Investigations are focusing on Q. petraea and Q. robur which are two widespread species growing throughout Europe from Spain to Russia and represent the largest economic resources among European broadleave species. Activities aim at the description of gene diversity of nuclear and cytoplasmic markers at different hierarchical and spatial levels (species, region, population) with particular emphasis on the mechanisms involved in the dynamics and evolution of genetic diversity including human factors. Different topics within this general frame required the development of molecular markers adapted to the field of interest.
Cytoplasmic DNA polymorphism in oaks and other temperate or Mediterranean species
Due to their maternal inheritance, chloroplasts and mitochondria migrate only at short distances, through seeds. This dispersion mechanism results in extremely high population differentiation for cytoplasmic genes. An additional consequence is the absence of recombination. Due to its (relatively) higher level of diversity, cpDNA (chloroplast DNA) is especially interesting for the study of phylogeography. Spatial distribution of cpDNA haplotypes is currently being analyzed on a European scale by sampling populations every 50 km in the frame of a EU supported project . The molecular technique that is used is a PCR-RFLP assay. Primers have been designed by aligning sequence data available in data banks and searching conserved region (genes) flanking polymorphic regions (non coding regions). A set of 17 pairs of chloroplast primers and 5 pairs of mitochondria primers has been developed so far (updated list). These primers were tested on different angiosperms (monocots and dicots) as well as in gymnosperms to test their universality. They amplify single copy regions in the chloroplast genome of most species making them broad scale tools for analysis of cpDNA polymorphism. These primer pairs allow the amplification of up to 40% of the cpDNA molecule. Current investigations aim at designing new primers in order to amplify most of the cpDNA molecule. Hence, the most polymorphic regions for a given species can eventually be detected. In the case of the oaks, a subset of four pairs of primers allowed the detection of 30 haplotypes in a large scale survey of about 500 populations originating from all over Europe.
Demesure B, Comps B, Petit RJ (1996) Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolution 50 : 2515-2520
Demesure B, Sodzi N, Petit RJ (1995) A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Mol Ecol 4: 129-131
Dumolin S, Demesure B, Petit (1995) Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theor Appl Genet 91: 1253-1256
El Mousadik A, Petit RJ (1996) Chloroplast DNA phylogeography of the argan tree of Morocco. Mol Ecol 5 : 547-555
Kremer A, Petit RJ, Zanetto A, Fougère V, Ducousso A, Wagner D, Chauvin C (1991) Nuclear and organelle diversity in Quercus robur and Q. petraea. In Genetic variation in European populations of forest trees. G. Müller-Starck and M. Ziehe (eds.) Sauerländer's Verlag, Frankfurt am Main
Kremer A, Petit RJ (1993) Gene diversity in natural populations of oak species. Ann Sci For 50: Suppl. 1, 186s-202s
Petit RJ, Demesure B, Pineau E (1996) Genetic differentiation at a local and continental scale in European oaks species: the importance of historical factors. In: Inter- and intraspecific variation in European oaks: evolutionary implications and practical consequences. Eds A. Kremer and H. Muhs, European Union, Brussels.
Petit RJ, Demesure B, Dumolin-Lapègue S (1996) CpDNA and plant mtDNA primers. In Molecular tools for screening biodiversity: Plants and Animals. A Karp, PG Isaac, D Ingram eds, Chapman & Hall (full paper)
Petit RJ, Kremer A, Wagner DB (1993) Geographic structure of chloroplast DNA polymorphisms in European oaks. Theor Appl Genet 87: 122-128
Petit RJ, Wagner DB, Kremer A (1993) Ribosomal DNA and chloroplast DNA polymorphism in a mixed stand of Quercus robur and Q. petraea. Ann Sci For 50: Suppl. 1, 41s-47s
Petit Rémy J, Pineau Emmanuel, Demesure Brigitte, Bacilieri Roberto, Ducousso Alexis, Kremer Antoine (1997) Chloroplast DNA footprints of postglacial recolonisation by oaks. Proc Natl Acad Sci USA 94 : 9996-10001
Dumolin-Lapègue Sylvie, Demesure Brigitte, Fineschi Silvia, Le Corre Valérie, Petit Rémy (1997) Phylogeographic structure of white oaks throughout the European continent. Genetics 146 : 1475-1487
Molecular organisation of species differentiation in closely related oak species
Q. petraea and Q. robur are two closely interfertile species, that exhibit phenotypic differences, but show only extremely low genetic differentiation for isozymes or chloroplast DNA, probably because genomic regions where interspecific differences are significant are rare. We therefore developed a systematic screening of the genome by using PCR with random primers. Frequencies of RAPD fragments were calculated for 2800 fragments, among which only 36 showed significant differences between the two species. 23 of these informative fragments were sequenced and transformed into SCARs by designing primers 20 bp long. These SCARs fragments appeared to be single copy. The comparison of their sequences with sequence data banks indicated similarities with a transposable element and other known genes. Since digestion of the SCARs with restriction enzymes did not reveal polymorphism in most cases, we developed SSCP to analyse the SCARs fragments.
Single strand conformation analysis could be adapted even on long fragments (up to 1000 bp) and revealed an extremely high number of alleles (from 13 to 30). There was no relationship between the number of alleles and the SCAR size. Mendelian inheritance of SSCP fragments were verified in controlled crosses. The informative discriminating regions are being localized on a genetic map in order to investigate their distribution pattern in the genome. Future prospects are also to sequence the different SSCP alleles to establish their gene tree and infer coalescence times.
Bodénès C, Laigret F, Kremer A (1996) Inheritance and molecular variations of PCR-SSCP fragments in Pedunculate oak (Quercus robur L.). Theor Appl Genet 93 : 348-354.
Bodénès C, Joandet S, Laigret F, Kremer A (1996) Detection of genomic regions differentiating two closely related oak species Quercus petraea (Matt.) Liebl. and Quercus robur L. Heredity 78 : 433-444
C. Bodénès, T. Labbé, S. Pradère and A. Kremer (1997). General vs. local differentiation between two closely related white oak species. Molecular Ecology 6, 713-724
T. Barreneche, N. Bahrman and A. Kremer (1996). Two dimensional gel electrophoresis confirms the low level of genetic differentiation between Quercus robur L. and Quercus petraea (Matt) Liebl. Forest Genetics 3(2) 89-92.
Inter- and intraspecific gene flow and paternity analyses in mixed oak stands using microsatellites
Preliminary investigations conducted with allozymes have shown that asymmetric hybridization occurs preferentially in mixed oak stands composed of Q. petraea and Q. robur, pollen grains of Q. petraea pollinating ovules of Q. robur. These experiments are now repeated by using microsatellites in order to subdivide the pollen cloud that pollinates a given tree into contribution of different male parents. The microsatellites motives in the oak genomes are identified by Austrian colleagues (J. Glössl and coworkers of the University of Vienna) in the frame of a EU (European Union) funded project. Primers flanking dinucleotide repeated sequences are designed by the Austrian colleagues and our group is verifying their mendelian inheritance before applying them to population genetics and paternity analyses. The routine procedure for scoring the microsatellite genotypes is an electrophoresis on sequencing gels followed by silver staining. Gels are then scanned for the identification of the genotypes. The first results obtained in a stand where approximately 200 trees of each species were scored for 6 microsatellite loci show that the number of alleles varies from 12 to 29 with the effective number of alleles varying between 5.6 and 15.
Steinkellner H. (e-mail), Flush S., Turetschek E., Lexer C., Streiff R., Kremer A., Burg K., Glossl J., (1997) Identification and characterization of (GA/CT)n microsatellite loci from Quercus petraea, Plant. Mol. Biol, 33, 1093-1096.
Streiff R., Labbe T., Bacilieri R., Steinkellner H., Glossl J., Kremer A., Within population genetic structure in Quercus robur L. and Quercus petraea (Matt.) Liebl. assessed with isozymes and microsatellites. Mol. Ecol. in press
Levels of diversity of tropical trees with contrasting life history traits
The distribution of genetic diversity in tropical rainforests is largely unknown. Our lab is involved in the analysis of gene diversity in the French Guyana rainforest in cooperation with a small group of population genticists of INRA and CIRAD installed at SYLVOLAB in Kourou. The project selected about 20 different species having contrasting life history traits as : spatial distribution patterns, pollen and seed dispersion system, phenology of flowering, successional status, sex expression. The objective is to estimate the level of diversity within each species with codominant (isozymes) or dominant markers (RAPD) available and to compare with its life history traits. Significant relationships between these two characters may allow to estimate the diversity existing in complex ecosystems composed of many tree species.
Mating systems and sexual reproduction in tropical trees
Simultaneously to the diversity analysis, mating system studies and phenological observations are conducted on the same species. These consist in comparing genetic structures of adult trees and their offsprings using codominant markers, mostly isozymes. Results obtained so far in Dicorynia guianensis showed that this species was mostly allogamous, but that the rate of outcrossing was extremely variable among trees. Analysis of mating systems are combined with observations on flowering biology conducted in situ or on grafts in a clone bank.
Spatial distribution of cpDNA polymorphism in tropical trees
The distribution of cpDNA polymorphism in the rain forest are conducted at the local and regional level. If chloroplasts are also maternally inherited in tropical trees, then cpDNA markers will be excellent tools to study the dynamics of regeneration. Preliminary results obtained in Dicorynia guianensis indicate that diversity of chloroplast genes is maintained in a population, contrary to temperate deciduous trees. The maintenance of cpDNA polymorphism is likely to be due to the rapid turnover of colonization-extinction phases.
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
Bahrman N, Petit RJ (1995) Genetic polymorphism in maritime pine (Pinus pinaster Ait.) assessed by two-dimensional gel electrophoresis of needle, bud and pollen proteins. J Mol Evol 41: 231-237
Bahrman N, Zivy M, Damerval C, Baradat Ph (1994) Organisation of the variability of abundant proteins in seven geographical origins of maritime pine (Pinus pinaster Ait.). Theor Appl Genet 88: 407-411
Estimation of diversity and differentiation measures
In cooperation with the Laboratory of Biometry of INRA (Jouy-en -Josas), analytical methods are being developed to calculate confidence intervals for classical diversity and differentiation measures (Nei's H and Gst) for haploid or diploid markers. Comparisons are made with bootstrap methods with special attention to the resampling procedures to be applied in case hierarchical sampling is used (for example, populations and individuals). Preliminary analysis were also conducted to estimate allelic richness by using techniques less sensitive to sample sizes as rarefaction methods used in ecology.
El Mousadik A, Petit RJ (1996) High level of genetic differentiation for allelic richness among populations of the argan tree (Argania spinosa L. Skeels) endemic of Morocco. Theor Appl Genet 92 : 832-839
Kremer A, Petit RJ, Pons O (1996) Measures of polymorphism within and among populations In Molecular tools for screening biodiversity: Plants and Animals. A Karp, PG Isaac, D Ingram eds, Chapman & Hall (in press)
Petit RJ, Kremer A, Wagner DB (1993) Finite island model for organelle and nuclear genes in plants. Heredity 71: 630-641
Pons O, Petit RJ (1995) Estimation, variance and optimal sampling of gene diversity. I. Haploid locus. Theor Appl Genet 90: 462-470
Pons O, Petit RJ (1996) Measuring and testing genetic differentiation with ordered and unordered alleles. Genetics 144 : 1237-1245
Petit RJ, Bahrman N, Baradat PH (1995) Genetic differentiation in maritime pine (Pinus pinaster Ait) estimated using isozyme, total protein and terpenic loci. Heredity 75: 382-389
Petit Rémy, El Mousadik Abdelhamid, Pons Odile. (1997) Identifying populations for conservation on the basis of genetic markers. Conservation Biology (in press).
Petit Rémy, Pons Odile (1997) Bootstrap variance of diversity and differentiation estimators in a subdivided population. Heredity (in press)
Simulation of postglacial recolonization
Stochastic simulation methods were developed to trace the colonization of
Europe by oaks after the last glaciation. The model considers populations
displayed on a grid every 50 km exchanging genes through pollen or seed flow.
Demography was included by the use of a logistic growth function for
population size. The first simulations attempted to obtain a dispersion
mechanism that would fit with the speed of colonization witnessed by the
pollen deposits which amounted to 500 meters per year. Results obtained showed
that the most likely colonization mechanisms corresponded to a stratified
dispersion: a large amount of seed being dispersed as a diffusion associated
to rare long distance dispersion. Rare and long dispersion events form founder
population that expand on their own in patchy structures. Stratified
dispersion is therefore the colonization mechanism that would account (1) for
the speed of dispersion and (2) for the present patchy structure of cpDNA
haplotypes.
Le Corre V., Machon N., Petit R. J., Kremer A.
(1997). Colonisation with long-distance seed dispersal and genetic structure
of maternally inherited genes in forest trees: a simulation study. Genetical
Research, Cambridge, in press.
Le Corre V., Dumolin-Lapègue S. and Kremer A.
(1997). Genetic variation at allozyme and RAPD loci in sessile oak Quercus
petraea (Matt.) Liebl. : the role of history and geography. Molecular Ecology,
in press.
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MAPPING AND TRAIT DISSECTION STUDIES