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1.TITLE
Synthetic maps of gene diversity and provenance performance for utilization
and conservation of oak genetic resources in Europe.
2. OBJECTIVES AND MILESTONES
The main objective is to provide geneticists, ecologists and
foresters with an integrated description of oak genetic resources in the form
of synthetic maps based on CpDNA polymorphism and provenance variation. A map
of genetic variation on a European scale is a prerequisite for defining seed
transfer rules, for identifying reproductive material of unknown origin. The
project will also afford basic knowledge on the structure of nuclear gene
diversity for decision makers in conservation biology.
Specific objectives are:
- to construct geographic maps based on the genetic differences of maternal lineages and pollen fossil deposits.
- to evaluate provenances raised in the different countries in geographic maps and to compare provenance performance with the geographic map of maternal lineages.
- to estimate the level of gene diversity and phenotypic variation in Q. petraea and Q. robur in different areas of the natural distribution
- to provide basic information for defining seed transfer rules among
different European countries.
The results and deliverables will be:
R1 - Synthetic geographic map of genetic types: the map that will be
produced at the end of the project will summarize the geographic distribution
of chloroplast cytotypes in Europe.
R2 - Historical maps of oak pollen
deposits in
Europe: the oak pollen data extracted from the European Pollen
Database will be compiled in a series of maps of pollen deposits dating from
15000 BP to present.
R3 - Database of oak
provenances: the compilation of data from various provenance tests
established in different countries can be summarized in a general data base
where passport data, performances, DNA genotyping are gathered.
R4 - Rapid DNA screening
techniques: the molecular methods that will be used with the same
protocols in different laboratories, will be available in the future for other
species of the Fagaceae.
R5- Bank of oak DNA extracts: The systematic sampling of populations
followed by the DNA extraction will result in a bank of DNA extracts, that may
be used in the future for any genetic survey.
R6- Patterns of provenance variation: The joint analysis of
provenance test installed in different countries, and the analysis of national
provenance test will reveal geographic patterns of intraspecific
variation.
R7- Reference populations for gene diversity : Pairs of natural
populations (Q. petraea and Q. robur ) with standardized
sampling stratregies will be selected for describing intra and interspecific
gene diversity.
R8- Levels and structure of gene diversity in natural stands: Levels
of nuclear diversity will be assessed in reference populations with codominant
markers.
R9- Levels of phenotypic variation: Phenotypic variation will be
assessed in the reference populations with quantitative genetic procedures and
compared with the levels of diversity obtained with molecular markers.
The expected benefits are:
Scientific
The geographic distribution of the cpDNA (chloroplast DNA) polymorphism
will allow to trace retrospectively the post glacial recolonisation routes.
The recolonisation pathways will be reinforced by the comparison with the
fossil pollen maps. Mechanisms of speciation in oaks and interspecific gene
flow will be approached by the analysis of the interspecific differentiation
with codominant markers.
Technical
The synthetic map will allow us to assign any population of
" unknown origin " to an alien or local source. This
information will be highly valuable for oak decline surveys and ecological
studies, since sensitivity to dieback or diseases may result from a non native
origin of the stand. For the first time, the indigenous origin of an oak
forest can be tested. Methodologies for gene conservation in oaks can be
identified from the results obtained on the within and between population
structure of gene diversity and phenotypic variation.
Economic
The comparison of the synthetic map with the provenance performances in
comparative plantations, will show whether there is a historical (maternal
origin) cause of provenance variation. If so, the choice of optimal material
for plantation can be indicated by these results. Depending on the resolution
of the map, the geographic origin of a provenance can be identified, which
will lead to a legal control of seed or seedlings lots in nurseries. Economic
benefits may be reduced in their ambition if (1)interprovenance variation
reveals to be more ecotypic than geographic and (2) if the geographic map
shows a " mosaic " rather than a clinbal pattern of
variation.
3 PARTICIPANTS
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4 RESEARCH PLAN AND DISTRIBUTION OF TASKS
Task 1 Construction of a geographic map of genetic types
Aim : The objective is to build a geographic map of C DNA
haplotypes. The map will be based on the analysis of chloroplast DNA
polymorphism according to a systematic sampling of populations every 50 km
throughout western Europe. But before analysing with this sampling,
populations present in provenance tests will also be analysed. The map of
genetic types will be compared with the map of pollen depositsto retrace
postglacial recolonization routes.
Subtask 1.1. Standardization of molecular techniques for analysis
Chloroplast DNA polymorphism
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The map can only be constructed if standard techniques
are used for the analysis of CpDNA polymorphism. Methods have been
developed and tested by partner P1 and will be shared with other
participants involved in Task 1 during a 1 week technical meeting.
The molecular technique used is a PCR-RFLP assay. For the time being, 28 different haplotypes can be identified from amplification products with 4 primer pairs, digested with 3 restriction enzymes. Scoring of haplotypesand construction of data files will be standardized. |
Subtask 1.2. Cp DNA analysis of populations established in provenance
tests (Table 1)
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For each provenance, buds or leaves will be collected
from 5 trees per provenance. DNA will be extracted and the PCR-RFLP
assay will be made on each sample to identify the haplotype(s) present
in the provenance.
Results will be stored in the provenance data base (Subtask 2.1). Efforts will be made during the project to obtain and analyze populations from eastern European countries especially from the balkan region, which is considered as a major glacial refugium. |
| P1 | Q.petraea |
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| P2 | Q.petraea
Q.robur |
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| P3 | Q. petraea |
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| P4 | Q.petraea
Q.robur |
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| P5 | Q.robur |
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| P7 | Q.robur |
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| P7 | Q.robur |
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| P10 | Q. petraea |
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| P10 | Q.robur |
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| P11 | Q.petraea+robur |
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Task 1.3. Cp DNA analysis of populations sampled on a grid of 50 km and construction of the CpDNA geographic map (Table 2)
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Samples will be collected throughout Europe on a grid system (50 km*50km). In the nearest oak forest of natural origin, buds or leaves will be collected on 5 trees per forest (on each species if both are present). The PCR-RFLP assay will be conducted as in subtask 1.2. Haplotypes present in each forest will be identified. |
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* : 70 geographic points on the grid system, but only 10 natural
stands.
Subtask 1.4. Analysis of pollen fossil deposits
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The current pollen database comprises data on pollen deposits collected in about 550 points distributed throughout Europe (Mostly France, Great Britain, Spain, Poland ..). Data on 300 additional points will be added through cooperative efforts with colleagues who have already collected the samples., mostly in Germany, Italy and in the Netherlands. Data relative to oak deposits will be extracted from the data base in order to construct isopollen maps for a series of ten-time slices since 15000 BP. |
Task 2 Provenance evaluation in experimental plantations
Aim : The objective is to analyze national and international
provenance tests on a range wide scale so that inferences may be drawn for
seed transfer rules. The provenance results will be compared with the CpDNA
results (subtask 1.1), in order to verify if there is any relationship between
the geographic variation of growth and adaptive traits and the maternal
lineage of the population.
Subtask 2.1 Data base of oak provenances in Europe
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Passport data of each provenance will be collected and stored in a data base, that will be accessible through internet. A standard data base package (ACCESS or PARADOX) will be used for that purpose. |
Subtask 2.2 Joint evaluation of the S. Madsen range wide provenance
test
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A set of 19 provenances from 9 different European countries was established in 4 different countries. This common set will allow a joint analysis of the provenance variation and the stability of the provenance response. |
Subtask 2.3 Analysis of national provenance tests and comparison of
results with the CpDNA map.
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Independent analysis of provenance tests raised in each country will be analyzed separately for their own characters of interest (see sub annex 1 for the details of the provenance tests). Assessments will mainly be made on growth, form and phenology (bud burst and bud set). Mean values of each provenance will be stored on the provenance data base and compared with the Cp DNA haplotypes. |
Task 3 Assessments and dynamics of levels of genetic diversity
Aim : Preliminary results obtained with isozymes in Q.
petraea have shown that there is a geographic trend of variation in the
level of allelic richness : populations of the central part of the natural
distribution (from the Loire to the Rhine) are less variable than populations
from the peripheral parts of the distribution. On the other hand Q.
petraea is more variable than Q. robur. Knowledge of the levels of
diversity in different species and different populations is of primary
importance for conservation strategies. The objective is to evaluate the level
of diversity in the two species and its geographic variation, by sampling
large size populations and using hypervariable markers (exhibiting numerous
alleles).
Subtask 3.1 Sampling of populations
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Pairs of populations of Q. petraea and Q. robur will be identified and selected for estimating gene diversity. The two populations of a pair should be in adult stands in close proximity with some mixed zones. Taxonomic identification will be made with leaf morphological characters . Populations size should be about 200 so that allelic richness can be measured. Trees will be mapped. Buds or leaves will be collected for DNA and allozyme analysis. |
Subtask 3.2. Standardization of molecular techniques for analysis of
nuclear polymorphism
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Techniques to monitor diversity will be shared during a two weeks technical meeting. These concern codominant markers allowing to identify the different alleles present in the populations: allozymes, microsatellites, PCR-SSCP fragments. Methods have already been developped for 13 enzyme-coding loci, and primers are available for 5 microsatellite loci, and 5 PCR-SSCP loci. Whereas, isozymes and microsatellites are anonymous markers, the PCR-SSCP fragments are specifically involved in the species differentiation. |
Subtask 3.3 Estimation of levels and structure of diversity
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The analysis of codominant markers in the sampled pairs
of population will be conducted to: - estimate the level of diversity in each population and species,
by assessing the number of alleles, and expected heterozygosity. - estimate the level of species differentiation by comparing
allelic frequencies between the two species in each pair - depict the within population structure of diversity by describing
the spatial distribution of alleles. - describe the overall interspecific and geographic organisation of diversity by a joint analysis of allele frequencies over all pairs. |
Subtask 3.4 Estimation of genetic variation of phenotypic traits
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Subtask 3.4 is hypothetical, in the sense that it depends on the seed crop produced during the project. If there is a successfull seed crop, open pollinated progenies will be collect on at least 60 trees per population and raised in the nursery. Assessments of juvenile characters (phenology, morphological traits) will permit to estimate the level of diversity for quantitative traits. These levels will be compared with those obtained with codominant markers (Subtask 3.3). |
Table 3. Distribution of activities in Task 3
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5 TIMESPAN OF TASKS
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6. COORDINATION
First joint coordination meeting and technical meeting.
(First year, first term)
Coordination meeting (2 days for Task 1, 2 and 3)
Task 1 : Sampling procedures for collecting the material. Sharing of the
collections points among the different countries. Comparisons of sampling for
the DNA analysis and fossil pollen analysis.
Task 2 : Establishment of passport data to be recorded (subtask 2.1),
standardization of data. Decisions on the assessments to be made in the S.
Madsen collection (Subtask 2.2)
Task 3 : Sampling of populations to be sampled and collected.
Technical meeting (2 weeks) for Task 1 (Subtask 1.1, 1.2, 1.3)
Sharing and standardization of molecular techniques for chloroplast DNA
analysis
Second joint coordination and technical meeting.
(Second year, first term)
Coordination meeting (2 days for Task 1, 2 and 3)
Task 1 : Comparison of results obtained in the provenance tests (Subtask
1.2.). Adjustments to be made in the sampling of populations according to the
grid system (Subtask 1.3). Presentation of first pollen data results.
Task 2 : Presentation of the provenance data base. Preparation of
assessments to be made in provenance tests.
Task 3 : Presentation of the sampled populations. Prospects for collecting
progenies
Technical meeting (2 weeks for Task3, Subtask 3.2)
Sharing and standardization of molecular techniques for nuclear
polymorphism (microsatellites, single copy regions) using PCR derived
techniques.
Third coordination meeting
(Second year, fourth term)
Task 1 : Comparisons of results, and sharing of data for the construction
of the CpDNA map
Task 2 : Presentation of the results of the S. Madsen collection. sharing
of data to prepare the comparison of CpDNA data and phenotypic traits of
provenance tests.
Task 3 : Presentation of first results obtained with nuclear markers.
Preparation of the progeny tests.
Final meeting
(Third year, third term)
General presentation of the results and preparation of the final report.
Prospects for future cooperation
It is expected during the course of the project that several visits among
individual participants will take place, since the project aims at applying
the same techniques on different populations or samples. Technical problems
may arise that will require visits by the coordinators of the different tasks,
or other scientists.
7 RESEARCH MILESTONES
Task |
Subtask |
Deadline (months) |
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1 |
1.1 |
4 |
Standardize protocols for CpDNA analysis |
| 1.2 | 24 | Identify the cytotypes of provenances | |
| 1.3 | 36 | Construct the geographic map of cytotypes | |
| 1.4 | 36 | Construct the geographic map of pollen deposits | |
| 2 | 2.1 | 18 | Create the data base of provenances planted in tests. |
| 2.2 | 36 | Evaluate range wide geographic variability (S. Madsen collection) | |
| 2.2 | 36 | Evaluate provenance performance in national provenance tests | |
| 2.3 | 36 | Correspondence between maternal lineage and provenance performance | |
| 3 | 3.1 | 12 | Identify and sample reference populations
Construct the map of the spatial distribution of trees |
| 3.2 | 15 | Standardize protocols for nuclear DNA analysis (microsatellites and single copy regions) | |
| 3.3 | 36 | Estimate Inter and intraspecific diversity in Q. petraea and Q. robur stands | |
| 3.4 | 36 | Estimate heritability values of juvenile traits |
Sub annex 1 List of provenance test analyzed for CpDNA analysis or phenotypic variation
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Populations in bold characters correspond to the S. Madsen collection
(Subtask 2.2)