Contributions of Retrotransposons to the Evolution of Quercus suber L. Filomena Nóbrega Rui Vidal COST Action E28 GenoSilva Meeting in Greece, 8th September 2002

FOREST SPECIES IN PORTUGAL Quercus ilex Castanea sativa Eucalyptus Other Hardwoods Other Softwoods Other Oaks Pinus pinaster Pinus pinea Quercus suber FOREST SPECIES IN PORTUGAL Presently, about one-third of the territory is covered by forests, totalling 3,467,000 ha (DGF 1998), but strong afforestation efforts have been made in the last decades, and it is considered that total forest area can reach 60%.

Main forest species in Portugal The main forest species is Pinus pinaster, followed by evergreen oaks (Quercus suber and Quercus ilex) and eucalyptus. Deciduous oaks, including Quercus robur, Quercus pyrenaica, Quercus faginea and recently introduced Quercus rubra, occur mainly in the north, and in high mountain areas in the central part of the country. Areas in 1,000 ha

Distribution of the forest areas by species Other Softwoods 1 % Pinus pinaster 31 % Pinus pinea 2 % Quercus suber 23 % Quercus ilex 14 % Eucalyptus 21 % Other Hardwoods 3 % Castanea sativa 1 % Other Oaks 4 %

The forest sector has a significant impact on the national economy of Portugal. Forestry products and their derivatives with the most impact are cork, pulp and paper, wood, gum, pine nut, and wood furniture. Portugal, which produces more than 50% of the world's cork, has been particularly careful with this resource. Today, cork represents a valuable resource for Portugal being one of the most important export products.

Distribution of the Cork Oak throughout the World Spain Portugal France Italy Tunisia Algeria Morocco Origin and expansion flux The Cork Oak is essentially found along the coastal strip of the western part of the Mediterranean and also in an area that includes part of the Lands, the Atlantic part of the Iberian Peninsula, and part of Morocco.

The Cork Oak (Quercus suber) in Portugal The Cork Oak is the second forest species in Portugal and can be found mainly in the southern part of the country. However, examples of Cork Oak can be found almost anywhere in the country.

The Cork Oak (Quercus suber) in Portugal The montados (cork oak forests) are extensive agro-forestry systems of great socio-economic and biological importance.

The Cork Oak (Quercus suber) in Portugal Cork Oak is a slow-growing species living for up to 300 years or longer. It is perennial of the Fagaceae family and of the Quercus gender, 10-20 m high and 4-5 m in CAP (circumference 1.30 m from the ground), with a wide and not very dense crown. The trunk has thick branches covered by a thick, cracked rhytidome, the CORK.

Cork Oak polymorphism Relating to cork, two characteristics also present a very high variability: thickness and porosity.

What is Cork? Each cork tree must be 20 to 25 years old before it can provide its first harvest of cork bark called "virgin". This type of cork has a hard and irregular structure. After extracting the virgin cork a new layer of cork starts generating. The first of these layers, extracted after 9 years is given the name "secondary cork" and the ones produced from subsequent extractions are "amadia". The first layer of reproduction cork is more even than virgin cork, but cannot match the perfect quality of amadia.

Phelogene Dead cells of cork What is Cork? CORK Cork is the outer bark of the Cork Oak tree. The bark is a vegetal tissue composed of an agglomeration of dead cells filled with air and lined with alternating layers of cellulose and suberin.

The average chemical composition of cork is: What is Cork? The average chemical composition of cork is: Suberin (45%) - the main component of the cell walls; responsible for the resilience of the cork. Lignin (27%) - the binding compound. Polysaccharides (12%) - components of the cell walls which help define the texture of the cork. Tannins (6%) - polyphenolic compounds responsible for colour. Ceroids (5%) - hydrophobic compounds that ensure the imperviousness of cork. Mineral water, glycerine, and others make up the remaining 4%. This extreme close-up reveals details about the suberin walls of cork cells. The image was obtained with an electronic microscope at CTCOR (2002).

Genetic improvement of Cork Oak and improvement of cork harvest. The importance for this ecosystem justifies special attention for the study of cork and respective tree in the its following diversified aspects Genetic improvement of Cork Oak and improvement of cork harvest. Determination of cork quality: factors affecting the quality, identification and evaluation. Studies on inter- and intra-macromolecular cork structural compounds. Influence of ecological conditions and sylvicultural practices on cork properties. Microbiological aspects related with cork product use, particularly stoppers. Evaluation of colour degradation of finished cork products. Cork extraction from pruned branches. New mechanized techniques. Development of fire resistant cork-based materials for building components. Study for new utilizations, recycling or treatment of cork processing wastes and LCA (Life Cycle Analysis) of cork products. Genetic improvement of Cork Oak and improvement of cork harvest. Determination of cork quality: factors affecting the quality, identification and evaluation. Studies on inter- and intra-macromolecular cork structural compounds. Influence of ecological conditions and sylvicultural practices on cork properties. Microbiological aspects related with cork product use, particularly stoppers. Evaluation of colour degradation of finished cork products. Cork extraction from pruned branches. New mechanized techniques. Development of fire resistant cork-based materials for building components. Study for new utilizations, recycling or treatment of cork processing wastes and LCA (Life Cycle Analysis) of cork products.

Our main research Cork quality, as expressed by its homogeneity and low porosity, is critical to the economy of the process. Because cork value is dependent on its quality and the result of some factors are controlled by the genetic constitution of the Quercus suber tree, it is necessary that the identification of potential molecular markers be used as a selection tool in future selection/breeding programmes in order to promote new plantations with recognised improved trees.

Our main research Identification of molecular markers associated with cork quality. Identification of proteic markers associated directly or indirectly with the suberin extracted from the cork to later identify the codifying genes of these proteins. Establish correlations between the genetic variability of this species and the variability of cork quality. Previous evaluation of cork quality and, consequently, to proceed to the juvenile selection of the trees with cork of high quality. Take appropriated steps for the production of more cork of a higher quality.

Our main research Since the retrotransposons have been recently exploited as DNA markers due to their wide distributions within several plant euchromatin domains, we have screened them for the first time on Quercus suber genomic DNAs extracted from some populations of cork trees, using PCR-based approach.

Our main research At this stage, we have already isolated and sequenced two Quercus suber retrotransposon fragments that have been registered in GenBank with the following accession numbers: AY099465, Quercus suber Melmoth retrotransposon-like RNAse H-like protein gene, partial cds. AF512588, Quercus suber reverse transcriptase (UGY-G1) pseudogene, partial sequence. Some important questions remain unanswered, such as the effects of these genetic elements on the cork quality. However, they have allowed the detection of genomic differentiations between the studied cork trees.

Application of genomic sciences to other species in Portugal The scientific community is working on a few species with great importance in the Portuguese forest: Pinus pinaster Eucalyptus globulus Pinus pinea Castanea sativa The application of genomic sciences to these species should improve an understanding of genes controlling important characteristics such as growth, wood quality, fruit quality and adaptation to biotic/abiotic stresses.

Application of genomic sciences to other species in Portugal Pinus pinaster Main research organizations involved in its study: Estação Florestal Nacional (INIA) Instituto de Tecnologia Química e Biológica (ITQB)

Application of genomic sciences to other species in Portugal Eucalyptus globulus Main research organizations involved in its study: RAIZ – Instituto de Investigação da Floresta e Papel Instituto de Tecnologia Química e Biológica (ITQB)

Application of genomic sciences to other species in Portugal Pinus pinea Main research organizations involved in its study: Estação Florestal Nacional (INIA)

Application of genomic sciences to other species in Portugal Castanea sativa Main research organizations involved in its study: Estação Florestal Nacional (INIA) Faculdade de Ciências da Universidade de Lisboa (UL)

Portuguese Delegates to Working Groups of the COST Action E28 GenoSilva WG 1 – Functional genomics of wood formation Filomena Nóbrega (EFN / INIA) Rui Vidal (FFUL) WG 2 – Functional genomics of tree maturation and reproduction Cristina Marques (RAIZ) ITQB representative WG 3 – Functional genomics of forest health Salomé Pais (FCUL) Rita Seabra (EFN / INIA)

This is what Portugal is betting on A Portuguese Network of five forest species has been established This is what Portugal is betting on