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Use of molecular markers to identify cryptic species or complexes of sibling species in invasive or potentially invasive conifer pest insects

Géraldine Roux-Morabito, Alain Roques, Marie-Anne Auger-Rozenberg

To cite this version:

Géraldine Roux-Morabito, Alain Roques, Marie-Anne Auger-Rozenberg. Use of molecular markers to identify cryptic species or complexes of sibling species in invasive or potentially invasive conifer pest insects. 23. International Congress of Entomology, Jul 2008, Durban, South Africa. 1 p. hal-

Use of molecular markers to identify cryptic species or complexes of sibling species in invasive or potentially invasive conifer pest insects

Géraldine Roux-Morabito, Alain Roques, Marie-Anne Auger-Rozenberg

INRA, Orleans, France

Introduction: Molecular genetic approaches are powerful tools for studying evolutionary aspects of biological invasions, especially for the identification of cryptic species or divergent lineages which can hinder a clear biological characterization of invasive species. Phytophagous insects show several examples of complexes of sibling species as well as successful invasion histories. We will present two case studies of phytophagous invaders.

The first case will consider species which have been extensively transported globally for decades along with the seed trade (Megastigmus spp. seed chalcids), and the second pine pitch weevil of unknown origin, Hylobitelus xiaoi, which is presently in outbreak and is expanding in southern China. We will present data showing tests of the efficiency of molecular techniques for assessing whether the insect species correspond to cryptic native species or invasive species.

Material and methods:

We use a phylogeographic approach by sequencing mitochondrial DNA genes (COI and/or COII). Individuals from different localities and from different host species were sampled in both the invaded and the presumed native range of the species.

Results and discussion:

The study revealed misidentifications in Megastigmus species which have been previously recorded either as invasive or native species. It validated the use of molecular tools to detect cryptic species in this genus. These results also highlighted the importance of studying genetics in native populations to confirm or invalidate species identification only based on morphological traits. It should allow both a clear identification of native lineages and the tracking of the source population for expansion in Hylobitelus.


S Minoia

J Vigouroux

G Mosca

G Sozio

A Petrozza

M F Devaux

F Piron

F Cellini

A Bendahmane

F Carriero

M Lahaye

Tomato TILLING mutants in Expansin1 gene Tomato TILLING mutants in Expansin1 gene show alteration in softening and cell wall show alteration in softening and cell wall metabolism during ripening metabolism during ripening

Plant material: Tomato M3 plants, including wild types (M82 and Red Setter) and genotypes homozygous for both the presence (+/+) and the absence (-/-) of the discovered mutations, were grown in greenhouse conditions according to standard tomato agronomic practice. TILLING molecular screening: PCR amplification was carried out using labelled primers (IRDye 700 and IRDye 800 dye). Mutation detection was performed with the mismatch specific endonuclease ENDO1 (8) and the LiCor 4300 DNA analyzer. After discovery, mutations were validated by sequence analysis. Fruit firmness: Penetrometric firmness was measured on whole tomato without peel by using a pressure tester (Forge Gauge, Lutron FG-5000-A) fitted with a cylindrical plunger of 6 mm in diameter. Tomato samples were collected at the breaker stage and analyzed in post-harvest conditions at fixed ripening stages. Pericarp tissue histology: Cell size distribution in pericarp sections of the different genotypes was established according to (9). Compression analysis: Compression was measured on pericarp tissue cylinders of 0.96 cm diameter taken at the equator. Compression test was realized at 20 mm/min using an Adamel Lhomargi DY34B (MTS) mechanical testing machine equipped with a 1 kN sensor. Cell wall polysaccharides chemical composition: Cell walls from pericarp tissue of 5 to 6 fruits per genotype were prepared as alcohol insoluble material. Neutral sugars were quantified by gas-liquid chromatography and uronic acid by colorimetry after sulfuric acid degradation (10). Neutral sugar composition was corrected for residual starch determined by amylolysis and HPLC. The degree of pectin-esterification by methanol (DM) and weight % of acetic acid ester (AA) was measured by HPLC after alkaline hydrolysis (11). Glucanase degradation of cell wall preparations was done as described (10). Oligosaccharides were identified by MALDI-TOF MS on M@ldi LR (Waters) spectrometer using DHB/6ATT matrix. Data treatment and statistic (ANOVA, pairwise t-test) were performed using R software or MATLAB (histology).

).

RESULTS and DISCUSSION

RESULTS and DISCUSSION

Tomato fruit ripening involves the expression of several types of enzymes and proteins targeted at the cell wall disassembly. Expansin1 gene (SlExp1) codes for the major expansin protein involved in fruit softening (1). This protein controls noncovalent interactions at the hemicellulosecellulose interface and regulates cell wall polysaccharides metabolism (2). To date, no non-GMO tomato genotypes modulated on expansin are available to realize controlled texture hybrids.

INTRODUCTION INTRODUCTION Figure 2. Figure 2. Firmness analysis on two TILLING

Firmness analysis on two TILLING mutants. mutants. Firmness was calculated from homozygous +/+ (striped bars), homozygous -/-(white bars) and from respective wild type (M82 or Red Setter) (black bars). Tomato samples were collected at the breaker (B0) stage and analyzed in post-harvest conditions at fixed ripening stages: breaker and zero (B0), five (B+5), ten (B+10), fifteen (B+15) and twenty-five (B+25) days after breaker. Bars represents standard errors, and asterisks indicate that homozygous +/+ are significantly different (P<0.05) from homozygous and wild type.

Table 2. Table 2. Cell wall polysaccharide composition.

Cell wall polysaccharide composition. Uronic acids (UA), total neutral sugars (NS) and total sugars (Total), acetic acid content (AA) (± standard deviation) on the weight basis of dry alcohol insoluble material and degree of methyl esterification of pectin (DM) in cell walls recovered from pericarp tissue of M82 (M) and Red Setter (R) wild type (W), control (0, -/-) and mutant (1, +/+) at the green (G), breaker (B), pink (P), red (R) and overripe (S) stages. P. value: results from a pairwise-t-test between data from sild type (M) or control (0) and mutant (1); in bold significantly different results (p<0.05); n=numbers of fruits. 3). Compression analysis on M82 and Q213* mutant. Modulus of deformation (E) at 20% strain ± standard deviation, analysis of variance between wild type and mutant (p value) and number of measures (n). In bold, statistically significant differences.

Genotype Ripening stage E (kPa) p value n

Table 3. Table 3. MALDI-TOF analysis.

MALDI-TOF analysis. Analysis of variance (p.values) and interaction (SxM) between ripening stages (S) and genotype (wild-type and mutants for M; 0 and mutant for Red Setter) for M82 and Red Setter genetic background. Oligosaccharide nomenclature: Hex: hexose based oligosaccharides (glucomannan) of degree of polymerization 4 (Hex4) to 9 (Hex9) bearing 1 to 3 acetic acid ester group (A1-A3); the nomenclature of xyloglucan type oligomers follows that of (7) and was extended by A1 or A2 following the number of acetyl esterification. Data in bold emphasize significant effects.

Figure 1 .Figure 1 .

 11 Figure 2. Figure 2. Firmness analysis on two TILLING Firmness analysis on two TILLING mutants. mutants. Firmness was calculated from homozygous +/+ (striped bars), homozygous -/-(white bars) and from respective wild type (M82 or Red Setter) (black bars). Tomato samples were collected at the breaker (B0) stage and analyzed in post-harvest conditions at fixed ripening stages: breaker and zero (B0), five (B+5), ten (B+10), fifteen (B+15) and twenty-five (B+25) days after breaker. Bars represents standard errors, and asterisks indicate that homozygous +/+ are significantly different (P<0.05) from homozygous and wild type. (A) Q213*; (B) W211S.

  composition on the dry weight of cell wall preparations is given in Table 2. M82 and Red Setter mutants at the green stage are poorer in uronic acids than the wild type content. On M82 mutant at the breaker stage neutral sugars and total sugar content of cell wall preparations are affected. Expansin being targeted to hemicellulose/cellulose interactions, the fine structure of hemicelluloses was followed during fruit ripening by β-glucanase degradation and MALDI-TOF MS analysis of the oligosaccharides produced. Two series of oligosaccharides ion were observed and corresponded to the two major xyloglucan and glucomannan hemicelluloses in tomato. The relative intensity of oligosaccharides attributed to glucomannan oligosaccharides (Hex) markedly decreased with the ripening stage. The fine structure of xyloglucan is also affected during ripening (6). Down-regulation of expansin affects significantly the proportion of xyloglucan fine structures particularly at the breaker stage (Figure 3, Table

  Figure 3. Figure 3. Evolution in the proportion of representative Evolution in the proportion of representative glucomannan glucomannan (Hex) (Hex) and xyloglucan fine structure (XXGA1, XXGGA1) in M82 and mutant and xyloglucan fine structure (XXGA1, XXGGA1) in M82 and mutant (Q213*) during ripening. (Q213*) during ripening. See legend ofTable 2 for sample nomenclature.

Table 1 . Table 1. Compression analysis on M82 and Q213* mutant.

 1

	Mutant green	1.9 ±0.6 0.6794 2 4
	Wild type green	2.0 ±0.8	3 6
	Mutant breaker	1.8 ±0.5 0.0048 2 3
	Wild type breaker	2.5 ±0.9	3 6
	Mutant pin k	0.6 ±0.5 0.0348 3 6
	Wild type pin k	0.8 ±0.3	3 4
	Mutant r e d	0.3 ±0.3 0.0005 2 6
	Wild type r e d	0.5 ±0.2	2 6
	Mutant overrip e	0.3 ±0.2 0.0002 2 8
	Wild type overrip e	0.5 ±0.2	3 1

Acknowledgements Acknowledgements


Julien Parelle
email: julien.parelle@univ-fcomte.fr

Erwin Dreyer
email: dreyer@nancy.inra.fr

Oliver Brendel
email: brendel@nancy.inra.fr

Genetic Variability and Determinism of Adaptation of Plants to Soil Waterlogging

Flooding or waterlogging, and associated soil hypoxia, affect severely the growth and fitness of plant species, from crops to forest ecosystems. An improved understanding of the intra-species genetic diversity of traits involved in hypoxia tolerance is a prerequisite for crop breeding programmes aimed at increasing the tolerance to waterlogging, as well as for assessing the adaptability of natural populations to waterlogging. Some genotypes within the species have developed adaptations to hypoxia, as shown by differences among populations in growth and fitness, and in traits conferring some degree of tolerance such as sequence, expression and activity of alcohol dehydrogenase, or the ability to develop adventitious roots, increased tissue porosity and hypertrophied lenticels. Genetic control has been estimated for a number of such traits. Overall, under waterlogging, specific tolerance traits show higher heritabilities compared to traits quantifying productivity, damage or overall performance. Genomic regions involved in the control of these traits (i.e., Quantitative Trait Loci QTL) have been detected for tolerance traits in a few species, and allow gaining some insight into the genetic basis of the observed natural diversity or may be a starting point for breeding purposes.

However, only for submergence tolerance in rice (sub-1) has a successful gene candidate approach resulted in the detection of alleles that are directly involved in the tolerance process.

Introduction

Excess soil water due to flooding or temporary waterlogging can be a major constraint on growth and yield of crops [START_REF] Tuberosa | Cereal genomics[END_REF] and forest stands [START_REF] Kozlowski | Responses of woody plants to flooding and salinity[END_REF]. It affects severely growth and probably also fitness and distribution of plant species in natural environments. Some species or genotypes within the plant species have developed adaptive responses to flooding and waterlogging. In the case of crops, the occurrence of some AU1 genetic diversity in tolerance traits is a prerequisite for breeding programmes. In natural ecosystems, due to the local occurrence of temporarily waterlogged soils (often called hydromorphic soils, Le ´vy et al. 1999), the frequency and severity of episodes of waterlogging or flooding act as a selective pressure and differences in tolerance can develop among species, or populations within species. To gain insight into the degree of inter-specific variability, we need a careful quantification of the tolerance to waterlogging in individuals and methods to assess it as objectively as possible.

Two major situations of excess water can be identified [START_REF] Colmer | Flooding tolerance: suites of plant traits in variable environments[END_REF]. Flooding, the partial, or in some cases the complete submergence of the shoot, can be permanent, such as in mangrove ecosystems, or temporary, such as in floodplains or in rice paddies. Waterlogging, due to excess water in the soil, usually occurs temporarily with a water level below or not much above the soil surface that affects primarily the root system and can occur in natural as well as in cultivated ecosystems, depending on soil type and water table dynamics.

In both cases, a temporal sequence of chemical changes occurs in the soil following the onset of waterlogging or flooding [START_REF] Setter | Review of prospects for germplasm improvement for waterlogging tolerance in wheat, barley and oats[END_REF]. Due to a reduced gas exchange between soil and atmosphere, changes in soil bacteria populations occur, oxygen concentration decreases rapidly (hypoxia), carbon dioxide and ethylene concentrations increase, reduced and toxic cations such as manganese (Mn ++ ) and iron (Fe ++ ) accumulate, and an intense de-nitrification occurs. In case of prolonged waterlogging, soils may be completely depleted of oxygen (anoxia) and hydrogen sulphide and methane are produced and diffuse into the U n c o r r e c t e d P r o o f atmosphere. Except the last, all of these steps occur usually within the first 20 days of waterlogging. In some soils, this sequence may even occur faster. In this review, we will concentrate on the hypoxia induced by waterlogging or total submergence with all the consequences it might have on respiration, metabolism and growth of affected plants.

To date, the processes conferring some degree of tolerance to waterlogging and hypoxia are still not fully understood despite accumulating information (e.g., [START_REF] Vartapetian | Plant anaerobic stress as a novel trend in ecological physiology, biochemistry, and molecular biology. 2. Further development of the problem[END_REF][START_REF] Voesenek | How plants cope with complete submergence[END_REF][START_REF] Colmer | Flooding tolerance: suites of plant traits in variable environments[END_REF][START_REF] Kawano | Morphological and physiological responses of rice seedlings to complete submergence (flash flooding)[END_REF][START_REF] Jackson | Evolution and mechanisms of plant tolerance to flooding stress[END_REF][START_REF] Parolin | Submerged in darkness: adaptations to prolonged submergence by woody species of the Amazonian floodplains[END_REF]. The degree of tolerance to a given level of waterlogging may be assessed: (1) indirectly through damage indices or the observed, usually negative, impact on growth, productivity and survival, or (2) directly by evaluating the occurrence of traits contributing to the acclimation to hypoxia (adaptive traits). These traits can be constitutive (i.e., they occur already in individuals growing under optimal conditions and provide some advantage during waterlogging) or induced (i.e., they appear only during episodes of waterlogging in response to a signalling cascade). Induced traits can be roughly grouped into short-term responses (e.g., metabolic adjustments) and long-term acclimations (e.g., development of aerenchyma). A typical short-term response of roots is a decrease of respiration and an increase of glycolytic flux and alcoholic fermentation [START_REF] Drew | Oxygen deficiency and root metabolism: injury and acclimation under hypoxia and anoxia[END_REF]. Some key enzymes in this process are alcohol dehydrogenase [START_REF] Mcmanmon | A metabolic theory of flooding tolerance: the significance of enzyme distribution and behaviour[END_REF][START_REF] Chan | Variation in alcohol-dehydrogenase activity and flood tolerance in white clover, Trifolium repens[END_REF][START_REF] Bailey-Serres | Flooding stress: acclimations and genetic diversity[END_REF], sucrose synthase or hexokinases [START_REF] Germain | The role of sugars, hexokinase, and sucrose synthase in the determination of hypoxically induced tolerance to anoxia in tomato roots[END_REF][START_REF] Ricard | Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant[END_REF]). Long-term responses are mainly related to growth, either of existing or of newly formed structures. In rice, where total submergence clearly poses a major problem for productivity [START_REF] Tuberosa | Cereal genomics[END_REF], the elongation of internodes is an important adaptive trait, resulting either in quiescence or an escape strategy [START_REF] Bailey-Serres | Flooding stress: acclimations and genetic diversity[END_REF]. The quiescence strategy consists in a lack of elongation [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF], whereas the escape strategy consists in an enhanced growth rate that maintains the top of the shoot above the water level [START_REF] Fukao | A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice[END_REF]. Adaptive morphological traits are slower to develop compared to purely physiological or metabolic adjustments. Assessment of such traits requires long-term experiments with the risk of an interaction between ontogenic development and stress response. A few anatomical traits, thought to allow transport of oxygen to roots and enable a partial maintenance of respiration, survival or even root growth, have commonly been measured in experiments on genetic variability.

They include the development of hypertrophied lenticels [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF]), of adventitious roots (Mano et al. 2005a, b) and of aerenchyma (porosity) in root or stem tissues [START_REF] Zaidi | Importance of secondary traits in improvement of maize (Zea mays L) for enhancing tolerance to excessive soil moisture stress[END_REF][START_REF] Mollard | Flooding tolerance of Paspalum dilatatum (poaceae: paniceae) from upland and lowland positions in a natural grassland[END_REF][START_REF] Mano | Qtl mapping of root aerenchyma formation in seedlings of a maize x rare teosinte Zea nicaraguensis cross[END_REF]Mano et al. , 2008;;Mano and Omori 2008;and see also Chap. 6 in this volume).

From an agronomic point of view, the maintenance of productivity, particularly yield, is of major importance. This can be evaluated by quantifying growth or biomass and also more indirectly by assessing, among others, leaf level gas exchange or photosynthetic capacity. Leaf gas exchange, for instance, has been used [START_REF] Dreyer | Compared sensitivity of seedlings from 3 woody species (Quercus robur L, Quercus rubra L and Fagus silvatica L) to waterlogging and associated root hypoxia: effects on water relations and photosynthesis[END_REF][START_REF] Wagner | Interactive effects of waterlogging and irradiance on the photosynthetic performance of seedlings from three oak species displaying different sensitivities (Quercus robur, Q petraea and Q rubra)[END_REF] to characterise the overall performance under waterlogging. Such traits bring no information about the U n c o r r e c t e d P r o o f 107 morphological and physiological mechanisms of tolerance; nevertheless, mainte-108 nance of productivity or photosynthesis contributes to fitness and survival of 109 individuals. Survival rate under hypoxia is, together with shoot dieback and other 110 fitness related traits (number of seeds produced, etc), an important means to assess 111 the degree of tolerance of populations. Leaf epinasty, the downward growth of leaf 112 petioles, is a specific response to root hypoxia in some species (Jackson and 113 Campbell 1976) and a direct indicator of the level of hypoxia stress perceived by 114 the individuals [START_REF] Vartapetian | Plant adaptations to anaerobic stress[END_REF]. All these traits may respond to 115 waterlogging with quite different intensities. On the other hand, some traits obvi-116 ously play a direct adaptive role, or at least are thought to do so. Such traits include 117 the development of hypertrophied lenticels, of adventitious roots, of aerenchyma or 118 the occurrence of physiological changes (switch from a respiratory to a fermenta-119 tive metabolism). All these traits contribute to mitigate the impact of hypoxia in the 120 soil, by maintaining a minimal supply of oxygen to roots.

121

The quantification of growth decline under stress provides a first indication 122 about the level of tolerance of a genotype. Traits that have been measured include 123 plant height, growth increment and shoot or root dry weight (see Table 12.1 for 124 examples from quantitative genetic studies). Yield, or the reduction thereof, 125 has also been quantified for crops under waterlogging [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF]Githiri t1:1 et al. 2006). However, the use of these traits to detect genetic differences in waterlogging tolerance requires a careful interpretation to identify adaptive traits involved in the tolerance to hypoxia, in contrast to nonadaptive traits indicating merely a genetic difference, for instance in growth potential.

The quantification of damage induced by waterlogging may also provide an estimation of tolerance. Some authors use visual ordinal scales of damage [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF][START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF][START_REF] Cornelious | Identification of QTLs underlying waterlogging tolerance in soybean[END_REF], others quantify leaf senescence [START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF], fraction of yellow leaves (Zhou et al. 2007a) or decline in leaf chlorophyll content [START_REF] Guangheng | QTL analysis of traits concerned submergence tolerance at seedling stage in rice (Oryza sativa L)[END_REF]). Damage indices have been successfully used to study the genetic determinism of tolerance in crops.

For example, the so-called sub-1 locus [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF] was identified in rice 10 years before the actual process controlled by the locus was understood (Xu et al. 2006). A large amount of damage eventually leads to mortality, which is a very simple approach to characterising tolerance [START_REF] Nandi | Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping[END_REF][START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF][START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF][START_REF] Martin | Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions[END_REF]. Despite the fact that survival is an ordinal trait, the approximately normal distribution within experiments allows using it for a genetic trait dissection in rice [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF] and soybean [START_REF] Cornelious | Identification of QTLs underlying waterlogging tolerance in soybean[END_REF]. The advantage of survival and damage traits is that the variability tested is directly related to the stress tolerance, and can therefore be fully attributed to genetic diversity.

To assess the genetic variability of tolerance to waterlogging or flooding, the most obvious procedure is the quantitative analysis of traits conferring directly or indirectly some level of tolerance to waterlogging. Prerequisites for suitable traits include: (1) the relevance and specificity of the trait as an indicator of adaptation;

(2) the repeatability of the measurement procedure and (3) the possibility of assessing a large number of individuals.

Genetic variability of tolerance traits can be studied in situ in natural populations only when detailed information on the environmental conditions and their spatial and temporal variability is available. Further, an already advanced knowledge of the genetic determinism of the trait studied is necessary, with a remaining risk of confusion between purely genetic differences and genotype and environment interactions. Therefore, all of the studies reviewed here were done using common conditions for all genotypes (vegetative copies or half-sib families), as provided by common-garden plantations (comparative plantations with a homogenized environment) or greenhouse experiments. However, even under such controlled conditions, statistical methods (such as complete or random blocks) should be used to minimise residual variations of environment or stress conditions. It is very difficult to control with large precision the level of soil hypoxia imposed to the different individuals, as the oxygen concentration in the soil depends also on rooting density, soil heterogeneity and the presence of soil microorganisms. One possibility to control more directly the amount of oxygen available to the root system is the use of hydroponic systems that are bubbled with a specific nitrogen/oxygen mixture (e.g. [START_REF] Ricard | Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant[END_REF]). However root growth; root anatomy and root system architecture differ widely between hydroponics and soils.

U n c o r r e c t e d P r o o f

Some degree of intra-specific genetic diversity of traits induced by root hypoxia has been shown for a number of species and traits. In the first section of this chapter, we review common garden comparisons of natural populations which are exposed to different levels of waterlogging at their sites of origin. The second section concentrates on offspring from controlled crosses of specific genotypes, often preclassified as tolerant or sensitive to waterlogging, used for quantitative analyses of the genetic determinism, and ultimately for the detection of Quantitative Trait Loci (QTL).

12.2 Diversity Among Populations: Adaptation to Water-Logged Soils?

Common garden comparisons of individuals grown from seeds collected in diverse populations were mainly published for noncrop species. The detected diversity was interpreted, with some caution, as revealing differences in adaptation due to natural selection.

Interest in intra-species variation of tolerance to waterlogging or to flooding emerged in the 1970s. Some examples include among-family variation in Veronica peregrina, an annual dicotyledon found on moist sites [START_REF] Linhart | Intra-population differentiation of physiological response to flooding in a population of Veronica peregrina L[END_REF]; population differences in Eucalyptus viminalis, from dry or wet forests [START_REF] Ladiges | Comparative effects of waterlogging on 2 populations of Eucalyptus viminalis Labill and one population of Eucalyptus ovata Labill[END_REF] or in E. ovata, Australian swamp gum [START_REF] Clucas | Variations in populations of Eucalyptus ovata Labill and the effects of waterlogging on seedling growth[END_REF]). These studies showed already that phenoypic differences could be detected among populations from sites differing in susceptibility to waterlogging and hypoxia.

Growth is strongly affected by waterlogging, and is usually significantly depressed in plants from both well-drained and hydromorphic soils. This was the case for Eucalyptus globulus and E. grandis families [START_REF] Marcar | Genetic variation among and within provenances and families of Eucalyptus grandis W Hill and E. globulus Labill. subsp globulus seedlings in response to salinity and waterlogging[END_REF], where at least E. globulus is know to be sensitive to waterlogging. Similarly, during a hypoxia experiment on Geum rivale (wetland species), Geum urbanum (dry habitat species) and hybrid populations, root dry weight was reduced in all families [START_REF] Waldren | Comparative studies of plant-growth and distribution in relation to waterlogging. 15. The effect of waterlogging on growth of various populations of and hybrids between geum-rivale l and geum-urbanum l[END_REF]. However, as examples span a large range of plant species, from monocotyledon grasses to forest trees, responses are very diverse. In some species, growth increased during waterlogging for populations from wet environments, such as in Panicum antidotale [START_REF] Ashraf | Relationships between leaf gas exchange characteristics and growth of differently adapted populations of blue panicgrass (Panicum antidotale retz.) under salinity or waterlogging[END_REF] or Paspalum dilatatum [START_REF] Loreti | Intraspecific variation in the resistance to flooding and drought in populations of Paspalum dilatatum from different topographic positions[END_REF] populations, whereas it decreased in other species. This differential growth response to waterlogging is an extreme case of environment and genotype interaction and can also be found to a lesser degree within species level. [START_REF] Marcar | Genetic variation among and within provenances and families of Eucalyptus grandis W Hill and E. globulus Labill. subsp globulus seedlings in response to salinity and waterlogging[END_REF] studied growth under waterlogging in different populations from two Eucalyptus species. A significant treatment and provenance effect was detected for shoot dry weight in E. globulus, a rather hypoxia-sensitive species, but none for E. grandis, a species growing on hydromorphic soils. Similarly, [START_REF] Waldren | Comparative studies of plant-growth and distribution in relation to waterlogging. 15. The effect of waterlogging on growth of various populations of and hybrids between geum-rivale l and geum-urbanum l[END_REF] found in G. rivale no population differentiation for growth during

U n c o r r e c t e d P r o o f

waterlogging, whereas G. urbanum and hybrid populations showed significant population and waterlogging interactions. Overall, these examples show that a diversity of adaptations to waterlogging can evolve in closely related species resulting in growth differences. This suggests a genetic differentiation among populations, and thus a genetic determinism of hypoxia tolerance. However, there are also examples of species where no specific adaptation has been detected for hypoxia-exposed populations that would result in growth differences. In Acer rubrum seedlings, no relationship was detected between population differences in response to controlled waterlogging and the maternal hydrologic conditions [START_REF] Will | Effects of rhizosphere inundation on the growth and physiology of wet and dry-site Acer rubrum (red maple) populations[END_REF].

Adaptive differences among populations have also been detected using net CO 2 assimilation rate and stomatal conductance as indicators of fitness on lowland (wet) and upland (dry) populations of P. dilatatum [START_REF] Mollard | Flooding tolerance of Paspalum dilatatum (poaceae: paniceae) from upland and lowland positions in a natural grassland[END_REF].

Flooded plants displayed higher net CO 2 assimilation and stomatal conductance compared to controls in lowland populations, and stomatal closure and reduced net CO 2 assimilation in upland populations: under similar hypoxia, lowland populations were able to maintain water absorption by roots, while upland populations were not.

Variability in growth and leaf gas exchange among genotypes during waterlogging is the result of anatomical or physiological adaptations, such as the ability to develop hypertrophied lenticels, adventitious roots and aerenchyma in root or stem tissue. Under waterlogging, a significant increase in the number and height of hypertrophied lenticels was found in populations of Luehea divaricata from temporarily waterlogged soils versus those from well drained soils (De Carvalho et al. 2008, C.F. Ruas, pers. comm.). The development of hypertrophied lenticels or adventitious roots is typically an induced adaptive response to hypoxia with quite a large genetic diversity. A genetic basis for diversity in adventitious root growth was detected among Carex flacca populations [START_REF] Heathcote | Phenotypic flexibility of Carex-flacca Schrebtolerance of soil flooding by populations from contrasting habitats[END_REF].

Continuous flooding increased adventitious root biomass in all populations to the same extent. Significant population differences and population and treatment interactions were detected during repeated transient episodes of flooding. This underlines that the modality of stress application may impact the degree of genetic diversity detected. Stress responses also often differ between organs, as in P. dilatatum, where porosity did not increase in roots during flooding, while it did in the leaf sheath [START_REF] Mollard | Flooding tolerance of Paspalum dilatatum (poaceae: paniceae) from upland and lowland positions in a natural grassland[END_REF]). However, there were strong treatments and population interactions: root porosity was different between lowland (wet) and upland (dry) populations under control conditions but not under flooding, whereas leaf sheath porosity was different under flooding but not under control conditions. The hypoxia-adapted lowland populations had constitutively higher root porosity, with little increase during flooding, whereas upland populations showed a larger response to flooding for leaf sheath porosity. Overall, these examples, covering a range of different plant types, suggest that genetic differences seem to have evolved for morphological adaptations to root hypoxia among natural populations exposed to different levels of soil hypoxia and that an adaptation to different environments has taken place.

U n c o r r e c t e d P r o o f

As described above, hypoxia induces changes in root metabolism. Genetic differences in the expression of alcohol dehydrogenase (ADH) have been studied intensively in a number of species. As early as the 1970s, ADH polymorphism has been shown to affect growth rate under waterlogging [START_REF] Marshall | Adaptive significance of alcohol dehydrogenase isozymes in maize[END_REF][START_REF] Brown | Adaptedness of variants at an alcohol dehydrogenase locus in Bromus mollis[END_REF]) and population differences were detected [START_REF] Torres | The early allele of alcohol dehydrogenase in sunflower populations[END_REF][START_REF] Brown | Isozymes, plant population genetic structure and genetic conservation[END_REF]. A genetic variability was also detected for the gene coding of ADH (locus ADH-B) among five European populations of Fraxinus excelsior, but was not related to flooding frequency at the sites of origin [START_REF] Ruedinger | Genetic structures of common ash (Fraxinus excelsior) populations in Germany at sites differing in water regimes[END_REF]. [START_REF] Herzog | Genetic structures of a flooded and a non-flooded oak (Quercus robur) population from the floodplains of the Rhein river[END_REF] studied 17 isoenzyme loci, of which some are thought to be involved in waterlogging or hypoxia tolerance. They found no evidence for a selection on these loci when comparing a frequently flooded and a dry-land population of Quercus robur. [START_REF] Chan | Variation in alcohol-dehydrogenase activity and flood tolerance in white clover, Trifolium repens[END_REF] found for Trifolium repens a strong population and treatment interaction for ADH activity in roots, with higher activities in populations from frequently flooded sites. ADH activity under waterlogging was positively correlated with relative growth rate, suggesting that a higher ADH activity contributes to a higher tolerance to hypoxia. This contradicts inter-specific comparisons, where more tolerant species displayed lower ADH activity [START_REF] Mcmanmon | A metabolic theory of flooding tolerance: the significance of enzyme distribution and behaviour[END_REF]. However, ADH activity varies with time during stress application: sensitive Brassica rapa L. plants displayed a higher ADH activity after 18 h of stress but not earlier or later [START_REF] Daugherty | Characterization of populations of rapid-cycling Brassica rapa L selected for differential waterlogging tolerance[END_REF]. Enzymes potentially involved in hypoxia tolerance have been studied in detail in diverse crop species, where genetically well-defined varieties or clones are available. Increased ADH activity was found for waterlogging tolerant compared to susceptible Zea mays genotypes [START_REF] Zaidi | Response of maize (Zea mays L) genotypes to excess soil moisture stress: morpho-physiological effects and basis of tolerance[END_REF]. Similarly, more tolerant Oryza sativa cultivars with a higher internode elongation rate under hypoxic conditions (escape strategy) showed also higher ADH and pyruvate decarboxylase (PDC) activities and ATP concentration (Kato-Noguchi and Morokuma 2007). This difference in ADH activity seems specific for roots [START_REF] Kato-Noguchi | Metabolic adaptation to flooding stress in upland and lowland rice seedlings[END_REF]. [START_REF] Fukao | Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia[END_REF] found with seeds of the weed Echinochloa crus-galli germinating under anoxic conditions, that aldolase, aldehyde dehydrogenase and PDC were more strongly induced in a tolerant compared to an intolerant variety, whereas sucrose synthase, enolase and ADH showed similar induction patterns for both. The occurrence of some genetic variation has been detected in the sequence, expression and activity of ADH, whereas less information is available for other enzymes involved in hypoxia responses. However, even for ADH, we still lack experimental support demonstrating that the genetic diversity that is observed results in variation in adaptation to waterlogging by natural populations. Such a demonstration could be provided, for example, by population genetic studies linking single nucleotide polymorphisms (SNP) within the ADH-gene or its promoter to survival and fitness in stressed environments. Further, theoretical population genetic models could then be applied (e.g., [START_REF] Beaumont | Evaluating loci for use in the genetic analysis of population structure[END_REF], testing whether nucleotide differentiation patterns of SNP within the ADH-gene could depart from neutral patterns and result from natural selection. This has been done, for example, with candidate genes for drought tolerance in Pinus pinaster populations [START_REF] Eveno | Contrasting patterns of selection at Pinus pinaster Ait drought stress candidate genes as revealed by genetic differentiation analyses[END_REF]).

U n c o r r e c t e d P r o o f 12.3 Genetic Control of Traits Related to Hypoxia Tolerance

A more direct approach to estimate genetic control of traits related to hypoxia tolerance is the estimation of heritability, which is, in the simplest case, the ratio between genetic and total variance within a given experimental set up [START_REF] Lynch | Identification of QTL controlling adventitious root formation during flooding conditions in teosinte (Zea mays ssp. huehuetenangensis) seedlings[END_REF]. The calculation of the genetic variance requires not only controlled conditions for trait estimations, but also an assessment of the relatedness of individuals within the experimental set up, such as multi-parental crossings (diallels, half-dialleles, clonal repetitions, etc). This approach is rarely possible with wild populations, however, it has been frequently used for crops. Heritability is difficult to compare among experiments, as it depends on environmental variance induced by the specific experimental set up. However, it provides an indication of the importance of the genetic control on a trait in a given experiment and can be used to predict results of artificial and natural selection [START_REF] Hartl | High-density rice genetic linkage map with 2275 markers using a single F2 population[END_REF], where narrow-sense heritability (ratio of additive genetic variance to total variance) is more important for population responses to individual selection than broad sense heritability (ratio of total genetic variance to total variance).

Significant levels of heritability have been detected under waterlogging or flooding in a number of species for biomass and yield (Collaku and Harrison 2005, Triticum aestivum;[START_REF] Silva | Inheritance of tolerance to flooded soils in maize[END_REF], Z. mays) as well as for traits assessing the sensitivity to hypoxia (e.g., the percentage of yellow leaf (Zhou et al. 2007b, Hordeum vulgare). However, without an estimate of heritability under controlled conditions, it is impossible to infer whether the observed genetic control refers to a constitutive or an induced trait. [START_REF] Marcar | Genetic variation among and within provenances and families of Eucalyptus grandis W Hill and E. globulus Labill. subsp globulus seedlings in response to salinity and waterlogging[END_REF] compared E. globulus (hypoxia sensitive) and E. grandis (tolerant) seedlings for shoot dry weight changes under water logging relative to control conditions. Narrow sense heritability for this trait was higher in the tolerant species and lower in the sensitive one. This species x environment interaction, suggests a larger genetic control of growth during hypoxia for the adapted plants. [START_REF] Kolodynska | Multivariate responses to flooding in Arabidopsis: an experimental evolutionary investigation[END_REF] observed during a threegeneration selection experiment with Arabidopsis thaliana that heritability changed in response to selection, and that morphological traits displayed increasing heritabilities compared to life-history traits. Selection did not alter the overall shape of reaction norms but lowered the phenotypic means of some traits. Hybrid families of G. rivale  urbanum (wetland x dryland species) showed no significant heritability for the response of shoot biomass to waterlogging [START_REF] Waldren | Comparative studies of plant-growth and distribution in relation to waterlogging. 15. The effect of waterlogging on growth of various populations of and hybrids between geum-rivale l and geum-urbanum l[END_REF]). However, the response of root dry weight or shoot/root ratio was under significant genetic control in this experiment. Thus, integrative traits with no direct link to hypoxia tolerance such as above-ground biomass yield or growth might, in some situations seem not to be under genetic control even though some genetic diversity was detected in adaptive traits. In such cases, differences in fitness during stress might be explained better by survival rate than by aboveground growth and biomass production.

A typical adaptive trait studied in rice is internode elongation during submergence. [START_REF] Nemoto | Inheritance of early elongation ability in floating rice revealed by diallel and QTL analyses[END_REF] studied the lowest elongated internode (LEI) for a U n c o r r e c t e d P r o o f diallel crossing of different Oryza sativa and O. rufipogon varieties. For this highly adaptive trait, high heritabilities were estimated (0.994 for broad sense and 0.962 for narrow sense) with a much larger additive than dominant variance, suggesting a high potential for individual selection. The heritability of adventitious root development was studied under flooding in Cucumis sativus [START_REF] Yeboah | Inheritance of waterlogging tolerance in cucumber (Cucumis sativus L)[END_REF]). Narrow-sense heritability was higher for this trait (0.74) than for the overall tolerance score (0.60). The heritability of total root dry weight was higher under waterlogging than in controls. In a large test with 436 Z. mays inbred lines, a low broad-sense heritability was found for root porosity under normal conditions, which increased significantly during waterlogging [START_REF] Zaidi | Importance of secondary traits in improvement of maize (Zea mays L) for enhancing tolerance to excessive soil moisture stress[END_REF]. In contrast, heritability of biomass and yield declined during waterlogging compared to control. The tight correlation between root porosity and grain yield under stress, and its absence in controls, stresses the importance of root porosity for hypoxia tolerance in this species. We found no estimate of heritability for the development of hypertrophied lenticels in the literature. Nevertheless, in general, whenever heritability was estimated for morphological adaptive traits, a rather tight genetic control was shown, and it often increased under stress. There are few estimates of genetic control of enzyme activities related to hypoxia tolerance. [START_REF] Chan | Variation in alcohol-dehydrogenase activity and flood tolerance in white clover, Trifolium repens[END_REF] showed a strong genetic control for hypoxia-induced ADH activity in T. repens populations (broad sense heritability 0.55AE0.13). Overall, tolerance traits seem to show higher heritabilities in stressed conditions compared to productivity or traits quantifying damage or overall performance.

Genetic Determinism of Tolerance to Waterlogging and Identification of the Involved Genome Regions

Once the occurrence of a genetic control of a trait has been established, the next step is to identify the underlying genetic determinism, that is, how many genes control the expression of the trait and to what extent each gene controls its variability. The classical approach to this question is QTL (quantitative trait loci) mapping, the resolution of quantitative traits into discrete mendelian inherited components [START_REF] Paterson | Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms[END_REF]). This requires a reference population screened for a high number ()100) of genetic markers. The recombination information produced by the progeny is then used to order the markers on a genetic map. The comparison of this genetic information across all individuals with their phenotype for a given trait allows identifying regions on the genetic map (QTL), that each determines a fraction of the observed phenotypic variability of the trait (called the phenotypically explained variance, PEV). The least likelihood (LOD), position on the genetic map, allelic substitution effect and PEV are estimated for each QTL. Depending on the statistical package used, the presence of a QTL is either determined by a LOD score threshold or a significance statistic calculated using permutation techniques. Further, bootstrap methods allow estimating standard deviations of all parameters.

U n c o r r e c t e d P r o o f

However, most parameters estimated during QTL analyses, including the number of QTL detected, depend heavily on the number of genotypes in the reference population. Simulations showed that PEV values are overestimated and that the number of QTL detected does not correspond to the number of loci involved when the sample size of the mapping pedigree (N) is below 1,000 [START_REF] Beavis | The power and deceit of QTL experiments: lessons from comparative QTL studies[END_REF]. QTL experiments for hypoxia-related traits never involved more that 300 genotypes (Table 12.2, range: 60-288), related to the large experimental set up, as for example, shown in Fig 12 .1. Some caution is therefore needed when attempting to infer the actual genetic determinism of specific traits as the number of detected QTL is likely to be smaller than the actual number of genes involved. QTL experiments with a relatively small number of genotypes will mainly detect major QTL with high allelic substitution effects and PEV.

QTL detection encompassed the whole range of traits and conditions described above, focusing on short-term responses of physiological processes, or long-term acclimation with the objective to investigate morphological adaptations. The environmental conditions used for QTL detection, ranged from waterlogging and flooding to total submergence.

Methodology of the Detection of QTL for Hypoxia

Tolerance: Caution and Strategies

Submergence Tolerance and Waterlogging Tolerance

To date, QTL detection for tolerance to total submergence concentrated on rice [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF][START_REF] Nandi | Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping[END_REF][START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF][START_REF] Xu | A high-resolution linkage map of the vicinity of the rice submergence tolerance locus sub1[END_REF][START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF][START_REF] Guangheng | QTL analysis of traits concerned submergence tolerance at seedling stage in rice (Oryza sativa L)[END_REF][START_REF] Hattori | A major QTL confers rapid internode elongation in response to water rise in deepwater rice[END_REF]. All these experiments detected a major QTL on chromosome 9 (see [START_REF] Chen | An integrated physical and genetic map of the rice genome[END_REF], for a detailed physical and genetic map of rice) and allowed the identification of the sub-1 locus (Xu et al. 2006). QTL have also been detected for several species during partial submergence and root hypoxia [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF][START_REF] Zheng | Mapping QTLs and candidate genes for rice root traits under different water-supply conditions and comparative analysis across three populations[END_REF][START_REF] Cornelious | Identification of QTLs underlying waterlogging tolerance in soybean[END_REF]Mano et al. 2005a, b;[START_REF] Cornelious | Yield potential and waterlogging tolerance of selected near-isogenic lines and recombinant inbred lines from two southern soybean populations[END_REF][START_REF] Githiri | QTL analysis of flooding tolerance in soybean at an early vegetative growth stage[END_REF][START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF][START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF]Zhou et al. 2007a). Duration of waterlogging as well as the height of the water table were highly variable, ranging from a few days [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF]) to several weeks [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF]Mano et al. 2005b). and from few centimetres (Mano et al. 2005b;[START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF]) to 10 cm above soil surface [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF][START_REF] Cornelious | Identification of QTLs underlying waterlogging tolerance in soybean[END_REF]. This diversity in experimental procedures may have contributed to the large variability in the number and localization of the detected QTL (Table 12.2). Phenotyping after variable stress durations and intensities may detect different tolerance processes and thus result in a QTL detection that varies with environment. QTL detection is a statistical process, whereby minor QTL with low allelic substitution effects and thus low PEV will often be below the detection or significance limit. [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF] QTL for epinasty of which the PEV varied from 2.8% to 11.6% depending on the observation date during permanent waterlogging. Furthermore, as discussed above, the estimation of PEV and allelic substitution effect of QTL also depends on the number of genotypes and of vegetative copies within genotypes. This, together with the variations in environmental conditions, makes it difficult to compare QTL among different experiments. However, comparing the position of major QTL across experiments clarifies their significance across conditions and genotypes.

One major QTL detected in rice during total submergence is the the sub-1 locus [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF], showing five to seven times higher LOD scores and at least two times higher PEV (Table 12.2) than QTL for any other adaptive trait despite the similar (AE20%) number of genotypes involved in the different experiments, e.g.: [START_REF] Mano | Qtl mapping of root aerenchyma formation in seedlings of a maize x rare teosinte Zea nicaraguensis cross[END_REF]Mano et al. ( , 2008) ) and Mano and Omori (2008) for aerenchyma; Mano et al. (2005a, b) for adventitious roots; [START_REF] Mano | Identification of QTL controlling flooding tolerance in reducing soil conditions in maize (Zea mays L) seedlings[END_REF] for leaf injury). Even when taking into account that the estimated LOD and PEV depend on the experiment, the sub-1 locus stands out among all QTL detected for hypoxia tolerance. This might be due to the fact that it controls internode growth, which is a trait controlled probably by only few genes; whereas strategies of tolerance to waterlogging are more complex, probably relying on multiple traits, and thus depending on many, interacting genes. This would result in the detection of more QTL with U n c o r r e c t e d P r o o f lower PEV. However, other than indicating effectively a stronger genetic determinism, the high LOD scores for sub-1 might also be due to the experimental control of the stress intensity. The high water table present during total submergence experiments homogenizes the hypoxic stress among plants, thus reducing within-experiment environmental variability; whereas hypoxic stress during waterlogging experiments also depends on the homogeneity of the soil and the rooting density.

As oxygen diffusion in water is slow, the actual oxygen deficiency in the soil depends on root and rhizospheric O 2 consumption, which creates a gradient from the soil surface to the root. This induces a large variability of the stress actually perceived among plants within an experiment, with evident effects on the phenotypes and on QTL detection. Indeed [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF] detected QTL for dissolved oxygen content in water in the vicinity of the roots of oaks submitted to waterlogging, revealing a clear problem of stress control among plants, with genotypes influencing their environment.

QTL Detection for Constitutive Traits of Tolerance

The most recently published experiments on maize [START_REF] Mano | Qtl mapping of root aerenchyma formation in seedlings of a maize x rare teosinte Zea nicaraguensis cross[END_REF](Mano et al. , 2008) ) detected QTL for aerenchyma formation, a key trait known to be highly related to hypoxia tolerance. This was achieved with an inter-specific cross between two species with different capacities of aerenchyma formation. They performed the analysis only under control conditions, which avoids stress heterogeneity among individuals (although not environmental variability). Aerenchyma formation is usually enhanced under stress, thereby showing genotype x environment interactions and a different genetic determinism than under control conditions. This could change allelic effects and PEV of the detected QTL. Genotype x environment interaction could also be a cause for the small number of co-localised QTL between the two different inter-specific crosses in the same experimental set-up: only one out of seven detected QTL co-localized.

Comparison with a Control Environment

QTL detection for induced adaptive traits does not necessarily require a control treatment. Nevertheless, QTL detection for growth and productivity needs a comparison between stress and control. This is necessary to distinguish constitutive QTL that influence growth independently of the applied stress, and induced QTL that control growth specifically in response to stress. [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF] and [START_REF] Githiri | QTL analysis of flooding tolerance in soybean at an early vegetative growth stage[END_REF] compared the phenotypes expressed by the same genotypes growing in water-logged versus control conditions. [START_REF] Githiri | QTL analysis of flooding tolerance in soybean at an early vegetative growth stage[END_REF] computed the ratio of seed production under waterlogging versus control, and used this ratio as a tolerance index. However, QTL for ratios between stress and control are difficult to interpret. This is mainly due to the fact that the condition that results in the highest among-genotype variability dominates the statistical analysis. Three situations (1) the variability is larger under stress, the QTL is then related to stress tolerance; (2) it is larger in controls (e.g. due to severe growth reduction under stress), and the QTL then describes the genetic variability of growth potential in the absence of stress, and not of tolerance; (3) the variability is similar in the two treatments and it is difficult to conclude whether the detected QTL is related to tolerance or not. Detecting QTL for each condition separately can provide support for the interpretation. This was for example used by [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF], who detected QTL for the ratio of growth parameters between waterlogging and control as well as for each treatment separately and used the resulting co-localisations to interpret QTL as either nonadaptive, constitutive or induced. Other than using stressed/ control ratios as traits, both datasets can be used within the same statistical analysis, such as in multi-environment QTL detection models [START_REF] Jansen | Genotype-by-environment interaction in genetic mapping of multiple quantitative trait loci[END_REF] which allow a direct computation of QTL x environment interactions. This has been used for example by [START_REF] Jermstad | Mapping of quantitative trait loci controlling adaptive traits in coastal Douglas fir. III. Quantitative trait loci-byenvironment interactions[END_REF] to detect QTL in a factorial experimental design using different winter chilling and spring flushing temperatures. Only one application of multi-environment QTL detection is known to the authors in the case of waterlogging. [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF] recorded epinasty, root collar diameter and leaf chlorophyll content in a Q. robur full-sib progeny and showed that the allelic substitution effects of the detected QTL varied significantly during the 4 weeks of waterlogging. This method described QTL by the temporal pattern of the corresponding allelic substitution effect, and compared such patterns among different QTL and traits. Interestingly QTL with correlated effect patterns were dispersed over the whole genome, suggesting a polygenic determinism of tolerance to hypoxia.

Major Loci Detected for Hypoxia Tolerance

One of the main tools for breeding crops for agriculture in areas submitted to waterlogging or submergence is the detection of genomic regions or genes for marker aided selection [START_REF] Vartapetian | Plant anaerobic stress as a novel trend in ecological physiology, biochemistry, and molecular biology: 1. Establishment of a new scientific discipline[END_REF]. On the other hand, QTL studies also aim at elucidating the molecular mechanisms of hypoxia tolerance. However, only few QTL experiments were performed with the aim to detect loci for which the genetic diversity is effectively selected in natural populations. Table 12.1 summarizes the traits and species for which QTL related to hypoxia response were detected.

12.4.2.1 QTL for Traits Submitted to Natural Selection Pressure in Hypoxic Environments Identifying candidate genes submitted to natural selection in water-logged or flooded soils would advance our understanding of speciation processes in such environments [START_REF] Lexer | Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (european aspen): the role of ecology and life history in gene introgression[END_REF]. [START_REF] Martin | Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions[END_REF] detected two QTL for survival in an Iris family. Their experiment was performed under water-logged 2007) who detected QTL for traits that are known to vary among natural population of two sympatric, hybridising oak species (Q. robur and Q. petraea). They detected two QTL for hypertrophied lenticel formation, five for the level of epinasty, but none for adventitious root development. Loci identified during these experiments could be a starting point in research strategies identifying candidate genes. Such genes could then be screened for genetic variability in natural populations.

12.4.2.2 QTL Detection for Breeding Purposes Many QTL were detected for a large range of traits of interest for the maintenance of productivity and growth under waterlogging (see Table 12.2 for details). Shoot growth was the main indicator of productivity during hypoxia, and QTL were detected for shoot biomass by [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF] or shoot height by [START_REF] Guangheng | QTL analysis of traits concerned submergence tolerance at seedling stage in rice (Oryza sativa L)[END_REF] and [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF]. [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF] used shoot growth during stress as a tolerance index, considering that eliminating the growth before stress would detect induced rather than constitutive QTL. As the root system is directly affected by hypoxia, some authors use it as an indicator of tolerance, for example [START_REF] Qiu | Mapping of QTL associated with waterlogging tolerance during the seedling stage in maize[END_REF] detected QTL for root length under hypoxic condition, or [START_REF] Zheng | QTLs and candidate genes for rice root growth under flooding and upland conditions[END_REF] for total root biomass. QTL detected for these traits might be used for marker aided selection or to produce inbred lines to improve crop performance under hypoxia [START_REF] Vantoai | Identification of a QTL associated with tolerance of soybean to soil waterlogging[END_REF], without necessarily having detailed information on the tolerance strategy that is controlled by the QTL.

QTL Detection for Tolerance to Hypoxia

The most recent studies on QTL detection of hypoxia tolerance were performed on hypoxia-induced morphological traits: 11 QTL were identified for aerenchyma formation in maize [START_REF] Mano | Qtl mapping of root aerenchyma formation in seedlings of a maize x rare teosinte Zea nicaraguensis cross[END_REF](Mano et al. , 2008) ) and two QTL for hypertrophied lenticel formation in oaks [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF]. Six QTL for adventitious root development were detected in maize (Mano et al. 2005a, b), but none in Q. robur despite a visible development of such roots. This lack of genetic determinism for adventitious root development in oak might be due to heterogeneity of soil hypoxia, which dominated the phenotypic variance of this trait, and therefore diluted the genetic variance [START_REF] Parelle | Quantitative trait loci of tolerance to waterlogging in a european oak (Quercus robur L): physiological relevance and temporal effect patters[END_REF]).

To characterise the tolerance of rice to total submergence, internode elongation was used to quantify the capacity of quiescence or stress escape. In consequence, this trait allowed the detection of two types of loci related to those two strategies.

This can be used in QTL detection studies when specific crosses are used, either combining two genotypes with different strategies or a tolerant and a non tolerant genotype. Indeed, the sign of the allelic substitution effect, combined with the knowledge of the strategy developed by the parents allowed to clearly attribute QTL to the two strategies. Several authors [START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF][START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF][START_REF] Nemoto | Inheritance of early elongation ability in floating rice revealed by diallel and QTL analyses[END_REF][START_REF] Tang | Comparison of QTLs for early elongation ability between two floating rice cultivars with a different phylogenetic origin[END_REF][START_REF] Hattori | A major QTL confers rapid internode elongation in response to water rise in deepwater rice[END_REF][START_REF] Kawano | Mapping of QTLs for floating ability in rice[END_REF] detected QTL related to the escape strategy of different rice varieties, and [START_REF] Xu | A major locus for submergence tolerance mapped on rice chromosome 9[END_REF] detected the sub-1 locus related to the quiescence strategy by an inhibition of the internode elongation. In Table 12.3 all traits are listed for which QTL were detected at the sub-1 locus. [START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF] and [START_REF] Hattori | A major QTL confers rapid internode elongation in response to water rise in deepwater rice[END_REF] performed multiple trait phenotyping of the early elongation ability, where the constitutive diversity included in each trait differed, whereas stress responses relating to the same tolerance mechanism would result in colocalisation of QTL. Both experiments resulted in the detection of the sub-1 locus, thus relating it clearly to low elongation ability (quiescence strategy). An important step for breeding was the successful introgression of the sub-1 locus into a rice variety of economic importance. [START_REF] Siangliw | Thai jasmine rice carrying QTL ch9 (subQTL) is submergence tolerant[END_REF] crossed three tolerant varieties of rice, containing the sub-1 allele conferring tolerance by quiescence strategy, with the hypoxia intolerant Thai jasmine rice. In the hybrid families, QTL were detected for hypoxia tolerance at the sub-1 locus and the alleles related to quiescence were always from the tolerant parent. Introgression increased survival from 1.6% in Thai jasmine to 23%-31% in the hybrid families. This example shows how the detection of a QTL for tolerance can be used directly for breeding purposes.

The sub-1 locus was the only QTL for which the underlying genes were clearly identified. It was reported for the first time by Xu andMackill (1995, 1996), and further detected during all QTL detection experiments in rice, including the tolerant variety F13A, in which elongation is inhibited in order to decrease energy demand during hypoxia (quiescence strategy) [START_REF] Nandi | Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping[END_REF][START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF]. [START_REF] Nandi | Mapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping[END_REF] demonstrated that a, cartography of the qualitative trait "surviving/not surviving to total submergence" was sufficient to detect the sub-1 locus. tolerance mechanisms, as for example the development of aerenchyma, adventitious roots and hypertrophied lenticels (Bailey-Serres and Chang 2005). To our knowledge no QTL experiment was performed directly for ethylene production or for other traits related to signalling of hypoxia stress.

Conclusions

Genetic diversity has been shown among populations or within mapping families, for indirect indicators of tolerance, such as growth and leaf level gas exchange, as well as for constitutive or induced adaptive traits. The genetic control was elucidated for only a small number of traits and even less gene candidates have actually been tested. At the enzyme level, some diversity was detected only for ADH. However, no signal transduction pathway has been put forward and related to the observed genetic differences. Further, the actual effect of genetic differences in ADH on survival and fitness in natural populations still lacks experimental support. The correlation between the observed genetic diversity of short term metabolic adjustments to hypoxia and long term morphological adaptations needs further investigation. The QTL detected for survival or traits known to vary among natural populations could be starting points for gene candidate approaches. Such gene candidates could then be screened for natural genetic variability, thereby generating knowledge on the adaptability of populations, especially with respect to environmental changes.

The major challenge for future QTL detection for traits conferring hypoxia tolerance is the definition of integrative traits (1) indicating different tolerance strategies and (2) well suited to high-throughput phenotyping required for quantitative genetic analyses. In addition to the sub-1 locus, a large number of minor QTL have been detected. The combination of several favourable alleles will determine the tolerance of an individual. However to decompose these processes, future approaches should combine large scale QTL experiments using complex traits and detailed studies on selected genotypes to decompose overall tolerance into elementary components.

  detected a BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09 U n c o r r e c t e d P r o o f t2:1Table 12.2 Number of QTL (N Q ) detected under hypoxic conditions (except Mano et al. 2007 and Mano et al. 2008) for different tolerance or growth traits; for each experiment (where n i is the number of genotypes of the tested family) o r r e c t e d P r o o f

Fig. 12 . 1

 121 Fig. 12.1 Example of the experimental procedure for QTL detection of waterlogging tolerance traits (Parelle et al. 2007). This photography shows 320 rooted cuttings of Quercus robur waterlogged for 4 weeks and phenotyped daily (photo Parelle)

  BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09 U n c o r r e c t e d P r o o f might occur:

  BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09 U n c o r r e c t e d P r o o f conditions in a common garden for 4 years. Another experiment was performed by Parelle et al. (

  BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09 U n c o r r e c t e d P r o o f

  For the LOD score and the R 2 values among repetitions of the QTL detection experiments are indicated BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09 U n c o r r e c t e d P r o o f

  Table 12.1 Traits tested during QTL experiments to identify hypoxia-tolerance related loci in

		different species
	t1:2	Stress type	Article	Genus	Trait types
	t1:3	Submergence	Xu and Mackill (1996)	Rice	Damage

  Table 12.3 Traits for which a QTL related to an allele of the F13 A variety of Oryza sativa ssp indica was detected at the Sub1 Loci

	t3:2	Trait	Article	Methods of	LOD score	R 2
				detection
	t3:3	Tolerance scrore	Xu and Mackill	Linear regression	36	0.69
		(visual scale)	1996
	t3:4	Surviving or not	Nandi et al.	Direct mapping of	-	-
			1997	the qualitative
				trait
	t3:5	Per cent plant survival Toojinda et al.	Composite interval	36.4-65.8	0.41-0.77
			2003	mapping
	t3:6	Relative shoot			16.2	9.7
		elongation
		compare to control
	t3:7	Tolerance score			26.6-38.3-52.6 0.38-0.61-0.63
		(visual scale)
		Leaf senescence after			29.3	0.53
		submergence

t3:1

BookID 182578_Mancuso_ChapID 12_Proof# 1 -25/11/09

U n c o r r e c t e d P r o o f

The sub-1 locus was not only detected in F13A, but also in other tolerant varieties. For example [START_REF] Sripongpangkul | Genes/QTLs affecting flood tolerance in rice[END_REF] detected a QTL for elongation ability on the sub-1 locus in a F8 RIL cross from two indica cultivars; the tolerant parent conferring the allele for a, faster elongation ability. This locus therefore seems to be also involved in the escape strategy developed by some deep-water varieties of rice. However, the main QTL for the fast elongation capacity was detected on a different chromosome than sub-1 [START_REF] Tang | Comparison of QTLs for early elongation ability between two floating rice cultivars with a different phylogenetic origin[END_REF][START_REF] Hattori | A major QTL confers rapid internode elongation in response to water rise in deepwater rice[END_REF][START_REF] Hattori | Mapping of three QTLs that regulate internode elongation in deepwater rice[END_REF].

Candidate gene approaches were used to identify the gene(s) beneath the sub-1 locus. [START_REF] Ruanjaichon | Small GTP-binding protein gene is associated with QTL for submergence tolerance in rice[END_REF] first mapped a small GTP-binding protein, belonging to a family known to be involved in signal transduction pathways and [START_REF] Kottapalli | In silico insight into two rice chromosomal regions associated with submergence tolerance and resistance to bacterial leaf blight and gall midge[END_REF] identified 1,473 (putative) genes. However the large confidence interval for the position of the QTL for sub-1 (6,4 cM for [START_REF] Kottapalli | In silico insight into two rice chromosomal regions associated with submergence tolerance and resistance to bacterial leaf blight and gall midge[END_REF] did not allow the identification of the gene(s) responsible for submergence tolerance. It was finally the sequencing of the rice genome (International Rice Genome Sequencing Project 2005), the construction of a high resolution genetic map [START_REF] Hartl | High-density rice genetic linkage map with 2275 markers using a single F2 population[END_REF][START_REF] Xu | A high-resolution linkage map of the vicinity of the rice submergence tolerance locus sub1[END_REF] and the comparison of genetic and physical maps [START_REF] Kamolsukyunyong | Mapping of quantitative trait locus related to submergence tolerance in rice with aid of chromosome walking[END_REF][START_REF] Chen | An integrated physical and genetic map of the rice genome[END_REF]) that allowed the identification of the sub-1 cluster of genes (Xu et al. 2006). To date, the physical structure of this locus is well known (see [START_REF] Fukao | A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice[END_REF]Xu et al. 2006 for details). This locus contains 13 genes, including three ethylene response factors called Sub1-A, Sub1-B and Sub1-C. Sub1-A is present only in O. sativa ssp indica, including the tolerant variety F13A (Xu et al. 2006;[START_REF] Fukao | Evolutionary analysis of the sub1 gene cluster that confers submergence tolerance to domesticated rice[END_REF]). This gene originates probably from the duplication of the Sub1-B gene, as the two genes display a large sequence homology, and as the presence of the Sub1-A gene was correlated with variation of Sub1-B alleles [START_REF] Fukao | Evolutionary analysis of the sub1 gene cluster that confers submergence tolerance to domesticated rice[END_REF]. Two alleles of Sub1-A, have been reported: the Sub1-A-1 and the Sub1-A-2. These alleles were correlated with variation of alleles of Sub1-C [START_REF] Fukao | Evolutionary analysis of the sub1 gene cluster that confers submergence tolerance to domesticated rice[END_REF]). The tolerant variety F13A possesses the Sub1-A-1 allele and the corresponding alleles of the Sub1-C and Sub1-B. Recombinant crossing experiments among the three genes (Xu et al. 2006;[START_REF] Septiningsih | Development of submergence-tolerant rice cultivars: the sub1 locus and beyond[END_REF]) demonstrated that variation of the two alleles of the Sub1-A locus modify the submergence tolerance of rice, independently of the effect of the alleles present in Sub1-B and Sub1-C. This suggests that the QTL is controlled by the allelic effect of one single gene, of which only two states were detected: presence / absence of the Sub1-A-1 allele or the presence / absence of the entire gene. Introgression of the Sub1-A-1 allele into other species than rice could improve productivity of crops under flooding. The Sub1-A-1 allele induces the quiescence strategy resulting from the inhibition of internode elongating [START_REF] Toojinda | Molecular genetics of submergence tolerance in rice: QTL analysis of key traits[END_REF]. Actually the Sub1-A-1 allele inhibited the effects of the Sub1-C gene on elongation initiation in response to ethylene [START_REF] Fukao | A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice[END_REF]). However, it is far from being clear whether the introgression of the Sub1-A-1 allele into other species actually confers a larger tolerance to total submergence, as interactions with other tolerance strategies need to be taken into account.

It is interesting to notice that the three sub1 genes (A, B, and C) are ethylene response factors. Ethylene is known to be involved in a large number of hypoxia

AU2

Following reference is not cited in text: [START_REF] Levy | Excess water: effects on growth of oak trees[END_REF]


domain: sdv sde

text:
Yvan Capowiez

Pierre Bouchand

Stéphane S Cadoux

Céline Pelosi

Jean Roger Estrade

Hubert H Boizard

Demonstration of the role of earthworms on the removal of highly compacted zones in field conditions

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The Symposium will be held in Poland in Kraków, at

Abstract:

By their excavation activities, earthworms are thought to contribute to the removal of highly compacted zones ("delta" zones) but the quantitative measurements of their impact is lacking.

A first experiment was carried out in semi-field conditions (caging experiment). Adult earthworm from the most common species (Lumbricus terrestris, Aporrectodea giardi, A. caliginosa and A. rosea) were in presence of different compacted zones: (i) horizontal, (ii) vertical layer and (iii) isolated zone representing different natural situations (under wheel or timmage compaction). Depending on the species (and their behaviour) we observed different impacts on compacted zones (L. terrestris is more prone to cross horizontal layer whereas A. caliginosa is more prone to colonize vertical ones). A second experiment was carried out in field conditions to characterise the dynamics of the recolonisation by earthworms after a compaction (through a 8t vehicle). Under and between wheels zones were followed during 2 years. We studied earthworm bulk density, earthworm community, 2D vertical macroporosity and water infiltration measurements were done. We observed that earthworm recolonisation is a rapid process (less than 6 months to recover similar abundances) whereas macroporosity and water infiltration are much slower processes. This illustrates the difficulty of using earthworm abundance as way to estimate the physical consequences of earthworm activity. Moreover, 3D soil cores were collected at different dates under and between wheels and analysed through Xray tomography. This enables us to visualize the (slow) recolonisation of the soil with earthworm macropores and to discuss the effect of the different species present in the field.

(oral communication)

  the Jagiellonian University at the Institute of Environmental Sciences.From 4 th to 9 th of September 2006

	Title: Demonstration of the role of earthworms on the removal of highly compacted zones in
	field conditions
	Authors: CAPOWIEZ Yvan(1), BOUCHAND Pierre(2), CADOUX Stéphane(2), PELOSI
	Céline(3), ROGER-ESTRADE Jean(3), BOIZARD Hubert(2)
	Address: (1) INRA/UAPV Ecologie des Invertebres-84914 AVIGNON cedex 09-France
	(2) INRA, Unité d'Agronomie Laon-Reims-Mons, Estrées-Mons, BP50136, 80203 Péronne,
	France
	(3) UMR INRA-INAPG, Laboratoire d'Agronomie, BP 01, 78850 Thiverval-Grignon, France