Maritime pine has a long tradition of genetic improvement at the INRA Cestas research station. It started with plus tree selection in the early fifties and has now reached the 4th generation of breeding. Pedigrees are currently available over 3 generations, providing excellent material for genetic mapping and QTL analysis. Investigations in Eucalypts are conducted in the frame of a cooperation with CIRAD-forêts involved in tree improvement and clonal forestry of Eucalypts in Congo. Genetic maps of these species were established in order to determine whether important quantitative traits are under oligogenic control with definable quantitative trait loci (QTLs) that could be manipulate by the breeder. Indeed, tree breeding could be accelerated if economically important traits are under simple genetic control and QTLs could be tracked in cycles of recombination and selection using molecular markers. Genetic maps of Oak (Q. robur) will serve as a basis for locating genomic regions involved in the differentiation of two Oak species (Q. robur and Q. petraea).
Linkage analysis and marker types comparison for linkage analysis in forest trees.
Incomplete linkage maps were first established with protein revealed by two-dimensional gel electrophoresis (2D-PAGE). However, this technique yields very limited number of polymorphisms making proteins not very powerful for applications that require a broad genome coverage. RAPD (random amplified polymorphic DNA) and AFLP (Amplified Fragment Length Polymorphism) were used to quickly saturate the genome of this species. Two relatively dense genetic maps were constructed by genotyping haploid megagametophytes of an F2 progeny. RAPDs were also assayed on diploid tissue of the same progeny and allowed to investigate recombination rate differences for pollen and seed parents. RAPDs are of limited value for characterizing the biological meaning of QTLs. Thus, as a complementary marker technique for constructing a map of expressed genes, 2-D PAGE is being used. A total of 44 protein extracted from megagametophytes and needles have been localized on the saturated map. The number of such markers could be substantially increased by analyzing of some physiologically contrasted organs (roots, bud, pollen, xylem...). Such translated regions of the genome might be very useful for the characterization of QTLs. In addition to proteins, PCR-based genetic markers corresponding to genes are being located on another map established with AFLP markers genotyped on a three-generation outbreed pedigree.
Bahrman N, Damerval C (1989) Linkage relationships of loci controlling protein amounts in maritime pine (Pinus pinaster Ait.). Heredity 63: 267-274
Gerber S, Rodolphe F, Bahrman N, Baradat Ph (1993) Seed-protein variation in maritime pine (Pinus pinaster Ait.) revealed by two-dimensional electrophoresis, genetic determinism and construction of a linkage map. Theor Appl Genet 85: 521-528
Gerber S, Rodolphe F (1994) Estimation and test for linkage between markers: a comparison of lod score and c2 test in a linkage study of maritime pine (Pinus pinaster Ait). Theor Appl Genet 88: 293-297
Gerber S, Rodolphe F (1994) An estimation of the genome length of maritime pine (Pinus pinaster Ait.). Theor Appl Genet 88: 289-292
Plomion C, O'Malley DM, Durel CE (1995) Genomic analysis in Maritime pine (Pinus pinaster). Comparison of two RAPD maps using selfed and open-pollinated seeds of the same individual. Theor Appl Genet 90,1028-1034
Plomion C, Bahrman N, Durel CE, DM O'Malley (1995) Genomic analysis in Pinus pinaster (Maritime pine) using RAPD and protein markers. Heredity 74: 661-668
Plomion C, O'Malley DM (1996) Recombination rate differences for pollen parents and seed parents in pine. Heredity 77 : 341-350
Plomion C, Liu BH, O'Malley DM (1996) Genetic analysis using trans dominant linked markers in an F2 family. Theor Appl Genet 93 : 1083-1089Plomion C, Liu BH, O'Malley DM (1996)
Plomion C, Costa P, Bahrman N, Frigerio JM (1997) Genetic analysis of needle proteins in maritime pine. 1. Mapping dominant and codominant protein markers assayed on diploid tissue, in a haploid-based genetic map. Silvae genetica 46,2-3 pp 161-165
C. Plomion, P. Hurme, J-M. Frigerio, M. Ridolfi, D. Pot, C. Pionneau, C. Avila, F. Gallardo, H. David, G. Neutelings, M. Campbell, F.M. Canovas, O.Savolainen, C. Bodénès, and A. Kremer (1998) Developing SSCP markers in two Pines species. Molecular Breeding, 5, 21-31
Costa P, Pot D, Dubos C, Frigerio J-M, Pionneau C, Bodénès C, Bertocchi E, Cervera M-T, Remington DL, Plomion C (2000) A genetic map of Maritime pine based on AFLP, RAPD and protein markers. Theor Appl Genet 100:39-48
In Eucalyptus, two maps were established with RAPD markers . A Eucalyptus urophylla x E. grandis inter-specific F1 family was used for that purpose. The Eucalyptus urophylla elite tree used as a female parent originated from Monte Lewotobi in Flores while the E. grandis elite tree used as a male parent originated from east of Atherton (Autralia). This particular cross was chosen because of its superior growth rate compared to other hybrid families. A total of 93 progenies were available for the mapping experiment. 269 and 236 RAPD markers were assigned to 11 linkage groups in E. urophylla and E. grandis, respectively. A total map length of approximately 1350 cM was obtained for both maps. Homologous linkage groups of both species were aligned with common RAPD markers. Genes are now being mapped in these maps.
Verhaegen D, Plomion C (1996). Genetic mapping in Eucalyptus urophylla and Eucalyptus grandis using RAPD markers. Genome 39 : 1051-1061
Gion J-M, Rech P, Grima-Pettenati J, Verhaegen D, Plomion C (2000) Mapping candidate genes in Eucalyptus with emphasis on lignification genes. Mol Breed (in press)
Two genetic maps were established with 96 individuals of an F1 progeny of Quercus robur (2n=2x=24) using RAPD (Random Amplified Polymorphism DNA), SCAR (Sequence Characterized Amplified Region), microsatellites, minisatellites, isozymes and rDNA markers . A total of 307 markers were assigned to the 12 paternal and maternal linkage groups. The objective of this study is to localise genomic regions which differentiate two oak species : Q. robur and Q. petraea: We want to know whether molecular markers that differentiate both species are clustered on particular linkage groups or dispersed all over the genome.
Barreneche T, Bodénès. C, Lexer C, Trontin J F, Fluch S, Streiff R , Plomion C, Roussel G, Steinkellner H, Burg K, Favre JM, Glössl J and Kremer. A. (1998) A genetic linkage map of Quercus robur L. (pedunculate oak) based on RAPD, SCAR, microsatellite, minisatellite, isozyme and rDNA markers.
Tree improvement is limited by the time needed to reach sexual maturity and the time lag required to evaluate growth performances. In addition, tree selection remains imprecise because environmental effects are rather high for the major economic traits. Heritabilities for height, diameter, volume, branching traits and straightness are in the range of 0.1-0.3, and only slightly higher for the specific gravity of wood. In that context, any tool directed toward selection process that improves the evaluation of genetic value and reduces the generation time would be of considerable value. Individual loci controlling the variation of quantitative traits (QTL: quantitative trait loci) have been detected and mapped on the genome of many plant and animal species. The manipulation of marker-QTL information has shown itself to be promising for increasing the selection efficiency.
The F1 family of E.grandis x E.urophylla was used to detect QTLs for traits of economic interest (wood density, stem growth and stem form). QTLs defined in such a narrow genetic background (a single pedigree) can readily be used to identify the ideotype to be propagated for clonal variety production. Marker-aided breeding could have a large impact on tree breeding through more precise identification of individuals.
D Verhaegen, C Plomion, J-M Gion, M Poitel, P Costa, A Kremer (1997) Quantitative trait dissection analysis in Eucalyptus using RAPD markers: 1- Detection of QTL in interspecific hybrid progeny, stability of QTL expression across different ages. Theor Appl genet 95 : 597-608
However, our main interest is to use favorable QTL allele (QTA) for breeding purpose. Thus, our research is directed towards the validation of these "specific" favorable QTAs in a broader genetic background. RAPD markers associated with "specific" QTLs were studied for marker:trait associations in a factorial design of E. grandis x E. urophylla (13 x 13). Some of them, show significant relationships with the additive variance estimated for the hybrid population. Whether this linkage disequilibrium would hold for the whole breeding population is not known yet but this result seems to be promising for implementation of marker-assisted selection in eucalypts.
D Verhaegen, C Plomion, M Poitel, P Costa, A Kremer (1998) Quantitative trait dissection analysis in Eucalyptus using RAPD markers : 2- Linkage disequilibrium in a factorial design between E. urophylla and E. grandis. Forest Genetics 5 : 61-69
We also develop a method aimed at estimating the breeding value of specific QTA, for the incorporation of marker-assisted selection in forest tree breeding programs. The proposed methodology expands on the pseudo-testcross QTL mapping strategy which is based on the selection of single dose markers present in one parent and absent in the other. It specifically exploits the fact that one of the parent of the full-sib is double null for the RAPD markers bracketing the QTL so that by looking at its half-sib family, "band present" allele frequencies of the two markers can be obtained at the population level. The half-sib of the other parent which is double heterozygous is then used to estimate the average effect of the two QTL alleles (i.e. the additive effect).
Plomion C, Durel CE, Verhagen D (1996) Utilisation des marqueurs moléculaires dans les programmes d'amélioration génétiques des arbres forestiers : exemple du pin maritime et de l'eucalyptus. Ann Sci For 53 : 819-848
Plomion C, Durel C-E (1996) Estimation of the average effects of specific alleles detected by the pseudo-testcross QTL mapping strategy.Gen Sel Evol 28 : 223-235
We used the pine mapping data to study the genetic architecture of total height at young age, by looking for QTLs of height growth elementary components, i.e. traits related to the initiation and the elongation of each shoot cycle. We also investigated the stability of height growth QTLs during the maturation of the trees. Other traits were analyzed such as d-3carene (absolute and relative content), and amount of protein revealed by 2D-PAGE.
Plomion C, Durel C-E, O'Malley D (1996) Genetic dissection of height in maritime pine seedlings raised under accelerated growth conditions. Theor Appl Genet 93 : 849-858
Plomion C, Yani A, Marpeau A (1996). Genetic determinism of δ3-carene in maritime pine using random amplified polymorphic DNA (RAPD) markers. Genome 39 : 1123-1127
Costa P, Plomion C (1999) Genetic analysis of needle protein in Maritime pine. 2. Quantitative variation of protein accumulation. Silvae Genet 48: 146-150
For reviews in plant proteomics see Thiellement et al and Thiellement et al
III.1. Two dimensional gel
electrophoresis of proteins
We use this technique mainly in maritime pine for : (i) genetic mapping of the expressed genome, (ii) population genetics studies, (iii) genome expression analysis under different abiotic stress. For a review on maritime pine proteome analysis see: Bahrman N, Plomion C, Petit RJ, Kremer A (1997).Contribution of two-dimensional electrophoresis of proteins to maritime pine genetics. Ann Sci For 54: 225-236.
III.2. Genetic mapping of protein markers (Map location of needle proteins)
Plomion C, Costa P, Bahrman N, Frigerio JM (1997) Genetic analysis of needle proteins in maritime pine. 1. Mapping dominant and codominant protein markers assayed on diploid tissue, in a haploid-based genetic map. Silvae genetica 46,2-3 pp 161-165
Costa P, Pot D, Dubos C, Frigerio J-M, Pionneau C, Bodénès C, Bertocchi E, Cervera M-T, Remington DL, Plomion C (2000) A genetic map of Maritime pine based on AFLP, RAPD and protein markers. Theor Appl Genet 100:39-48
III.3 Water stress responsive proteins
The development and growth of plants are greatly dependent on water availability. Drought can reduce growth capacity or induce mortality of plants. Understanding the genetic basis and the molecular mechanisms of drought stress adaptation, as well as other stress, is of particular importance for long lived forest tree species given the prospect of possible rapid climatic changes. This information could be useful for the management of genetic resources and for the genetic improvement of drought stress tolerance of cultivated forest tree species. Elucidation of the mechanism of drought stress adaptation and characterisation of genes involved are a prerequisite for application of biotechnology to plants.
We are currently undertaking a molecular, genetic and physiological analysis of drought responses in maritime pine in order to elucidate the genetic control and physiological processes that determine adaptability of this species to dry sites. Maritime pine (Pinus pinaster Ait.) is widely used in intensive monospecific stands and covers more than 4 million hectares in southwest Europe (Spain, Portugal, Italy and France) where contrasting climatic environments are prevailing. Its natural distribution also extends to Morocco and Algeria. Selection and improved sylvicultural practices of this species has led to a considerable improvement in productivity. A breeding programme for southwest France began in the early 1960s and has now reached its third generation. Genotypes that performed well under current environmental conditions will form the genetic basis for future forest regenerations. Therefore, the genotypes used in the breeding program should be selected for their adaptation to present and future climatic conditions if major losses are to be avoided. Different studies have shown contrasting behaviours facing to drougth between provenances of maritime pine. Variability between provenances grown under the same conditions of water availability has been found for different physiological parameters involved in drought responses, i.e. height growth, transpiration and sap pressure, osmotic regulation and root growth, or more recently in water-use efficiency assessed by carbon isotope discrimation. However, drought resistance features have not been considered so far in the breeding program, mainly because of the difficulty in defining precise qualitative or quantitative criteria of drought adaptation.
Our first objective is to identify and characterize proteins whose expression is modified by water deficit in maritime pine seedlings. We examined and quantified needle proteins using two-dimensional gel electrophoresis.
Costa Paulo, Bahrman Nasser, Frigerio Jean-Marc, Kremer Antoine, Plomion Christophe. (1998) Water-deficit responsive proteins in Maritime pine, Plant Molecular Biology, 39, 587-596
Costa P, Pionneau C, Bauw G, Dubos C, Bahrman N, Kremer A, Frigerio J-M, Plomion C (1999) Separation and characterization of needle and xylem maritime pine proteins. Electrophoresis 20: 1098-1108
Our second objectif is to map Quantitative Trait Locus (QTL) for physiological traits (predawn water potential, water consumption, stomatal conductance, predawn needle osmotic potential, relative water content of needle, gaz exchange (maximum photosynthesis), height growth, phenology notation, stress visual symptums, ABA, carbon isotope discrimination) and Protein quantity locus (PQL) for drought responding proteins, and compare their location in the same genetic map constructed with molecular makers genotyped on 200 F2 seedlings.
A third objectif is to investigate the response at the transcriptome level
III.4 Proteins involved in wood formation
Costa P, Pionneau C, Bauw G, Dubos C, Bahrman N, Kremer A, Frigerio J-M, Plomion C (1999) Separation and characterization of needle and xylem maritime pine proteins. Electrophoresis 20: 1098-1108
Christophe Plomion, Cédric Pionneau, Jean Brach, Paulo Costa and Henri Baillères. (2000) Compression wood-responsive proteins in developing xylem of maritime pine (Pinus pinaster Ait.). Plant Physiol, 9, 959-969
IV - GENOME ANALYSIS
V - BREEDING AND TREE IMPROVEMENT
V.1 Maritime pine plant material certification
Chloroplastic and nuclear microsatellites are being developped for diversity analysis with a final objective of plant material certification
Ribeiro MM, Plomion C, Petit R, Vendramin GG, Szmidt AE (2000) Human impact upon the genetic structure of forest species: a case study of maritime pine in Portugal. Theor Appl Genet (in press)
Mariette S, Chagne D, Decroocq S, Vendramin GG, Lalanne C, Madur D, Plomion C (2000) Microsatellite markers for Pinus pinaster Ait. Ann For Sci (in press)
V.2 Yellow poplar (Tulipier de Virginie, in french)
Previous is : I - RESEARCH ACTIVITIES IN POPULATION AND
EVOLUTIONARY GENETICS