Individuals were selected for resistance against the imperfect fungus Seiridium cardinale Wag in six populations of Mediterranean cypress (Cupressus sempervirens L.). The collections of resistant clones and their base populations were surveyed at several isozyme gene loci. A total of 140 adult trees and of 109 clones were genotyped at six isozyme gene loci. The comparison yielded information on changes in genetic variation due to artificial selection. The genetic structure of most clone collections were similar to their base populations. Nevertheless, the number of rare alleles among the resistant clones had consistently decreased, although the numbers of investigated trees were similar to those of the clones. Possible implications for breeding strategies are discussed. Presentation during the international conference: "Dynamics and conservation of genetic diversity in forest ecosystems", Strasbourg, 2-5 December 2002

1. Loss of alleles due to resistance
breeding
The case of cypress
Strasbourg 2002

2. Aristotelis C. Papageorgiou
Department of Forestry, Environment and Natural Resources,
Democritus University of Thrace, Greece
Sotirios Xenopoulos
Institute of Mediterranean Forest Ecosystems and Forest
Products, National Agricultural Research Foundation, Greece
Reiner Finkeldey, Hans H. Hattemer
Institute of Forest Genetics and Forest Tree Breeding,
Georg August University Göttingen, Germany
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3. The Mediterranean cypress
• Occurs naturally from
Afghanistan to Greece
• Cultivated all over the
world – amenity tree and
plantations
• Typical element of the
Mediterranean landscape
• Stress tolerant
• Endangered by the fungus
Seiridium cardinale
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4. Breeding for resistance
• AGRIMED (1980-1988)
• CAMAR (1991-1994)
• AIR II (1993-1997)
• FAIR (1997-2000)
Research Group: INRA, University of
Montpellier (France); CNR, University of
Florence (Italy); MAICH, NAGREF,
University of Thessaloniki (Greece);
University of Lisbon and Vila Real
(Portugal); IVIA (Spain); University
Göttingen (Germany).
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5. Outcome of the research
• Mechanism of resistance
described
• Genetic variation for
resistance discovered
• Cypress clones resistant
against the disease were
produced and tested
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6. Resistant clones in Greece
• 1981: Seeds harvested from 6 base
populations (15 trees in each stand)
• 1982: Nursery; 15 families per stand,
40 trees per family
• 1984: Inoculation
• 1987: Trees were evaluated and
classified as resistant (5%) and
susceptible (95%)
• 1988: Material from resistant trees was
propagated in several clone plantations
in Greece (grafting).
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7. Questions
• What is the genetic diversity of the new populations
that will be founded with the resistant material?
• What are the changes in genetic variation due to
artificial selection?
• Are there any correlations between genetic markers
and resistance?
• What is the best way to treat the resistant material,
in order to maintain high levels of adaptability?
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8. Base populations
• Samos, Rodos, Symi and
Kos – Aegean islands
• Alepohori, Mystras –
Peloponnese
• Aegean populations are
considered natural, while
those from the Pelopon-
nese are domesticated
• Genetic studies revealed a
founder effect in the
Peloponnese populations
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9. Material used for genetic analysis
Base Number of trees in Number of
Populations base population clones
AL 28 12
MY 29 17
SA 21 20
KO 21 16
RO 21 21
SY 20 23
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10. Material & Methods
• Megagametophytes (2n)
and needles from clone
ramets analyzed
• Clones were genotyped
• Plantations: Tatoi,
• Horizontal starch gel Megalopolis, Lappa
electrophoresis
• Six polymorphic enzyme
loci
• PGI-B, PGM-A, PGM-B,
NDH-A, GDH-A, LAP-A
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11. Allele frequency change: PGI-B
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12. Allele frequency change: NDH-A
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13. Allele frequency change: PGM-B
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14. Allele frequency change: LAP-A
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15. Allelic structures
• Clone collections showed – in general - no
major differences in comparison with the
base populations
• Most “typical” structures of populations
remained unchanged in the clones
• A few changes were observed, without any
specific trend in favor of a specific variant
• Exceptional changes in PGM-B and LAP-A
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16. Expected heterozygosity
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17. Observed heterozygosity
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18. Changes in allelic multiplicity
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19. Which alleles are lost?
• AL: PGM-B3 (0,220), GDH-A3 (0,018),
LAP-A3 (0,036)
• MY: GDH-A1 (0,017), LAP-A2 (0,069)
• SA: PGM-B1 (0,025), GDH-A3 (0,071),
LAP-A2 (0,024)
• KO: GDH-A3 (0,111), LAP-A2 (0,075)
• RO: GDH-A3 (0,095)
• SY: PGM-B1 (0,026)
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20. Diversity and heterozygosity
• Loss of alleles was observed in 3
loci and 5 clone collections; The
alleles lost have low frequencies in
the base populations
• Diversity was less in almost all
clone collections; differences were
not significant
• Observed heterozygosity remained
more or less unchanged
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21. “Man made” bottleneck
• Alleles are lost during the
breeding procedure
• Despite the large breeding
population (15 families, 40
trees / family) in each
population, strong selection
intensity after the first
inoculation caused a
bottleneck
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22. Gene pool of populations vs. clones
Number of Observed Expected
alleles heterozygosity Heterozygosity
Populations 19 0,36 0,43
Clones 18 0,36 0,40
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23. Genetic distances between the total clone set
and the base populations (d0 )
AL MY SA KO RO SY
PGI-B
0.135 0.080 0.093 0.044 0.121 0.068
PGM-A
0.242 0.332 0.049 0.076 0.056 0.060
PGM-B
0.219 0.215 0.186 0.136 0.323 0.219
NDH-A
0.281 0.247 0.172 0.219 0.169 0.065
GDH-A
0.108 0.080 0.234 0.155 0.163 0.069
LAP-A
0.343 0.298 0.130 0.152 0.130 0.114
Mean
0.221 0.208 0.144 0.131 0.160 0.099
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24. Total clone collection
• When all resistant clones are considered as a set,
only one allele is lost (PGM-B1), which is very rare
in two base populations (SA and SY)
• Large differentiation among populations and clone
arrays; a rare allele in a given set may be frequent in
another
• Similar structures of pooled data for populations
and clones
• The totality of the clones is not representative any
base population
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25. Literature review
• Almost all comparisons consider pooled data
• No differences in genetic structures between pooled data
from populations and clones – variation “captured”
• Private and locally rare alleles are lost in the population
level: Picea sitchensis (Chaisurisri & El Kassaby 1994), Pseudotsuga
menziesii (El Kassaby & Ritland 1996)
• No alleles are lost – even increased – in pooled data: Thuja
plicata (El Kassaby et al. 1993), Picea sitchensis (Chaisurisri & El
Kassaby 1994), Pseudotsuga menziesii (El Kassaby & Ritland 1996)
• Alleles are lost in pooled data: Picea glauca (Cheliak et al. 1988),
Picea abies (Bergmann & Ruetz 1991), Picea glauca X engelmanni
(Stoehr & El Kassaby 1997) – low differentiation (?)
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26. Breeding strategies
• To avoid allele loss, selection
pressure should be lower
• The mixture of clones from
different origins can reduce allele
loss and increase adaptability
• However, adaptedness is probably
reduced if a mixture of clones is planted
• In species with little differentiation, mixed
sets of clones may still have less alleles
• More research is needed for the detection
of possible associations between markers
and resistance
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27. Acknowledgements
• Eric Teissier du Cros for his advice, support and
coordination of the cypress projects
• C. Andreoli, F.A. Aravanopoulos, G. Brofas,
A. Doulis, B. Fady, E. Gillet, L. Leinemann,
G. Lyrintzis, G. Mantakas, K.P. Panetsos,
C. Pichot, P. Raddi, S. Raddi, P. Ramos,
A. Santini, P. Tsopelas, M. Ziehe (and many
more) for their assistance and advice
• A. Drouzas and L. Iliadis for useful comments
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