) increase
in intensity with GS, (
) decrease in intensity
with GS.Résumé
Les flux de gènes par graines et par pollen sont à l'origine de la dynamique de l'évolution de la diversité végétale. Nous avons étudié cinq espèces de biologie et d'histoire contrastées : espèce sociale (chênes), espèces dispersées (alisier, frêne, angélique), espèce introduite (cèdre), espèces monoïque ou dioïque, espèces des forêts tempérées et tropicales. Pour chacune de ces espèces, nous avons mis en place une recherche de parenté (père et mère) à partir de jeunes plants récoltés sur le terrain. Nous nous sommes pour cela appuyés sur des marqueurs moléculaires codominants ou dominants et avons conçu des outils d'analyse communs conçus spécifiquement pour la recherche de parenté avec ces marqueurs (logiciel FaMoz). La capacité à reconstituer des filiations à l'intérieur du dispositif d'étude et les flux de gènes extérieurs déduits apparaissent assez différents selon les espèces, avec néanmoins des constantes. Les dispersions observées sont toujours plus faibles qu'en cas de liens aléatoires entre parents et descendants, suggérant des flux réduitsfavorisés dans les sites étudiés. La comparaison entre les dispersions mâles (pollen) et femelles (graines) souligne les particularités des modes de pollinisations ainsi que des divers types de graines et de mode de dispersions associés.
Mots clés : flux de gènes / marqueurs / arbres forestiers / filiations
Abstract
- Direct measures of gene flow in forest
Gene flow through seeds and pollen are major processes shaping genetic diversity in plants. In this project we studied gene flow in five tree species of contrasting life history traits : social species (oaks), scattered species (wild service tree, ash tree, angelique), introduced species (cedar), monoecious and dioecious species, species from temperate and tropical forests (symphonia). For each species, we performed a parentage analysis ( in order to assign male and female parents to saplings sampled in the stands. We used codominant or dominant molecular markers and the same analytical methods for parentage studies (FaMoz software). Our ability to reconstruct parent-offspring relationships inside stands and to estimate the gene flow stemming from outside the studied area differed markedly among species. Observed dispersions were always shorter than expected at random, reduced gene flow inside the stands was thus favoured. Contrasted results between male (pollen) and female (seeds) dispersions are likely due to particular pollinationsystems and diverse seed dispersion mechanisms.
Key Words: gene flow / markers / forest trees / parentage
FAMOZ (an acronym for father/mother) is a software useful in reconstructing parentage for dominant, codominant and uniparentally inherited markers. It is written in C and TclTk languages and is available for Unix, Linux and Windows systems. Parameters and assumptions used in the calculations are few and simple. Exclusion and identity probabilities, log-likelihoods of any genetic relationship, potential father and parent or parent pair, half- and full-sibship are calculated based on real or simulated data. Error rates for genotypic mistyping can be introduced. Simulations can be done to build statistical tests for parentage assignment.
Reaction wood formation is a complex developmental process in trees facing a gravitational stimulus, allowing their stem to maintain a vertical position. Its formation occurs during the whole growing season and during the whole life of a tree. In order to detect genes involved in reaction wood formation and to dissociate seasonal effects, we sampled differentiating xylem from compression and opposite wood on four replicated clones of the same maritime pine (Pinus pinaster Ait.) genotype after 8, 35, 66 and 120 days of mechanical bending of their trunk. To follow gene expression high-density filters were constructed with 875 spotted ESTs and these were hybridised with cDNA probes constructed from the xylem samples. ANOVA was used to detect genes showing seasonal, gravitational and interaction effects. Transcript accumulation of 70% (612 ESTs) of the genes was found to be affected by the season, whereas only 14% (125 ESTs) were found to be affected by bending. Of the genes affected by bending, 11 displayed a two-fold change in expression level between opposite and compression wood, compared to 171 that overshot this ratio across the growing season. This result suggests that the transcriptome of wood forming tissue is under greater seasonal than gravitational control. a two-fold change in expression level between opposite and compression wood, compared to 171 that overshot this ratio across the growing season. This result suggests that the transcriptome of wood forming tissue is under greater seasonal than gravitational control.
Additionnal data : Table 2 - Table 3
Root adaptation to soil environmental factors is very important to maritime pine, the main conifer species used for reforestation in France. The range of climates in the sites where this species is established varies from flooded in winter to drought-prone in summer. No studies have yet focused on the morphological, physiological or molecular variability of the root system to adapt its growth to such environment. We developed a strategy to isolate drought-responsive genes in the root tissue, in order to identify the molecular mechanisms that trees have evolved to cope with drought (the main problem affecting wood productivity), and to exploit this information to improve drought stress tolerance. In order to provide easy access to the root system, seedlings were raised in hydroponic solution. Polyethylene glycol was used as an osmoticum to induce water deficit. Using the cDNA-AFLP technique, we screened more than 2500 transcript derived fragments, of which 33 (1.2%) showed clear variation in presence/absence between non stressed and stressed medium. The relative abundance of these transcripts was then analysed by reverse northern. Only two out of these 33 genes showed significant opposite behavior between both techniques. The identification and characterization of water-deficit responsive genes in roots provide the emergence of physiological understanding of the patterns of gene expression and regulation, involved in the drought stress response of maritime pine.
Growth, development and productivity of long-lived organisms such as forest trees are continuously challenged by abiotic stresses, and may also be greatly affected by the possibility of rapid climatic changes in the near future. As a first step toward creating resistant maritime pine (Pinus pinaster Ait.) varieties by marker-assisted breeding, we describe the identification and characterization of water deficit-inducible genes in hydroponically grown seedlings. The cDNA-AFLP technique was used to identify genes regulated by water deprivation obtained by addition of polyethylene glycol. Approximately 4000 transcript derived fragments (TDFs) were screened, of which 48 showed clear "presence/absence" variation between well watered (- 0.08MPa) and osmotically stressed (- 0.45MPa) conditions. The accumulation of 28 and 20 TDFs were found to increase or decrease in the stress medium, respectively. Of these, 62.6% corresponded to known function proteins, indicating the main mechanisms involved in osmotic stress response (photosynthesis, carbohydrate metabolism, cell wall synthesis and plant defence). While 16.6% were similar to Arabidopsis thaliana gene products, 10.4% were similar to Pinus taeda ESTs and 10.4% presented no hit in public databases. The relative abundance of these transcripts was then quantitatively analysed by reverse northern, in both aerial and root tissues, confirming the effectiveness of the cDNA-AFLP technique in detecting differentially expressed genes. The identification and characterization of water deficit-responsive genes provide new insights into the nature of the machinery involved in response to water deprivation in a forest tree.
Key words : frost tolerance, genetic mapping, QTL, quantitative trait loci, Scots pine.
Two-year-old cuttings of five genotypes of maritime pine (Pinus pinaster Ait.) were subjected to a four-month treatment of slowly decreasing water availability in pots. Final predawn needle water potential varied from 0.82 to 1.48 MPa, and was negatively correlated with plant size. Osmotic adjustment was estimated as the slope of the regression line fitted between relative water content (RWC) and osmotic potential () assessed during the drying cycle, and as the value of the relative water content for a given level of (i.e. = -1.7 MPa). Under the experimental conditions, the genotypes showed a high capacity for osmotic adjustment in needles, and one genotype distinguished itself with a smaller capacity. The results are discussed in terms of protocol and interpretation when ranking genotypes for osmotic adjustment.
We constructed a high-density linkage map of maritime pine (Pinus pinaster Ait.) based on AFLP (Amplified Fragment Length Polymorphism) markers using a three-generation outbred pedigree. In a first step, male and female maps were established independently with test-cross markers segregating 1:1 (presence : absence of the amplified fragment in the full-sib progeny). In a second step, both maps were merged using intercross markers segregating 3:1 in the progeny. A combination of MAPMAKER and JOINMAP softwares was used for the mapping process. A consensus map was obtained and is available at URL http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. It covers 1441 cM and comprises a total of 620 AFLP markers on 12 linkage groups. The physical size of the maritime pine genome (51.5 pg/2C) was measured by flow cytometry, providing a physical / genetic size ratio of 13.78 Mb/cM. This map will be used to dissect the genetic architecture of economically (growth, wood quality) and ecologically (water-use efficiency) important traits into mendelian inherited components (QTLs: Quantitative Trait Loci). It will also provide a framework to localize more informative markers (ESTs: Expressed Sequence Tags) to be used as candidate genes in QTL detection experiments. The location of orthologous markers (ESTs and SSRs: Simple Sequence Repeats) will also allow the study of the genome structure of closely related conifer species using a comparative genome mapping approach.
Wood is one of our most important natural resources and has been exploited for many hundreds of years as fuel, building material and a source of paper. Its composition is variable among and within species. The ability to monitor the intra-specific variability is a prerequisite to improve wood and end-products properties. This paper describes a study of the genetic control of a large set of wood properties, including growth, timber quality traits, wood chemical composition, kraft pulp production parameters and pulp properties, in a 12 x 12 half diallel of maritime pine (Pinus pinaster Ait.). While relatively high (hns² > 0.3) narrow-sense heritabilities were observed for density heterogeneity, lignin content, alpha-cellulose content and coarseness, no significant genetic effect was detected for hemi cellulose, water extractives, kraft pulp production parameters and pylodin. Slightly lower heritabilities (0.15<hns²<0.3) were also obtained for wood density and fibre properties (length, width, curl, zero span). As a consequence and considering the phenotypic coefficient of variation obtained for these traits, improvement by selection of trees with outstanding wood quality is feasible. Nevertheless, it seems obvious that wood quality breeding can not be done without taking into account growth, and the only way to manage this constraint (negative correlation between growth and density) will be the constitution of elite "wood quality" populations in a already growth improved genetic population.
Maritime pine (Pinus pinaster Ait.) is the first conifer used for reforestation in France and now covers 2.4 Million ha of the Iberian Peninsula. In order to preserve the genetic resources of this economically and ecologically important species prior knowledge of the distribution of genetic diversity is needed. In this paper, a genetic diversity study was performed using nuclear simple sequence repeats (SSRs or microsatellites). Classical parameters of diversity (allelic richness and heterozygosity) and differentiation were estimated for 47 populations of P. pinaster. Most of the populations (40) were collected in France, six populations were also collected in the Iberian Peninsula and one Moroccan population was also included in the study. The population genetic parameters indicated that some populations should be a focus of conservation efforts (higher level of diversity, higher allelic richness and presence of rare alleles). A diagnost test for sample origin was developed to distinguish Corsican from Landes populations.
Classical quantitative genetics and quantitative trait dissection analysis (QTL) approaches were used in order to investigate the genetic determinism of wood cellulose carbon isotope composition (δ13C, a time integrated estimate of water use efficiency) and of diameter growth and their relationship on adult trees (15 years) of a forest tree species (maritime pine). We used a half diallel experimental set-up to (1) estimate heritabilities for δ13C and ring width and (2) to decompose the phenotypic δ13C / growth correlation into its genetic and environmental components. We found considerable variation for δ13C (range of over 3 ‰) and for ring width (range of over 5 mm) and significant heritabilities (narrow sense 0.17 / 0.19 for δ13C and ring width, respectively, 100% additivity). The significant phenotypic correlation between δ13C and ring width was not determined by the genetic component, but was attributable to environmental components. Using a genetic linkage map of a full-sib family, four significant and four suggestive QTLs were detected for δ13C, the first for δ13C in a forest tree species, as far as known to the authors. Two significant and four suggestive QTLs were found for ring width. No co-location of QTLs was found between δ13C
We compared the genetic variation of Pinus pinaster populations using amplified fragment length polymorphism (AFLP) and chloroplast simple-sequence repeat (cpSSR) loci. Populations' levels of diversity within groups were found similar with AFLPs, but not with cpSSRs. The high inter-locus variance associated with the AFLP loci could account for the lack of differences in the former. Although AFLPs revealed much lower genetic diversity than cpSSRs, the levels of among-population differentiation found with the two types of marker were similar, provided that loci showing less than four null-homozygotes, in any population, were pruned from the AFLP data. Moreover, the French and Portuguese populations were clearly differentiated from each other, with both markers. The Mantel test showed that the genetic distance matrix calculated using the AFLP data was correlated with the matrix derived from the cpSSRs. Due to the concordance found between markers we conclude that gene flow was indeed the predominant force shaping nuclear and chloroplastic genetic variation of the populations within regions, at the geographical scale studied.
A full length cDNA encoding a PR-10 protein was isolated from maritime pine drought stressed seedlings. The predicted protein contained 150 amino acids, has a molecular mass of 16.7 kDa and an isoelectric point of 5.28. The transcript level of PR-10 displayed a transient accumulation in needles of drought stressed plants, and was not detectable in root and stem tissues.
Twenty-three populations of Pinus pinaster (13 Aquitaine populations and 10 Corsican populations) were analysed at three microsatellite loci and 122 AFLP loci. The aims of the study were: (i) to compare levels of within-population and among-population diversity assessed with both kinds of markers; (ii) to compare Aquitaine and Corsican provenances of P. pinaster; and (iii) to know if both markers gave the same information for conservation purposes. Classical population genetics statistics were estimated and the ranking of populations obtained using different markers and/or parameters were compared by computing Spearman's rank correlations. Even though microsatellites showed a higher within-population diversity, they showed the same level of differentiation as AFLP markers. Moreover, both markers also showed a higher genetic diversity in the Aquitaine provenance and a higher differentiation among Corsican populations. AFLPs and microsatellites gave different population diversity rankings. Consequently, the results do not support the potential population identification within each provenance for conservation purposes.
Abstract : This study reports the cloning and characterization of nine microsatellite primer pairs in a scattered woody species (Sorbus torminalis), and shows their potential for further use in 36 species of the Maloideae, a Rosaceae sub-family containing important fruit crop and ornamental species. These primers were designed through the construction from Sorbus torminalis genomic DNA of microsatellite library enriched for CA and GA repeats. Genotyping 48 Sorbus torminalis of a natural population with the six best markers yielded a mean of 10.7 alleles per locus, and an expectation of exclusion probability for paternity analysis greater than 0.993.
Abstract : Maritime pine seed-lots from northwestern Iberian regions (Portugal and Galicia) were introduced in the 1950s to the southwest of France (Aquitaine region), and the stands they formed suffered considerable frost damage. In the mid 1980s, a biochemical test was developed to test the putative origin of adult stands in Aquitaine, before seeds could be distributed for commercial purposes in France. In this paper, we describe a new test employing chloroplast simple-sequence repeats (cpSSRs) to facilitate identification of stand origin based on randomisation tests. The origin of five stands of unknown origin was determined with both the cpSSR and biochemical (terpene profile analysis) tests. The results from the two tests were concordant, but the DNA-based test gave faster and more accurate results. Use of this test should help when determining the origin of maritime pine stands in the Aquitaine region of France.
Résumé : Des lots de graines du nord ouest de la péninsule ibérique (Portugal et Galice) ont été introduits dans les années 1950 dans le sud-ouest de la France (Aquitaine), et les peuplements issus de ces graines ont fortement souffert des gelées. Un test variétal basé sur les marqueurs terpéniques fut développé dans les années 1980 afin d'identifier l'origine géographique des peuplements adultes en Aquitaine sur lesquels des graines étaient récoltées puis commercialisées. Dans cet article nous décrivons un nouveau test qui utilise des marqueurs microsatellites chloroplastiques (cpSSRs) et simulations pour identifier l'origine des peuplements. Une étude comparative des tests biochimique (terpènes) et cpSSR a été menée sur cinq peuplements adultes. Les résultats obtenus sont identiques, mais le test ADN s'est avéré plus rapide et plus précis. L'utilisation de ce nouveau test devrait permettre de garantir l'origine géographique des peuplements de pin maritime du sud-ouest de la France (Aquitaine).
Abstract : A novel concept of seed orchard was developed by INRA for the maritime pine breeding programme: the polycross seed orchard (PSO). The expected genetic gain of the PSO can only be reached if the fathers used in the pollen mix contribute equally to the next generation (i.e. to the base material of the PSO), and if the pollen contamination from the surrounding stands is limited. Using chloroplast simple-sequence repeats (cpSSR), we showed that the cpDNA was uni-paternally inherited in Pinus pinaster, and verified that the chloroplast haplotype composition of the megagametophyte tissue corresponded to the chloroplast haplotype of the female parent. As a practical application, a statistical test based on cpSSR markers and simulation was established to verify the PSO origin of maritime pine seed lots. As a result of the cpSSR test, it was observed that: (i) departure from even distribution of the fathers in the PSO was barely significant, (ii) the minimum pollen contamination rate in the PSO was 36%, and (iii) the contamination was not evenly distributed in the PSO. As a consequence, the expected genetic gain will range between 50 and 82% of what was initially foreseen.
Résumé : Dans le cadre du programme d'amélioration génétique du pin maritime, un nouveau concept de verger à graines, le verger à graines de familles polycross (VGFP), a été développé par l'INRA. Pour installer un VGFP, des arbres élites sont croisés selon un schéma polycross, puis les descendants sont plantés à densité définitive. La variété améliorée est finalement produite par pollinisation libre entre les arbres du verger. Le gain génétique espéré d'un VGFP ne peut être réalisé que si les parents mâles contribuent également à la génération suivante (le matériel de base du verger), et si la pollution pollinique due aux arbres extérieurs au VGFP est limitée. En utilisant des marqueurs microsatellites chloroplastiques (cpSSR), nous avons tout d'abord montré que le génome chloroplastique présentait une hérédité paternelle. Dans un second temps, nous avons pu vérifier que les mégagamétophytes d'un même arbre possédaient le même haplotype chloroplastique que l'arbre mère. Un test statistique empirique construit par simulation a alors été établi pour décider de l'appartenance ou non d'un lot de graines quelconque au VGFP. Il a permi de montrer que l'écart à l'égale participation des pères dans le VGFP était tout juste significatif, et que le taux de pollution était au minimum de 36.5% et réparti de façon hétérogène dans le VGFP. En conséquence, le gain génétique espéré sera compris entre 50% et 82 % de celui annoncé.
Abstract: Simple sequence repeats (SSRs) or microsatellites are valuable tools for genome mapping and population genetic studies for as they are codominant and highly polymorphic markers. Seventy-six SSR primer pairs from four Pinus species were tested to amplify microsatellites in Pinus pinaster. Twenty-six primer pairs were stemmed from a microsatellite library on P. pinaster and the other primer pairs were obtained in other species of the same genus (P. radiata, P. strobus and P. halepensis). Only three out of the 76 SSR primer pairs amplified at a single polymorphic locus in P. pinaster. The mendelian inheritance of those three primer pairs was studied and their genetic map position was determined. The number of alleles and the level of heterozygosity were assessed in an analysis of a sample of 196 trees. The development of microsatellites in Pinus species has been reported to be a difficult task because of the size and complexity of their genome. The results of this study showed that cross-species amplification was quite unsuccessful.
The genetic variation of twelve Pinus pinaster (maritime pine) populations spanning most of the distribution range of the species in Portugal was evaluated using six polymorphic chloroplast microsatellite (cpSSR) loci. Thirty-two haplotypes were found. There were indications of very weak differentiation among populations (Weir’'s Theta coefficient, 0.023), and the RST value, derived from the stepwise mutation model (SMM), was not significantly different from zero. The pattern, in which similarities in allele size do not contribute to the genetic structure, may be due to the recent mixing of genetic material from different stands through plantations. Overall, high level of haplotypic variation within populations was detected. Using the SMM estimator, mean genetic distance of individuals within populations, the populations were divided into two groups, with above and below average values. The first group contained five populations, mainly from the central part of the country, which possess, in general, high levels of haplotypic diversity. Among them, two populations were divergent from the others, based on the pair-wise Nei’s distance. The results indicate that there is little or no geographic genetic pattern in Portuguese populations of P. pinaster. The history of expansion of the species range in Portugal during this century (mainly due to human activity), and extensive gene flow among populations associated with the expansion, might explain this finding.
Pine seedlings contain two cytosolic glutamine synthetase (GS1) isoforms: GS1a is predominant in green tissue whereas GS1b is a minor represented enzyme whose relative amount is increased following phosphinotricin (PPT) treatment. We have used the PPT response of pine seedlings to identify and clone a cDNA for cytosolic glutamine synthetase GS1b which hybridizes to an mRNA that accumulates transitorily 8h after herbicide treatment. A comparison of the GS1b sequence with the previously reported GS cDNA sequence from pine GS1a showed that they correspond to separate cytosolic GS genes encoding distinct protein products. The phylogenetic analysis with other GS sequences, including GS1a revealed that the newly reported sequence is closer to cytosolic angiosperm GS sequences rather than to the other cytosolic pine GS, suggesting therefore that GS1a could be a divergent gymnospermous GS1 gene. The gene mapping using a F2 family of maritime pine showed co-localization of both GS genes on group #2 of the genetic linkage map. This result agrees well with the proposed origin of different members of the GS1 family by a gene duplication phenomena. The implications of this fact in gymnosperm genomes organization are discussed.
Summary AFLP (Amplified Fragment Length Polymorphism) is a high throughput molecular marker technique that is used increasingly in a variety of genetic analyses. Here, the conditions for carrying out AFLP analysis have been established for different tree species, including both angiosperm and gymnosperm trees, with genome sizes ranging from 0.54 to 38 pg DNA/2C. Specific parameters have been determined to provide informative and reproducible AFLP fingerprints of peach, eucalypt, oak, poplar, and loblolly pine. Typically, 80 to 130 amplified DNA fragments (i.e. loci analyzed per primer combination) were obtained. Subsequently, these AFLP conditions were evaluated for intra and interspecific genetic variability studies as well as for genome mapping purposes of woody species. This work demonstrates that AFLP is a powerful tool in forest tree genetics.
Résumé L'AFLP (polymorphisme de longueur de fragments amplifiés) est une technique de marquage moléculaire à haut débit de plus en plus utilisée dans de nombreuses analyses en génétique. Dans cet article, les conditions de mise en oeuvre de cette technique ont été établies pour différentes espèces darbres, comprenant des angiospermes et des gymnospermes, et dont la taille du génome varie de 0.54 à 38pg dADN/2C. Des paramètres ont été déterminés pour obtenir des empreintes AFLP informatives et reproductibles chez le pêcher, leucalyptus, le chêne, le peuplier et le pin taeda. En général, 80 à 130 fragments dADN amplifiés ont été obtenus par combinaison damorces. Ces conditions ont ensuite été utilisées pour étudier la variabilité génétique intra et inter-spécifique, ainsi que pour la cartographie génétique chez les espèces ligneuses. Ce travail démontre que lAFLP est un outil danalyse puissant pour effectuer des analyses génétiques chez les espèces forestières.
We have applied a two-way pseudo-testcross strategy in an analysis of Pinus sylvestris for genetic mapping and detection of quantitative trait loci (QTLs) associated with economically important traits targeted in the Swedish tree-breeding program. Based on 94 full-sib progeny of a cross between two plus-trees from northern Sweden we generated two parental maps using AFLP markers. The female map was comprised of 94 markers assigned to 15 linkage groups giving a size of 796 cM. On the male map 155 markers were assigned to 15 linkage groups, giving a total size of 1335 cM. The recombination frequency was found to be sex-dependent, being 29.3% higher in male than in female gametes. The number of QTLs detected for the male and female parents also differed. On the female map, 12 QTLs were detected (but none for branch diameter or wood density). Three QTLs for tree height accounted for 25.8% of the total phenotypic variation of this trait. When the QTLs detected for all the traits were taken independently, the percentages of phenotypic variance ranged from 9.3% to 22.7%. The highest value was observed for frost hardiness, an important trait in northern Sweden for which a major gene seemed to be involved. A cluster of QTLs for tree height, trunk diameter and volume was located on one linkage group. On the male map, four QTLs for trunk diameter and volume were detected. Due to the reduced number of individuals under study, the results are preliminary and have to be validated on more trees.
Since the description of the technique of two-dimensional gel electrophoresis (2DE) of proteins, by O’Farrell (1975) and Klose (1975), the plant biologists have used it for various purposes, either for genetical or for physiological studies. The opportunity given to reveal several hundreds of gene products on one single gel (see Figure 1) indeed permitted to examine the differences in protein patterns between genotypes: genetic distances have been estimated and phylogenetic relationships have been established. Variations in gene expression have been studied too, according to the development and in response to various treatments, leading to the identification of regulated proteins. Mutants have been characterized at this protein level and pleiotropy estimated this way. The genes encoding the revealed proteins can be mapped on the chromosomes, as far as their products show a variation in isoelectric point or molecular mass (i.e. position shift or PS variants, Figure 2): expressed genes can be added to the genetic maps currently established with nucleic acid markers. In addition, thanks to devoted softwares (review in Appel et al. 1997) a protein amount can be treated as any quantitative trait amenable to QTL (Quantitative Trait Loci) analysis procedures, that localize on the genetic map the factors controlling the variability of this amount. Technological advances in sorting out and characterizing the spots from 2DE gels are currently applied in plant biology. Several plant protein databases are in progress. This will be of the foremost importance since the first plant genome(Arabidopsis thaliana), will be entirely sequenced within the next two years. The recently developed «transcriptome» tools, such as high-density cDNA filters, cDNA microarrays or DNA chips (Castellino, 1997; Lemieux et al., 1998; Marshall and Hodgson 1998), will be usefully complemented by proteomics, since the amounts of a protein and of its mRNA are not always correlated (Anderson and Seilhammer, 1997; Haynes et al., 1998; Anderson and Anderson 1998), and since proteins turn over and posttranslational modifications cannot be studied at the cDNA level. Several reviews on plant proteomics have recently been published (de Vienne et al. 1996; 1999; Thiellement et al. 1999) and in the following pages the various uses of proteomics in plant biology will be exemplified, with a special emphasis on its contributions to the progress of plant genetics.
We used the single-strand conformation polymorphism (SSCP) technique to map eight genes on Eucalyptus urophylla and Eucalyptus grandis linkage maps. These included four genes involved in the common phenylpropanoid pathway (Caffeic acid 3-O-methyltransferase, Caffeoyl CoA 3-O-methyltransferase, 4-Coumarate CoA ligase and phenylalanine ammonia-lyase), two genes involved in the “ lignin specific ” pathway (Cinnamoyl CoA reductase and Cinnamyl alcohol dehydrogenase), and two symbiosis regulated genes (EgHypar and EgTubA1). A novel source of variation which affects the SSCP pattern, i.e. presence or absence of electrophoresis buffer upon loading the samples into the polyacrylamide gel, was found. The placement of these genes on the Eucalyptus maps was carried out using an interspecific hybrid mapping population. This will further facilitate the identification or exclusion of “ positional ” candidate genes for characterizing quantitative trait loci (QTL) for wood quality and vegetative propagation related traits.
The AFLP (amplified fragment length polymorphism) technique was adapted to carry out genetic analysis in maritime pine, a species characterized by a large genome size (24pg/C). A genetic linkage map was constructed for one F1 individual based on 239 AFLP and 127 RAPD (randomly amplified polymorphic DNA) markers. Markers were scored on megagametophytes (1n) from 200 germinated F2 seedlings. Polymorphism rate, labour time and cost of both AFLP and RAPD techniques were compared. The AFLP technique was found to be twice as fast and three times less costly per marker than the RAPD technique. Thirteen linkage groups were identified with a LOD score ³ 6 covering 1873cM, which provided 93.4% of genome coverage. Proteins were extracted from needles (2n) of the F2 progeny and revealed by 2-DE (two-dimensional electrophoresis). Thirty-one segregating proteins were mapped using a QTL detection strategy based on the quantification of protein accumulation. Two framework maps of the same F1 individual are now available. The first map (Plomion et al. 1996) uses RAPD markers and the second map, presented in this study, uses mostly AFLP markers. Although the total genetic length of both maps was almost identical, differences among homologous groups were observed.
Woody plant species, among which trees, are generally long-lived organisms presenting a high level of adaptation to different types of environment and a very high level of genetic variability. Often lately-fertile, their long generation time slows the pace of genetic studies and breeding processes compared to that of annually cultivated crops. Notwithoutstanding these difficulties, breeders have developed different tools which have contributed to intensive plantation and market driven agricultural production. In recent years increasing interest in biodiversity studies around the world has raised the need for more tools applicable to species identification. Genetic and biodiversity studies of woody plant species have greatly benefited from the development of molecular techniques which allow nowadays to differentiate segregating individuals using hundreds of so-called molecular genetic markers. To date, genetic linkage maps have been constructed for several woody plant species. Such genetic maps provide information about the structure, organization and evolution of their genomes. Availability of dense genetic linkage maps increases the efficiency of quantitative trait loci (QTL) mapping and facilitates positional gene cloning strategies. We will review currently available techniques enabling molecular markers and point out their advantages and limitations for species identification and linkage map construction. Genome map applications and mapping strategies will be discussed in light of the latest results obtained with woody plant species.'
This study compares the properties of dominant markers, like AFLPs, to those of codominant multiallelic markers, like microsatellites, in reconstructing parentage. These two kind of markers were used to look for both parents of an individual without prior knowledge of their relationships, by calculating likelihood ratios based on genotypic data, including mistyping or not. Experimental data on 89 oak trees genotyped for 6 microsatellite markers and 159 polymorphic AFLP loci were used as a starting point for simulations and tests. Both sets of markers produce high exclusion probabilities, and among dominant markers those with dominant allele frequencies in the range 0.1 to 0.4 are more informative. Such codominant and dominant markers can be used to construct powerful statistical tests to decide whether a genotyped individual (or two individuals) can be considered as the true parent (or parent pair). Gene flow from outside the study stand (GFO) inferred from parentage analysis with microsatellites overestimates the true GFO whereas with AFLPs, it is underestimated. As expected, dominant markers are less efficient than codominant markers to achieve this task, but can still be used with a good confidence, especially when loci are purposely selected according to their allele frequencies.
When a conifer shoot is displaced from its vertical position, compression wood (CW) is formed on the under side, and can eventually return the shoot to its original position. Changes in cell wall structure and chemistry associated with CW are likely to result from differential gene/protein expression. Two-dimensional PAGE of differentiating xylem proteins was combined with the physical characterisation of wooden samples to identify and characterise CW-responsive proteins. Differentiating xylem was harvested from a 22-year-old crooked maritime pine (Pinus pinaster Ait.) tree. Protein extracted from different samples were revealed by high-resolution silver stained two-dimensional PAGE and analysed with a computer-assisted system for single spot quantification. Growth strain (GS) measurements allowed xylem samples to be classified quantitatively from normal wood to CW. Regression of lignin and cellulose content on GS showed that an increase in % lignin and a decrease of % cellulose corresponded to increasing GS values, i.e. CW. Out of the 137 studied spots, 19% were significantly associated with GS effect. Up-regulated proteins included 1-aminocyclopropane-1-carboxylate oxidase (an ethylene forming enzyme), a putative transcription factor, two lignification genes (C-OMT and CCoAOMT), members of the SAM-synthase gene family, enzymes involved in nitrogen and carbon assimilation (glutamine synthetase and fructokinase). A clustered-correlation analysis was performed to study simultaneously protein expression along a gradient of gravistimulated stressed xylem tissue. Proteins were found to form expression clusters that could identify: (i) gene product under similar control mechanisms, (ii) partner proteins, or (iii) functional groups corresponding to specialized pathways. The possibility of obtaining regulatory correlations and anti-correlations between proteins provide us with a new category of homology (regulatory homology) in tracing functional relationships.
Figure 4. Distance
dendrogramme, constructed using the Euclidian distance as similarity metric,
between the 137 Pinus pinaster xylem proteins, mechanical (growth strain) and
chemical (lignin content) wood properties. The axis next to the tree
indicates the average distance (inverse of similarity) between members of the
two branches joined at each node. () increase
in intensity with GS, () decrease in intensity
with GS.
(Postscript 63.5 KB -- POWER POINT 108 KB)
Figure 5. Ordered spot
x spot correlation matrix » for Pinus pinaster. Each entry of the matrix is a
Pearson correlation coefficient that indicates the degree of similarity
between pattern of spots expression across the xylem samples. Growth strain
and lignin content are also added to the matrix. The matrix was ordered
according to the cluster tree (figure 4). Groups of spots positively
correlated (P<0.05) are indicated by red patches, whereas negatively
correlated spots are in green. () increase in
intensity with GS, () decrease in intensity
with GS.
(Postscript 1.4 MB -- POWER POINT 187.4 KB)
Two-dimensional gel electrophoresis (2-DE) and image analysis are currently used for proteome analysis in maritime pine (Pinus pinaster Ait.). We present in this study a database of expressed proteins extracted from needles and xylem, two important tissues for growth and wood formation. Electrophoresis was carried out by isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second. Silver staining made it possible to detect on average 900 and 600 spots on 2-DE gels from needles and xylem respectively. A total of 28 xylem and 35 needle proteins were characterized by internal peptide microsequencing. Out of these 63 proteins, 57 (90%) could be identified based on amino acid similarity with known proteins, of which 24 (42%) have already been described in conifers. Overall comparison of both tissues indicated that 29% and 36% of the spots were specific to xylem and needles respectively, while the others spots were of identical molecular weight and isoelectric point. The homology of spot location in 2-DE patterns was further validated by sequence analysis of proteins present in both tissues. A proteomic database of maritime pine is accessible on the internet (http://www.pierroton.inra.fr/genetics/2D/).
Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology, for deciphering the function and role of the genes that are or will be sequenced. In this review we focus on the various, mainly genetical, applications of the proteomic tool that have been developed in the last years: characterization of individuals or lines, estimation of genetic variability within and between populations, establishment of genetic distances that can be used in phylogenetic studies, characterization of mutants and localization of the genes encoding the revealed proteins. Improvements of specifically devoted softwares have permitted to precisely quantify the variation in amounts of the proteins, leading to the concept of “ Protein Quantity Loci ” which, combined with the “ Quantitative Trait Loci ” approaches, results in testable hypotheses regarding the role of “ candidate proteins ” in the metabolism or phenotype under study. This new development is exemplified by the reaction of plants to drought, a trait of major agronomic interest. The accumulation of data regarding genomic and cDNA sequencing will be connected to the protein databases currently developed in plants.
Experimental variation and genetic background effects in protein accumulation were studied in a three-generation F2 inbred pedigree of maritime pine (Pinus pinaster Ait.). Proteins extracted from needles were revealed by high-resolution silver-stained two-dimensional polyacrylamide gel electrophoresis and analysed with a computer-assisted system for single spot quantification. The integrated intensity of 77% of the studied spots showed a linear relationship with the total amount of protein loaded into the gel. A significant difference of integrated intensity was found among both parents and their hybrid for 31% of the studied proteins, from which 78% followed a non-additive mode of inheritance. The extent of the observed non-additivity is discussed and compared with results found in similar experiments in pea, maize and wheat. Finally, QTL mapping allowed the detection of PQL (Protein Quantity Loci) that explained part of the quantitative variation of protein accumulation.
Based on two polymorphic chloroplast microsatellites that had been previously identified and sequence characterised in the genus Abies, genetic variation was studied in a total of 714 individuals from 17 European silver fir (Abies alba Mill.) populations distributed all over the natural range. We found 8 and 18 different length variants at each locus respectively, which combined into 90 different haplotypes. Genetic distances between most populations were high and significant. There is also evidence for spatial organisation of the distribution of haplotypes, as shown by permutation tests, which demonstrate that genetic distances increase with spatial distances. A large heterogeneity in levels of diversity across populations was observed. Furthermore, there is good congruence in the levels of allelic richness of the two loci across populations. The present organisation of levels of allelic richness across the range of the species is likely to have been shaped by the distribution of refugia during the last glaciation and the subsequent recolonisation processes.
The scale insect Matsucoccus feytaudi is a specific pest of maritime pine, but the damages inflicted by the insect to its host tree are very heterogeneous, ranging from no apparent damage to severe decays of the maritime pine stands. Rangewide variation of mitochondrial DNA among M. feytaudi populations was analysed by PCR-RFLP-SSCP and the results compared with the genetic information already available for its host. Three main non-overlapping lineages can be distinguished in M. feytaudi. The phylogeography of the pest population is clearly related to the history of its host. Most local associations could result from common evolution while others must be interpreted as intraspecific host shifts. Because the distribution of cultivated tree species is greatly influenced by man, much may be learned concerning their genetic structure from the indirect study of their specific pests.
Extensive introgression of cytoplasmic genomes across oak species is now a well-established fact. To distinguish between ancient hybridization events and ongoing introgression, a direct test for the existence of local exchanges is proposed. Such local exchanges must be comparatively recent, that is, contemporaneous with or later than the last postglacial recolonization. The test is applied to an extensive set of data comprising 377 pure or mixed populations (1744 individuals) of four white oak species in southern France. After demonstrating that local exchanges have occurred frequently between all species pairs, another test is performed to check if species status does nevertheless play some role in restricting cytoplasmic gene flow. The results vary according to the species pairs considered, and the observed pattern may be related to the ecology and/or compatibility of interspecific crosses. It is also shown that, for some of these oak species, the presence of related species in a population significantly influences the intraspecific diversity. Altogether, the results demonstrate that (1) intraspecific cytoplasmic gene flow varies according to the species, (2) interspecific cytoplasmic gene flow varies according to the species pair, and (3) both components of gene flow are at least partly related.
Previous studies have established that chloroplasts are inherited paternally in Actinidia interspecific crosses. However, fertilisation problems in interspecific crosses may affect the transmission of organelles. Six female clones, i.e. Abbott, Bruno, Greensill, Hayward, Jones, Monty, and four male clones were used to identify cpDNA polymorphisms within the cultivated kiwifruit species A. deliciosa. The restriction patterns by HpaII of a chloroplast fragment amplified by PCR with a pair of universal primers revealed a polymorphism at the intraspecific level. The inheritance of cpDNA in 143 seedlings from three intraspecific crosses in kiwifruit (Actinidia deliciosa) was studied. All offsprings displayed the restriction pattern of the paternal parent, indicating that maternal inheritance of cpDNA in kiwifruit is rare at best. Strict maternal inheritance of mtDNA was confirmed in the same crosses used to investigate cpDNA transmission. Studies of cytoplasmic inheritance in the Actinidia genus represent to date the best documented report of differential organelle inheritance of cpDNA and mtDNA in angiosperms.
This study demonstrates the feasibility of generating sequence-based markers in Pinus species, from data available in electronic data bases. Nucleotide sequences from 23 partially or fully characterized cDNAs or genomic sequences of pines were used to design PCR primers for amplifying targeted fragments of genomic DNA from Maritime and Scots pine. Various template DNA and MgCl2 concentrations, annealing temperatures, and buffer compositions were used to optimize the PCR amplifications. The polymorphism of 16 sequences was then investigated in a tree-generation inbred pedigree of Maritime pine and in a two-generation pedigree of Scots pine, using single-stranded DNA conformation polymorphism (SSCP) on polymerase chain reaction products. The level of polymorphism was shown to be independent of (i) fragment size, (ii) presence vs. absence of introns in the amplified product and (iii) temperature during electrophoresis. Mendelian segregation was tested for 5 SSCP markers in each species. Chromosomal locations of five genes were identified by linkage analysis with previously mapped markers in a genetic map of Maritime pine. The use of SSCP is recommended for constructing a transcriptional map for comparative mapping studies among pines and to provide useful " candidate genes " for characterizing quantitative trait loci.
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To select candidate populations of wild species to be given priority for conservation, genetic criteria gained from the study of molecular markers may be useful. Traditionally, diversity measures such as expected heterozygosity or percentage of polymorphic loci are considered. For conservation, we propose instead that priority should be given to measures of allelic richness. To standardize the results across populations, the technique of rarefaction is used. This technique allows evaluation of the expected number of different alleles among equal-sized samples drawn from several different populations. We also show how the contribution of each population to total diversity can be partitioned into two components. The first one is related to the level of diversity of the population, and the second one to its divergence from the other populations. For conservation purposes, the uniqueness of a population (in term of its allelic composition) may be at least as important as its diversity level. These new descriptors are illustrated using isozyme and chloroplast DNA data obtained for an endangered tree species, the argan tree of Morocco (Argania spinosa (L.) Skeels). With these analyses, the conservation value of the argan tree populations, especially those of two isolates present in the north of the country, can be better appreciated. The methods proposed to identify priority areas for conservation of the genetic resources of the argan tree are compared to those sometimes advocated in the case of reserve design, where one of the goals is to maximize species richness.
Software : CONTRIB
Eighteen maritime pines (pinus pinaster AIT.) were genotyped at 84 loci using two-dimensional electrophoresis of total protein contained in their haploid megagametophyte. These trees were also characterised for megagametophyte weight and for 49 traits related to young and adult growth of their progenies. Three loci were associated to megagametophyte weight. The genotype of the trees at 17 loci was significantly associated to different growth traits. Among these 17 loci, there were more loci responsible for quantitative variation of polypeptide spots than what would have been expected by chance only. The significance of these associations is discussed and the relevance of two-dimensional electrophoresis of protein for the study of quantitative traits genetics is outlined.
Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees evenly sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with SSCP (Single Strand Conformation Polymorphism), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 combined cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination, leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes), is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. Second, in southern France, the groupings based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization took place in this area, without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this time scale. Such results confirm and expand our earlier conclusions based on controlled crosses.
Two-year-old maritime pine seedlings were submitted to a progressive water deprivation by withholding water in a staggered fashion during the vegetative growth. Needles were sampled before, during and after the stress and drought-responsive proteins were detected from silver stained 2-D PAGE patterns. Out of approximately 1000 spots that were quantified using a computer analysis system, 38 responded during stress. Some proteins seemed to be expressed de novo while others accumulated or were suppressed. One to three internal microsequences were obtained for 11 proteins, from which 10 were identified on the basis of sequence homologies. We discussed their putative roles during stress, regarding their identity and behaviour.
A genetic map of Pedunculate oak (Q.robur) was constructed based on: 271 RAPDs, 10 SCARs, 18 microsatellites, 1 minisatellite 6 isozymes and 1 5S rDNA marker. A total of 94 individuals from a full-sib family were genotyped. Two maps, including 307 markers, were constructed according to the "two-way pseudo-testcross" mapping strategy. Testcross markers segregating in the 1:1 ratio were first used to establish separate maternal (893.2 cM , 12 linkage groups) and paternal (921.7 cM , 12 linkage groups) maps. Both maps provided 85%-90% of genome coverage. Then, homologies between male and female linkage groups were identified based on 74 intercross markers segregating in the 3:1, 1:2:1 and 1:1:1:1 ratios (RAPDs, SCARs, SSRs, 5S ADNr and isozymes) in the hybrid progeny. In each map, approximatively 18 % of the studied markers showed segregation distorsions. More than 60 % of skewed markers were due to an excess of heterozygote genotypes. This map will be used for: i) studying molecular organisation of genomic regions involved in inter and intraspecific differentiation in oak, and ii.) identification of QTLs for adaptive traits.
A 13 x 13 factorial design between E. urophylla and E. grandis, comprising 87 full-sib families, was used to assess the relationships between RAPD marker frequency classes obtained from parental genotypes and the interspecific additive mean (IAM) of the hybrid progeny. For any marker showing a significant association, the cumulative number of the « present band » allele in the parents was significantly correlated either positively or negatively, with the IAM of the traits studied: i.e. volume, stem taper and wood quality. We discuss the potential origin of such correlations in terms of linkage disequilibrium between QTL allele and marker allele. We also examine the possible use of such information, firstly in order to select the parents for further generations of breeding, and secondly in order to choose the hybrid families in which QTAs of specific value could be detected and used to identify the best trees to be vegetatively propagated for the production of clonal variety.
We have recently proposed new estimators of the parameters of genetic diversity and differentiation and of their variances for a haploid locus in a population subdivided into a large number of subpopulations, with a two-stage sampling of populations and individuals (Pons & Petit, 1995). Here they are compared with bootstrap estimators. Several resampling methods are evaluated : sampling of populations only, individuals within populations only, or both. Theoretical results and a numerical example show that the most appropriate bootstrap variance estimators are obtained by resampling the populations alone and not both populations and individuals. However, some bias is apparent in the bootstrap methods, and the direct estimators proposed previously should therefore be preferred.
Five mitochondrial fragments amplified by PCR with consensus primers were cut with restriction enzymes and analysed by SSCP (Single Strand Conformation Polymorphism analysis) in 21 individuals of Quercus robur L. The addition of the SSCP step allowed the detection of 10 polymorphisms against 2 with the classical PCR-RFLP technique alone. The SSCP analysis combined to PCR-RFLP appears very useful for the intraspecific studies of mitochondrial DNA in plants where the principal problem is to detect the rare sequence polymorphisms usually present in this genome. The simplicity and rapidity of the PCR-RFLP-SSCP technique will be particularly interesting in population genetic studies.
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[No summary; first paragraph:]
Recently, there has been a growing interest for the use of the chloroplast (cpDNA) genome in population genetic studies of plants. Indeed, despite its low substitution rate (Wolfe et al., 1987), its typically uniparental mode of transmission, and hence clonal mode of evolution is a unique feature of cytoplasmic genomes of great interest for evolutive studies. The plant mitochondrial (mtDNA) genome shares these characteristics, with however an even slower substitution rate, but is much less studied since it varies enormously in size and gene arrangement (Palmer, 1992). Although the low substitution rate of the organelle genomes is interesting in reconstructing plant phylogenies at high taxonomic levels, it is a serious problem in studies at low taxonomic levels. Therefore, consensus primers which are homologous to the most conserved coding regions but amplify the more variable non-coding regions are very useful. In particular, the conservation of the arrangement of the genes in cpDNA has allowed the design of such consensus chloroplast primers that had a great impact in population genetics and phylogenetic studies. Here, we enlarge a previous list published in this journal (Demesure et al., 1995). A total of 16 pairs of cpDNA primers and 12 pairs of mtDNA primers (respectively 7 and 9 of which are described for the first time) are listed here. Their degree of conservation is tested on 5 species, 3 angiosperms (two dicotyledons and one monocotyledon), a pine (gymnosperm) and a fern (pteridophyte). Hence, a wider taxonomic range is represented as compared to the previous study.
Several investigations undertaken on maritime pine genetics using two-dimensional gel electrophoresis of megagametophyte, collected from germinated or non germinated seed, needle, bud, and pollen proteins are reviewed in the present paper. Different extraction methods adaptable to each tissue or organ are used to obtain reproducible protein patterns. Genetic studies deal with rangewide analysis of genetic diversity, differential genome expression, and genome mapping. Using 16 protein loci, the allelic frequencies were scored and the mean genetic diversity and differentiation were estimated in 192 individuals from six different origins of maritime pine. Proteins whose expression is restricted to a single organ were shown to be more variable than unspecific proteins in a study comparing needle, bud and pollen proteins from eighteen unrelated trees. The level of the variability was slightly more in the bud than that in the needle or in the pollen. Moreover, larger proteins were shown to display more allelic diversity than proteins having a lower molecular weight. Ninety protein loci were found polymorphic in megagametophyte (haploid tissue) and were used to construct a linkage map containing 12 linkage groups. Twenty seven and seventeen proteins showing Mendelian segregation in germinated seed megagametophyte and in needles of an F2 progeny respectively were introduced in another linkage map containing 436 RAPDs markers. These studies outline the usefulness of the two-dimensional gel electrophoresis technique in genetic studies of Conifers.
A genetic analysis of proteins was performed in a three-generation inbred pedigree of maritime pine (pinus pinaster Ait.). Proteins were extracted from needles (2n) and revealed by two-dimensional gel electrophoresis. A total of 17 qualitative variants (presence/absence variations and position shifts) were observed and conformed to Mendelian inheritance patterns. These markers were localized in a previously reported genetic map based on RAPD markers assayed on megagametophytes (1n). To achieve this integration, evenly spaced RAPD markers were genotyped on diploid tissue. The internal amino acid sequences of three proteins were determined, and two of them corresponded to genes of known function. We discuss the use of proteins separated in two-dimensional gels for constructing a genetic map of expressed genes.
After the last glacial period, forest trees have expanded to their present range very rapidly, with rates up to 500 m year-1 for oaks in Europe, which can be explained only by the dispersion of acorns over long distances. We used a stratified dispersal model, including both diffusive and long distance dispersal of seeds, to simulate the colonisation of a 100 km by 300 km grid by populations of oak trees. An appropriate rate of spread is obtained with rare dispersal at distances of the order of tens of kilometers. We simulated the effect of stratified versus diffusive dispersal of seeds on the spatial genetic structure at a maternally inherited locus. Founding events associated with stratified dispersal generate a high amount of genetic differentiation among populations, which is likely to persist over a long time after colonisation. Using autocorrelation methods, we show that diffusive and stratified dispersals create quite different spatial patterns of variation for the maternally inherited locus. Stratified dispersal create patchy patterns, that are concordant with a previous experimental investigation of chloroplast DNA variation at a regional scale in the oaks Quercus petraea and Quercus robur. For plant populations which have passed through recent episodes of range expansion, long distance dispersal events are probably the most important factors of spatial genetic structuring of maternally inherited genes at small or medium geographic scales.
Molecular EcologyThe nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at 8 allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and post-glacial history on patterns of nuclear variation. Because of its small population size and maternal transmission, the chloroplast genome is a good marker of populations history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations, and, second, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation (Gst) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. A possible divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci, the RAPD method probably provided a less biased picture of the relative role of geography and history.
In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q.petraea and Q.robur. 17 out of 52 tested primers pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q.robur trees Mendelian inheritance could be shown for five loci.
The objective of this study was to use random amplified polymorphic DNA (RAPD) to determine the genetic location and effects of genomic regions controlling wood density, stem growth and stem form in two species of Eucalyptus. Two hundred F1 trees generated from an interspecific cross E. urophylla x E. grandis between two elite trees were used. Genetic maps were constructed for each parent with markers segregating in the 1:1 ratio in FS progeny. A total of 86 and 92 markers distributed among 11 linkage groups covered 1295 cM and 1312 cM for the E. urophylla and E. grandis parent, respectively. Traits were measured three times up to selection age (38 months). The magnitude of the phenotypic variation explained by the joint action of the segregating quantitative trait alleles indicate that genetic factors of large effect were involved in the control of the studied characters. Several regions controlling part of the variation for the studied traits were identified by interval mapping. Some regions of the genome exerted effects on more than one trait, providing a genetic explanation for at least some of the correlation between the traits. Based on an age by age analysis, a partial stability of QTL expression was observed with 68% of the QTL being expressed at two ages and 32% being age specific. No QTL were significant for all three ages. Taking advantage of repeated measurements on the same material across different ages, we investigated with a maximum statistical power, the effect of marker genotype on traits, age and QTL x age interaction effects being removed. A two-way analysis of variance made it possible to detect significant marker-trait associations over the period studied. Most of them were already detected in the annual analysis. This result is very encouraging for the application of marker information for early selection of hybrid trees to be vegetatively propagated for the production of clonal varieties.
Recolonisation of Europe by forest tree species after the last glaciation is well documented in the fossil pollen record. This spread may have been achieved at low densities by rare events of long-distance dispersal, rather than by a compact wave of advance, generating a patchy genetic structure through founder effects. In long-lived oak species, this structure could still be discernible using maternally transmitted genetic markers. To test this hypothesis, a fine scale study of chloroplast DNA (cpDNA) variability of two sympatric oak species was carried out in western France. The distribution of six cpDNA length variants were analyzed at 188 localities over a 200 300 km area. A cpDNA map was obtained by applying geostatistics methods to the complete data set. Patches of several hundreds square kilometers exist which are virtually fixed for a single haplotype for both oak species. This local systematic interspecific sharing of the maternal genome strongly suggests that long-distance seed dispersal events followed by interspecific exchanges were involved at the time of colonization, about 10,000 years ago.
Patterns of chloroplast DNA (cpDNA) variation were studied in eight white oak species by sampling 345 populations throughout Europe. The detection of polymorphisms by restriction analysis of PCR-amplified cpDNA fragments allowed the identification of 23 haplotypes that were phylogenetically ordered. A systematic hybridization and introgression between the eight species studied is evident. The levels of subdivision for unordered (GST) and ordered (NST) alleles are very high and close (0.83 and 0.85). A new statistical approach to the quantitative study of phylogeography is presented, that relies on the coefficients of differentiation GST and NST and the Mantel's test. Based on pairwise comparisons between populations, the significance of the difference between both coefficients is evaluated at a global and a local scale. The mapped distribution of the haplotypes indicate the probable routes of postglacial recolonization followed by oak populations that had persisted in southern refugia, especially in the Iberian peninsula, Italy and the Balkans. Most cpDNA polymorphisms appear to be anterior to the beginning of the last recolonization. A subset of the preexisting haplotypes have merely expanded north, while others were left behind in the south.
The geographical distribution of species differentiation throughout the natural range of two sympatric, closely related species of oaks, Quercus petraea (Matt) Liebl. and Quercus robur L., was investigated. By sampling species in pairs in different European regions, from Spain to Poland and Romania, the differentiation between the two species could be subdivised into general and local differentiation. Nine sequences characterized amplified region (SCAR) markers corresponding to genomic regions which discriminate the two species were analysed using single-strand conformation polymorphism (SSCP) or classical electrophoresis (double-stranded DNA on agarose gel) on PCR products providing, respectively, codominant and dominant markers. Similar levels of gene diversity (HE) within the two species were observed, varying generally from 0.3 to 0.5 for dominant markers and from 0.5 to 0.86 for codominant markers. SSCP loci exhibited numerous alleles that were differently involved in species differentiation. The geographical distribution of species differentiation is heterogeneous between the regions, the north-east populations exhibiting higher differentiation than the others. For most loci, general differentiation was higher than local differentiation and was interpreted as the result of historical causes, selection pressures, and intra- and interspecific gene flow.
The sessile (Quercus petraea [Matt.] Liebl.) and pedunculate (Quercus robur L.) oaks are two closely related species having a wide sympatric distribution over Europe. Under natural conditions, they frequently form mixed forests, where hybridization is suspected to occur. In this paper, two different approaches have been applied to the study of the mating system and the interspecific gene flow in a mixed stand formed by the two species. The mating systems of both species have been studied separately by means of the mixed-mating model. The relative contribution of the parental species to the progenies have been estimated with two different methods. The first uses the admixture model. The second is an extension of the mixed-mating model and subdivides the outcrossing rate into intra- and interspecific components. The two species were almost completely outcrossing. This high level of outcrossing and interspecific gene flow could play an important role in the maintenance of the genetic diversity in these long-lived forest tree species. The contribution of the sessile oak to the pedunculate oak progenies varied from 17% to 48%. In contrast, ovules of sessile oak trees appear to be preferentially fertilized by other extreme sessile genotypes. We suggest that interspecific and directional gene flow was responsible for such patterns. Pedunculate oak is considered as a pioneer species and is progressively replaced by sessile oak. Our present findings add a further genetic component to this succession scheme, suggesting that unidirectional gene flow reinforces succession between the two species.
The genetic structure of temperate, wind-pollinated forest trees characterised by large geographic range is particurlarily interesting to describe since a great deal is known of their postglacial history from the analysis of fossil pollen deposits. The common beech (Fagus sylvatica L., Fagaceae) is widespread in most parts of Europe. It is a monoecious, anemophilous and allogamous species (Schaffalistzky de Muckadell 1955, Merzeau et al., 1994) which can be found under very contrasted climates (Comps 1972). However, previous studies using isozymes markers have revealed little population differentiation for the loci analysed (Cuguen et al. 1988, Comps et al., 1987, 1990). This study investigates the cpDNA (chloroplast DNA) diversity and the geographic structure of the common beech in Europe using a PCR technique which takes advantage of the numerous universal cytoplasmic primers recently described (Demesure et al., 1995). We show that the high level of differentiation for cpDNA together with the highly structured geographic variation contrast with the low level of nuclear genetic differentiation measured in previous studies with isozymes, indicating a low level of gene flow by seeds. The northernmost populations are genetically uniform, suggesting a bottleneck at the time of postglacial recolonisation, a scenario which fits with palaeobotanical reconstructions.
Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemic species of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of twelve chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) fragments were amplified and digested with a single restriction enzyme (HinfI). Polymorphisms were identified in six of the cpDNA fragments, whereas no mtDNA polymorphisms were detected in a survey of 95 individuals from 19 populations encompassing most of the natural range of the species. The cpDNA polymorphisms allowed the identification of eleven haplotypes. Two lineages, one in the south-east and the other in the north-west, divide the range of the argan tree into two distinct areas. The level of genetic differentiation measured at the haplotype level (GSTc = 0.60) (i.e., with unordered haplotypes) was smaller than when phylogenetic relationships were taken into account (NSTc = 0.71-0.74) (ordered haplotypes), indicating that population history must be considered in the study of the geographic distribution of cpDNA lineages in this species. If contrasted with the level of nuclear genetic differentiation measured in a previous study with isozymes (GSTn = 0.25), the results indicate a relatively high level of gene flow by seeds, or conversely a relatively low level of gene flow by pollen, as compared with other tree species. Goats and camels could have played an important role in disseminating the fruits of this tree.
At the end of the last glaciation, 13.000 years ago, the geographic range of pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea (Matt.) Liebl.) was limited to southern Spain and Portugal, Italy and the Balkans (Bennett et al., 1991, Huntley and Birks, 1983). Hence, most oak forests located at higher latitudes are comparatively recent and originate from these so-called refugia. Moreover, during the long glacial periods which were predominant during the quaternary, the potential for genetic divergence between oak populations was great. Indeed, these refugia were isolated from each other from west to east by the presence of the Mediterranean sea. Whether the initial genetic substructure set out during the period of recolonisation of Europe, which lasted from 13.000 BP (Before Present, ie. 1950) to 7.000 BP has persisted up to the present is unknown. This is indeed dependent on the amount of genetic mixing experienced since the populations originating from the different refugia came into contact in northern Europe (Hewitt, 1993). It is the goal of this contribution to examine the importance of these historical factors in shaping the present genetic constitution of European oak forests at a continental but also at a regional scale.
Single Strand Conformation Polymorphism (SSCP) profiles of six PCR amplified fragments (250 to 800 bp) were analyzed in three full sib families of pedunculate oak (Quercus robur L.) and their parents. Among the 6 fragments, 4 were polymorphic and 1 exhibited complex patterns that were not changed by varying SSCP conditions. The number of bands for the analyzed fragments varied between 2 and 4 among individuals regardless of the fragment size. As shown by segregation data, the variation in the number of bands between trees could only be attributed to the allelic composition (homozygotes vs heterozygotes). A genotype that exhibited two bands was presumably homozygous, whereas a genotype exhibiting 3 or 4 bands was heterozygous. Mendelian proportions were observed in all crosses for each polymorphic fragment. In one cross, we could clearly identify a null allele due to a possible mutation on a primer site. Single base mutations and short insertion-deletion were shown to be the molecular causes of the SSCP polymorphism observed between different alleles. The use of SSCP as a technique to identify codominant markers of PCR fragments (up to 800 bp) is recommended for gene diversity studies or gene mapping.
Genomic regions differentiating Quercus petraea and Quercus robur were detected by screening 2800 PCR amplification products using random primers on 22 trees of each species sampled in 11 natural populations. Two percent of the amplified fragments only exhibited significant frequency differences between the two species and none of them were specific to a species. The nucleotide divergence between the two species estimated with RAPD data was 0.5% in the overall genome and increased to 3.3% in the discriminant regions. Twenty three informative fragments were cloned and partially sequenced. New primers were derived from these sequences to obtain Sequence Characterized Amplified Region (SCAR) fragments. Southern blot experiments indicated that the SCARs were generally in low copy number in the genome. A search for similarity between SCARs sequences and sequences contained in data banks revealed that three of them corresponded to known DNA sequences.
Genetic diversity at nine isozyme loci was surveyed in an endangered tree species endemic of south-western Morocco, the argan tree. The species is highly diverse (3.6 alleles/locus) and the populations are strongly differentiated from each other (FST = 0.25). This example is used to illustrate a method to standardize measures of allelic richness in samples of unequal sample sizes. This method was developed in the ecological literature for the estimation of the number of species and relies on the technique of rarefaction. Furthermore, it is shown that the measure of subdivision, RhoST, obtained when allelic richness is used in place of h (Nei's index of diversity), is much larger than the FST (e.g., RhoST (40) = 0.52, where (40) indicates the specified sample used to estimate the allelic richness). This suggests that rarer alleles (which influence more strongly measures of allelic richness) have a more scattered distribution than more frequent ones, a result which raises special conservation issues for the argan tree.
Polymorphism is created by the existence of variants in a given set of samples. Variants can be identified at different interlocked levels of the genetic background: genotypes, alleles, haplotypes, nucleotides, whereas a set of samples may be as well subdivided in hierarchical levels: species, population, individuals. In most studies, genetic polymorphism was investigated at either the allelic level (proteins, allozymes, RFLPs), or the nucleotide level (sequence data, restriction analysis). We will therefore describe the methods employed for allelic and nucleotide data. Both methods rely on the same basic concepts that will be outlined in the first paragraph.
Genetic diversity studies have generally two major objectives: (1) the analysis of the level of polymorphism at a given hierarchical level (most usually populations) and (2) the study of the distribution of polymorphisms among the different hierarchical levels (among populations), that are investigated. We will focus our review on the parameters corresponding to the two objectives. The definitions of the parameters will be given, followed by their estimates with their variances (when available) and comments on sampling strategies inferred from experimental data. All parameters reviewed here are descriptive, i.e. they do not depend on evolutionary interpretations. F-statistics of Wright (1943), which were further studied by Weir and Cockerham (1984) and interpreted in terms of coancestry, and number of migrants Nm derived for such estimates of differentiation will therefore not be described.
Estimates and variances of diversity and differentiation measures in subdivided populations are proposed which can be applied to haplotypes (ordered alleles such as DNA sequences, which contain a record of their own histories). Hence, two measures of differentiation can be compared for a single data set: one (GST) which makes use only of the allelic frequencies and the other (NST) for which genealogical relationships are taken into account in addition. Tests are proposed to compare NST and GST with zero and with each other. The difference between NST and GST can be caused by several factors, including sampling artefacts, mutation rates and phylogeographic structure. The method presented is applied to a published data set where a nuclear DNA sequence had been determined from individuals of a grasshopper distributed in 24 regions of Europe (Cooper et al., 1995). Additional insights into the phylogeographic differentiation of these populations are obtained by progressively combining related haplotypes and reanalysing the data each time.
Significantly less recombination was observed for the female gametes than for the male gametes in one individual of maritime pine (Pinus pinaster Ait.). Two segregating mapping samples of the same hybrid tree were used for genomic mapping. One sample consisted of diploid tissues from 192 F2 seedlings. The other sample consisted of megagametophytes (haploid maternally derived nutritive tissue) extracted from 156 germinated F2 seedlings. A total of 94 RAPD markers covering 65% of the genome were used for constructing the maps with both samples. Comparison for the total length of the two maps shows that meiotic recombination differs between the haploid and the diploid mapping samples. Map distances in the diploid mapping sample were on average 14% larger than in the megagametophyte sample, corresponding to a 28% greater rate of recombination in the pollen parent. Departure from homogeneity of recombination rate was also tested by marker interval using a likelihood-based method. Inference for greater meiotic recombination during male gametogenesis was verified analytically. A genome-wide reduction in the female gamete's recombination rate in gymnosperms presents interesting evolutionary implication, since this is exactly the opposite trend than in the angiosperms studied so far. Direct consequences for pine tree breeding are also discussed.
Trans dominant linked markers pairs (trans referring to the repulsion linkage phase) provide a model for inferring the F2 progeny genotype based upon both the conditional probabilities of F2 genotypes given the F2 phenotype, and prior information on marker arrangement. Prior information of marker arrangement can be readily obtained from a linkage analysis performed on marker segregation data in a family resulting by crossing the F1 individual to a "tester" parent or obtained directly from the gametes of the F1, or from recombinant inbred lines. We showed that a Trans Dominant Linked Markers pair (TDLM) can be recoded as a "codominant megalocus" when the recombination fraction, r1, for a pair of TDLM is less than 0.05. We obtained a maximum likelihood estimator (MLE) of recombination frequency, r2, between a TDLM pair and a codominant marker in an F2 family using the EM algorithm. The MLE was biased. Mean bias increased as r1 and r2 increased, and decreased as sample size increased. The information content for r2 was compared to the information content of dominant and codominant markers segregating in an F2 family. It was almost identical with two codominant markers when r1 < 0.01 and r2 > 0.05. For larger values of r1, (0.05 < r1 < 0.15) a TDLM pair provided 75% to 66% of the information content of two codominant markers. Although dominant markers can be converted to codominant markers by a laborious process of cloning, sequencing, and PCR, TDLM pairs could easily substitute for codominant markers to detect quantitative trait loci (QTL) and estimate gene action in an F2 family.
Two single-tree linkage maps were constructed for Eucalyptus urophylla and E. grandis , based on segregation of 480 random amplified polymorphic DNA (RAPD) markers in a F1 interspecific progeny. A mixture of three types of single-locus segregations was observed: 244 1:1 female, 211 1:1 male and 25 markers common to both, segregating 3:1. Markers segregating in the 1:1 ratio (testcross loci) were used to establish separate maternal and paternal maps, while markers segregating in the 3:1 ratio were used to identify homology between linkage groups of the two species-maps. An average of 2.8 polymorphic loci were mapped for each arbitrary ten-mer primer used in the polymerase chain reaction. The mean interval size beween framework markers on the maps was 14 cM. The maps comprised 269 and 236 markers covering 1331 cM and 1415 cM in 11 linkage groups, for E. urophylla (2n=2x=22) and E. grandis (2n=2x=22), respectively. A comparative mapping analysis with two other E. urophylla and E. grandis linkage maps showed that RAPDs could be reliable markers for establishing a consensus species-map. RAPDs markers were automatically and quantitatively scored with an imaging analyzer. They were classified into four categories based on their optical density. A fragment intensity threshold is proposed to optimize the selection of reliable RAPD markers for future mapping experiment.
(French English)
Les arguments émis contre l'application de la sélection assistée par marqueurs (SAM) chez les arbres forestiers, notamment les problèmes potentiels liés au manque de déséquilibre de liaison entre locus dans les populations panmictiques, sont reconsidérés en raison de la baisse des coûts et surtout de l'automatisation des techniques de marquage moléculaire issues de la technique RAPD (Random Amplified Polymorphic DNA). La construction de cartes génétiques saturées pour les individus d'une population d'amélioration élite devrait permettre de contourner ce problème. Cette proposition est présentée et discutée. Les stratégies de cartographie génétique et de détection des facteurs génétiques controlant les caractères quantitatifs (QTL) s'appuyant sur des pedigrees couramment rencontrés dans les programmes d'amélioration forestiers (familles de demi-frères et de plein-frères) sont envisagées. Afin d'illustrer la faisabilité de la sélection assistée par marqueurs, des propositions sont faites pour les programmes de sélection du pin maritime et de l'eucalyptus. Les gains génétiques et le coût de la SAM sont évalués dans des cas concrets et comparés à d'autres stratégies permettant une efficacité de la sélection identique.
Marker assisted selection in forest tree breeding programs as illustrated by two examples: maritime pine and eucalyptus
The arguments raised against the feasibility of marker-assisted-selection (MAS) in forest trees, especially the expected absence of linkage disequilibrium between markers and quantitative trait loci in large random mating populations are reconsidered. Decrease in costs and automation of the RAPD (Random Amplified Polymorphic DNAs) technique make possible to construct single-tree maps for every individual of an elite breeding population: an extreme alternative to deal with linkage equilibrium. Mapping strategies and quantitative trait loci (QTL) detection using existing pedigrees in forestry breeding programs (half-sib and full-sib progenies) are presented and discussed. The feasibility of MAS is illustrated for the maritime pine and eucalyptus breeding program. Genetic gains and costs associated with the use of molecular markers are evaluated and compared to other strategies that aim to obtain a similar selection efficiency.
In a full-sib progeny between heterozygous parents, the analysis of marker-trait associations using dominant RAPD (random amplified polymorphic DNA) markers allows the detection of specific quantitative trait loci (QTL) for each parent of the cross. Here we developed a method aimed at estimating the breeding value of such QTL alleles in the population, for the incorporation of marker-assisted selection in operational 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 family 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 family of the other parent which is double heterozygous is then used to estimate the average effect (i.e. additive effect) of the two QTL alleles.
Random Amplified Polymorphic DNAs (RAPDs) were used to investigate quantitative trait loci (QTL) for traits related to height growth on 126 F2 seedlings of maritime pine (Pinus pinaster Ait). The haploid megagametophyte was used to determine the maternal genotype of each F2 individual. The seedlings were raised for two years in a greenhouse under accelerated growth conditions consisting of intense fertilization combined with continuous light treatments. Total height was measured at different developmental stages and height growth components were measured after the second growth period. QTLs were identified for each trait. For total height, QTLs of different developmental stages were located on distinct linkage groups. However, rather than a complete temporal change in QTL expression, our results showed that maturation may induce a progressive shift of the genetic control of height growth. This may provide an explanation for a low juvenile-mature phenotypic correlation previously reported for height. Height growth components related to initiation (controlled by the apical meristem) and elongation of shoot cycles (controlled by the subapical meristem) were mapped to different chromosomes, suggesting that the activity of these meristems is controlled by separate genetic mechanisms.
An F2 progeny of maritime pine (Pinus pinaster Aïton) was used to investigate the mode of inheritance of the d3-carene using a quantitative and a qualitative approach. A previously reported genetic map constructed with random amplified polymorphic DNA (RAPD) markers made it possible to locate on linkage group 5, one major quantitative trait locus (QTL) accounting for most of the phenotypic variation of this trait. In the qualitative approach, the "C" locus that controls the relative quantity of d3-carene (C+ for the richness allele and C- for the poorness allele) was found to be strongly associated with RAPD markers in the same genomic region of linkage group 5. The co-location between the QTL and the "C" locus suggests that a major gene or closely linked loci affect the variation in d3-carene.
The genetic differentiation between Quercus petraea and Quercus robur was investigated using two dimensional electrophoresis. Twenty three oaks from six European countries covering partly the natural geographic range of white oaks in europe, were studied. Five hundred and thirty polypeptide spots were scored, among which 101 were polymorphic. We did not find any spot specific to Quercus petraea or Quercus robur, however 3 spots shown sigificant frequency differences between the two species. Interspecific and intraspecific dissimilarities were very close. Total proteins confirms the results obtained with other molecular markers (isozymes, RAPD and cpDNA) concerning the low level of genetic differentiation between Quercus petraea and Quercus robur.
[No summary; first paragraph:]
The genetic information present in the plant mitochondrial DANN (mtDNA) and chloroplast DANN (cpDNA) is of great interest in phylogeny and in population genetics, largely because of the non-Mendelian mode of inheritance of these genomes. The circular cpDNA molecule has been shown to be highly conserved in structure (Palmer & Stein 1986). This has lead to the design of pairs of universal primers which can amplify non-coding regions separating two coding sequences in most land plants (Taberlet et al. 1991). Such non-coding sequences can then be explored (through sequencing or simply by using a variety of restriction enzymes) in order to detect informative polymorphisms at a variety of taxonomic levels. The efficiency of this approach has already been demonstrated in phylogenetic and population genetic studies (e.g. Arnold et al. 1991).
However, only a limited number of such universal chloroplast primers seem to have been described. Though the substitution rate of plant mitochondrial DANN genes is even smaller than that of chloroplast genes (Wolfe et al. 1987) , making it easy to identify conserved coding sequences tobanchor primers, the amazing lability of the organisation and size of this genome (Palmer 1992) is a serious limitation when non-coding sequences need to be amplified.
Here we describe mitochondrial universal primers, together with additional chloroplast universal primers, which we expect to be useful in a variety of studies of the genetic variability of both organelle genomes of land plants.
Using two-dimensional polyacrylamide gel electrophoresis, the genetic variation of proteins was examined in three organs (needle, bud, and pollen) from 18 trees of maritime pine. Three types of variation were noted : presence/absence, staining intensity, and position variation of the spots. Of the 902 polypeptides scored in the three organs, 245 (27.2%) were polymorphic. Moreover, among these variable spots, 117 were found in a single organ, demonstrating an increased polymorphism of the organ-specific polypeptides (56.0% vs 18.4% for the organ-unspecific polypeptides). Finally, a positive correlation was found between variability level and subunit molecular weight for spots showing position variation but not for spots showing presence/absence or staining intensity variations. Possible explanations for this observation are discussed.
An extension of Nei's analysis of diversity in a subdivided population is proposed for a haploid locus. The differentiation GST becomes a natural extension of Wright's FST and generalizes Weir and Cockerham's parameter of co-ancestry by relaxing the assumption of identical correlation for all the alleles. Inter- and intrapopulation variances of the estimated diversities and differentiation are derived. Finally, the optimal sampling strategy for measuring GST when a fixed number of individuals can be analysed is considered. It is shown that, at a given locus, there is a unique sample size per population which yields the smallest variance of GST, regardless of the number of populations studied. These theoretical developments are illustrated with an analysis of chloroplast DNA diversity in a forest tree. The results emphasize the necessity of sampling many populations, rather than many individuals per population, for an accurate measurement of the subdivision of gene diversity at a single locus.
Thirty genes belonging to three classes of biochemical markers (8 isozymes loci, 16 protein loci revealed by two dimensional gel electrophoresis, and 6 terpenic loci) were scored in six populations of maritime pine. The purpose of the study was to compare the level of genetic differentiation (GST) among the populations of this pine in order to test whether differential selective pressures are acting on these markers.
However, each class of loci was found to display different levels of average diversity. Although this should not affect the comparison of the level of differentiation (theoretically a diversity-independent genetic measure) we find here that single locus values of differentiation significantly depend on the values of diversity. This result is explained analytically by showing that the sampling of a limited number of populations results in GST taking maximal possible values lower than unity, especially when the level of diversity is low. By removing the less polymorphic loci, measures of differentiation independent of the level of diversity can be obtained. They turned out to be very close for each class of markers indicating the absence (or similar level) of selection acting on the three classes of loci and a high level of differentiation in this pine (GST = 0.17) typical of species having highly fragmented range.
Two genomic maps were constructed for one individual tree of maritime pine, Pinus pinaster Ait.. The maps were constructed using a common set of 263 RAPD markers (random amplified polymorphic DNA). The RAPD markers were chosen from a larger number of polymorphic RAPD fragments based on repeatability and inheritance in a 3-generation pedigree. The maps were constructed from 2 independent mapping samples of 62 megagametophytes (1n) from a self cross and from an open-pollinated cross. The markers were grouped (LOD > 4 ; q < 0.25) and assigned to thirteen major and five minor linkage groups. Two framework maps were constructed using the ordering criterion of interval support > 3. Comparison of the 2 framework maps suggested that the locus order was incorrect for 2% of the framework markers. A bootstrap analysis showed that this error rate was representative for our data set. The results showed that framework maps constructed using RAPD markers were repeatable and that differences in locus order for maps of different genotypes or species could result from chance. The total map distance was 1380 cM, and the map provided coverage of approximately 90% of the genome.
A detailed genomic map was constructed for one F1 individual of maritime pine, using randomly amplified polymorphic DNA (RAPD) and protein markers scored on megagametophytes of germinated seeds. Proteins allowed to localise exclusively coding DNA in the large genome of this Pinus species, mapped with RAPD markers that essentially fall within repetitive (i.e. mostly non coding) DNA. Dot blots experiments of 53 RAPD fragments showed that 89% amplified from highly repetitive chromosomal regions. The map comprised 463 loci, including 436 RAPDs amplified from 142 ten-mer oligonucleotide primers and 27 protein loci. Twelve major and one minor linkage groups were identified using a LOD score > 5 and a recombination fraction q < 0.30. A framework map was ordered with an interval support > 4, covering 1860 cM which provided almost complete coverage of the maritime pine genome. The average distance between two framework markers was 8.3 cM; only one interval was larger than 30 cM. Protein loci were well distributed throughout the map. Their potential use as anchor points to join RAPD-based maps is discussed. Finally, the genomic maps of Arabidopsis and maritime pine were compared. Linkage groups were shown to have similar total map lengths on a chromosomal basis, despite a 57-fold difference in DNA content.
Restriction patterns of two chloroplast and one mitochondrial DNA fragments amplified by PCR with universal primers were studied to determine the mode of inheritance of these organelles in 143 progenies of 5 intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that chloroplast DNA but also mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.
The first step in the construction of a linkage map involves the estimation and test for linkage between all possible pairs of markers. The lod score method is used in many linkage studies for the latter purpose. In contrast with classical statistical tests, this method does not rely on the choice of a first-type error level. We thus provide comparison between the lod score and a c2 test on linkage data from a gymnosperm, the maritime pine. The lod score appears to be a very conservative test with the usual thersholds. Its severity depends on the type of the data used.
The genome length, in units of Morgans or centimorgans, is a fundamental feature of a species. It can be calculated from a complete linkage map. However, the genome size can be estimated with partial linkage data. Using linkage data obtained by the analysis of a two-dimensional electrophoresis of the proteins contained in an haploid organ, the megagametophyte, we suggest an estimation and a confidence interval of the genome length of a gymnosperm, the maritime pine (Pinus pinaster Ait.). The results indicate an important gap between the physical and the genetic maps.
The comparison of 42 two-dimensional protein patterns from megagametophytes of maritime pine from seven geographical origins enabled the analysis of the genetic variability of abundant proteins. More than 84% of the polypeptides were variable. The intra- and inter-origin variability levels were of a similar magnitude. Correspondence analysis and a dendrogram computed using a dissimilarity index between individuals showed three main groups. The first group included the individuals from Landes (France, Portugal, eastern Spain, and Corsica, without individualising the provenances. The second group was composed of accessions from Italy and Sardinia, and the individuals of each location were separated. The third group included all of the individuals of Moroccan origin. This clustering was in agreement with the Atlantic, Mediterranean and North African structuration of maritime pine established from terpene data.
Proteins from haploid megagametophytes from 18 trees were studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A total of 222 seeds, an average of 12 per tree, were analysed individually. 150 protein spots appeared to segregate on the polyacrylamide gels in at least one tree. Genetic interpretations were made to define the number of loci responsible for the presence versus absence, staining differences or position variation of the segregating spots. The complete covariation observed between some spots could be the result of either the separation of a single gene product into two or more constituents, very close linkage, or the action of pleiotropic gene. Human genetics techniques were used to map the 84 putative loci detected. Sixty-five loci were organised in 17 linkage groups, whereas 19 remained unlinked.
More than 70 trees belonging to the morphologically distinguishable species Quercus robur L. and Quercus petraea (Matt.) Liebl. were sampled in a mixed stand located in western France. The ribosomal DNA repeat was characterized by a high level of length polymorphism; while chloroplast DNA in our sample was nearly fixed at 2 previously identified polymorphic regions. Overall, very little differentiation was found between species using both markers. The implications for our understanding of this complex of species are discussed.
Recurrence equations for genetic diversities and differentiation were developed for hermaphrodite plant species in an island model of population structure. This was made possible by the definitions of diversities at all hierarchical levels from gamete to total population and by the definition of migration rates specific to plants for both nuclear and cytoplasmic genomes. Mating system was also incorporated. Numerical computations were used to compare equilibrium values of differentiation obtained with our equations with those predicted by classical formulas. We show that the differences (sometimes high) result from the interpretations of the definition of gene diversity in a population of finite size. We interpret it as the probability that two genes sampled with replacement are different alleles (instead of without replacement). The effects of several parameters (ploidy level, mode of inheritance, outcrossing rate, population size) on genetic subdivision were evaluated. Contrary to the situation in animals, plant migration is intrinsically asymmetrical because a gene transmitted to the next generation through the male gamete may migrate in the pollen grain and in the seed, whereas a gene transmitted through the female gamete can migrate only in the seed. As a consequence, mode of inheritance (in the case of cytoplasmic genes) and outcrossing rate have strong impacts on subdivision, especially when pollen migration is larger than seed migration (a likely situation in many plant species). Parameters estimated in a survey of oak populations (Quercus robur L.) were used to examine whether our understanding of a real situation could be improved by the model. In particular, the rate of return to equilibrium was studied after a perturbation, i.e. a temporary decrease of population sizes (a bottle-neck).
Chloroplast DNA polymorphisms have been detected by the conventional Southern-blotting hybridization method in four species of European oaks (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica). Three polymorphisms, shared by at least three of these species, can be scored directly in ethidium bromide-stained gels and were used in a broad survey of the level of differentiation of the oak species and of their pattern of genetic structure in western Europe. The highly significant geographic variation and the high genetic differentiation (Gst = 0.895, sGst = 0.025) indicate a low level of cytoplasmic flow. We conclude that cytoplasmic genomes are well suited for the reconstruction of past migrational routes of such a complex of species.
This contribution reviews studies of nuclear and organelle gene diversity in oak species. Studies of allozymes were reported for 33 species belonging to the sections Erythrobalanus, Lepidobalanus and Mesobalanus of the genus Quercus. The extent and organization of gene diversity were investigated at 3 hierarchical levels: complex, species and population. Total diversity at the species and population level varies greatly among species (from 0.06 to 0.40). The range of variation among species is as large as that observed in other plant genera. Life history characteristics and evolutionary history are the main explanations for these results. Species with large and continuous distributions such as Q. petraea and Q. rubra exhibit high levels of gene diversity. Within a complex, most of the nuclear gene diversity is distributed within populations (74%). The remaining diversity is mainly due to species differentiation (23%), while the between-population component is low (3%). Organelle gene diversity has been investigated recently in 2 species complexes in the section Lepidobalanus (one in North America and one in Europe). Compared to nuclear genes, organelle gene diversity is strikingly different. Contributions of within-stand variation, species differentiation and population differentiation to total diversity, are respectively 13%, 11% and 76%. Trees of a given population generally share the same chloroplast genome. Moreover, trees of different species (with reported introgression) occupying the same stand exhibit a high degree of similarity.
Levels and structure of gene diversity in the nuclear (allozymes) and organellar (chloroplast DNA, cpDNA) genome were studied within the complex of two European white oaks, Q. robur and Q. petraea. The data are based on 15 loci for allozymes and 3 loci for cpDNA. No species difference was observed for levels of gene diversity, whether parameters were estimated on a population or on a species level. Both species exhibit high levels of gene diversity. Within population expected heterozygosity was 0.275 for Q. petraea and 0.264 for Q. robur. Gene diversity was further partitioned in different components: species, population and individual. The first and second components comprised 5% and 4%, respectively, of the total diversity, whereas the within population components accounted for 91% of the diversity. At the organellar level, these estimates were strikingly different: 88% of the total diversity was due to the differentiation between populations. Geographic organisation of the gene diversity was further studied in Q. petraea. On a monolocus basis, differentiation between geographic regions or between populations (within regions) accounted for less than 1% of the total diversity. However, on a multilocus basis, multivariate analysis resultede in a geographic pattern of variation.
The comparison of two-dimensional protein patterns of 56 haploid megagametophytes from a single maritime pine tree allowed the analysis of genetic determinism of qualitative and quantitative variation. One hundred and nineteen loci were localized in 12 linkage groups while two other loci were unlinked. Among the 81 qualitative variants, 58 could be interpreted as allelic products of structural genes. Their distribution in the linkage groups appeared to be non-random, since most of them clustered at a common point in two groups. The remaining 23 qualitative and 40 quantitative variants could best explained by assuming monogenic determinism of protein amounts. Pleiotropic effects could be responsible for some of the variations in amounts of proteins which cosegregate.
