Akchheta Karki

Joachim Vollbrecht

Alana L Dixon

Nora Schopp

Max Schrock

Manjunatha Reddy
email: gnm.reddy@univ-lille.fr

Thuc-Quyen Nguyen

G N Manjunatha Reddy

Understanding the High Performance of over 15% Efficiency in Single-Junction Bulk Heterojunction Organic Solar Cells

Keywords: organic photovoltaics, recombination, charge extraction, low voltage losses, solid-state NMR

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

Introduction

Polymer:non-fullerene acceptor (NFA) based single-junction organic solar cells (OSCs) have recently attained record-breaking power conversion efficiencies (PCEs) of over 16%. [1] Numerous This article is protected by copyright. All rights reserved.

3 recently reported studies attribute the high performance of polymer:NFA-based OSCs to an improvement in the open-circuit voltage (V OC ) without significantly impeding the charge generation efficiency. [2][3][4] Such improvements in the V OC can be credited to a rise in the number of systems with relatively low energetic offsets (ΔG S1-CT ), which is defined as the energy difference between the charge transfer (CT) state and the singlet exciton state (S 1 ) of the lower bandgap component in the blend. [2,5,6] While blend systems with low energetic offsets are sought for achieving high open-circuitvoltages, such blends commonly suffer from modest short-circuit current (J SC ) and fill-factor (FF) values, which can limit the PCE. [5][6][7][8][9][10] In this study, the recently reported high performing blend system PM6:Y6, was examined to obtain an in-depth understanding of the voltage losses, as well as the charge recombination and extraction dynamics. Remarkably, the PM6:Y6 blend is simultaneously able to achieve high V OC (0.825V) with exceptional FF (74%) and J SC (25.2 mA/cm 2 ) values, which is likely linked to a favorable morphology. Therefore, to gain detailed insights into the morphology of the blend, three different morphology characterization techniques were used in combination to visualize the photoconductive donor and acceptor phase-separated regions on the surface of the blend film, characterize the ordering in the bulk of the blend film, and probe the D:A inter-and intramolecular interactions. This work provides key insights on the device physics and detailed morphology of the highly efficient single-junction OSC blend, while unravelling the underlying mechanisms for achieving PCEs of over 15 %.

The chemical structures of the donor and acceptor materials, along with the energy levels and device structure of the PM6:Y6 blend system are shown in Figure 1. Devices with PEDOT:PSS as the bottom electrode and solution processed PDINO layer capped with evaporated Aluminum electrodes This article is protected by copyright. All rights reserved.

4

were optimized as reported in the previous study. [11] The J-V characteristics of a device at 1 sun illumination (100 mW cm -2 AM 1.5) is shown in Figure S1a. The average PCE in Table S1 was obtained from testing 20 devices, and the best performing device exhibited a PCE of up to 15.35%.

An integrated J SC from the external quantum efficiency (EQE) spectra of 24.9 mA/cm 2 was obtained which is within 5 % of the average J SC measured via the J-V characteristics of the devices at 1 sun illumination (Figure S1b). A combined simulation and experimental approach [12] was used to obtain an exceptional internal quantum efficiency (IQE) of over 95% over the relevant wavelength regime (500-800 nm) in this blend, suggesting highly efficient charge generation and extraction (Figure S1b).

Furthermore, the preliminary results by Neher et. al. [13] obtained via a combination of internal generation efficiency (IGE) using time-delayed collection field (TDCF) and IQE measurements suggest that losses due to exciton harvesting and geminate recombination are minimal in this blend system.

To understand what makes this blend system achieve over 15% PCE, we examine the individual components that make up the PCE (V OC , FF, and J SC ) in detail. To understand the high V OC , we begin by delving deeper into understanding the voltage losses in this blend. For evaluating the losses limiting the V OC in this blend, we evaluate the energy loss, E loss as:

𝐸 𝑙𝑜𝑠𝑠 = 𝑆 1 -𝑞𝑉 𝑂𝐶 , (1)
where, S 1 is the singlet exciton energy of the lower bandgap component in the blend and q is the elementary charge. A recently published paper by Vandewal el. al. [14] dissuades the use of ill-defined absorption onsets or HOMO and LUMO energies as reference points for evaluating voltage losses.

Therefore, we adopt the optical method described in the paper [14] for a precise measurement of the singlet exciton energy. To obtain the S 1 , the intersection point of the emission spectra (from electroluminescence measurements, EL) and absorption spectra (from photovoltaic external quantum efficiency measurements, EQE PV ) of the lower band-gap component in the blend (acceptor, This article is protected by copyright. All rights reserved.

Y6

) is determined (Figure S2). The S 1 obtained from this method was 1.36 eV.

Losses limiting the V OC can be further divided into two parts: losses due to charge transfer as defined by the difference between the S 1 and the energy of the CT state (E CT ), and losses due to recombination defined by the difference in E CT and V OC . Commonly, to identify the CT state in a blend, the EQE PV is measured, and the CT state is identified as the absorption feature visible at energies lower than the bandgap of either the donor or acceptor. The E CT is then determined by fitting an expression derived from Marcus theory (Equation S1) to the tail of the blend EQE PV spectrum. [7,15] However, the CT state is not always pronounced in the low-energy tail of the EQE PV spectra, especially in blends where the energetic offsets between the donor and acceptor are low (i.e., low HOMO-HOMO or LUMO-LUMO offsets) [2,5,8,16] , and it can therefore be difficult to distinguish the energy of the CT state from the donor or acceptor singlet state. It is possible, however, to significantly reduce the degrees of freedom in the fitting by 1) performing a simultaneous fit to both the EQE PV (Equation S1) and the EL (Equation S2) spectra [3,9,[17][18][19] using equations derived from Marcus theory, as was first demonstrated by Vandewal et. al. [15] and 2) calculating the EQE PV down to 10 -7 using sensitively measured EL data and the relationship shown in Equation S3. [20] The added sensitivity of four orders of magnitude in the measurements allows for the deconvolution of the Gaussian-shaped CT absorption which was previously absent from the sharp absorption tail of the measured EQE data. In fact, a recently published paper [19] has shown that this method significantly lowers errors in the fitting parameters (E CT , 𝜆, 𝑓) in low energetic offset systems. Using this analysis, the E CT in the PM6:Y6 blend was determined to be 1.31±0.003 eV (Figure 2), further confirming the low offset in this system with a difference between S 1 and E CT of only ~ 0.05 eV. It is worth noting, however, that despite the reduction in degrees of freedom in the Marcus fitting, the E CT value obtained here serves only as an estimate due to the significant uncertainty that arises in separating the CT state emission and the singlet exciton emission in low energetic offset systems. Therefore, we report the CT energy in this blend -as has been done in some recently reported papers [9,19] -with a caveat that the obtained E CT value is an estimate only.

Next, losses due to recombination (E CT to V OC ), which can be divided into radiative (ΔV rad ) and non-radiative losses (ΔV non-rad ), are quantified (Equation S4). As derived from a detailed balance analysis, about 200-250 meV of radiative recombination is needed to establish thermodynamic equilibrium. [17] In recent years, an increasing number of non-radiative recombination loss-related studies have led us to understand the nature and origin of this recombination in more detail, with studies showing that this recombination can be attributed to Similar to this blend, the suppression of non-radiative recombination losses with a decreased energetic offset has been described in several recently reported studies. [3,5,9,23] This observation has This article is protected by copyright. All rights reserved. 8 been explained by the idea that a decreased overlap of the vibrational wave function of the CT and ground state leads to the suppression of the non-radiative recombination pathway. [23] In addition, the recent paper by Qian et. al. [5] suggests that in low energetic offset systems, hybridization of the CT state with the highly emissive S 1 state will increase the radiative ability of the CT state through the intense borrowing mechanism. [30,31] In such a case, an efficient transition from the CT state back to the S 1 state can be possible, which opens up an additional radiative relaxation pathway through the highly emissive S 1 state. From the modeling of excitonic and CT states, it was found that if the radiative relaxation channel from the highly emissive S 1 state can be made efficient in this way, the non-radiative voltage losses via the CT state should synchronously decrease. [5] To further confirm that the S 1 state (Y6) is in fact highly emissive, we measured the EQE EL of the Y6 only devices (Figure S2f), confirming the lower non-radiative recombination (ΔV non-rad = 0.242 ± 5 eV) in the acceptor-only device compared to that of the blend (ΔV non-rad = 0.286 ± 3 eV eV). than or compatible to the recombination time, charge recombination can be reduced. [6,32,33] As a first step, the photocurrent density (J ph ) of the device is calculated using the following equation:

𝐽 𝑝ℎ = 𝐽 𝑙𝑖𝑔ℎ𝑡 -𝐽 𝑑𝑎𝑟𝑘 , (2)
where J light is the current density measured under illumination and J dark is the current density measured in the dark. The photocurrent density is plotted against the effective voltage

V eff = V 0 -V cor (Figure S4 a,b),
where V 0 is the voltage at which J ph = 0 (V 0 = 0.842 V). The corrected voltage V cor can be obtained by taking into account the voltage losses over the series resistance:

𝑉 𝑐𝑜𝑟 = 𝑉 𝑎𝑝𝑝 -𝐽 • 𝑅 𝑠𝑒𝑟𝑖𝑒𝑠 , (3)
where V cor is the corrected voltage, J is the current density, and R series is the series resistance which is equal to the saturated differential resistance at forward bias (i. e. ∂V app /∂J = const.). [34,35] Relatively high photocurrents were achieved for the PM6:Y6 solar cells and the value of J ph remains high even at comparatively low effective voltages (V eff = 0.1 V). Subsequently, the probability of charge collection (P C ) can be estimated by analyzing the ratio between the saturated photocurrent density J ph,sat and the values for J ph at different biases [36] :

𝑃 𝐶 = 𝐽 𝑝ℎ 𝐽 𝑝ℎ,𝑠𝑎𝑡 . ( 4
)
Figure 3a shows that the P C retains very high values under short-circuit (P C,SC = 97.7 %, blue circle), and maximum power conditions (P C,MP = 91.8 %, red cross), followed by a considerable drop has been used to calculate the exponent of  = 0.92 ± 0.02. Since the devices show exceptional charge extraction under short-circuit conditions (P C,SC = 97.7 %), it can be hypothesized that the influence of bimolecular recombination on the J SC is limited. Furthermore, it should be noted that deviations in the exponent ( < 1) can also be caused by space charge effects. [37,38] Overall, these considerations constrain the predictive abilities of this type of measurement on the recombination dynamics. To better understand the recombination processes, the relationship between the V OC and the light intensity I was also determined. [39] The V OC -ln(I)-plot exhibits a slope of s = 1.02 ± 0.03 kT/q,

where k is the Boltzmann constant, T is the absolute temperature (T = 300 K), and q is the elementary charge. In order to rule out the effect of leakage on the slope of the V OC -ln(I)-plot, the J-V curves at different light intensities were plotted and analyzed (Figure S5c,d). These results indicate that bimolecular recombination is the dominant recombination mechanism. However, other types of recombination such as bulk (s > 1 kT/q) and surface trap-assisted (s < 1 kT/q) recombination should also be taken into account as loss mechanisms, most notably because the opposite influence of surface and bulk trap-assisted recombination on the slope s could cause slopes of s ≈ 1 kT/q, which This article is protected by copyright. All rights reserved. could be falsely attributed to pure bimolecular recombination. [40] Therefore, a more in-depth analysis based on capacitance spectroscopy was carried out to obtain quantitative results. [35,41,42] The capacitance of the PM6:Y6 BHJ, C b , was used to determine the charge carrier density n and the effective mobility  eff of the studied solar cell under any operating conditions (Figures S6 andS8;

a detailed description of obtaining the effective mobility is provided in the SI). [33,43] Under forward bias conditions, a carrier density of n > 10 17 cm -3 was observed, placing the carrier density in this system at the upper end of high performing NFA blends. [44] Additionally, the effective mobility ( eff ≈ 1 × 10 -4 cm 2 V -1 s -1 ) determined using the procedure described in the SI is comparable to other high performing NFA as well as fullerene blend systems. [33] As a starting point in the quantitative analysis of the non-geminate recombination dynamics, it is assumed that the recombination current density (

(J rec = J ph,sat -J ph )
) 5
Here L is the active layer thickness,  is the charge carrier lifetime, and k is the recombination coefficient of the three different recombination mechanisms (bm: bimolecular; t,b: bulk trapassisted; t,s: surface trap-assisted). It was possible to obtain the recombination coefficients by reconstructing the recombination current density obtained using the J-V characteristics from the charge carrier density (n) and the effective mobility ( eff ) (Figure S8). The carrier density, effective mobility, voltage, and dielectric constants were used as input parameters, while the reduction factor (, bulk-trap density (N t,b ), and surface-trap density (N t,s ) were the fitting parameters. Based on these results, it can be confirmed that bimolecular recombination is the dominant non-geminate recombination mechanism, while bulk trap-assisted recombination is negligible, and surface trap-This article is protected by copyright. All rights reserved.

assisted recombination has only a very limited contribution at high forward biases. [45] Overall, the dependence of bimolecular recombination on the applied bias and the charge carrier density is less pronounced and the values for the respective coefficients are relatively small (k bm = 3.0 -5.8 × 10 - 13 cm 3 /s) in comparison to other high performing NFAs and up to one order of magnitude lower than that for high performing fullerene solar cells. [33,46] In addition, open-circuit voltage decay (OCVD) measurements (also referred to as transient photovoltage decay) were conducted as a second

technique to analyze and confirm the recombination dynamics, since other approaches relying on the electron and hole mobility determined via SCLC are not viable due to the questionable electron mobility values in the blend (Figure S9). [47,48] This technique yields a higher value for the bimolecular recombination coefficient when compared to the results obtained from the capacitance spectroscopy analysis (k bm,OCVD = (4.54 ± 0.15) • 10 -12 cm 3 /s vs. k bm,CS = 3.0 -5.8 • 10 -13 cm 3 /s).

However, such a trend (i.e. k bm,OCVD > k bm,CS ) between these two types of measurement techniques has been observed and reported in previous studies for fullerene and NFA solar cells. [33,42] Nonetheless, the extraction of charge carriers has to also be quantified to obtain a comprehensive understanding of the processes in the studied devices.

To gain quantitative insights into the charge carrier extraction dynamics, the effective extraction time,  ex , was determined under the assumption that a charge carrier needs to traverse, on average, half of the PM6:Y6 active layer thickness until it reaches one of the electrodes. Furthermore, the active layer is treated as an effective medium [32] and the following relationship was used:

𝜏 𝑒𝑥 = 𝑞𝐿𝑛 𝐽 , ( 6
)
This article is protected by copyright. All rights reserved.

14

where L is the active layer thickness, q is the elementary charge, n is the charge carrier density, and J is the current density obtained from the J-V curves (details are given in the SI, Equations S22-S24).

The effective extraction time  ex can then be directly compared to the effective charge carrier lifetime  rec , which can be accessed by re-arranging Equation 5 (Figure 3b). [42] Interestingly, extraction is at least two orders of magnitude faster than recombination over most of the operating conditions. Only at high forward biases, approaching V OC , do the effective extraction and charge carrier lifetimes converge. This observation correlates well with the high photocurrents J ph and collection probabilities P C retained under forward bias, as displayed in Figure 3a. In addition, the voltage dependent competition factor  was calculated by taking the ratio of the effective extraction and recombination times ( =  ex / rec ) (Figure 3c). [32] A smaller competition factor has been shown to correlate to higher FF. [32,33] In essence, the collection probability P C and the quantitative analysis based on capacitance spectroscopy show that the PM6:Y6 BHJ device exhibits exceptional extraction coupled with moderate bimolecular recombination losses, which is the basis for the high FF and J SC observed. While the detailed analyses of extraction and recombination processes in PM6:Y6 provide sufficient evidence on the bulk electronic characteristics, much of these favorable charge carrier properties are expected to originate from the BHJ morphology. To confirm this, we carried out detailed morphological and structural characterizations of the PM6:Y6 blend at different length scales. This article is protected by copyright. All rights reserved.

16

The BHJ morphology has a significant influence on the device performance. [6,[49][50][51] To gain detailed insights into the morphology of the PM6:Y6 blend at different length scales, three different techniques were used: photo-conductive atomic force microscopy (pc-AFM, sub-m to m) for visualizing the photoconductive hole and electron rich domains on the film surface, Grazing-Incidence Wide-Angle X-ray Scattering (GIWAXS) for probing the long-range structural order (ca.

100's nm) in the bulk of the blend film, and one and two dimensional (1D, 2D) solid-state-NMR for elucidating the D:A inter-and intramolecular interactions at sub-nm to nm distances. In a pc-AFM measurement, an electrically conductive Platinum-Chromium coated tip was used to scan the surface of photoactive layers using a white light source, which allows to map out the photoresponsive features in the PM6:Y6 blend films. The topography images (Figure S10a,b) show features amounting to small root-mean-square (RMS) roughness values (~ 0.8 nm) and the photocurrent image (Figure S10c,d) reveals small phase separated domain sizes (ranging from 20-30 nm) of hole (PM6) and electron rich (Y6) regions on the film surface in comparison to other reported blends. [6,[51][52][53][54] Such a small degree of phase separation can explain the efficient charge separation and moderate recombination in the blend. [39,49,[55][56][57][58] Next, in order to characterize the ordered regions in the PM6:Y6 blend film, we investigated the GIWAXS of the blend film (Supporting Information, Figure S11). As was also previously reported for this blend [11] , the GIWAXS pattern of PM6:Y6 showed that the blend displays face-on orientation which can be beneficial for charge transport in the direction normal to the substrate surface. [59,60] A detailed discussion of the GIWAXS analyses in the blend can be found in the SI.

Several theoretical and experimental studies have suggested that the nature of the D:A interactions can affect the rates of charge transfer and recombination [24,61,62] , electronic coupling [18] , charge generation [18,61] , and charge delocalization [63] -all of which can in turn influence the loss mechanisms in an OSC. While the correlation between the D:A interactions in a blend and bulk optoelectronic properties can be highly useful to understand the device performance, it is rarely studied for polymer:NFA-based BHJs. Here, solid-state 19 F MAS NMR spectroscopy has been employed to obtain atomic-level insights about the inter-and intramolecular interactions of the PM6:Y6 BHJ films. Solid-state NMR spectroscopy provides information on local structures and intermolecular interactions at sub-nm to nm distances, which could be related to both ordered and disordered regions of OPV blends. The isotropic chemical shifts and dipole-dipole couplings are sensitive to inter-and intramolecular interactions and relative orientations of donor and acceptor molecules in the BHJ morphologies. These interactions can be used, in particular, to measure internuclear distances in the absence of long-range structural order by measuring and analyzing heteronuclear dipolar couplings in heterogeneous soft matter materials. [51][52][53] For example, quantitative insights into the ordered and disordered regions in organic semiconductors and interfacial contacts in BHJs have been previously attained by solid-state NMR analyses in conjunction with X-ray scattering measurements and DFT calculations. [58,[66][67][68][69] The 1D and 2D 19 F MAS NMR spectra of PM6, Y6, and the PM6:Y6 blend were analyzed and compared in order to understand the changes in the local environments of 19 F sites in PM6 and Y6 upon BHJ formation. The intrinsic high sensitivity due to 100% natural abundance and large chemical shift range associated with 19 F MAS NMR enabled the local environments of 19 F sites in PM6 and Y6

to be distinguished and assigned (Figure 4). The 19 F signal at -131 ppm (Figure 4a) was attributed to local environments of 19 F sites in fluorinated thiophene groups of PM6 backbone moieties. In contrast, the 19 F MAS NMR spectrum of Y6 exhibited different distributions of signals that are only This article is protected by copyright. All rights reserved.

18

partially resolved in the range between -115 ppm and -130 ppm (Figure 4b). These distributions of 19 F chemical shifts and signal intensities were attributed to the 19 F sites in different Y6 backbone structures, which is consistent with the presence of distinct structural orders that co-exist in the Y6 material as previously characterized by Yuan et. al. using GIWAXS measurements. [11] To confirm this analysis, 2D 19 F{ 19 F} spin-diffusion (SD) experiments [START_REF] Elena | Unraveling the High Performance of over 15 % Efficiency in Single-Junction Bulk Heterojunction Organic Solar Cells[END_REF] were carried out in order to identify and distinguish whether or not these 19 F pairs are spatially proximate to each other at sub-nm to nm distances (Figure S12). In a 2D 19 F{ 19 F} spin-diffusion experiment, the 19 F magnetization is allowed to exchange between different 19 F sites of spatially close 19 F nuclei for a given mixing period, which leads to self-and cross-correlation intensities. The self-correlation intensities at the diagonal of the 2D 19 F{ 19 F} correlation spectrum of Y6 acquired using shorter mixing period of 100 ms (Figure S12)

indicate that this mixing time is not adequate enough for the magnetization exchange to occur between different 19 F sites. At a relatively longer mixing time of 1 s (Figure S12b), the crosscorrelation intensities are observed between 19 F signals at -120 and -124 ppm confirming the exchange of magnetization between different 19 F sites in Y6 backbone moieties. These crosscorrelation intensities at very long mixing times (> 500 ms) were expected to originate from weak 19 F- 19 F dipole-dipole interactions. This further confirms the spectral assignments of signals at -120 and -124 ppm to 19 F sites in different endgroups of Y6 rather than two 19 F sites in ortho position to each other within the same endgroup. Otherwise strong cross-correlation intensities at much shorter mixing times due to efficient magnetization exchange between 19 F sites that are close to each other would be observed. To further corroborate this analysis, the 19 F chemical shifts of neat PM6 and Y6 moieties were compared with analogous chemical shifts in the PM6:Y6 blends (Figure 4c). Interestingly, the 19 along the diagonal for the spectra recorded using short and long mixing times (100 ms, 1 s) confirming that there is no magnetization exchange between 19 F sites in PM6 and Y6. This rules out, at least in part, the likelihood of Y6 backbone moieties placed in between the  stacked PM6 polymer chains and confirms the lack of close contact between the acceptor moieties on the Y6 and the PM6 backbones.

From a combined investigation of the morphology using the aforementioned three different techniques, we can deduce important insights about the structural arrangement of the PM6:Y6 blend. Firstly, the phase separation of donor and acceptor regions at smaller length scales, as revealed by pc-AFM, results in moderate recombination and efficient charge separation in the blend.

Secondly, GIWAXS measurements show that the blend displays good π-π stacking, which has been shown to be beneficial for charge transport. [59,60] . Although X-ray scattering and pc-AFM techniques are capable of unveiling such structural details at m to 10-nm length scales, these methods do not have sufficient spatial resolution to identify atomic-level information. To this end, thirdly, solid-state 19 F MAS NMR analyses provide information about atomically resolved inter-and intramolecular D:A interactions. The main finding from ssNMR is that there is no close contact between the acceptor This article is protected by copyright. All rights reserved. 20 moieties on the Y6 and the PM6 backbones. This was confirmed from the 2-D spin diffusion correlation studies, where there is clear evidence that the spins of the F atoms in the donor and acceptor do not interact with each other within a 1 nm distance. This alludes to a well-defined D:A interface. Several previously reported studies have shown that an enhanced intermixing of the donor and acceptor in a blend can be detrimental to the device performance by increasing charge recombination. [6,41,55] Therefore, the moderate recombination rates in this blend can be explained by the beneficial phase separation between the polymer and NFA, as visualized on a micron-scale by pc-AFM, and confirmed on a sub-nm to nm scale by ssNMR. The exceptional extraction observed in the devices can be attributed to two things. First, in the neat Y6, two different packing motifs were resolved with the 1D 19 F ssNMR. In the blend, only the signal from one of these two motifs dominates, which indicates a more uniform and structured packing of the Y6 molecules in the blend.

Second, the 1D 19 F ssNMR results indicate that the PM6 packing remains unaffected with the addition of Y6 confirming that the order of the PM6 observed in the neat film is also retained in the blend. It is worth noting that the π-π stacking peaks of PM6 and Y6 obtained from GIWAXS, as reported in the previous literature, [11] are broad and quite close to each other to definitively distinguish if the π-π stacking peak observed in the blend is coming from PM6 or Y6. In this regard, ssNMR analyses which are sensitive to short range structures and interactions, are more informative about the details of the blend morphology. Ultimately, the exceptional extraction in the devices is due to the unencumbered pathways for sweeping out charge carriers via the retention of multiple unhindered and organized PM6 and Y6 domains.

Figure 1 .

 1 Figure 1. (a) Chemical structures of PM6 and Y6, and (b) energy levels of the different layers used in

Figure 2 .

 2 Figure 2. (a) Simultaneous fitting of the reduced EQE PV and EL spectra; reciprocity relationship between EQE PV and EL from Equation S3 was used to calculate the EQE PV down to 10 -7 shown by the red solid line, the fitting parameters used were: 𝜆 = 0.10 eV, E CT = 1.31 eV, f = 3.0 × 10 -3 eV 2 . (b)

  once the voltage approaches open-circuit conditions (P C,OC = 7.03 %), which is indicative of an exceptional charge extraction and at the same time, moderate charge carrier recombination losses. It can be deduced that geminate recombination has a negligible influence on the device performance as evidenced by the significant collection probability even at comparatively low voltages. In the next step, the PM6:Y6 solar cells were tested under varying light intensities I by measuring light intensity dependent short-circuit current density and open-circuit voltage in order to qualitatively determine the dominant type of non-geminate recombination (Figure S5). The light intensities were decreased by neutral density filters. The relationship between the J SC and I (J SC ∝ I  )

  is a superposition of the three aforementioned recombination mechanisms: 𝐽 𝑟𝑒𝑐 = 𝐽 𝑏𝑚 + 𝐽 𝑡,𝑏 + 𝐽 𝑡,𝑠 = 𝑞𝐿 ( (𝑘 𝑏𝑚 𝑛 2 + 𝑘 𝑡,𝑏 𝑛 + 𝑘 𝑡,𝑠 (𝑉 𝑂𝐶 )).

Figure 3 .

 3 Figure 3. (a) Collection probability P C , (b) charge carrier lifetime  rec and extraction time  ex , as well as

19 F{ 19 F}

 1919 F signals of Y6 at -120 ppm displaced towards a lower frequency and at -124 ppm displaced towards a slightly higher frequency that leads to a relatively narrow distribution of signal intensities centered at -123 ppm. This indicates the improved structural order of Y6 molecules accompanied by subtle differences in the local environments of19 F sites in the fluorinated endgroups of Y6 moieties in the PM6:Y6 blend. By comparison, the19 F chemical shift of fluorinated thiophene groups in the PM6 polymer remains at -131 ppm, which reveals the retained organization of the thiophene endgroups in the PM6 polymer upon blend formation with Y6. In addition, the 2D correlation spectra of PM6:Y6 (FigureS12c,d) exhibited self-correlation intensities exclusively

Figure 4 . 22 (

 422 Figure 4. Solid-state 19 F MAS NMR spectra of (a) PM6, (b) Y6 and (c) PM6:Y6 blend acquired at 60 kHz and at room temperature. The distributions of 19 F chemical shifts in Y6 centered at -120 and -124 ppm were attributed to the distribution of different 19 F sites in different Y6 backbone structures. A

Acknowledgements A.K. and J.V. contributed equally to this work. This work was supported by the Department of the Navy, Office of Naval Research Award No. N00014-14-1-0580. A.K. acknowledges funding by the Schlumberger foundation. J.V. acknowledges funding by the Alexander-von-Humboldt Stiftung. G. N. M. R. gratefully acknowledges the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 795091. Financial support from the IR-RMN-THC FR-3050 CNRS France for conducting solid-state NMR measurements is acknowledged. The authors would like to thank Liuyang Zhou and Professor Yingping Zou for advice This article is protected by copyright. All rights reserved. 23 with device optimization and Dr. Viktor Brus and Dr. Martin Seifrid for helpful discussions. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231.

SHORT ABSTRACT:

The high performing single-junction organic solar cell blend, PM6:Y6, was examined to obtain an indepth understanding of the voltage losses, and charge recombination and extraction dynamics. The devices exhibit remarkable extraction coupled with moderate recombination losses. This behavior can most likely be credited to a beneficial morphology as evidenced by atomically resolved 19 Please do not use any other dimensions))


Hélène Pidon
email: pidon@ipk-gatersleben.de

Neele Wendler

Antje Habekuβ

Anja Maasberg

Brigitte Ruge-Wehling

Dragan Perovic

Frank Ordon

Nils Stein
email: nilssteinstein@ipk-gatersleben.de

Antj Habek

High-resolution mapping of Rym14Hb, a wild relative resistance gene to barley yellow mosaic disease

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

Viruses are an increasing threat to crops worldwide. The soil-borne barley yellow mosaic disease, caused by a complex of two Bymoviruses (Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV)) is one of the most important diseases of winter barley. Widespread in central Europe and East-Asia, it causes severe yield losses up to even total crop failure [START_REF] Plumb | The effects of infection by barley yellow mosaic virus on the yield and components of yield of barley[END_REF][START_REF] Jianping | Progress and prospects of studies on Polymyxa graminis and its transmitted cereal viruses in China[END_REF][START_REF] Kühne | Soil-borne viruses affecting cereals-known for long but still a threat[END_REF]). As chemical control of those viruses, transmitted by the plasmodiophorid Polymyxa graminis [START_REF] Kanyuka | Polymyxa graminis and the cereal viruses it transmits: a research challenge[END_REF], is not possible, only the use of resistant varieties can preserve yield in infected fields.

To date, 20 barley resistance genes have been identified, almost exclusively conferring recessive resistance [START_REF] Jiang | Bymovirus-induced yellow mosaic diseases in barley and wheat: viruses, genetic resistances and functional aspects[END_REF]. Two of these loci have been cloned: the EUKARYOTIC TRANSLATION INITIATION FACTOR Communicated by Kevin Smith.

Electronic supplementary material

The online version of this article (https ://doi.org/10.1007/s0012 2-020-03733 -7) contains supplementary material, which is available to authorized users.

1 3 4E gene (eIF4E), [START_REF] Stein | The eukaryotic translation initiation factor 4E confers multiallelic recessive bymovirus resistance in Hordeum vulgare (L.)[END_REF] of which several allelic forms providing resistance are described, including rym4 and rym5, [START_REF] Hofinger | An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations[END_REF][START_REF] Perovic | Genetic analyses of BaMMV/BaYMV resistance in barley accession HOR4224 result in the identification of an allele of the translation initiation factor 4e (Hv-eIF4E) exclusively effective against barley mild mosaic virus (BaMMV)[END_REF][START_REF] Yang | Sequence diversification in recessive alleles of two host factor genes suggests adaptive selection for bymovirus resistance in cultivated barley from East Asia[END_REF][START_REF] Shi | Bulked segregant RNA-sequencing (BSR-seq) identified a novel rare allele of eIF4E effective against multiple isolates of BaYMV/BaMMV[END_REF], and the PROTEIN DISULFIDE ISOMERASE LIKE 5-1 (PDI5-1) gene which is also represented by a handful of alleles providing resistance, including rym1 and rym11 [START_REF] Yang | Sequence diversification in recessive alleles of two host factor genes suggests adaptive selection for bymovirus resistance in cultivated barley from East Asia[END_REF]. The rym4 allele provides a recessive resistance to BaMMV and to the common BaYMV pathotype BaYMV-1, but not to pathotype BaYMV-2, which emerged in Europe at the end of the 1980s [START_REF] Adams | Occurrence of two strains of barley yellow mosaic virus in England[END_REF][START_REF] Huth | Ein weiterer Stamm des barley yellow mosaic virus aufgefunden[END_REF][START_REF] Adams | The distribution of barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) in UK winter barley samples, 1987-1990[END_REF][START_REF] Graner | RFLP mapping of the ym4 virus resistance gene in barley[END_REF][START_REF] Steyer | Characterization of a resistance-breaking BaYMV isolate from Belgium[END_REF]. The spectrum of rym5 covers also BaYMV-2, however, resistance-breaking isolates of BaMMV and BaYMV have emerged [START_REF] Kanyuka | Biological and sequence analysis of a novel European isolate of barley mild mosaic virus that overcomes the barley rym5 resistance gene[END_REF]Habekuß et al. 2008;[START_REF] Li | Barley yellow mosaic virus VPg is the determinant protein for breaking eIF4E-mediated recessive resistance in barley plants[END_REF]. Facing the prospect of boom-and-bust cycles for known resistance genes [START_REF] Brown | Plant-parasite coevolution: bridging the gap between genetics and ecology[END_REF], it is critical to continue searching for alternative resistance loci to underpin resistance breeding and to allow pyramiding of disease resistance loci. In particular, sources of non-host resistance, e.g. resistance exhibited from a plant species against all isolates of a pathogen which is not coevolutionary adapted, are particularly promising as they are thought to cover a larger resistance spectrum and to be more durable [START_REF] Ayliffe | Plant nonhost resistance: paradigms and new environments[END_REF]. Bulbous barley (Hordeum bulbosum L.), a perennial wild relative and representative of the secondary gene pool of cultivated barley (Hordeum vulgare L.), has been described as source of resistance to numerous barley pathogens, including barley leaf rust [START_REF] Johnston | Rph22: mapping of a novel leaf rust resistance gene introgressed from the non-host Hordeum bulbosum L. into cultivated barley (Hordeum vulgare L.)[END_REF][START_REF] Yu | Genetic mapping of a barley leaf rust resistance gene Rph26 introgressed from Hordeum bulbosum[END_REF]) and barley powdery mildew [START_REF] Xu | Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)[END_REF][START_REF] Pickering | The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)[END_REF][START_REF] Shtaya | The resistance to leaf rust and powdery mildew of recombinant lines of barley (Hordeum vulgare L.) derived from H. vulgare × H. bulbosum crosses[END_REF]. So far, all H. bulbosum accessions investigated exhibited resistance to BaMMV and BaYMV [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF], suggesting that the species is probably a non-host to those viruses. Two major dominant resistance genes from H. bulbosum to both BaMMV and BaYMV have been described: Rym14 Hb [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF] and Rym16 Hb [START_REF] Ruge-Wehling | Mapping of Rym16Hb, the second soil-borne virus-resistance gene introgressed from Hordeum bulbosum[END_REF]. Rym14 Hb was introgressed to barley by translocation of a H. bulbosum segment to barley chromosome 6HS [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF]). In the past, a lack of suitable markers, alongside severely reduced recombination in the target region between the barley and H. bulbosum fragments, rendered precise mapping of Rym14 Hb elusive. Thanks to the development of genetic and genomic resources for H. bulbosum [START_REF] Wendler | Unlocking the secondary gene-pool of barley with next-generation sequencing[END_REF][START_REF] Wendler | Bulbosum to go: a toolbox to utilize hordeum vulgare/bulbosum introgressions for breeding and beyond[END_REF], it is now possible to fine-map loci from this species in a H. vulgare background.

We aimed to map Rym14 Hb at high resolution, and to provide markers for its introgression into elite barley, ideally without linkage drag, using large populations and high-throughput genotyping to overcome the lack of recombination.

Materials and methods

Plant material

A first round of low-resolution genetic mapping was performed using four F 6 families derived from F 5 plants heterozygous at the Rym14 Hb locus from the BAZ-4006 family of the population described in [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF] and obtained from the cross between the susceptible winter barley cv 'Borwina' and the resistant H. bulbosum accession 'A42'.

To achieve a population size suitable for fine mapping, an additional eight F 2 families were generated by crossing an Rym14 Hb /Rym14 Hb F 6 plant (derived from F 5 4006/337) to either (i) var. 'KWS Orbit' or (ii) var. 'KWS Higgins', both missing the Rym14 Hb resistance locus (-/-). In the purpose of instant pyramiding of disease resistance loci, both cultivars carry rym4-based resistance (rym4/rym4) to BaMMV and BaYMV.

DNA extraction

Genomic DNA of plants from the low-resolution mapping population was isolated as described by [START_REF] Stein | A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum[END_REF]. Genomic DNA of plants from the fine mapping population was extracted according to the guanidine isothiocyanatebased protocol described by [START_REF] Milner | Genebank genomics reveals the diversity of a global barley collection[END_REF].

Genotyping-by-sequencing and data analysis

Genotyping-by-sequencing (GBS) libraries for the low-resolution mapping were prepared from genomic DNA digested with PstI and MspI (New England Biolabs) as described by [START_REF] Wendler | Bulbosum to go: a toolbox to utilize hordeum vulgare/bulbosum introgressions for breeding and beyond[END_REF]. Between 93 and 153 barcoded samples were pooled in an equimolar manner per lane and sequenced on the Illumina HiSeq 2500 for 107 cycles, single-end reads, using a custom sequencing primer.

The GBS reads were processed, aligned, and used to generate variant calls as described by [START_REF] Milner | Genebank genomics reveals the diversity of a global barley collection[END_REF]. Alignment was performed against the TRITEX genome assembly of barley cultivar 'Morex' [START_REF] Monat | TRITEX: chromosome-scale sequence assembly of triticeae genomes with open-source tools[END_REF]. Individual variant calls were accepted wherever the read depth exceeded four. Variant sites were retained if they presented a minimum mapping quality score (based on read depth ratios calculated from the total read depth and depth of the alternative allele) of 20, a maximum fraction of 40% of missing data, a fraction of heterozygous calls between 30 and 70%, and between 10 to 40% of each homozygous call. Individuals with more than 40% missing data were excluded.

3

Marker development

Exome capture data of the introgression line '4006/163', described in [START_REF] Wendler | Unlocking the secondary gene-pool of barley with next-generation sequencing[END_REF] (accession number ERP004445), were mapped to the TRITEX genome assembly of barley cultivar 'Morex' [START_REF] Monat | TRITEX: chromosome-scale sequence assembly of triticeae genomes with open-source tools[END_REF] together with the exome capture data of the H. bulbosum genotype 'A42' and of eight barley varieties: 'Bonus', 'Borwina', 'Bowman', 'Foma', 'Gull', 'Morex', 'Steptoe', and 'Vogelsanger Gold', described in Mascher et al. (2013b) (accession number PRJEB1810). Read mapping and variant calling were performed as described by [START_REF] Milner | Genebank genomics reveals the diversity of a global barley collection[END_REF]. The variant matrix was filtered for the following criteria: heterozygous and homozygous calls had to be covered by a minimum depth of three and five reads, respectively, and have a minimum quality score of 20. Single nucleotide polymorphism (SNP) sites were retained if they had less than 20% missing data and less than 20% heterozygous calls. SNPs that were carrying the reference call in all eight barleys and the alternate call in 'A42' and '4006/163' was selected as candidates to design Kompetitive Allele Specific PCR (KASP) markers, either using KASP-by-design (LGC Genomics, Berlin, Germany) or 3CR Bioscience (Essex, UK) free assay design service. Those markers are latter designated as KASP and PCR Allele Competitive Extension (PACE) markers, respectively. Since no suitable SNPs were identified in the first 500 kbp of chromosome 6HS on the 'Morex' reference genome, the exome capture data were additionally mapped to the genome assembly of cultivar 'Barke' (Jayakodi et al. in press). The SNP at coordinate 241,723 bp on chromosome 6H of the 'Barke' genome assembly was retrieved and used to design the telomeric marker Rym14_Bar241723. Furthermore, in order to control the genetic state at the segregating rym4 resistance locus, the diagnostic SNP for the resistance conferring allele [START_REF] Stein | The eukaryotic translation initiation factor 4E confers multiallelic recessive bymovirus resistance in Hordeum vulgare (L.)[END_REF] was also used to design a KASP marker. Further information on KASP and PACE markers is provided in supplementary tables 1 and 2, respectively.

Genotyping

Genotyping assays with KASP markers were carried out in a final volume of 5 μl consisting of 0.7 μl genomic DNA (50-100 ng/µL), 2.5 μl of KASP V4.0 2X Master Mix High Rox (LGC Genomics, Berlin), 0.07 μl KASP assay mix (KASP-by-design, LGC Genomics, Berlin) containing the primers, and 2.5 μl of sterile water. PCR amplifications were performed using the Hydrocycler 16 (LGC Genomics, Berlin) with cycling conditions as follows: 94 °C for 15 min, followed by a touchdown profile of 10 cycles at 94 °C for 20 s and 61 °C for 1 min with a 0.6 °C reduction per cycle, followed by 26 cycles at 94 °C for 20 s and 55 °C for 1 min. Genotyping assays with PACE markers were carried out in a final volume of 5 μl consisting of 0.7 μl genomic DNA (50-100 ng/µL), 2.5 μl of PACE Master Mix High Rox (3cr Bioscience, Essex, United Kingdom), 0.07 μl primer mix containing the primers (12 µM of each allele specific primers and 30 µM of the common reverse primer), and 2.5 μl of sterile water. PCR amplifications were performed using the Hydrocycler 16 (LGC Genomics, Berlin) with cycling conditions as follows: 94 °C for 15 min, followed by a touchdown profile of 10 cycles at 94 °C for 20 s and 65 °C for 1 min with a 0.8 °C reduction per cycle, followed by 30 cycles at 94 °C for 20 s and 57 °C for 1 min.

For both marker types, the genotyping results were read out using the ABI 7900HT (Applied Biosystems) using an allelic discrimination file. Readings were made before and after PCR, and the data were analyzed using SDS 2.4 Software (Applied Biosystems).

Phenotyping

Resistance to BaMMV was tested under greenhouse conditions as described by Habekuß et al. (2008). After sowing, the plants were grown in a greenhouse (16 h day/8 h night, 12 °C). The susceptible barley variety 'Maris Otter' was systematically included to monitor success of infection. At the 3-leaf stage (around 2 weeks after sowing), the plants were mechanically inoculated twice at an interval of 5-7 days with the isolate BaMMV-ASL1 (Timpe and Kühne 1994) using the leaf-sap of BaMMV-infected leaves of susceptible cv. 'Maris Otter', mixed in K 2 HPO 4 buffer (1:10; 0.1 M; pH 9.1) containing silicon carbide (caborundum, mesh 400, 0.5 g/25 ml sap). Five weeks after the first inoculation, the number of infected plants with mosaic symptoms were scored, and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal BaMMVspecific antibodies produced by the Serum Bank of the Institute of Epidemiology and Pathogen Diagnostics (JKI Quedlinburg, Germany) was carried out in parallel according to published protocols [START_REF] Clark | Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses[END_REF]. Virus particles were estimated via extinction at 405 nm using a Dynatech MR 5000 microtiter-plate reader. Plants with an extinction E 405 > 0.1 were qualitatively scored as susceptible.

Results

Low-resolution mapping

A population of 427 F 6 from the cross 'Borwina' x 'A42' was genotyped by GBS and phenotyped for resistance to BaMMV. Data for 389 plants and 77 SNPs passed the quality filters (supplementary table 3). On chromosome 6H, 73 plants were homozygous for the 'Borwina' allele, 92 were homozygous for the 'A42' allele, 220 were heterozygous, 1 3 and four recombined. The infection rate was low with only 10% of plants infected, compared to an expected 25% when resistance is controlled by a single dominant gene. Two susceptible barley cultivars were tested: 92.5% and 72.2% of Maris Otter and Igri plants, respectively, were infected. It is known that the penetrance of infection in such experiments is never complete, and that the genetic background of the plant plays a role in this phenomenon, Maris Otter being the most susceptible cultivar tested [START_REF] Adams | The susceptibility of barley cultivars to barley yellow mosaic virus (BaYMV) and its fungal vector, Polymyxa graminis[END_REF][START_REF] Adams | The effect of cereal break crops on barley mild mosaic virus[END_REF]. Despite that, among the 39 plants phenotyped as susceptible to BaMMV, 38 were homozygous for the 'Borwina' allele and one recombined on chromosome 6H, indicating a strong association of phenotype and genotype.

To further confirm this association, 26 lines were phenotyped on progenies of 12-20 plants (Fig. 1a, Table 1, supplementary table 4). These included (i) 17 lines with the susceptible genotype on chromosome 6H but scored as resistant, (ii) five heterozygous lines, and (iii) the four recombinant lines. Progenies of lines presenting the susceptible genotype displayed infection rates between 50 and 95%, while those of heterozygous lines displayed rates between 5 and 20%. The progeny of line 5204-58 displayed an intermediate level of susceptibility, with 35% of infected plants. However, this line had been phenotyped as susceptible in F 2 generation, and was therefore classified as susceptible.

These results support the low penetrance of the infection in this experiment, with only half of the expected susceptible plants successfully infected, as well as the association of the chromosome 6H locus with resistance to BaMMV. Moreover, the phenotypes of the four recombinant progenies defined Rym14 Hb interval between the telomere of chromosome 6HS and the marker position at base pair 4,553,134.

A B

Fine mapping

The population of 7500 F 2 was genotyped at the Rym14 Hb locus with four KASP markers (Rym14_Bar241723, Rym14_2370223, Rym14_3087282, and Rym14_5003183, supplementary table 1). The recessive resistance gene rym4 to BaMMV and BaYMV-1, located on chromosome 3HL, also segregated in the population. Therefore, in order to properly assign the resistance to the control of Rym14 Hb , the segregation of rym4 was monitored with the rym4_SNP KASP marker (supplementary table1). We identified 28 recombination events, corresponding to a genetic distance of ~ 0.2 cM, between the markers Rym14_Bar241723 and Rym14_5003183. These results confirmed the strongly reduced recombination rate between the H. bulbosum and the H. vulgare fragments on chromosome 6HS. In cultivated barley, the syntenic 5 Mbp Rym14 Hb interval on chromosome 6HS corresponds to a genetic distance of 4 cM (Mascher et al. 2013a), implying a 20-fold reduction in recombination frequency between the H. bulbosum and the H. vulgare fragment.

All recombinants were genotyped with seven PACE markers (Fig. 1b, supplementary tables 2 and 4). Among the recombinants, ten plants were homozygous for the rym4 allele, nine were heterozygous, and the remaining nine were homozygous wildtype at the rym4 locus (supplementary table 5). As plants homozygous for the rym4 allele would be resistant to BaMMV, irrespective to their genotype at Rym14 Hb , only F 3 families derived from the 18 Rym14-recombinants heterozygous or homozygous for the susceptible allele at rym4 were phenotyped using 30 and 20 F 3 siblings, respectively (Table 2). The infection rate during 6). The Rym14 Hb phenotypes of the recombinant lines were deduced from the segregation of infection in F 3 progenies that does not carry rym4 at homozygous state, controlled by a χ 2 test of goodness to fit the expected ratio of 3R:1S (three resistant plants for one susceptible plant expected for a dominant locus) for segregation of a single dominant resistance gene (Table 2). The cosegregation of the phenotype with the genotype at the three markers in the F 3 progenies largely confirmed the analysis (supplementary table 6). The only discrepancy was the significant χ 2 result for line Rym14_49/7_149, that was explained by a distortion of segregation of the susceptible allele in the small number of plants tested: a single plant was homozygous for H. vulgare allele in Rym14 Hb interval.

Based on this analysis, the Rym14 Hb target region was reduced to a 2 Mbp interval on the 'Morex' reference genome, between the telomere of chromosome 6HS and Rym14_2066975 (Fig. 1b, Table 2).

Candidate genes

In the absence of a genomic sequence for a Rym14 Hb plant, we cannot precisely define the genes present in the Rym14 Hb interval. However, as synteny between the two Hordeum species is high [START_REF] Wendler | A high-density, sequence-enriched genetic map of hordeum bulbosum and its collinearity to H. vulgare. plant[END_REF], it is still relevant to assess the genes annotated in the orthologous interval of the H. vulgare reference genome as a proxy for suggesting Rym14 Hb candidate genes. In the respective interval of the 'Morex' V2 reference sequence, 30 high-confidence (HC) (Table 3) and 17 low-confidence genes [START_REF] Monat | TRITEX: chromosome-scale sequence assembly of triticeae genomes with open-source tools[END_REF] are annotated. In addition, all HC gene models were checked for homology with other genes by a BLASTx (v2.9.0, default parameters) homology searches against the non-redundant protein sequence database [START_REF] Camacho | BLAST+: architecture and applications[END_REF]

and for

Table 2 Phenotype of the progeny that are not homozygous for the rym4 allele of the 18 F 2 recombinants from high-resolution mapping population. F 3 plants that are homozygous for rym4 allele were excluded from this table a Specified as the recombinant (rec) haplotype name in Fig. 1 b Resulting P-value from a χ 2 test of goodness to fit the expected ratio of 3R:1S (three resistant plants for one susceptible plant expected for a dominant locus) c Resistant (R), susceptible (S) or segregating (seg) presence of conserved domains in NCBI conserved domains [START_REF] Lu | CDD/SPARCLE: the conserved domain database in 2020[END_REF]). Among the HC genes, HORVU.MOREX. r2.6HG0448010 is annotated as a TIR-NBS-LRR gene in [START_REF] Monat | TRITEX: chromosome-scale sequence assembly of triticeae genomes with open-source tools[END_REF], however, our analysis reveals that it does not contain any of the major domains of nucleotidebinding and leucine-rich repeat domain (NLR) genes (TIR or coiled-coil, NB-ARC and LRR), and monocotyledons so far have not been shown to contain TIR-NLR genes [START_REF] Jacob | Evolution and conservation of plant NLR functions[END_REF]). This gene is therefore interpreted as a pseudogene. HORVU.MOREX.r2.6HG0448100, annotated as a dirigent protein, is a jacalin-related lectin, while HORVU. MOREX.r2.6HG0448250, annotated as part of the protein kinase protein family, displays the highest homology with a wall-associated receptor kinase, and HORVU.MOREX. r2.6HG0448290 codes for a papain-like cysteine protease (PLCP). Interestingly, the interval also contains no less than 14 HC genes annotated as thionins, sharing with each other at least 88% of their coding sequence. In addition to these annotated genes in the 'Morex' genome, additional candidate genes could be unique to the resistant genotypes.

Discussion

Resistance genes deployed in breeding and in the field are often overcome by new pathogen variants after only a few years [START_REF] Brown | Plant-parasite coevolution: bridging the gap between genetics and ecology[END_REF]. Pyramiding several resistance genes has proven to increase the resistance durability, however, this strategy requires the availability of several independent resistance loci [START_REF] Werner | Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2)[END_REF][START_REF] Riedel | Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate[END_REF][START_REF] Kim | Evaluation and verification of barley genotypes with known genes for resistance to barley yellow mosaic virus and barley mild mosaic virus under field conditions in South Korea[END_REF]. In light of these facts, non-adapted resistance genes from wild crop relatives are precious, since they are assumed to confer more durable resistance than genes originating from within the diversity of the cultivated species, owing to co-evolution between the cultivated host and pathogen genotypes [START_REF] Fonseca | Genes involved in nonhost disease resistance as a key to engineer durable resistance in crops[END_REF]. Until recently, the fine mapping of genes from crop wild relative species was impractical, owing to strong suppression of recombination with the cultivated species [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF][START_REF] Kakeda | Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild selfincompatible species related to cultivated barley[END_REF][START_REF] Wijnker | Managing meiotic recombination in plant breeding[END_REF][START_REF] Prohens | Introgressiomics: a new approach for using crop wild relatives in breeding for adaptation to climate change[END_REF]. The results of this study demonstrate that high-throughput genotyping coupled with large mapping populations can overcome this limitation, by constraining the interval of the Rym14 Hb viral resistance gene to the telomeric 2 Mbp of chromosome 6HS, and providing markers suitable for marker-assisted-selection.

Rym14 Hb was described as providing resistance against both BaMMV and BaYMV [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF]. However, phenotyping for resistance to BaYMV is only feasible in infested fields, and is not well adapted to gene mapping. Therefore, in this study, we only mapped BaMMV resistance. Among cloned by movirus resistance genes, the resistance alleles rym4 and rym5 of the eIF4E gene, and the alleles rym1 and rym11 of PDI5-1 gene provide resistance against isolates of both virus species [START_REF] Kanyuka | Biological and sequence analysis of a novel European isolate of barley mild mosaic virus that overcomes the barley rym5 resistance gene[END_REF][START_REF] Stein | The eukaryotic translation initiation factor 4E confers multiallelic recessive bymovirus resistance in Hordeum vulgare (L.)[END_REF][START_REF] Ordon | Molecular assessment of genetic diversity in winter barley and its use in breeding[END_REF][START_REF] Habekuss | Identification of barley mild mosaic virus Isolates in Germany breaking rym5 resistance*[END_REF]). Thus, the two viruses are genetically similar enough for a gene to provide resistance against isolates of both viruses. But the possibility of the described Rym14 Hb BaYMV resistance being provided by a closely associated, but distinct, locus cannot be excluded at this point and will require further testing.

While genes coding for NLR are the usual suspects for dominant resistance to pathogens, including viruses [START_REF] De Ronde | Dominant resistance against plant viruses[END_REF][START_REF] Boualem | The battle for survival between viruses and their host plants[END_REF], only a pseudogene presenting similarities with this gene family is annotated in the Rym14 Hb interval on the barley reference genome. However, it is not rare that susceptible genotypes do not possess a functional copy of the resistance gene. NLRs are overrepresented in regions displaying presence/absence variation [START_REF] Xu | Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes[END_REF][START_REF] Bush | Presenceabsence variation in A. thaliana is primarily associated with genomic signatures consistent with relaxed selective constraints[END_REF]. Therefore, some NLR resistance genes, like RPM1 and RPS5, are only present in the resistant genotype [START_REF] Grant | Independent deletions of a pathogen-resistance gene in brassica and arabidopsis[END_REF][START_REF] Henk | A new Ac-like transposon of arabidopsis is associated with a deletion of the RPS5 disease resistance gene[END_REF]). In the case of wheat leaf rust resistance gene Lr21, it was shown that the gene is a chimera of two non-functional alleles that probably evolved via a recombination event [START_REF] Huang | Evolution of new disease specificity at a simple resistance locus in a crop-weed complex: reconstitution of the <em>Lr21</em> gene in wheat[END_REF]).

Among the other annotated genes at the Rym14 Hb locus, two are very good candidates. Wall-associated protein kinase-like HORVU.MOREX.r2.6HG0448250 is described resistance genes in plant-bacteria and plant-fungus pathosystems [START_REF] Li | A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance[END_REF][START_REF] Li | CsWAKL08, a pathogen-induced wallassociated receptor-like kinase in sweet orange, confers resistance to citrus bacterial canker via ROS control and JA signaling[END_REF][START_REF] Dmochowska-Boguta | TaWAK6 encoding wall-associated kinase is involved in wheat resistance to leaf rust similar to adult plant resistance[END_REF]. Their role in plant-virus pathosystems is less clear but it has been suggested that a cell wall-associated protein kinase was involved in the repression of plasmodesmal transport of the Tobacco mosaic virus by phosphorylating its movement protein [START_REF] Citovsky | Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase[END_REF][START_REF] Waigmann | Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein[END_REF]. A second promising candidate is HORVU.MOREX. r2.6HG0448100. It codes for a jacalin-related lectin and is thus part of the family that includes the Arabidopsis thaliana genes RTM1 and JAX1 that provide dominant major resistance against poty-and potexviruses, respectively [START_REF] Chisholm | Cloning of the arabidopsis RTM1 gene, which controls restriction of longdistance movement of tobacco etch virus[END_REF][START_REF] Yamaji | Lectin-mediated resistance impairs plant virus infection at the cellular level[END_REF].

However, other genes in the Rym14 Hb interval, even if less likely candidates, might also play a role in resistance. For example, HORVU.MOREX.r2.6HG0448290 codes for a PLCP. PLCPs are known to play a major role in programmed cell death triggered by NLR genes. Interestingly, CYP1, a tomato PLCP, is targeted by the Tomato yellow leaf curl virus V2 protein, suggesting that V2 could downregulate CYP1 to counteract host defenses [START_REF] Bar-Ziv | The tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1[END_REF]. Rcr3, a tomato papain-like cysteine protease gene, is required for the function of the resistance gene Cf-2 to Cladosporium fulvum [START_REF] Krüger | A tomato cysteine protease required for <em>Cf-2</em>-dependent disease resistance and suppression of autonecrosis[END_REF], while NbCathB, from Nicotina benthamiana, is requested for the HR triggered by the non-host pathogens Erwinia amylovora and Pseudomonas syringae [START_REF] Gilroy | Involvement of cathepsin B in the plant disease resistance hypersensitive response[END_REF]). The high level of thionin duplication at this locus also raised our attention. Thionins are part of common anti-bacterial and antifungal peptides [START_REF] Bohlmann | The role of thionins in plant protection[END_REF], conferring enhanced resistance to several pathogens. Thionins were also found to exhibit increased expression in resistant compared to susceptible pepper genotypes during infection by the Chili leaf curl virus [START_REF] Kushwaha | Chilli leaf curl virus infection highlights the differential expression of genes involved in protein homeostasis and defense in resistant chilli plants[END_REF], suggesting a possible role in basal defense. Additionally, the cytochrome P450 superfamily has been associated with resistance to the Soybean mosaic virus [START_REF] Cheng | Polymorphisms of soybean isoflavone synthase and flavanone 3-hydroxylase genes are associated with soybean mosaic virus resistance[END_REF][START_REF] Yang | Proteomics-based analysis of novel genes involved in response toward soybean mosaic virus infection[END_REF]. Some subtilisin proteases are induced by pathogens and involved in programmed cell death [START_REF] Figueiredo | Subtilisin-like proteases in plant-pathogen recognition and immune priming: a perspective[END_REF], and GDSL lipases were found to be either negative or positive regulators of plant defense mechanisms [START_REF] Hong | Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance[END_REF][START_REF] Kwon | GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in arabidopsis[END_REF].

The feasibility of further reducing the target interval by recombination through additional fine mapping is low and would require the screening of tens of thousands of additional F 2 plants for the chance of finding one additional recombinant in the smallest target region. Therefore, a candidate gene approach may be a more fruitful strategy for continued progress. Despite the presence of promising candidate genes like HORVU.MOREX.r2.6HG0448250 and HORVU. MOREX.r2.6HG0448100 in the haplotype of the susceptible cultivar 'Morex', the resistance conferring gene may be present only in the haplotype of the resistant H. bulbosum. Therefore, deciphering the resistant haplotype, most likely though a high-quality chromosome-scale genome assembly of the interval in H. bulbosum, is an essential prerequisite to the prioritization of candidate genes for further functional testing.

The markers identified in this study are tightly linked to Rym14 Hb and therefore are of prime importance to barley breeding. These markers will allow the reliable introgression of this resistance into barley elite lines with a minimum of 1 3 linkage drag compared to the previously established markers [START_REF] Ruge | Mapping of Rym14Hb, a gene introgressed from Hordeum bulbosum and conferring resistance to BaMMV and BaYMV in barley[END_REF]. This is essential for introducing this gene into new cultivars. As the prevalence of resistance-breaking isolates of rym4 and rym5 will increase in the barley growing area in Europe and Asia [START_REF] Kühne | Soil-borne viruses affecting cereals-known for long but still a threat[END_REF], introgression of Rym14 Hb into new elite varieties together with other resistance loci represents a critical opportunity to improve the durability and spectrum of barley resistance to BaMMV and BaYMV.

Fig. 1

 1 Fig. 1 Physical map of the Rym14 Hb locus. a Low-resolution mapping of the Rym14 Hb locus. Graphical genotype and phenotype of the four recombinant F 6 lines. H. vulgare, H. bulbosum, and heterozygous allelic states are represented as orange, blue, and yellow bars, respectively. Coordinates on 'Morex' reference genome (Monat et al. 2019) of strategic markers are displayed. Names of the haplotypes are displayed on the left, and phenotypes deduced from the phenotyped F 7 progenies are shown on the right (R resistant, S susceptible, seg seg-

Table 1

 1 Phenotype of 26 F 2 lines from the cross 'Borwina' x 'A42' and of their F 3 progenies a Either the recombinant haplotype name in Fig.1, or, for non-recombinant lines, 'heterozygous' or 'H. vulgare'

	b Resistant (R), susceptible (S) or segregating (seg)

Table 3

 3 Genes annotated with high confidence in Rym14 Hb interval on the 'Morex' genome[START_REF] Monat | TRITEX: chromosome-scale sequence assembly of triticeae genomes with open-source tools[END_REF]

	Name	Start	Stop	Gene type
	HORVU.MOREX.r2.6HG0447840	195,540	196,334	Thionin
	HORVU.MOREX.r2.6HG0447850	220,610	221,213	Thionin
	HORVU.MOREX.r2.6HG0447860	256,998	259,999	Thionin
	HORVU.MOREX.r2.6HG0447880	373,994	438,209	Thionin
	HORVU.MOREX.r2.6HG0447890	460,556	461,157	Thionin
	HORVU.MOREX.r2.6HG0447900	461,856	462,457	Thionin
	HORVU.MOREX.r2.6HG0447910	497,194	497,795	Thionin
	HORVU.MOREX.r2.6HG0447920	597,800	598,403	Thionin
	HORVU.MOREX.r2.6HG0447930	625,302	625,905	Thionin
	HORVU.MOREX.r2.6HG0447940	691,184	707,575	Thionin
	HORVU.MOREX.r2.6HG0447950	749,829	776,991	Thionin
	HORVU.MOREX.r2.6HG0447960	792,195	827,832	Thionin
	HORVU.MOREX.r2.6HG0447980	958,137	958,736	Thionin
	HORVU.MOREX.r2.6HG0447990	1,004,017	1,004,618	Thionin
	HORVU.MOREX.r2.6HG0448010	1,259,976	1,260,591	TIR-NBS-LRR class disease resistance protein
	HORVU.MOREX.r2.6HG0448020	1,300,107	1,300,565	Dimeric alpha-amylase inhibitor
	HORVU.MOREX.r2.6HG0448100	1,493,250	1,493,945	Dirigent protein
	HORVU.MOREX.r2.6HG0448110	1,574,160	1,575,749	Cytochrome P450 family protein, expressed
	HORVU.MOREX.r2.6HG0448120	1,578,752	1,580,023	Aspartic proteinase nepenthesin-1
	HORVU.MOREX.r2.6HG0448130	1,598,418	1,600,649	Subtilisin-like protease
	HORVU.MOREX.r2.6HG0448140	1,605,306	1,610,732	Fatty acyl-CoA reductase
	HORVU.MOREX.r2.6HG0448160	1,753,412	1,756,451	Glycerol-3-phosphate acyltransferase 3, putative
	HORVU.MOREX.r2.6HG0448200	1,792,383	1,794,963	Transposon protein, putative, CACTA, En/Spm sub-class
	HORVU.MOREX.r2.6HG0448210	1,796,825	1,804,280	O-acyltransferase WSD1
	HORVU.MOREX.r2.6HG0448220	1,840,897	1,842,376	GDSL esterase/lipase
	HORVU.MOREX.r2.6HG0448230	1,853,483	1,854,626	Short-chain dehydrogenase/reductase
	HORVU.MOREX.r2.6HG0448250	1,945,996	1,952,442	Protein kinase family protein
	HORVU.MOREX.r2.6HG0448260	1,954,346	1,955,384	zinc finger MYM-type-like protein
	HORVU.MOREX.r2.6HG0448290	2,061,596	2,062,919	Cysteine protease-like protein
	HORVU.MOREX.r2.6HG0448300	2,066,856	2,067,293	Proteinase inhibitor type-2

Acknowledgements

We gratefully acknowledge the excellent technical support by Manuela Kretschmann in DNA extraction and KASP genotyping, Dörte Grau in BaMMV resistance phenotyping and Susanne König in GBS library preparation. We thank Axel Himmelbach for his valuable support in next generation sequencing, Klaus Oldach, Viktor Korzun and Jörg Grosser for their valuable inputs and Timothy Rabanus-Wallace for language editing.

Funding Open Access funding enabled and organized by Projekt DEAL. This work was supported as part of the collaborative projects "TransBulb" (grant 0315966 from the German Federal Ministry of Education and Research (BMBF)) and "BulbOmics" (grant 2818201615 from the German Federal Ministry of Food and Agriculture (BMEL)).

Availability of data and material

The GBS dataset generated and analyzed in this study is deposited at EMBL-ENA under the project ID PRJEB39211.

Code availability Not applicable.

Compliance with ethical standards

Author contributions statement NS, FO, and DP concepted the project and acquired the funding. BRW and AM designed and constructed the mapping populations. HP and NW performed the genotyping experiments. AH carried out the phenotyping experiments. HP processed the experimental data, performed the analysis and drafted the manuscript. NS supervised the project. All authors provided critical feedback and helped shape the manuscript.

Conflict of interest NW and AM are employed at KWS SAAT SE &

Co and KWS LOCHOW, respectively. The other authors declare no conflict of interest.

Ethics approval Not applicable.

Consent to participate Not applicable.

Consent for publication Not applicable.

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