyledons of somatic embryos at late developmental stages along with transcripts for photosynthesis genes and arginase. These findings suggest that germination was initiated in maturing somatic embryos. • GS1b transcripts were found mainly in procambial cells in both zygotic and somatic embryos. Expression of the GS1b in procambial cells before the differentiation of mature vascular elements indicated that this gene could be useful as a molecular marker for early stages of vascular differentiation in pine. Accordingly, a correlation was found between the quality of somatic embryos generated from three different cell lines and the pattern and level of GS1b expression. • Our data suggest that GS1a and GS1b genes play distinct functional roles in the biosynthesis and mobilization of seed nitrogen reserves. Furthermore, the results presented may have potential application for improving conifer somatic embryogenesis.

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.

We developed 20 polymorphic nuclear microsatellites from Pinus pinaster using genomic libraries enriched with repeated motifs. A total of 25 individuals from five different populations were used to estimate genetic diversity parameters. © 2005 Blackwell Publishing Ltd.

International audience; Cryopreservation of a core collection of 444 elm (Ulmus spp.) clones in liquid nitrogen was carried out by two laboratories participating in a European project of conservation of elm genetic resources. Plant material, collected in nine European countries, represented a large sample of the genetic diversity within three European elm species and their hybrids. The cryopreservation technique used in both laboratories involved the stepwise freezing of cryotubes containing dormant buds. Comparisons with fresh buds showed that the cryopreservation treatment had no negative effect on the viability and regrowth potential of frozen buds. Tests on a random sample of 26 clones showed that direct regrowth of cryopreserved buds (i.e., through propagation by microcuttings) of Ulmus minor and Ulmus laevis was possible; conversely, Ulmus glabra could only be regrown through micrografting. Most thawed explants from all 26 clones survived through the whole cultivation phase and were successfully transferred to the field. A calculation of costs indicates that cryopreservation of elm buds is economically competitive to field clonal archives.; Deux laboratoires ont réalisé conjointement la cryoconservation d'un noyau (core collection) de 444 clones issu de la collection d'orme (Ulmus sp.) réunis dans le cadre d'un projet européen de conservation des ressources génétiques. Le matériel végétal collecté dans 9 pays européens est représentatif de la diversité génétique naturelle de 3 espèces européennes d'orme et de leurs hybrides. La technique de cryoconservation utilisée est basée sur la descente progressive en température de cryotubes contenant des bourgeons dormants. La comparaison avec des bourgeons en végétation a établi que la technique utilisée n'a pas d'effet délétère sur la viabilité et le potentiel de reprise de croissance des bourgeons congelés. Des tests réalisés sur un échantillon aléatoire de 26 clones montrent que la réactivation de bourgeons cryoconservés d' U. minor et U. laevis est possible par microbouturage direct; contrairement à U. glabra qui ne peut être réactivé que par microgreffage. La plupart des explants dégelés ont produit des plantes transférées avec succès en parcelles expérimentales pour l'ensemble des 26 clones. Un calcul de coût montre que la cryoconservation de bourgeons d'orme est économiquement compétitive en comparaison avec la conservation au champ.

Some progress has recently been made in establishing a system enabling somatic embryos to be initiated from old elite trees. We report here the first results demonstrating the molecular conformity of somatic embryos initiated from increasingly old Norway spruce (Picea abies (L.) Karst.), as indicated by an analysis of six nuclear microsatellites that showed an extremely high tendency to mutate during in vitro culture. No allelic difference was detected at these loci among plants regenerated from somatic embryos or between the former and mother plants. Moreover, phenotypical data acquired on the same 3- to 9-year-old plants growing in the field sampled for molecular analyses were totally in accord with the results on molecular conformity.

The trnK gene has been identified on a cloned plastid DNA fragment of potato (Solanum tuberosum cv Désirée). This gene codes for a tRNA-Lys and is interrupted by a 2.5-kb intron belonging to the group II organellar introns. In addition, this intervening sequence contains a long open reading frame potentially coding for a 509 amino-acid polypeptide (ORF509) related to mitochondrial intron-encoded maturases from fungi. The translational capacity of the trnK intron was first demonstrated in vitro in a prokaryotic DNA-directed expression system. In order to examine the expression of the intron in the potato plant, a synthetic peptide corresponding to the last nine amino acids of the predicted ORF509 product was used to raise antibodies. Western-blot experiments on chloroplast protein extracts, using a sensitive chemiluminescent detection system, identified polypeptides similar to in-vitro products. These results suggest that the trnK intron is expressed at the protein level in the plant. This is the first report of the in-vivo expression of an intron-encoded polypeptide in higher plant plastids.

Qu'elle utilise la transgenese ou se cantonne aux methodes classiques de croisements et selection, l'amelioration genetique des arbres progresse tres lentement. L'embryogenese somatique, une technique qui arrive a maturite chez les resineux, va sans doute lui donner un coup de fouet.

An efficient procedure has been developed for DNA extraction from cotton by modifying the original CTAB method. The major improvement concerns the use of activated charcoal to bind resinous and coloured compounds which copurify with the DNA. The efficiency of amplification by RAPD was used as a criterion to evaluate the action of activated charcoal. Twenty-five random decamers from Operon Technologies were used to compare DNA samples extracted in the presence or absence of activated charcoal. The results in terms of amplification suggest that the use of activated charcoal in DNA extraction enhances RAPD amplification. This technique was initially developed for cotton but it has been applied successfully to other recalcitrant plants such as coffee, rubber tree, cassava and banana.

International audience

International audience

The influence of carbon sources and polyethylene glycol combined with 0.45 and 0.9% (w/v) of gellan gum on the maturation of maritime pine somatic embryos was tested. The effect of the carbon source and polyethylene glycol varied widely between lines. One out of the five lines tested showed a striking response to polyethylene glycol (PEG) treatment; the addition of this osmoticum limited the embryonal-suspensor mass (ESM) proliferation while it enhanced the maturation rate. Conversely, the ESM proliferation was stimulated by PEG in the other lines without subsequent improvement of the maturation rate. The use of a high concentration of gellan gum (0.9%) improved the maturation of the five ESM lines. It was concluded that the most efficient culture medium to recover cotyledonary embryos from all lines is one supplemented with sucrose at 6% (w/v) and gellan gum at 0.9% (w/v) without PEG. The determining factor in the maturation of maritime pine somatic embryos is the genotype and/or the quality of ESM. The possible relationship between maturation performances and ESM morphology, particularly the suspensor organization, is discussed.

International audience; The progress made in the conservation of European elm genetic resources since the 1st International Elm Conference is reviewed, and the complementarity of in situ and ex situ methods is discussed. The financial support of the European Union to RESGEN project CT96-78 has permitted to co-ordinate and rationalize the ex situ conservation of elms. The project, which involved 17 partner institutes in nine west European countries, aimed at a better evaluation, conservation and utilisation of the existing collections of native elm clones. Main achievements are: establishing a common database of about 2000 clones; characterizing over 500 clones through RAPDs and chloroplast DNA PCR-RFLPs molecular markers; completing and rationalizing the existing collections; establishing a long-term core collection of 850 clones; cryo-preserving a subset of 444 clones; and identifying clones of interest for breeding and prudent use in the reconstruction of countryside hedges. The 'Noble Hardwoods' network of the pan-European program EUFORGEN groups members representative of 31 countries, and promotes the dynamic conservation of the genetic resources of several genera of broadleaf forest trees, including Ulmus spp. Strategies for the conservation of the adaptive potential of elm resources were defined and will be disseminated among foresters and conservationists through "Guidelines" leaflets. Some countries have already started implementing conservation measures for U. laevis, associating in situ preservation and the establishment of seed orchards. Others are undertaking inventories, or acquiring genetic knowledge on target populations. At a broader scale, the 'Noble Hardwoods' network facilitates the rationalisation of the conservation populations network in a range-wide perspective.