S Niang

M Charlton

J J Choi

M Chung

P Cladé

P Comini

P Crivelli

P-P Crépin

O Dalkarov

P Debu

L Dodd

A Douillet

P Froehlich

J Gafriller

S Guellati

J Heinrich

P-A Hervieux

L Hilico

A Husson

P Indelicato

G Janka

S Jonsell

J-P Karr

B H Kim

E-S Kim

S K Kim

A Kleyheeg

Y Ko

T Kosinski

N Kuroda

B Latacz

H Lee

J Lee

A M M Leite

E Lim

L Liszkay

T Louvradoux

D Lunney

K Lévêque

G Manfredi

B Mansoulié

M Matusiak

G Mornacchi

V V Nesvizhevsky

F Nez

R Nishi

S Nourbaksh

K H Park

N Paul

P Pérez

B Radics

C Regenfus

S Reynaud

J-Y Roussé

A Rubbia

J Rzadkiewicz

Y Sacquin

F Schmidt-Kaler

M Staszczak

B Tuchming

B Vallage

D P Van Der Werf

A Voronin

A Welker

S Wolf

D Won

S Wronka

Y Yamazaki

K-H Yoo

Gbar Collaboration

C J Baker

Accumulation of positrons from a LINAC based source

Accumulation of positrons from a LINAC based source

Introduction

The GBAR collaboration's aim is to measure the acceleration of antihydrogen atoms in the Earth's gravitational field [START_REF] Chardin | Proposal to measure the Gravitational Behaviour of Antihydrogen at Rest[END_REF]. In the GBAR scheme, one first produces H

+ ions [START_REF] Comini | Antihydrogen ion production from collisions between positronium and keV antiprotons for GBAR[END_REF], which are subsequently sympathetically cooled using D r a f t laser cooled Be + ions [START_REF] Walz | A proposal to measure antimatter gravity using ultracold antihydrogen atoms[END_REF]. When the anti-ion is cold enough, the extra positron is removed using a laser pulse and the acceleration is derived by timing the subsequent annihilation products of the H. The antiprotons are obtained from the ELENA decelerator at CERN [START_REF] Oelert | The ELENA project at CERN[END_REF], while positronium is produced by implantation of about 10 10 positrons into a mesoporous silica film at a kinetic energy of 4 keV [START_REF] Crivelli | Measurement of the orthopositronium confinement energy in mesoporous thin films[END_REF].

To obtain the necessary positrons, a 9 MeV LINAC accelerates electrons into a tungsten target equipped with a mesh moderator biased at 50 V. The resulting low energy positrons are then accumulated in a buffer gas trap (BGT) and transferred in a high field trap (HFT).

The BGT is a Penning-Malmberg trap based on the Greaves-Surko scheme [START_REF] Surko | Positron plasma in the laboratory[END_REF], and was developed at CEA Saclay [START_REF] Leite | Development of a buffer gas trap for the confinement of positrons and study of positronium production in the GBAR experiment[END_REF]. It uses N 2 as the trapping gas and CO 2 as a cooling gas. The trap is constructed from 3 sets of electrodes. In the first stage, inelastic collisions occur under a typical N 2 pressure of 10 -3 mbar, whilst the second one is used for the accumulation at a typical N 2 pressure of 10 -4 mbar, with the final stage used to store positrons for a dozens of seconds with a typical N 2 pressure of 10 -6 mbar. The magnetic field in this trap is ∼ 0.05 T and an electric potential between -140 V and 140 V can be applied to the electrodes, as appropriate.

The HFT [START_REF] Grandemange | Trapping and accumulation of positrons from a pulsed beam produced by a linear accelerator for gravitational interaction of antimatter study[END_REF] is a 5 T Penning-Malmberg trap with 27 electrodes each able to hold potentials between -1 kV and 1 kV. The base pressure is lower than 10 -9 mbar. This trap is used for long time storage.

In this article we present the outcome of optimisation studies to trap a large cloud of positrons. The final goal is to accumulate 10 10 positrons in 100 s.

Studies of the trapping conditions

After an accumulation time t, using the potential profile shown in Figure 1a, the positron cloud is axially compressed, and then dumped on a target at the exit of the trap (Figure 1b-c). The positron number is estimated by detecting the γ rays resulting from their annihilations on the target using a CsI detector. The signal is proportional to the number of accumulated positrons, which evolves as

N (t) = Rτ (1 -e -t τ
), with N the number of positrons, τ the lifetime and R the trapping rate. Some accumulation curves for e + in the second stage are presented in Figure 2a for different N 2 pressures as measured at the injection point of the system. If t τ , N (t) ≈ Rt, and the only parameter to optimise is then R. For the rest of the experiment we keep P N 2 = 6 mbar, with τ = 0.67(1) s and we accumulate for 100 ms before transferring to the third stage (Figure 1c-d). Currently, the energy spread in the longitudinal direction of the incoming positrons is σ = 8.8(3) but a spread of energy smaller than 3 eV [START_REF] Surko | Positron plasma in the laboratory[END_REF] would increase R and therefore the overall efficiency.

In the third stage, the e + lifetime is 16.5(5) s, as shown in Figure 3a, and since this is much greater than 100 ms a stacking procedure of the positrons from the second stage can be done. Figure 3b shows that, using a static potential, only 6 stacks can be accumulated in the well in a linear way, beyond which the stacks are lost. It also shows that by lowering the bottom of the well by 0.2 V per stack, a dozen stacks can be accumulated without any loss. Therefore 10 stacks are accumulated in 1 s (10 × 100 ms) before being transferred to the HFT.

To stack positrons from the BGT third stage in the HFT, one first uses a potential well made of 3 electrodes with -10 V as bottom of the well as shown in Figure 4a. Figure 4b shows that the well is saturated after 80 D r a f t stacks. To accumulate positrons in a linear way, a sequence is used in which the well is enlarged and deepened each time we approach a saturation limit.

In the first well, 50 stacks are accumulated, after which the bottom of the well is lowered. Then 150 stacks are added, and again the bottom of the well is lowered and then a further 200 stacks are added. Finally we lower the bottom of the well to reach a maximum, so in total, we can accept 560 stacks of e + from the third stage corresponding to 3.8(4) × 10 8 e + . We are planning to extend this process to trap even more positrons. With the knowledge that the LINAC based positron source provides 1.54(6) × 10 5 e + per pulse at a frequency of 200 Hz, the current overall efficiency is 2.3%.

Conclusion

We succeeded in trapping 3.8(4) × 10 8 e + in 560 s, which is a first important step for the development of the GBAR experiment. Improvements will be D r a f t made in the coming months to reach our next step of 10 10 positrons in a few hours, after which the goal will be to decrease this accumulation time, by increasing the e + flux at the entrance, and by improving efficiencies at the interfaces of the system.

Figure 1 :

 1 Figure 1: On-axis potentials used in the Buffer Gas Trap. (a) Accumulation in the second stage. (b) Axial compression of the cloud. (c) Transfer from the second to the third stage. (d) Re-trapping. (e) Preparation for transfer / dumping. (f) Dumping.

Figure 2 :

 2 Figure 2: (a) Number of e + as measured by the CsI detector, fit with N (t)+c. (b) R vs N 2 pressure. (c) Positron annihilation rate (λ = 1/τ ) vs N 2 pressure. Pressures in mbar as measured at the injection point of the system.

Figure 3 :

 3 Figure 3: (a) Lifetime of positrons in the third stage. (b) Measurement of positron signal for different accumulation conditions in stage 3 (see text for details). The bottom of the well is lowered by dV between each stack.

Figure 4 :

 4 Figure 4: (a) Subsequent on-axis potentials in the HFT. (b) Accumulation signal from HFT, for respectively a 1-, 2-, 3-and 4-step sequence.

Acknowledgement

This research was partially supported by CNES under convention N • 5100017115, the French National Research Agency (grant ANR-14-CE33-0008), the Swiss National Science Foundation (grant 20FL21 173597) and the ETH Zurich (grant ETH-46 17-1).


B H Kim

J J Choi

M Chung

P Cladé

P Comini

P Crivelli

P-P Crépin

O Dalkarov

P Debu

L Dodd

A Douillet

P Froehlich

S Guellati

J Heinrich

P-A Hervieux

L Hilico

A Husson

P Indelicato

G Janka

S Jonsell

J-P Karr

E-S Kim

S K Kim

Y Ko

T Kosinski

N Kuroda

B Latacz

H Lee

J Lee

A M M Leite

E Lim

L Liszkay

T Louvradoux

D Lunney

K Lévêque

G Manfredi

B Mansoulié

M Matusiak

G Mornacchi

V V Nesvizhevsky

F Nez

S Niang

R Nishi

S Nourbaksh

P Lotrus

K H Park

N Paul

P Pérez

B Radics

C Regenfus

S Reynaud

J-Y Roussé

A Rubbia

J Rzadkiewicz

Y Sacquin

F Schmidt-Kaler

M Staszczak

B Tuchming

B Vallage

D P Van Der Werf

A Voronin

A Welker

S Wolf

D Won

S Wronka

Y Yamazaki

K-H Yoo

Gbar Collaboration

Development of a PbWO 4 detector for single-shot positron annihilation lifetime spectroscopy at the GBAR experiment September 30, 2019

published or not. The documents may come

Development of a PbWO 4 detector for single-shot positron annihilation lifetime spectroscopy at the GBAR experiment

Introduction

The GBAR (Gravitational Behaviour of Antihydrogen at Rest) experiment [?, ?] aims to measure the acceleration of antihydrogen atoms in the terrestrial gravitational field. A first milestone of the experiment, is to produce (anti) hydrogen atoms and (anti) hydrogen ions (antihydrogen ion is formed by an antiproton and two positrons) with keV or less kinetic energy. First, an antihydrogen atom is produced in the collision between an antiproton and an o-Ps. Then, an antihydrogen ion is produced in the collision between the antihydrogen atom and a second o-Ps. These two collisions occur in a dense o-Ps cloud (up to 10 12 Ps/cm 3 ) created inside a small cavity (typically 1×1×10 mm). These processes are equivalent to the production of hydrogen atoms and ions by proton-positronium and hydrogen-positronium charge exchange.

A PWO detector has been developed to measure the density of o-Ps in the target region using the method of single shot positron annihilation lifetime spectroscopy (SSPALS) [?]. PWO is a dense crystal scintillator that can absorb all the energy of a γ ray from the decay of o-Ps without the Compton edge made by the γ ray escaping with partial energy loss in the crystal. The assembly of a PWO crystal and a photomultiplier tube (PMT) generates only a few photo-electrons per γ ray. This allows a large dynamic range by changing the PMT gain. The PWO detector can measure the quantity of o-Ps and the intensity of the positron beam at the same time due to its short decay time. To understand the systematic uncertainties of the measurement, a simulation based on Geant4 with the Penelope library (Geant v4.10.03) [?, ?] and a positronium library [?] has been performed. Experimental methods to reduce the systematic uncertainties have been developed to achieve less uncertainty than in a previous cross section measurement of the hydrogen atom production [?].

In this report, the development of the PWO detector system is described.

Experimental details

A positron beam is generated by an electron linac. The particles are cooled and accumulated in Penning-Malmberg traps [?]. After extraction from the trap, the positron pulse is accelerated by a switched drift tube and focused by Einzel lenses to hit a positron/positronium converter made of mesoporous silica [?, ?] as shown in the schematic view in Fig. ??. The target region of the collision between the o-Ps cloud and the antiproton beam is located at the center of the reaction chamber. For the first tests, performed at relatively low beam intensity compared with the required intensity for antihydrogen ion production, a flat positronium converter with size 2×2 cm 2 is used. An assembly of a microchannel plate (MCP) and a phosphor screen of 1 cm diameter is used for the measurement of the positron beam profile at the target position. The PWO detector measures the γ rays from the annihilation of positrons and the decay of o-Ps at the same time. The PWO detector is placed 10 cm behind the positronium converter. It is assembled from a PWO crystal of 4×4×3.8 cm 3 size segmented to 4 pieces and a PMT (H7195, Hamamatsu). A plastic scintillator is installed at the backside of the reaction chamber as a reference detector.

Beam test and Compton background measurement

The raw signal from the PWO detector is shown in Fig. ?? when the positron beam hits the positronium converter (solid line) or the MCP (diagonal filled).

The FWHM of the positron annihilation signal is less than 20 ns which is sharp enough to distinguish it from the o-Ps decay signal that appears as an exponential decay curve with 142 ns lifetime. The two lines show a clear indication of o-Ps production and decay. The small peak near 0.8µs is expected as the after pulse from PMT [?]. The switching noise from the switched drift tube appears near 0.1µs.

The simulation shows that the most important uncertainty comes from Compton scattered γ rays. A Compton background fraction about 16 -21% of the total signal is expected when the distance between the positronium converter and the PWO detector is 15 -25 cm. In the SSPALS method, the deposited energy of single γ rays is not measured. Thus, the Compton background must be subtracted properly from the measured intensity. The simulation shows that the measured beam intensity with the current setup is over-estimated by up to 21%. To understand the Compton background, a tungsten block of 4×4×4 cm 3 is installed in front of the PWO detector at a distance 11 cm from the positronium converter. The γ rays originating from the annihilation of positrons and the decay of o-Ps at the target position can not reach the PWO detector other than by Compton scattering on obstacles such as the chamber itself. The measured γ intensity is reduced by 21% when the tungsten block is mounted and the distances between the positronium converter and the PWO detector is 20 and 25 cm, as compared in the absence of the tungsten block, while the simulation gives 19% for same distances. We use a cross-shaped assembly of the four small PWO detectors to determine the amount of escaped o-Ps as shown in Fig. ??. The four small PWO detectors are mounted along the four sides of a tungsten block (2×2×4 cm 3 ). In front of the detector assembly, another tungsten block of 2×4×4 cm 3 is attached in such a way that the detectors at the top and bottom have higher acceptance to γ rays originating from a decay position outside of the cavity. Detectors at the left and right sides have less dependent sensitivity on the decay position. Figure ?? shows the time distributions of the signal in the simulation for the detectors at the top/bottom sides of the tungsten block (top panel) and at the left/right sides (bottom panel). This time distribution is fitted using a likelihood method with a Gaussian probability density function (PDF) for positron annihilation and a Gaussian convoluted with an exponential decay PDF for o-Ps decay. The fitted lifetime is 152±2 ns for the left and right detectors and 263±12 ns for the top and bottom side detectors when the angular spread of o-Ps emission and reflection is the isotropic distribution. For the cosine distribution, the fitted lifetime is 150±2 ns for the left and right side detectors and 195±10 ns for the top and bottom detectors. Thus, for both angular distribution models, the observed lifetime is different for the two detector positions. We can then use the relation between the two apparent lifetime values to reduce the uncertainty of the positronium density measurement.

Detection system development

Conclusion

A PWO detector has been developed and tested to measure the positron beam intensity and the o-Ps density using SSPALS method for the GBAR experiment. We optimized the design in order to measure the positronium density in a cavity. We confirmed that the system for the positronium production and measurement operates properly. An accurate knowledge of the o-Ps density will allow one to extract a precise measurement of the antihydrogen and antihydrogen ion formation by antiproton-positronium, antihydrogenpositronium charge exchange cross sections.

Figure 1 :

 1 Figure 1: Schematic view of the target area. The tubes with gray color show the Einzel lenses for the positron beam focusing. The circular line outlines the reaction chamber. The positronium converter and the MCP assembly are translated by a linear drive to place them at the center of the chamber. A camera is placed downstream of the MCP assembly to image the positron beam profile. The PWO detector and a plastic scintillator (PS) are installed at the backside of the target region for the γ ray measurement.

Figure 2 :

 2 Figure 2: The raw signal distribution from the PWO detector (averaged data sample). The diagonal filled histogram shows the signal from the PWO detector when the e + beam hits the MCP and the solid line histogram shows the signal when the e + beam hits the positronium converter.

  When the cavity shaped target is used instead of a flat target, another source of uncertainty comes from the amount of o-Ps that decays outside the target cavity. The present simulation is based on a 20×2×2 mm 3 inner size. It has a 10×2 mm 2 entrance window made of 30 nm thick Si 3 N 4 . The top and bottom walls are made of SiO 2 . The wall opposite the entrance window is coated with a mesoporous silica film [?, ?]. The two other 2×2 mm 2 sides are free, allowing o-Ps to escape. The o-Ps is generated inside the target cavity at about 50 meV kinetic energy and moves during its lifetime with several reflections at the cavity surface. The angular spread of o-Ps emission [?] and reflection is not very well known. The angle with regard to the normal to the emission/reflection plane is generated with an isotropic or cosine distribution, resulting in different amounts of o-Ps escaping.

Figure 3 :

 3 Figure 3: The drawing of the cross-shaped detector assembly is shown in the top of the figure. The four PWO crystals are mounted along the four sides of a tungsten block. Each PWO crystal is assembled with cylindrical PMT. The target cavity drawn as a thin box is at the bottom of figure. The tungsten block is installed between the detector assembly and the target cavity.

Figure 4 :

 4 Figure 4: The simulated raw signal distribution of the detector assembly. The positron pulse is simulated by a Gaussian distribution and the angular distribution at emission or reflection of o-Ps is isotropic. The raw signal distributions at the left and right PWO detectors (top panel) and the top and bottom PWO detectors (bottom panel) are shown. The solid line shows the fitted PDF.

Acknowledgement

This work is supported by the Ministy of Education of the Republic of Korea and the National Research Foundation of Korea NRF-2016R1A6A3A11932936, NRF-2016R1A2B3008343 and French National Research Agency grant ANR-14-CE33-0008.


Elsa Ballini
email: elsa.ballini@supagro.fr

Muriel Tavaud
email: muriel.tavaud@supagro.fr

Aurélie Ducasse
email: aurelie.ducasse@inra.fr

Etienne Paux
email: etienne.paux@inra.fr

Jonathan ; Kitt
email: jonathan.kitt@inra.fr

Gilles Charmet
email: gilles.charmet@inra.fr

Delphine Audigeos
email: d.audigeos@arvalis.fr

Pierre P Roumet
email: pierre.roumet@inra.fr

Jacques David
email: jacques.david@supagro.fr

Jean-Benoit Morel
email: jean-benoit.morel@cirad.fr

Ballini

Ducasse Aurélie

PAUX Sanchez Dimitri
email: dimitri.sanchez@supagro.fr

Kitt Jonathan

Charmet Gilles

Audigeos Delphine

DAVID Roumet Pierre

Jean -Benoit

Morel Jean

Genome wide association mapping for resistance to multiple fungal pathogens in a panel issued from a broad composite cross-population of tetraploid wheat Triticum turgidum

Keywords: Triticum turgidum, wheat blast, leaf rust, septoria tritici blotch, septoria nodorum blotch, resistance inducers, GWAS, chitin

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

Introduction

Durum wheat (Triticum turgidum subsp durum) is an important cereal in Mediterranean diets, mainly used for semolina and pasta. Because it is relatively well-adapted to arid climates, more than 50% of world production is in the Mediterranean region [START_REF] Royo | and 2. Durum wheat breeding. Current approaches and future strategies[END_REF]. Fungal diseases of durum wheat cause significant crop loss, and the use of fungicides to control infection can have harmful effects on the environment and farmers [START_REF] Figueroa | A review of wheat diseases-a field perspective[END_REF]. In France, the main fungal diseases infecting durum wheat are septoria (Fones and Gurr 2015) and leaf rust [START_REF] Goyeau | Low diversity and fast evolution in the population of Puccinia triticina causing durum wheat leaf rust in France from 1999 to 2009, as revealed by an adapted differential set[END_REF]. European durum wheat is also facing a new fungal threat from the emergence of wheat blast [START_REF] Ceresini | Wheat Blast: Past, Present, and Future[END_REF]. The use of varieties harboring particular resistance genes can be an effective means of managing fungal disease threats. However, it is often not sustainable [START_REF] Priyamvada | Durable resistance in wheat[END_REF] as fungal pathogens evolve rapidly to overcome host resistance [START_REF] Mcdonald | Pathogen population genetics, evolutionary potential and durable resistance[END_REF]. The development of more effective and sustainable use of resistant varieties requires a wider range of resistance genes, and markers associated with allelic forms of these genes, for exploitation in breeding programs [START_REF] Royo | and 2. Durum wheat breeding. Current approaches and future strategies[END_REF]. Genetic mapping of resistance genes to these diseases has mostly been done in the hexaploid bread wheat (Triticum aestivum) because of its greater economic interest. However, fungal diseases of wheat are often host specific, meaning that results obtained in studies of bread wheat are often not applicable to durum wheat [START_REF] Karisto | Genetic variation for virulence and resistance in the Wheat-Mycosphaerella graminicola Pathosystem I. Interactions between pathogen isolates and host cultivars[END_REF][START_REF] Goyeau | Distribution of Pathotypes with Regard to Host Cultivars in French Wheat Leaf Rust Populations[END_REF]. Durum wheat breeders, therefore, lack the specific markers of resistance alleles allowing for better-designed breeding programs to combat these diseases [START_REF] Ghaffary | Practical breeding strategies to improve resistance to Septoria tritici blotch of wheat[END_REF].

Disease etiology of septoria can be confusing because two different pathogens cause similar necrotic symptoms and can co-infect the same plant: Septoria tritici blotch (STB) caused by Zymoseptoria tritici and Septoria nodorum blotch (SNB) caused by Parastagonospora nodorum. There are an estimated €1.7 bn in economic losses annually due to septoria diseases on durum and bread wheat in Europe (Fones and Gurr 2015). Although STB appears to have gradually replaced SNB in France, SNB remains an important threat for durum wheat [START_REF] Bearchell | Wheat archive links long-term fungal pathogen population dynamics to air pollution[END_REF].

At least 21 STB resistance genes have been identified in bread wheat as well as 89 Meta-QTL [START_REF] Brown | Genetics of resistance to Zymoseptoria tritici and applications to wheat breeding[END_REF].

However, most likely due to host specificity, very few of these markers are useful in durum wheat breeding programs [START_REF] Ghaffary | Practical breeding strategies to improve resistance to Septoria tritici blotch of wheat[END_REF]. Recently, the bread wheat resistance gene Stb6, which codes for a wall-associated receptor kinase (WAK), and mapped to chromosome 3A [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF], was shown to be locked in a genefor-gene coevolutionary interaction with the associated Z. tritici avirulence gene AvrStb6 [START_REF] Zhong | A small secreted protein in Zymoseptoria tritici is responsible for avirulence on wheat cultivars carrying the Stb6 resistance gene[END_REF].

Resistance to the necrosis phenotype caused by STB has been a focus of research. However, resistance may independently arise from reducing pathogen reproduction, as measured by a reduction in the number of fungal fruiting bodies called pycnidia [START_REF] Karisto | Genetic variation for virulence and resistance in the Wheat-Mycosphaerella graminicola Pathosystem I. Interactions between pathogen isolates and host cultivars[END_REF]Fones et al. 2015).

Both Z. tritici and P. nodorum produce toxins during infection, partly explaining necrotic symptoms and host susceptibility, with the absence or modification of proteins targeted by the toxins conferring resistance. Toxins can also be neutralized by detoxification mechanisms. However, few loci associated with toxin resistance have been identified in durum wheat [START_REF] Virdi | New Insights into the Roles of Host Gene-Necrotrophic Effector Interactions in Governing Susceptibility of Durum Wheat to Tan Spot and Septoria nodorum Blotch[END_REF]). In the case of SNB, nine toxin/host pairs have been identified [START_REF] Francki | Improving Stagonospora nodorum Resistance in Wheat: A Review[END_REF][START_REF] Haugrud | Genetics of Variable Disease Expression Conferred by Inverse Gene-For-Gene Interactions in the Wheat-Parastagonospora nodorum Pathosystem[END_REF]. The wheat gene Snn1, coding for a WAK on chromosome 1B, has recently been cloned [START_REF] Shi | The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease[END_REF]) and the associated S. nodorum effector toxin Tox1 isolated [START_REF] Liu | The Cysteine Rich Necrotrophic Effector SnTox1 Produced by Stagonospora nodorum Triggers Susceptibility of Wheat Lines Harboring Snn1[END_REF]. The absence of Snn1 reduces necrosis and strengthens resistance. Thus, toxin infiltration, instead of classical spore inoculation, is an efficient way of identifying resistance to SNB in wheat [START_REF] Shi | The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease[END_REF].

Another important disease of durum wheat is leaf rust (LR), caused by the biotroph Puccinia triticina. While durum wheat appears more resistant to leaf rust than bread wheat, aggressive strains of leaf rust have recently appeared in Europe that are detrimental to durum wheat production [START_REF] Goyeau | Distribution of Pathotypes with Regard to Host Cultivars in French Wheat Leaf Rust Populations[END_REF]. Moreover, none of the 100 representative French elite lines are resistant to the leaf rust recently collected in a durum wheat production area in France (personal data). Leaf rusts infecting durum wheat are genetically different from those infecting bread wheat [START_REF] Goyeau | Distribution of Pathotypes with Regard to Host Cultivars in French Wheat Leaf Rust Populations[END_REF], and only twelve of the 68 genes associated with LR resistance in bread wheat appear important in durum wheat infections [START_REF] Aktar-Uz-Zaman | Genetic analysis of rust resistance genes in global wheat cultivars: an overview[END_REF][START_REF] Aoun | Genome-Wide Association Mapping of Leaf Rust Response in a Durum Wheat Worldwide Germplasm Collection[END_REF]). Thus, it is important to identify durum wheat resistance genes and QTL specifically associated with durum wheat LR isolates for wheat improvement [START_REF] Ordonez | Virulence Phenotypes of a Worldwide Collection of Puccinia triticina from Durum Wheat[END_REF].

Wheat blast (WB) is an emerging hemibiotrophic disease caused by the Magnaporthe oryzae Triticum (MoT) pathotype (Anamorph Pyricularia oryzae Triticum) [START_REF] Ceresini | Wheat blast: from its origins in South America to its emergence as a global threat[END_REF]. The disease emerged in Brazil in 1985 and has recently spread to Bangladesh [START_REF] Ceresini | Wheat blast: from its origins in South America to its emergence as a global threat[END_REF]. Fungicides are relatively ineffective against WB, and the nine resistance genes that have been identified lack fine-mapping with markers easily exploitable by breeders [START_REF] Anh | Rmg8 and Rmg7, wheat genes for resistance to the wheat blast fungus, recognize the same avirulence gene AVR-Rmg8[END_REF][START_REF] Krattinger | The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice[END_REF][START_REF] Tagle | Rmg7, a New Gene for Resistance to Triticum Isolates of Pyricularia oryzae Identified in Tetraploid Wheat[END_REF]. Thus, these four fungal diseases are emerging as major threats to durum wheat production, they present different types of trophic development, and further genetic information is required to develop durable resistance management in the field [START_REF] Figueroa | A review of wheat diseases-a field perspective[END_REF].

Induction of plant defenses, for instance, through the use of chitin, is an alternative or complementary technique used to protect plants from some fungal diseases, including rust [START_REF] Bohland | Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate[END_REF][START_REF] Popova | Influence of Chitosan Hybrid Derivatives on Induced Wheat Resistance to Pathogens with Different Nutrition Strategies[END_REF][START_REF] Vander | Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves[END_REF]. Indeed such induction increase basal immunity [START_REF] Vander | Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves[END_REF][START_REF] Naz | Pathogenesis-Related Protein Expression in the Apoplast of Wheat Leaves Protected Against Leaf Rust Following Application of Plant Extracts[END_REF], and a positive effect of prophylactic induction in bread wheat has been reported [START_REF] Cruz | Foliar application of products on the reduction of blast severity on wheat[END_REF]. However, to our knowledge, no study on wheat has examined genetic variation in resistance following prophylactic induction treatment.

In recent years with the full sequence availability of the wild emmer (T. turgidum subsp dicoccoides Thell.) and durum wheat genomes (Appels et al. 2018;[START_REF] Avni | Wild emmer genome architecture and diversity elucidate wheat evolution and domestication[END_REF][START_REF] Maccaferri | Durum wheat genome highlights past domestication signatures and future improvement targets[END_REF][START_REF] Ling | Genome sequence of the progenitor of wheat A subgenome Triticum urartu[END_REF], there is a shift towards genomics-assisted breeding. Genome-wide association studies (GWAS) have already been used to map resistance to these four diseases in bread wheat [START_REF] Crossa | Association analysis of historical bread wheat germplasm using additive genetic covariance of relatives and population structure[END_REF][START_REF] Gurung | Genome-Wide Association Study Reveals Novel Quantitative Trait Loci Associated with Resistance to Multiple Leaf Spot Diseases of Spring Wheat[END_REF][START_REF] Jighly | Genomic regions conferring resistance to multiple fungal pathogens in synthetic hexaploid wheat[END_REF][START_REF] Kertho | Genome-Wide Association Mapping for Resistance to Leaf and Stripe Rust in Winter-Habit Hexaploid Wheat Landraces[END_REF][START_REF] Tagle | Rmg7, a New Gene for Resistance to Triticum Isolates of Pyricularia oryzae Identified in Tetraploid Wheat[END_REF][START_REF] Naz | Pathogenesis-Related Protein Expression in the Apoplast of Wheat Leaves Protected Against Leaf Rust Following Application of Plant Extracts[END_REF][START_REF] Gao | Genome Wide Association Study of Seedling and Adult Plant Leaf Rust Resistance in Elite Spring Wheat Breeding Lines[END_REF][START_REF] Adhikari | Association Mapping of Quantitative Resistance to Phaeosphaeria nodorum in Spring Wheat Landraces from the USDA National Small Grains Collection[END_REF][START_REF] Juliana | Genome-wide association mapping for resistance to leaf rust, stripe rust and tan spot in wheat reveals potential candidate genes[END_REF][START_REF] Muqaddasi | Genome-wide Association Mapping and Prediction of Adult Stage Septoria tritici Blotch Infection in European Winter Wheat via High-Density Marker Arrays[END_REF]. However, fewer studies of the genetic architecture of disease resistance in tetraploid durum wheat have been carried out [START_REF] Letta | Association Mapping Reveals Novel Stem Rust Resistance Loci in Durum Wheat at the Seedling Stage[END_REF][START_REF] Liu | Genome-wide association mapping reveals a rich genetic architecture of stripe rust resistance loci in emmer wheat (Triticum turgidum ssp. dicoccum)[END_REF][START_REF] Maccaferri | Association mapping of leaf rust response in durum wheat[END_REF][START_REF] Kidane | Genome-Wide Association Study of Septoria tritici Blotch Resistance in Ethiopian Durum Wheat Landraces[END_REF][START_REF] Aoun | Genome-Wide Association Mapping of Leaf Rust Response in a Durum Wheat Worldwide Germplasm Collection[END_REF]. While elite populations have often been used, the severe genetic bottleneck occurring during domestication [START_REF] Haudry | Grinding up wheat: a massive loss of nucleotide diversity since domestication[END_REF]) may have left these populations with fewer resistance alleles than wild and primitive accessions. Composite cross-populations have been proposed as an effective alternative to remobilize diversity by crossing a large number of parents and allowing recombination [START_REF] Enjalbert | Dynamic management of crop diversity: From an experimental approach to on-farm conservation[END_REF].

Thus, the EPO durum wheat lines, derived from an Evolutionary Pre-breeding pOpulation, has the advantage of being highly diversified and weakly structured, making it an ideal panel for GWAS [START_REF] David | Genotyping by sequencing transcriptomes in an evolutionary prebreeding durum wheat population[END_REF].

In this study, we used the EPO panel in a series of GWAS with the goal of identifying resistance loci, potentially rare or absent in modern domesticated populations, to three main fungal pathogens of durum wheat (STB, SNB, and LR) as well as to emerging WB disease. Moreover, associations with resistance to LR was evaluated both with and without prophylactic induction by chitin, an inducer of immunity, in order to evaluate the impact of this treatment on resistance.

Material and Methods

Plant Panels

The Evolutionary Pre-breeding pOpulation (EPO) is a genetically diverse, outcrossed tetraploid wheat population founded in 1997. It has since been cultivated with an outcrossing rate of 10%, artificially managed by maintaining sterile male plants in the population [START_REF] David | Genotyping by sequencing transcriptomes in an evolutionary prebreeding durum wheat population[END_REF]. In brief, genetic diversity in this population arises from the first generation artificial crossing of accessions from several subspecies of Trititum turgidum (subspecies dicoccoides, dicoccum, turgidum, polonicum, carthlicum, and durum). In 2009, a set of 180 lines was derived from the EPO and fixed by five generations of single seed descent. These lines are a subset of 425 original lines, and were chosen to eliminate genetic redundancy arising from sister lines derived from the same lineage.

A durum wheat collection of seventy-eight commercial lines provided by French private companies and genotyped on the 420 K Affymetrix Axiom genotyping array, TaBW280K [START_REF] Rimbert | High throughput SNP discovery and genotyping in hexaploid wheat[END_REF], was screened for allelic diversity at two of the detected QTL (STB_Nec3A and STB_Pyc3A). Out of them, fourteen lines were selected for phenotyping based on their genotypes at these QTL.

All experiments were carried out in a glasshouse under 16 hours of light at 24°C/20°C, as described by [START_REF] Ballini | Diversity and genetics of nitrogen-induced susceptibility to the blast fungus in rice and wheat[END_REF]. All wheat plants were grown in Neuhaus S soil with the addition of poudzolane (5L/70L soil) and slowreleasing fertilizer (Flocoat 90g/70L soil). Each pot was sown with six seeds of individual lines to ensure the growth of at least four seedlings.

Molecular Data

The EPO lines were genotyped using a 420 K Affymetrix Axiom genotyping array, TaBW280K, developed for the BreedWheat project [START_REF] Rimbert | High throughput SNP discovery and genotyping in hexaploid wheat[END_REF]. We used 168,725 high-resolution, publicly available reliable SNPs.

The genomic position for each SNP was identified by BLAST onto both the wild emmer (accession Zavitan) genome sequence (v2.0) and the Svevo sequence (v1.0) [START_REF] Maccaferri | Durum wheat genome highlights past domestication signatures and future improvement targets[END_REF][START_REF] Zhu | Improved genome sequence of wild emmer wheat Zavitan with the aid of optical maps[END_REF]. Missing data, occurring at a low rate of 3.84%, were imputed by binomial sampling of alleles using allele frequencies as parameters.

The Stb6 locus was genotyped using two previously published primer pairs and PCR conditions: 8311-16 (forward GCGACATGGTAGCTCAATCAAA; reverse TTCCTTCCATGGTCGGTAACTT) and 13609 (forward CTGAAAAAAAATACGAGGCCATGA: reverse GGCTTGTCTCCTCCGATCTTGT) [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF].

AvrStb6 was sequence based on the available sequence MG018996 using following primers designed for this project (TCCACCTGTCGCCGAATC and CTAGGAAAGTGGGCGCAAAA).

Fungal Inoculations and Symptoms

The 180 lines from the EPO population were phenotyped for resistance to the different pathogens following the same adapted experimental design. For each genotype, six seeds were sown per pot, and each plant that grew was phenotyped. According to limited space in the glasshouse, we maximised the number of independent lines to increase our chance to detect QTLs [START_REF] Korte | The advantages and limitations of trait analysis with GWAS: a review[END_REF]. Nevertheless, to control for potential spatial variability in the greenhouse and to get an estimate of the error variance (evaluation of the broad sense heritability and computation of BLUPs), we chose to apply a p-rep design [START_REF] Cullis | On the design of early generation variety trials with corrected data[END_REF]) instead of repeating control checks. Thus, 20% of the genotypes were grown twice and these replicates distributed at random in the different sublocks (pots ware assemble in 16 pots trays). Pots were randomly divided and arranged in the greenhouse. All inoculations or infiltrations were conducted at seedling stage on 2-weeks old plants.

SNB resistance was evaluated using two complementary phenotyping methods: infiltration with Tox1 toxin and spore inoculation with an isolate not producing Tox1. The TOX1 toxin and the SNB fungal isolate were both obtained from Bioger (UMR INRA-AgroParisTech, Thiverval-Grignon, France). The SNB isolate SE15017 was collected by Arvalis from durum wheat in 2015 in Gomel (France). The WB isolates BR43 and BR32 were selected from the CIRAD strain collection (Cirad, Montpellier, France) and cultured and harvested as previously described [START_REF] Ballini | Diversity and genetics of nitrogen-induced susceptibility to the blast fungus in rice and wheat[END_REF]. STB and LR fungal strains were isolated in 2015 in Montpellier (France) by INRA-BGPI from durum wheat Pescadou (STB P1a and LR AD2015) and Miradoux (STB M1a). LR isolate AD2015 was genotyped (H. Goyeau, INRA Bioger) and identified as a haplotype currently circulating in France (data not shown).

The Tox1 assay was conducted as previously described [START_REF] Liu | Genetic and Physical Mapping of a Gene Conditioning Sensitivity in Wheat to a Partially Purified Host-Selective Toxin Produced by Stagonospora nodorum[END_REF]). Briefly, a 1-ml needleless syringe was used for infiltration on the last ligulated leaf. The boundaries of the infiltrated area were marked on the leaf with a nontoxic pen. Seven days after infiltration, disease reactions were scored on a scale from 1 to 4: 1 -a plant without any symptoms, 2 -a plant with some chlorosis, and 3 and 4 for different levels of necrosis. SNB inoculation with SE15017 involved dipping a paintbrush into a leaf spore suspension, made by adding one drop of Tween 20 per 10 mL of inoculum containing 10 6 conidial spores, and applying it to the last ligulated leaf with six strokes of the brush. Symptom severity was evaluated seven days after inoculation on a 0 to 5 scale as described previously, where 0 is highly resistant [START_REF] Liu | Genetic and Physical Mapping of a Gene Conditioning Sensitivity in Wheat to a Partially Purified Host-Selective Toxin Produced by Stagonospora nodorum[END_REF]).

For STB inoculations with P1a or M1a, an inoculum of 10 6 spores per ml was prepared and applied using a paintbrush as described for SNB. We scored STB symptoms three weeks after inoculation. For each leaf, total necrotic leaf areas were automatically recorded from a scanned image using the image analysis program LeafTool, available on GitHub (https://github.com/sravel/LeAFtool). Total necrotic areas with pycnidia were then manually recorded. From these measurements, we calculated two phenotypes used for QTL analysis: host damage (% of necrotic leaf area) and pathogen reproduction (density of pycnidia per unit area of lesion).

Wheat blast resistance was screened using two different fungal isolates from Brazil: BR43 and BR32. The isolates were cultured as in Berruyer et al. (2003). To establish infection, an inoculum solution of 50,000 spores/ml was prepared and sprayed onto the plants. A week after inoculation, disease symptoms were scored on a scale of 1 to 4: 1 -a plant without any symptoms, 2 -a plant with some chlorosis, and 3 and 4 for different levels of sporulating lesions on the leaf surface.

LR inoculation with strain AD2015 was conducted by spray with a solution containing: gelatin 0.5%, one drop of Tween 20 per 10 mL, and 8mg of spores per 150 plants. To test for the effect of prophylactic induction, four hours prior to inoculation plants were either mock-treated (gelatin 0.5%) or treated with chitin (100 µg/mL and gelatin 0.5%) following a previously established protocol [START_REF] Cruz | Foliar application of products on the reduction of blast severity on wheat[END_REF]. The quantitative level of susceptibility was recorded ten days after inoculation using image analysis to evaluate the percentage of leaf surface with pustules using the image analysis program LeafTools.

Statistical Analysis

All inoculations were done on at least three independent plants and the mean values used as the susceptibility score for each line. To take into account potential variation arising from environmental differences within the glasshouse, we used two-dimensional P-spline mixed models implemented in the SpATS R package [START_REF] Velazco | Modelling spatial trends in sorghum breeding field trials using a two-dimensional P-spline mixed model[END_REF]).. Broad sense heritabilities based on the 1.2 repetitions per line were estimated using the SpATS model. We then used the best linear unbiased predictions (BLUPs) as genotypic values to perform the GWAS.

Genome-wide associations (GWA) were performed using GEMMA software [START_REF] Zhou | Genome-wide efficient mixed-model analysis for association studies[END_REF]. QQplots were used to determine the most appropriate correcting method for each analysed phenotype. As the EPO panel is weakly structured, the association model including only a kinship matrix has been used for all the traits.

We filtered SNPs for a minor allele frequency (MAF) of 5%. Kinship coefficients were calculated on R, using the Identity by State equation 2.3 [START_REF] Astle | Population structure and cryptic relatedness in genetic association studies[END_REF]. Manhattan plots were made on R (qqman package), using SNP locations derived from the BLAST on the Zavitan wild emmer genome. The proportion of variance explained (PVE) by each QTL was calculated, following the GEMMA author's recommendations [START_REF] Zhou | A unified framework for variance component estimation with summary statistics in genome-wide association studies[END_REF], as the ratio between variance on beta for the SNP considered, and the phenotypic variance for the trait analysed. We identified significant QTL using a threshold of 10e-5. The confidence interval was evaluated by keeping all SNP with a threshold above 10e-2 before and after significant SNP.

For gene annotation, the reference genome Zavitan v2.0 was used on GrainGenes database (https://wheat.pw.usda.gov/GG3/genome_browser) [START_REF] Zhu | Improved genome sequence of wild emmer wheat Zavitan with the aid of optical maps[END_REF]. To search for colocalization with previously mapped genes, published markers were BLASTed onto the Zavitan v2.0 genome sequence.

Results and discussion

Mapping the SNB resistance gene Snn1 validates the usefulness of EPO lines for disease resistance studies Resistance to Tox1 and to plant damage after inoculation by spores in the absence of Tox1 were evaluated independently. Indeed, the presence of Tox1 can sometimes hide the effect of other toxins due to epistatic interactions [START_REF] Phan | Differential effector gene expression underpins epistasis in a plant fungal disease[END_REF]). These two phenotypes were highly heritable (0.81 and respectively 0.53) and we identified four QTL (Fig1, Table 1, and online resource 1).

Two loci were associated with resistance to Tox1. The SNB_Tox1B locus maps to a 1,8 Mbp region at the beginning of chromosome 1B. The resistance allele is relatively frequent in the EPO lines (33%). The SNB_Tox1B peak (log(Pval) = 9,2) is located 66 kb from Snn1, a known resistance gene in bread wheat (Liu et al. 2012) (Fig2).

It is thus very likely that SNB_Tox1B corresponds to Snn1 [START_REF] Shi | The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease[END_REF]. This result validates using the EPO lines in a GWAS to identify resistance genes with strong phenotypic effects and alleles segregating at somewhat balanced frequencies. Its also further demonstrate that only replicating 20% of the lines can produce relevant mapping information. Another QTL for Tox1 resistance, SNB_Tox7A, was also identified in a 1,9 Mbp region on chromosome 7A. This region contains nine candidate genes and in particular, a cluster of five WAKs (online resource 3), suggesting that Tox1, which is known to target another WAK [START_REF] Shi | The hijacking of a receptor kinase-driven pathway by a wheat fungal pathogen leads to disease[END_REF], may broadly target this gene family.

In the second assay using spore inoculation, two QTLs were identified: SNB_Nec4B and SNB_Nec7B (Fig1, Table1), that were associated with resistance to an SNB isolate that does not produce Tox1. SNB_Nec4B is located in a 1,8 Mbp region on chromosome 4B containing fifteen candidate genes, including two receptor kinases (online resource 3). A QTL was previously mapped in the same region when screening for resistance to the toxin Tox3 in the absence of Tox1 [START_REF] Phan | Differential effector gene expression underpins epistasis in a plant fungal disease[END_REF].

Identification of Wheat Blast resistance QTL

Resistance to each of the fungal isolates BR43 and BR42 was highly heritable (0.81 and 0.68 respectively) and we identified five QTL (Fig1, Table1, and online resource 1). Two QTL associated with resistance to BR43, WB_BR43_2A, and WB_BR43_2B, mapped closely to regions on chromosome 2 containing Rmg7 and Rmg8, previously identified as resistance genes in bread wheat [START_REF] Anh | Rmg8 and Rmg7, wheat genes for resistance to the wheat blast fungus, recognize the same avirulence gene AVR-Rmg8[END_REF]. The 450 kbp region of WB_BR43-2A co-localizes with the 2 Mbp locus of Rmg7 (Fig3). If the two loci are allelic, this study suggests that finer mapping of this resistance gene is possible. Two chitinases, involved in biotic defense and present in this genomic region, could also be good candidates for WB43 resistance (online resource 3).

The QTL WB_BR32_7A maps to a 17 Mbp region on the same chromosome as Rmg2, which has not been finely mapped [START_REF] Zhan | Identification of two genes for resistance to Triticum isolates of Magnaporthe oryzae in wheat[END_REF]). The SNP with the strongest association with BR32 resistance is located 75 kbp away from a Nod-like receptor (NLR) gene (TRIDC7AG077360). NLRs are known to confer resistance to rice blast [START_REF] Saitoh | Rice blast resistance-inducing mechanisms <i>via</i> effector recognition by NLR immune receptors[END_REF], and TRIDC7AG077360 is thus a good candidate for WB_BR32_7A.

Identification of STB resistance QTL EPO lines were screened for the resistance to the STB isolate P1a. Two resistance phenotypes were scored (online resource 4): 1) resistance to primary infection, evaluated as the percentage of leaf surface with necrosis (STB_Nec), and 2) resistance to septoria reproduction, evaluated as the percentage of necrotic surface with pycnidia (STB_Pyc). The heritability of these two traits was high (0.69 and 0.68, respectively). We found two QTL associated with resistance to primary infection. STB_Nec5A mapped to a 4,7 Mbp region on chromosome 5A containing 51 candidate genes, among which six code for receptor kinases and three for pathogenesis-related proteins (online resource 3). STB_Nec3A maps to a 327 kbp region on chromosome 3A containing a cluster of six WAK genes, one of which is homologous to the known resistance gene Stb6 (online resource 3; see below). We found one QTL associated with pycnidia density. Notably, the QTL STB_Pycn3A mapped to a 326 kbp region on chromosome 3 containing nine candidate genes. Among these candidates is a wheat homolog of the rice negative regulator of defense NRR [START_REF] Chern | Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1[END_REF]) and a tryptophan synthase orthologous to Chinese spring TraesCS3A01G041800 (Fig6 and online resource 3). A reduction in pycnidia density could be due to a higher basal defense level [START_REF] Ors | Cultivar-dependent partial resistance and associated defence mechanisms in wheat against Zymoseptoria tritici[END_REF], and in this case, the NRR gene located in this region would be an excellent candidate. Tryptophan synthase is also a good candidate since this enzyme is involved in the biosynthesis of serotonin, a key player for defense in rice and wheat [START_REF] Ishihara | Involvement of Tryptophan-Pathway-Derived Secondary Metabolism in the Defence Responses of Grasses[END_REF][START_REF] Fall | The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat[END_REF][START_REF] Erland | Serotonin: An ancient molecule and an important regulator of plant processes[END_REF]. Reductions in pycnidia may be linked to restricted access to nutrients [START_REF] Zhan | Variation for neutral markers is correlated with variation for quantitative traits in the plant pathogenic fungus Mycosphaerella graminicola[END_REF][START_REF] Bancal | Zymoseptoria tritici development induces local senescence in wheat leaves, without affecting their monocarpic senescence under two contrasted nitrogen nutrition[END_REF]). However, annotations of the nine genes in the region do not suggest a role in nutrient access.

The QTL STB_Nec3 and STB_Pycn3A are only 3,3 Mbp on chromosome 3 (Fig1, Table1, and online resource 2). We did not find a single EPO line in the panel carrying resistance alleles at all three of these loci (online resource 5A). Indeed, all the lines containing the STB_Pyc3A resistance allele developed extensive necrosis (Fig4), demonstrating that these two QTL are linked but distinct.

A recent GWAS in bread wheat reported a resistance QTL on chromosome 2 associated with STB reproduction [START_REF] Yates | Precision Phenotyping Reveals Novel Loci for Quantitative Resistance to Septoria Tritici Blotch[END_REF], suggesting that this type of indirect resistance through controlling pathogen reproduction is common in wheat. In order to reduce the candidate gene number and to find recombinant lines at this locus, it could be worthwhile to come back to the still recombining EPO population. Due to the outcrossing rate in the population, the range of linkage disequilibrium in the 28 th generation should be shorter, and we may find accessions with recombinant alleles at the target locus.

In order to validate the effect of STB_Pyc3A locus, we looked for the presence of the resistance allele in a French elite durum wheat population of 74 lines, and four lines were identified. It is noteworthy that because of the resistance allele's low frequency, a GWAS of this population would not have had sufficient power to identify this QTL. A total of 14 lines, four lines carrying the resistance allele at STB_Pyc3A and ten with the susceptibility allele, was screened using two different STB isolates, P1a and M1a (Fig5). All of the lines carrying the resistance allele completely inhibited pycnidia production whereas nine of the ten lines carrying the susceptible allele displayed a high level of pycnidia formation (Fig5A and B and online resource 5B). Thus, we established the reliability of the STB_Pyc3A marker and the presence of genetic resistance to STB in four elite lines: Coussur, Miradoux, Nefer and Plussur. These lines could be used in breeding programs designed to increase the durability of STB resistance. Indeed, a reduction of the production of pycnidia not only limits epidemic spread, by reducing the number of spores produced, but also reduces the inoculum size of a virulent strain that may appear during the season.

A possible relation between Stb6 and STB_Nec3A

The same set of 14 elite lines was used for validation of the locus STB_Nec3A. From the two isolates used for phenotyping, only P1a triggered resistance to necrosis in EPO lines; thus, this locus seems to be isolate-specific (Fig5C and D) in a similar way than Stb6 [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF]. However, none of the three elite lines carrying the resistant allele at the peak SNP for the STB_Nec3A locus were resistant to either isolated (Fig5A and B). Thus, the STB_Nec3A marker found in the EPO population is not a good predictor of resistance in the elite durum wheat population.

The resistance allele at Stb6 identified in bread wheat is present in about 15% of European cultivars in this species [START_REF] Brown | Genetics of resistance to Zymoseptoria tritici and applications to wheat breeding[END_REF]. Three durum wheat lines carrying an orthologous Stb6 resistance allele found in bread wheat have been previously described [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF]), but resistance to STB strains adapted to durum wheat has not been evaluated. In order to evaluate the possible relationship with STB_Nec3A, Stb6 was genotyped in 14 durum elite lines and 23 EPO lines (online resource 5C and 6). Three different alleles for Stb6 were detected and compared to previously described alleles (online resource 6) [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF]. One allele is similar to the published resistance-associated Stb6-1 allele. The other two alleles are similar to the alleles Stb6-7 and Stb6-12, for which no information is available on resistance in bread wheat. Only one of the 27 lines with either the Stb6-1 or Stb6-7 allele was resistant to the P1a isolate (online resource 5C). By contrast, the Stb6-12 allele is present in all EPO lines carrying the resistance allele at the STB_Nec3A locus (online resource 5C), suggesting that the presence of Stb6-12 allele at this candidate gene is highly predictive of resistance. However, the only elite line (TOMCLAIR) carrying both the Stb6-12 allele and the resistance allele at STB_Nec3A was susceptible to both STB isolates tested here, suggesting that Stb6-12 is not a good predictor in elite durum wheat.

The Stb6-12 allele (and STB_Nec3A) afforded only partial resistance in our study (Fig4), whereas previous research on this locus reported associations with complete resistance [START_REF] Saintenac | Wheat receptor-kinase-like protein Stb6 controls gene-for-gene resistance to fungal pathogen Zymoseptoria tritici[END_REF]. In order to gain further insight, we sequenced AvrStb6, the cognate avirulence gene to Stb6 [START_REF] Brunner | Evolutionary analyses of the avirulence effector AvrStb6 in global populations of Zymoseptoria tritici identify candidate amino acids involved in recognition[END_REF], in the P1a and M1a isolates. Neither isolate carried the AvrStb6 allele known to be recognized by Stb6 (online resource 7). The allele present in P1a isolates was similar to the virulent virStb6 haplotypes 2 and 3 that are not recognized by the resistance allele Stb6-1 [START_REF] Brunner | Evolutionary analyses of the avirulence effector AvrStb6 in global populations of Zymoseptoria tritici identify candidate amino acids involved in recognition[END_REF]. The M1a haplotype corresponded to another uncharacterized allele. Thus, strain-specific differences in cognate avirulence genes to Stb6 in durum wheat could explain the observed interactions. Altogether, we cannot definitively conclude with our analysis if the observed partial resistance associated with STB_Nec3A is conferred by the Stb6-12 allele or by another paralogous WAK in this region that would recognize yet unknown alleles of AvrStb6. Because of the high recombination rate in EPO population [START_REF] David | Genotyping by sequencing transcriptomes in an evolutionary prebreeding durum wheat population[END_REF] further genetic analysis of the STB_Nec3A is possible using these lines.

Alternatively, durum wheat and STB strains could be genetically transformed with known alleles of Stb6 and AvrStb6 in order to directly test for gene-for-gene interactions affecting resistance.

Identification of a QTL for leaf rust resistance in the presence or absence of prophylactic induction EPO lines were inoculated with a leaf rust isolate and a complete resistance phenotype was detected with a heritability of 0.49 (online resource 8A). In order to test the effect of chitin treatment on LR resistance, the experiment was repeated with prophylactic induction by chitin 4 hours prior to LR inoculation. Chitin treatment did not affect either complete resistance or partial resistance symptoms (online resource 8B). Two independent GWAS were conducted using LR symptoms on chitin treated and untreated plants. In total, 26 lines were completely resistant and this phenotype was confirmed in both experiments. Importantly, because none of the 78 French elite lines demonstrate resistance to this LR isolate (personal data), these 26 lines are potentially valuable resources for breeders. Four QTL were identified in both experiments (Fig1, Table 1, and online resource 2). The QTL LR_6B11 on chromosome 6B conferred the strongest resistance and was identified in both experiments (Fig7). The locus is relatively large (3,24 Mbp), and contains 33 candidate genes (online resource 3), among which is a cluster of LRR-Kinases and a cluster of NLRs. There is a lack of SNPs between 10,15 and 11,15 Mbp near the NLR cluster; thus, we are not able to identify the best candidate based on the proximity with the most significant SNP. Three other QTL were identified on chromosome 2B, 6A, and 6B (Fig1, Table 1 andonline resource 2). Several LR resistance genes have already mapped on 6BS chromosome: Lr36 (Dvořák and Knott 1990), Lrac104 [START_REF] Hussein | Chromosome locations of leaf rust resistance genes in selected tetraploid wheats through substitution lines[END_REF], Lr53 [START_REF] Dadkhodaie | Mapping genes Lr53 and Yr35 on the short arm of chromosome 6B of common wheat with microsatellite markers and studies of their association with Lr36[END_REF]), Lr61 (Kthiri et al. 2018;[START_REF] Herrera-Foessel | Molecular mapping of a leaf rust resistance gene on the short arm of chromosome 6B of durum wheat[END_REF][START_REF] Maccaferri | Association mapping of leaf rust response in durum wheat[END_REF][START_REF] Qureshi | Genetic and Molecular Characterization of Leaf Rust Resistance in Two Durum Wheat Landraces[END_REF][START_REF] Loladze | Genetic analysis of leaf rust resistance in six durum wheat genotypes[END_REF]) and two QTL [START_REF] Maccaferri | Association mapping of leaf rust response in durum wheat[END_REF]. Lr36 and Lr53 originate from wild grasses relatives (T turgidum ssp. speltoides and ssp. dicoccoides respectively). Lr61 has been mapped in five different publications, originally in the Chilean durum wheat cultivar Guayacan INIA.

Lr61 can also be found in two ICARDA lines (Geromtel_3 and Tunsyr_2) and a Portuguese landrace (Aus26582).

Based on marker position, it is difficult to conclude if LR_6B11 is new compared to Lr36 and Lr61. The short arm of chromosome 6B also contains several resistance genes, but lack of fine mapping makes comparisons difficult. Indeed, two resistance genes have been identified in Geromtel_3: one is probably allelic to Lr61, but a second could be tightly linked to LR_6B11 [START_REF] Kthiri | Characterization and mapping of leaf rust resistance in four durum wheat cultivars[END_REF]. Our mapping of LR_6B11 has finer resolution in this genomic region and EPO lines could be useful to define this resistance locus further. Three other QTL have been identified but not further investigated: LR_6B135 on the short arm of the chromosome 6B, LR_2B on chromosome 2B, the latter colocalizing with Lr50 [START_REF] Brown-Guedira | Performance and Mapping of Leaf Rust Resistance Transferred to Wheat from Triticum timopheevii subsp. armeniacum[END_REF]) and LR_6A on chromosome 6A which colocalizes with previously identified resistance QTL [START_REF] Maccaferri | Association mapping of leaf rust response in durum wheat[END_REF][START_REF] Aoun | Genome-Wide Association Mapping of Leaf Rust Response in a Durum Wheat Worldwide Germplasm Collection[END_REF].

Conclusion

Our ability to easily map the SNB Snn1 resistance gene demonstrates the utility of the EPO lines for finding resistance QTL using a GWAS model and that this panel holds promise for discovering the genetic architecture of other phenotypes. In total, we identified sixteen QTL associated with resistance to the main fungal threats facing duram wheat growers. Among these were two QTL associated with resistance to Wheat Blast, possibly corresponding to finer positions of the Rmg2 and Rmg7 genes, that could provide valuable markers for durum wheat breeders for combatting this emerging disease. For STB, we identified a QTL associated with pycnidia density as well as a resistance QTL that may be allelic with the Stb6 resistance gene that was previously found in bread wheat. Once combined, these two STB QTL hold promise for breeders in producing durable resistance against this major fungal pathogen. Finally, a resistance QTL associated with LR was detected on chromosome 6B, near Lr61. Finer mapping of these loci could easily be done using the source EPO population that is still evolving and recombining in the field.

Figure and table captions

Fig1 Physical map of fungal resistance QTL identified in durum wheat. The map distance is given on the left in megabase pairs (Mbp) based on the genomic position in the Zavitan map v2.0. QTL names are located on the right of the chromosome and are represented by a black segment on the chromosome. QTL are designated according to Table1. QTL positions were defined by a support interval with a pvalue <0.00001. QTL were identified from association mapping in the EPO durum wheat panel of 180 lines for leaf rust (LR), septoria tritici blotch (STB), septoria nodorum blotch (SNB), wheat blast symptoms (WB). When previously mapped genes were identified at the same locus, they are represented with a skeleton segment on the right of the chromosome. Cloned Snn1 and Stb6 genes are shown as markers on chromosomes 1B and 3A respectively. The most relevant SNP markers are represented on the right of the chromosome for each QTL. ESM_8 A Distribution of leaf rust severity evaluated as the percentage of leaf surface covered with pustules after prophylactic induction with chitin treatment (dark grey) or following mock treatment (light grey). B Boxplot of leaf rust severity for completely resistant and partially resistant lines. Complete resistance was considered when no pustules were observed on the leaf surface.

Fig2Fig3Fig4Fig5Fig6

  Fig2 Mapping of resistance to SNB toxin, Tox1 in EPO durum wheat population. A Manhattan plot of the QTL SNB_Tox1B. SNP positions are given in megabase pairs based on genomic position in the Zavitan map v2.0. The y-axis is plotted on a log scale. Snn1 maps at 1,165 Mbp on Zavitan. B Boxplot for Tox1 phenotype based on the genotype at SNB_Tox1B locus. A scale from 1 to 4 was used for evaluation: 1 is complete resistance and 4 is leaf with 100 % necrosis symptoms. The number of lines carrying each allele is in parenthesis.Fig3 Mapping of resistance to wheat blast in EPO durum wheat population. A Manhattan plot of the QTL WB_BR43-2A associated with resistance to WB isolate BR43. SNP positions are given in megabase pairs based on genomic position in the Zavitan map v2.0. The y-axis is plotted on a log scale. Rmg7 is located between 771,35 and 773,37 in a 2,02 Mbp locus, based on published marker position blasted on Zavitan. B Boxplot for BR43 phenotype based on the genotype at WB_BR43-2A locus. A scale from 1 to 4 was used for evaluation, where 1 was complete resistance and 4 representing susceptibility. The number of lines carrying each allele is in parenthesis. Fig4 Evaluation of quantitative resistance to STB in the EPO panel. A Boxplot for STB disease severity (necrotic area) based on the genotype at QTL STBNec_3A and STBNec_5A. S_S are lines without any alleles of resistance. S_STBNec_5A are lines without resistant allele on chromosome 3 but a resistant allele on chromosome 5. STBNec_3A_S are lines without resistant allele on chromosome 5 but a resistant allele on chromosome 3. B Boxplot for STB density of pycnidia based on the genotype at QTL STBPyc_3A. The number of EPO lines carrying the resistance allele are shown in parentheses.Fig5 Evaluation of quantitative resistance to STB in a validation panel. STB resistance was evaluated in Elite lines and a subset of EPO lines using the isolate used for GWA (P1a) and a second isolate (M1a) A Boxplot of disease severity (necrotic area) in Elite lines and EPO lines inoculated with isolate M1a and P1a. Lines carrying a susceptible allele at STBNec_3A are in white, lines carrying the resistant allele are in black. B Boxplot of density of pycnidia in Elite lines and EPO lines inoculated with isolate M1a and P1a. Lines carrying a susceptible allele at STBPyc_3A are in white, lines carrying the resistant allele are in black. Significance was evaluated using a Wilcoxon test; *** pvalue <0.01.Fig6 Manhattan plot on chromosome 3A for STB resistance: STB_Pyc3A. EPO lines were inoculated with STB isolate P1a and density of pycnidia was evaluated. The QTL position was defined by a support interval with a pvalue <0.00001. The SNP position is given in megabase pairs based on genomic position in the Zavitan map v2.0.The y-axis is plotted on a log scale.

Fig7A

  Fig7 A Manhattan plot on chromosome 6B for LR resistance: LR_6B11. EPO lines were inoculated with LR isolate and the surface with pustule was evaluated. The QTL position was defined by a support interval with a pvalue <0.00001. The SNP positions are given in megabase pairs based on genomic position in the Zavitan map v2.0. The y-axis is plotted on a log scale. B Boxplot for LR phenotype based on the genotype at the LR_6B11 locus. The number of lines carrying each allele is in parenthesis.

Acknowledgments

Acknowledgments of people, grants, funds, etc. should be placed in a separate section on the title page. The names of funding organizations should be written in full.

Highbred, CASDAR-BURRITOS, FSOV-WEAB and in particular Richard Oliver (Curtin University, Australia) for providing purified Tox1.