Gaucheron Frédéric
email: frederic.gaucheron@rennes.inra.fr

Biodiversity of milk and dairy products all over the world -Some examples

Milk and dairy products are present in countries all over the world. They exist under several forms depending on type of milk, climate, level of milk production, type of transformation, social and cultural developments, local economy and food habit of people.

The objective of this presentation is to show some examples describing the diversity in biochemical compositions of milks from different species, milk management and technological practices which ultimately lead to a huge diversity in dairy products.

Diversity in biochemical composition of milks

Milk is generally described as a white liquid with a pH close to the neutrality, with a neutral to sweet flavor containing water, proteins, lipids, carbohydrates, minerals and vitamins. Among all the milks, cow milk is well known for its biochemical composition and technological transformation. For these reasons, it is always taken as reference. In this presentation, we will focus on the composition of seven most described milk species and compare them with cow's milk. As described in Table 1, the milk compositions are not the same and depend on the specie. Milks from buffalo, sheep, reindeer and yak have high dry matter because the milk from them has high lipids and protein contents. Thus, reindeer milk is described as dense and "thick" in relation to its high contents in proteins and lipids. By contrary, mare milk is considered poor for dry matter so it is not suitable for transformation into cheeses or other some products succesfully.

Simpósio Internacional de Ciência e Tecnologia do Leite, Universidade Federal de Viçosa-UFV, Brazil -31 october-2 november 2013 2 Table 1 -Global composition of milks from different species. Concentrations are expressed in g/l These compositions corresponding to average compositions, can vary depending on the race, stage of lactation, nutrition, herd management, nutrition and health. More details on the composition of different milks are described especially in handbook dedicated to milk of non-bovine mammals (Handbook of milk of nonbovine mammals, Ed YW Park, GFW Haenlein, Blackwell Publishing Oowa, USA, 2006). The "structural organisations" of these milks are very similar; all are solution containing dissolved molecules (minerals, whey proteins, lactose, etc), suspension with casein micelles, and emulsion with fat globules. However, quantitative and/or qualitative differences between each milk exist. To illustrate these differences in term of milk composition between species, we will discuss few examples like:

-All milks contain whey proteins but in different amounts. Moreover, their amino-acid compositions and tridimensionnal structures are different;

-The casein/whey protein ratios (w/w) are different e.g. 80/20 for cow milk against 60/40 for mare milk;

-The amino acid compositions of casein molecules are not the same; thus, there is only 60% of homology between caseins from cow and camel milks;

-All milk contains minerals like calcium, magnesium, sodium, potassium, chloride, inorganic phosphate, citrate and trace elements (iron, copper, zinc). However, their amounts are not the same as a function of the considered specie. In each milk, calcium is partly associated to casein molecules to form micellar calcium phosphate but the amount of calcium associated to casein depends on the specie; for example, casein micelles from buffalo milk are more mineralised than casein micelles from cow milk;

β-lactoglobulin is not present in camel milk;

-Lactose is always present as main sugar but its concentration is variable (mare > cow > reindeer);

-The size of casein micelles from camel milk is bigger than those from cow milk;

Simpósio Internacional de Ciência e Tecnologia do Leite, Universidade Federal de Viçosa-UFV, Brazil -31 october-2 november 2013 3 -The size of fat globules is smaller in camel milk compared to cow milk and they have low creaming properties;

-The goat milk contains more fatty acids with short chains than cow milk.

Due to qualitative and quantitative differences in milk composition, the transformations of these milks in dairy products are not the same. Sometimes, it is necessary to adapt the technological treatment to the specific type of milk. In other cases, it is impossible to do the treatment. For example, due to low content of dry matter, it is impossible to make cheese with mare milk. In case of camel milk, it is very difficulty to make UHT milk and cheese. The rennet coagulation of this milk is not easy except when camel rennet is used. The gel is fragile and soft leading to a difficulty in mechanical treatment. The possible causes are the low level of κ-casein. With buffalo milk, the curds have strong firmness after acid and rennet coagulations. Due to its richnesss in proteins and minerals, its resistance to acidification (buffering capacity) is more important than cow milk. Due to its richness in lipids, the yield in the manufacture of butter are better for buffalo than cow milks; thus the production of one kg of butter requires 14 kg of cow milk against only 10 kg for buffalo milk.

It is different examples illustrating some qualitative differences between milk from different species; in the reality the number of differences existing are more important.

Diversity in the "management" of milk: breeding conditions, milking and transportation

The milk "management" vary and depending on the countries. In industralised/developed countries, milk is considered as a very precious liquid and a lot of attentions are brought to preserve its quality. These precautions are taken at different time between the milking in the farm and the transformation into different products and their commercialisation. Special attentions are paid to maintain the cold chain to avoid proliferation of micro-organisms which can be dangerous for the health of consumers and can also alter the transformation and the final quality of the dairy products. In developing countries, for mutiple reasons, these precautions concerning the milking conditions, storage of milk before transformation, quality of water, cleaning of equipments, transportation are not always taken into account. In these cases, the risks of alteration of milk and dairy products increase. The oral presentation will show different cases.

Diversity in technological practices: traditional methods to industrial methods

Milk is considered as "ideal medium" for the proliferation of micro-organisms because of its richness in nutrients. To limit the negative effects of microbiological development, human consumes milk immediately after milking, or after different technological operations. These technologies, used at different scales (i.e. few liters to several thousands liters per day), preserve microbiological quality of milk but alter biochemically the dairy constituents. Thanks to these technological treatments, the dairy products can be consumed during periods of time ranging from few days (pasteurized milk), months (UHT milk), or years (some cheeses or dairy powders). In addition to the improvement of the conservation and sanitary guarantees, the dairy technologies are also used to produce or isolate different compounds with specific biological potential.

Figure 1 sumarizes some technological operations used to make different dairy products. The important number of these different types and levels of transformation contribute to the diversity of dairy products. This presentation will show different cases observed in several countries in the world.

Diversity of micro-organisms used in dairy technology

Micro-organisms can participate in positive way to the elaboration of dairy products. In general, the microbiological flora is complex and contributes to the quality and typicity of some products especially cheeses. These micro-organisms play roles in acidification, texture, flavor, gas production, color, appearance, nutritional properties, etc. These micro-organisms can be bacteria, yeasts, or molds depending on the dairy products. They produce different enzyms which have specific actions modifying biochemically proteins, lipids and lactose. In this sense, they are considered as "biological tools" used in the manufacture of specific dairy products (Table 2) and contribute to the diversity. In dairy products, the concentrations of micro-organisms are ranged from 10 7 to 10 9 cells per g. In general, these micro-organisms are together in the dairy products. Thus, the species Streptococcus thermophilus and Lactobacillus bulgaricus live together in yoghurt. In Camembert cheese, there are 8-10 different species including Penicillium camemberti. Penicillium roqueforti is present in the blue-veined cheese Roquefort whereas Brevibacterium linens is responsible for the orange colored rind in Livarot or Munster.

Diversity of dairy products

Thanks to the knowledge acquired on the composition of milk and development of different dairy technologies, the Human can manufacture a large variety of dairy products with different properties, tastes and forms. Today, in some parts of the world, the consumer has at his disposal, thousands of different dairy products. The majority of dairy products in the world are made from cow's milk but there are also products from other milk depending on the considered region in the world. These products are milk which are heated, microfiltered, enriched in proteins, vitamins, minerals or fiber, flavoured, reduced in fat and lactose. They are also cheeses with different compositions, textures and original taste (fresh, soft, pressed, cooked or melted ...), creams more or less rich in fat, type of butter (hard, soft, more or less salty, ...), fermented milk with different micro-organisms, dairy specialities, infant formula, functional ingredients, powders, etc (Figure 1 and Table 3).

Buffalo

-The dairy products manufactured are those found for cow milk but with a lesser variety and diversity -Butter, butter oil (named ghee), milks more or less heated (pasteurised to sterilised), concentrated and spray-dried milks, ice creams, acidified milks (Paneer) and fermented (Lassi and Dahi) and some classical cheeses type Cheddar, Domiati, Ricotta, Ras,

Gouda and Mozzarella

-Use of buffalo milk as ingredient in the manufacture of sweet desserts (Khoa, Kheer, Chhana,...)

Goat

Washing of curd

Milks of different species

Treatments of cheese : washing, pulverisation, … properties). Thus, these fractions are used in different food formula more or less elaborated like sauces, soupes, cakes, etc. Beyond of these techno-functionnal properties, there are also intense researches to highlight positive activities of milk proteins and peptides on the biological functions and human health. Indeed, some of these constituents are benefical especially on digestion, blood coagulation, blood pressure and immunity. Some products, to improve health state of consumers, are developed thank to these recent results.

Dairy constituents can also be integrated in non-food applications. For examples, caseins are used as ingredients in the composition of glue and paint, lactose in pharmaceutical products, milk minerals in toothpaste. Recent works describe the use of bovine milk proteins as components of biodegradable packaging and textiles. By their dermatological properties, milk of mare and donkey are used in the manufacture of soap.

Preserve the diversity: milk and dairy products for future

From the recent knowledge, it is possible to produce variety of milk which can be consumed raw or transformed into other products more or less elaborated, with a more or less long time of preservation with various tastes (neutral to typical). It is essential to preserve, protect and increase this diversity by increasing our knowledge on the fine composition and organisation of milk and dairy products (bovine and non-bovine). On the other hand, a best knowledge of the milk transformation is also important to consider for the development of new products.

The research projects and innovations depend on the considered country. We can classify the new projects in two main categories. For developing countries, it is essential to improve the quality, especially the microbiological quality to manufacture and diversify dairy products according to consumer expectations in terms of nutritional and sensorial properties. In the developed countries where the qualitative and quantitative production/transformation are optimised, attentions should be paid on the manufacture of sophisticated dairy products with typical organoleptical properties, given functionalities and in some cases with demonstrated biological properties.

Figure 1 -

 1 Figure 1 -Examples of technological operations used in dairy transformation. In this simplified figure, the operations indicated in italic are "in cascade" to have a best valorisation of the generated coproducts. For the part "cheese technologies" (top left of this figure), see Figure 2 for details.

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  Consumed as raw milk or after different technological treatments commonly used for cow milk -Whole, skimmed, fortified, flavoured milks, fermented products like yoghurts, ice creams -Concentrated and spray dried products -Several cheeses having original and typical organoleptic properties (Rocamadour, Banon, Chabichou, Pélardon, Picodon, …) Sheep -Consumed as raw milk -Skimmed, heated, dried milks -Fermented milk -Several cheeses (Roquefort, Manchego and Feta,….) Mare -Consumed as raw milk by local population in Central Asia -Fermented especially with a complex flora composed of yeasts (Torula, Kluyveromyces, Candida, Brettanomyces), lactobacilli and streptococci in alcoholic drinks named Koumiss -Lyophilisate (Germany, France and Italia) to be used in dietary or food supplements -Used for hydrating properties in cosmetics Camel -Consumed as raw milk -Low level of transformation (even in countries where significant dairy production is significant) Difficulties to be transformed in cheese or UHT milk -Pasteurized milks (Mauritania, Saudi Arabia), fermented milks (Zrik in Mauritania, Shubat in Kazakhstan, Airag in Mongolia, Oggtt in Saudi Arabia), different cheeses (fresh, soft cheese called "Camelbert" or "Caravan" in Mauritania, and pressed cheeses) and butter (Kenya, Sudan) Reindeer -Consumed fresh, diluted in water, tea or coffee -Low level of transformation -Frozen or fermented milks, few cheeses and butter -Used for the production of cosmetics by local population Yak -Mostly consumed boiled and mixed with tea -Low level of transformation -Yak butter used as a food ingredient, glossing agent and in religious ceremonies -Fermented and transformed into Gruyere and cheeses type Chhurpi and Chura made from heated whey in Tibet or Tarag produced in Mongolia. Simpósio Internacional de Ciência e Tecnologia do Leite, Universidade Federal de Viçosa-UFV, Brazil -31 october-2 november 2013 8 The best examples illustrating the diversity of dairy products are cheeses. If we consider the technological flow chart to manufacture cheese (Figure 2), human can made a wide range of products by playing with different variables concerning milk preparation, curd fabrication and ripening conditions (see the book Le Fromage, Ed Tec & Doc, Lavoisier, Paris, France, 1997). Thanks to the multiple practices, which are performed at artisanal or industrial scale, and which are often accompanied by a transfer of knowledge from generation to generation, there are thousands different types of cheeses all over the world. The cheese diversity concerns the biochemical and microbiological compositions, nutritional properties, tastes, odors, textures, forms, weights, conditions of storage, stories….

Figure 2 -

 2 Figure 2 -Simplified technological diagram for cheese manufacture with some possible variations of parameters of manufacture. These variations are responsible for the cheese diversity.

Table 2 -

 2 Some examples of micro-organisms used in different fermented dairy products(In : Dairy Starter Cultures. Ed Cogan TM & Cogan JP, VCH Publishers Inc., New York, USA, 1996).

	Types of micro-organisms

St: Streptococcus thermophilus -Lh: Lactobacillus helveticus -Ll: Lb.delbtueckki lactis ssp.lactis (Lb lactis) -Lb: Lb.delbrueckkii ssp. Bulgaricus -La: Lb.acidophilus -Lca: Lb.casei -Lc: lactococci -Ln: Leuconostoc mesenteroides ssp.cremoris

Table 3 -

 3 Examples of dairy products manufactured from milk of differents species

	Cow	-Several thousands of dairy products (see following paragraph)


Marie Foulongne-Oriol

Nathalie Minvielle

Jean-Michel Savoie

QTL for resistance to Trichoderma lytic enzymes and metabolites in Agaricus bisporus

Keywords: competitor, green mould disease, adaptation, fitness, breeding

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The fungal competitor Trichoderma aggressivum is the cause of the green mould disease in the cultivated button mushroom Agaricus bisporus. It develops a dense hyphal network in mushroom compost or casing materials, followed by sporulation and an almost complete lack of mushroom production. The T. aggressivum species compete for space and nutrients and are adapted for growth in Agaricus mushroom compost by resisting the inhibiting effects of bacteria in this cultivation substrate [START_REF] Savoie | Factors influencing the competitive saprophytic ability of Trichoderma harzianum Th2 in mushroom (Agaricus bisporus) compost[END_REF]. The North American Trichoderma aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroom A. bisporus which results in drastic crop losses [START_REF] Krupke | The North American mushroom competitor, Trichoderma aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroom Agaricus bisporus[END_REF]. This threat affects an ever larger part of producing areas all over the world and becomes a major disorder in commercial mushroom production [START_REF] Hatvani | Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma Species[END_REF]. Prophylactic management and disinfectant treatments may limit the epidemic but the severe level of chemical residue in food product together with the increasing risk of fungicide resistance led us to consider the cultivation of resistant A. bisporus varieties as a promising alternative.

Little is known about the resistance to T. aggressivum and its mechanism in A. bisporus. Although brown line formation and laccases production were described when Lentinula edodes or Pleurotus species are confronted to T. aggressivum, no such defense reactions of A. bisporus cultivars to T. aggressivum attack have been observed [START_REF] Mamoun | Interactions between the pathogen Trichoderma harzianum Th2 and Agaricus bisporus in mushroom compost[END_REF][START_REF] Mumpuni | Effect of metabolites produced by Trichoderma harzianum biotypes and Agaricus bisporus on their respective growth radii in culture[END_REF][START_REF] Savoie | Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2[END_REF]. Wide variations within A. bisporus strains in response to substrate infestation by T. aggressivum have been demonstrated. A few strains appeared slightly susceptible but no absolute resistance was observed [START_REF] Kerrigan | Trait diversity in wild Agaricus bisporus[END_REF][START_REF] Mamoun | Green mould disease in France: Trichoderma harzianum Th2 and other species causing damages on mushroom farms[END_REF].

The evaluation of green mould resistance appeared to be quite difficult and mostly influenced by the environment [START_REF] Savoie | Factors influencing the competitive saprophytic ability of Trichoderma harzianum Th2 in mushroom (Agaricus bisporus) compost[END_REF]. Many components are involved in the interaction between A. bisporus and T. aggressivum. Some of them are due to abilities of the mushroom strains to counteract growth of moulds by producing fungistatic compounds and to resist to growth limiting compounds (enzymes, volatile compounds, small non-volatile molecules) produced by Trichoderma sp. In a previous study, an in vitro test using as media supplement a commercial product (Lysing Enzyme®) containing cell wall lytic enzymes and undefined metabolites allowed to identify strains of A. bisporus able to resist to this growth limitation [START_REF] Savoie | Susceptibility of European populations of Agaricus bisporus to Trichoderma metabolites[END_REF]. For exploiting such abilities as a component of resistance in breeding programs, the knowledge of its genetic basis is a prerequisite.

The objective of the present work was to investigate the genetic control of the resistance to Trichoderma lytic enzymes and metabolites in an A. bisporus progeny using QTL analysis. Progresses in the understanding of this particular fungal-fungal interaction and prospects for mushroom breeding are discussed.

MATERIALS and METHODS

Offspring. The population used in our study consisted of 103 second generation hybrids obtained by crossing the homokaryotic offspring (H i ) of the hybrid JB3-83 x U1-7 (H) with the homokaryon U1-2 [START_REF] Callac | Bsn-t alleles from French field strains of Agaricus bisporus[END_REF][START_REF] Imbernon | BSN, the primary determinant of basidial spore number and reproductive mode in Agaricus bisporus, maps to chromosome I[END_REF][START_REF] Kerrigan | The heterothallic life-cycle of Agaricus bisporus var burnettii and the inheritance of its tetrasporic trait[END_REF]. The strains JB3, U1, and the first generation hybrid H were used as controls in the experiments.

Experimental design and phenotypic evaluation. The experiment was performed according to a methodology adapted from Savoie et al. [START_REF] Savoie | Susceptibility of European populations of Agaricus bisporus to Trichoderma metabolites[END_REF] and described in Fig. 1.

Cristomalt (1%) and agar (1.5%) was used as the basic media. Lysing Enzyme® (LE) from T. harzianum (Sigma, L1412) is a lyophilized powder obtained from cultures of T. harzianum, containing about 80% protein, with cellulase, chitinase and protease activities. Supplementation of Cristomalt Agar with LE was made by diluting the product in water (0.75 g in 10 mL), sterilizing by filtration (0.22 μm), and adding this solution to 1 L of autoclaved media. Un-supplemented controls contained 10 mL of sterile water.

For each strain of A. bisporus, a mycelium agar plug of pre-culture (5 mm-∅) was placed in the centre of Petri dishes containing malt-agar with or without LE supplementation. The inoculated media were incubated at 25 °C in the dark for 14 days and at the end of this period, the diameters of mycelial colonies were recorded (two perpendicular diameters per colony). The mean of these two values stood for mycelial growth rate parameter expressed in mm. The measures obtained from this first set of supplemented (L) or unsupplemented malt agar plates were designated as Culture 1 growth rates (C1). Culture 2 mycelial growth rates (C2) were obtained as above after taking inoculation disks from the margin of 14-day old Culture 1 plates incubated under the same conditions. Four replicate Petri dishes were inoculated for each strain in both Culture 1 and Culture 2 sets. Two additional variables were calculated: C2L/C1 and C2L/C1L [START_REF] Savoie | Susceptibility of European populations of Agaricus bisporus to Trichoderma metabolites[END_REF].

Strains maintained in collection plates

Pre-culture on malt-agar media Culture on malt-agar unsupplemented (mycelial growth rate)

C1

Culture on malt-agar with Lysing-Enzyme supplementation (mycelial growth rate)

C1L

Culture on malt-agar with Lysing-Enzyme supplementation (mycelial growth rate) C2L, C2L/C1, C2L/C1L The genetic linkage map used for QTL mapping was previously developed by Foulongne-Oriol et al. [START_REF] Foulongne-Oriol | An expanded genetic linkage map of an intervarietal Agaricus bisporus var. bisporus x A. bisporus var. burnettii hybrid based on AFLP, SSR and CAPS markers sheds light on the recombination behaviour of the species[END_REF]. QTL detection was performed by composite interval mapping (CIM) with QTL Cartographer software [START_REF] Basten | QTL Cartographer, version 1.17[END_REF], using B1 design. A LOD threshold of 2.5 was used to declare a QTL significant. The most likely position of each QTL was defined by the LOD score peak and a LOD-1 confidence interval. MapChart software [START_REF] Voorrips | MapChart: software for the graphical presentation of linkage maps and QTLs[END_REF] was used to produce visualisations of chromosomes carrying QTLs. The percentages of phenotypic variation explained by each QTL (R²) and their individual additive effects (a) were given by the model. The phenotypic variation explained by all detected QTLs (R²T in %) was determined by multiple regression analysis, using the closest markers to the LOD score peak for each putative QTL as explanatory variables.

RESULTS and DISCUSSION

Traits evaluation. The C1 variable represented the behaviour of the mycelium in control conditions. The LE supplementation significantly affected the mycelia growth rate (Fig. 2A). Among the 103 hybrids progeny, 6% showed no significant difference between C1 and C2L, suggesting that these strains are highly tolerant to LE effects. Conversely, no lethal effect was observed. The level of tolerance of a strain could be estimated as the ratio C2L/C1 [START_REF] Savoie | Susceptibility of European populations of Agaricus bisporus to Trichoderma metabolites[END_REF]. Twenty percent of the strains had similar mycelium growth rates for Culture 2 + LE and Culture 1 + LE, depicting a potential capacity of adaptation to LE effects [START_REF] Savoie | Susceptibility of European populations of Agaricus bisporus to Trichoderma metabolites[END_REF]. This trait was assessed using the ratio C2L/C1L. Six percent of the strains showed higher significant mycelial growth rate on Culture 2 + LE than on Culture 1 + LE but lower than controls as illustrated in Fig. 2B. Phenotypic variation. Each trait under consideration showed a continuous distribution (Fig. 3) suggesting a quantitative inheritance. This confirmed also that the trait evaluation procedure used was reliable to assess variability in the behaviour of the hybrids progeny facing Trichoderma lytic enzymes and metabolites. For C1L and C2L, we observed a shift of the distribution towards the lowest values of the traits (Fig. 2, Table 1), demonstrating that the LE supplementation affects mycelium growth rates. In control condition C1, 77% of the hybrids showed a mycelium growth higher than 55 mm. This percentage decreased to 38 % and 30 % for C1L and C2L respectively.

The ANOVAs revealed significant genotype effects for each trait. Post-hoc tests of ANOVAs showed that for all the traits except C2L/C1L, the two parents U1 and Jb3 were not significantly different from each other. The first generation hybrid H was found significantly better than the best parent for each trait, suggesting a strong heterosis effect. Transgressive segregants towards lowest but also in some cases highest values of trait were found among hybrids (Table 1). The broad-sense heritabilities based on genetic variance were high, ranging from 0.60 (C2L/C1) to 0.90 (C2L) indicating that the observed variation between strains is mostly under genetic control. This result was consistent with the in vitro experiment which allowed quite controlled environmental conditions.

The mycelial growth rates on LE supplemented media (C2L, C1L) were strongly correlated with the mycelial growth rate on control C1 (Fig. 3). The parameter of tolerance (C2L/C1) strongly correlated with the parameter of adaptation (C2L/C1L) but these two parameters were not found correlated with C1. QTL results. A total of 3, 2 and 2 QTLs were detected using the 2.5 LOD threshold for C1, C2L/C1, C2L/C1L respectively (Table 2, Fig. 4) with individual R² ranging from 5.4 (QC1-I) to 15.6 % (QC1-IV). The total phenotypic variation explained by all the QTLs (R²t) ranged from 23.4% to 26.7%. The remaining phenotypic variation which could not be explained by the detected QTL might come from other source such as QTL with too small effect to be detected in this experiment or QTL masked by differential interaction effect between segregating nuclei and the constant nucleus [START_REF] Larraya | Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus[END_REF]. The number of QTL and their effects range for the mycelium growth rate in control condition (C1) were quite comparable to those found for Pleurotus ostreatus [START_REF] Larraya | Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus[END_REF]. (a) nearest marker upstream to the LOD score peak (b) LOD score value at the LOD score peak (c) parental allele that contributes to increase the trait Although 7 QTLs were detected for all the traits, only 4 genomic regions were highlighted (Fig. 4). Thus, the overlapping of confidence intervals revealed 2 clusters of QTLs that governed distinct traits, on LGIV and LGXIII. A genomic region of approximately 20 cM around the marker PR041 was detected for each trait (C1, C2L/C1, C2L/C1L), with the highest LOD score value. Two other genomic regions on LGI and LGVI were found each one specific to one trait, C1 and C2L/C1 respectively. These results, together with the correlation parameters suggested that either the tolerance or the adaptation were tightly linked to the growth rate ability. No significant epistatic interaction was detected.

The favourable allele that contributes to increase the trait came from either U1-7 or Jb3-83. This result was not surprising regarding the phenotypic value of each parent U1, JB3 and the H hybrid (Table 1). It suggested a complex genotypic makeup of the tolerance to Trichoderma lytic enzymes and metabolites. It confirmed also that potential source of resistance to green mould could be found in both wild and cultivated strains [START_REF] Mamoun | Green mould disease in France: Trichoderma harzianum Th2 and other species causing damages on mushroom farms[END_REF].

As the cap colour is a segregating trait in the studied progeny [START_REF] Callac | Evidence for PPC1, a determinant of the pilei-pellis color of Agaricus bisporus fruitbodies[END_REF], our results also confirmed that the adaptation to Trichoderma lytic enzymes and metabolites, a trait contributing to the green mould resistance, was not related to the brown cap colour [START_REF] Mamoun | Green mould disease in France: Trichoderma harzianum Th2 and other species causing damages on mushroom farms[END_REF][START_REF] Guthrie | Chitinase production during interaction of Trichoderma aggressivum and Agaricus bisporus[END_REF]. Indeed, no QTL related to the traits studied here was found in the vicinity of the PPC1 locus on LGVIII.

CONCLUSIONS

This was the first study dealing with QTLs related to resistance to the green mould disease in basidiomycetes. The procedure used here can be applied to other Trichoderma-fungus interaction to identify genomic regions involved in the resistance to this competitor causing significant damages during cultivation of many mushroom species.

We have demonstrated that the ability to resist to T. harzianum lytic enzymes and metabolites in A. bisporus was quantitatively inherited and under oligogenic control. Besides, we have identified one robust QTL on LGIV that lets presume a key role played by this particular part of the genome. The recent release of the A. bisporus genome sequence will help to go further in the understanding of the growth rate control.

Our results suggested that the genetic factors involved in the ability to resist or adapt to Trichoderma lytic enzymes and metabolites are linked to the fitness of the A. bisporus strains. Both traits might be involved in resistance to the green mould disease. The incidence of severity of the disease might be reduced if A. bisporus successfully colonizes the compost before T. aggressivum develops [START_REF] Mamoun | Interactions between the pathogen Trichoderma harzianum Th2 and Agaricus bisporus in mushroom compost[END_REF][START_REF] Beyer | Evaluation of epidemiological factors and mushroom substrate characteristics influencing the occurrence and development of Trichoderma green mold[END_REF]. During infection, at the contact point between the two fungi, the strains of A. bisporus having the ability to resist or to adapt to the lytic enzymes and metabolites with antibiotic effects produced by T. aggressivum are better armed to be less affected than others.

Further work is needed to correlate closely the present laboratory test to the resistance to T. aggressivum in compost based cultivation systems, but our results offer opportunities to breed and select for A. bisporus strains with increased potentials to outcompete the green mould.

Figure 1 .=

 1 Figure 1. Methodology used to evaluate the effects of Trichoderma metabolites (Lysing-Enzyme) on mycelial growth rate of Agaricus bisporus strains, adapted from Savoie and Mata [13].

Figure 2 .

 2 Figure 2.A-Box plot distribution of mycelial growth rate under the three tested conditions: control (C1), Culture 1 + LE (C1L), Culture 2 +LE (C2L) B-Behaviour of some contrasted hybrids, together with the parental strains for C1, C1L and C2L

Figure 3 .

 3 Figure 3. Frequency distribution histograms and scatter-plot matrix of Trichoderma metabolites resistance traits in the second generation hybrids progeny. Pearson correlation values between each pair of traits are indicated in the upper left corner of the scatter-plots (ns non significant, * significant at p<0.05, *** significant at p<0.001)

Figure 4 .

 4 Figure 4. Map positions of the significant QTLs on the A. bisporus linkage map. A dash indicates LOD score peak position. The vertical bars represent the confidence intervals of the QTL (LODmax-1). QTLs for C1, C2L/C1, C2L/C1L are indicated in black, red and green respectively.

Table 1 .

 1 Means, standard deviation (sd), range and heritability for Trichoderma metabolites resistance traits in the parental strains U1 and JB3, the H hybrid, and the second generation hybrids progeny (n=103).

	____________________________________________________________________________
		Parents			Offspring
	Trait	U1	JB3	H	mean	sd	range	h²
	C1	54.25 B	52.12 B	69.75 A	59.07	10.68	16.5 C -77.16 A	0.84
	C1L	30.50 B	28.87 B	66.00 A	41.27	12.43	16.12 C -67.83 A	0.88
	C2L	18.00 C	21.25 C	54.75 B	33.74	14.38	11.25 D -64.25 A	0.90
	C2L/C1	0.33 C	0.41 C	0.79 B	0.56	0.19	0.22 C -1.03 A	0.60
	C2L/C1L	0.59 C	0.74 B	0.84 B	0.79	0.17	0.33 D -1.22 A	0.63
	Capital letter in exponent indicates significant means differences revealed by post-hoc ANOVA tests (p<0.05).

Table 2 .

 2 Summary of QTL analyses for Trichoderma metabolites resistance traits

	____________________________________________________________________________
	Trait	QTL name	LG	Marker (a)	LOD (CIM) (b) additive effect	parental allele (c)	R² (%)	R²T (%)
	C1	QC1-I	I	PR007	2.9	5.33	U1-7	5.4	26.7
		QC1-IV	IV	EAAMAGt	5.3	10.3	Jb3-83	15.6
		QC1-XIII	XIII	PR003	3.2	7.2	U1-7	8.3
	C2L/C1	QC2L/C1-IV	IV	PR041	3.7	0.2	Jb3-83	13.9	23.4
		QC2L/C1-VI	VI	ECGMCCs	2.9	0.33	Jb3-83	10
	C2L/C1L	QC2L/C1L-IV	IV	EAAMAGt	4.3	0.14	Jb3-83	15.3	23.8
		QC2L/C1L-XIII	XIII	ECGMACf	3.1	0.12	U1-7	9.4

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