Screening of Wheat Genotypes for Nitrogen Deﬁciency Tolerance Using Stress Screening Indices

: An increased awareness of environmental protection and sustainable production raise the necessity of incorporating the selection of low nitrogen-tolerant winter wheat cultivars for high yield and quality in the breeding process. This selection can be assisted by using stress screening indices. Our study aimed to evaluate and compare a number of stress screening indices and to determine and select the most nitrogen deﬁciency-tolerant winter wheat cultivars for further breeding. The experiment included forty-eight winter wheat cultivars from eight different countries that were grown for two consecutive years at three different locations under low-nitrogen (LN) and high-nitrogen (HN) conditions. The results emphasized the importance of applying the appropriate stress screening indices in evaluating and selecting nitrogen deﬁciency-tolerant wheat cultivars. The promising stress screening indices were the mean productivity index (MP), geometric mean productivity index (GMP), harmonic mean index (HM), stress tolerance index (STI) and yield index (YI). They identiﬁed cultivars Sofru, BC Opsesija and MV-Nemere as the most tolerant cultivars to LN conditions for grain yield. The same indices classiﬁed U-1, OS-Olimpija, Forcali, Viktoria and BC Tena cultivars as the most tolerant to LN conditions for the grain protein content. Using the tolerance index (TOL), yield stability index (YSI) and relative stress index (RSI), the Katarina and Ficko cultivars were denoted as LN-tolerant cultivars in terms of the grain yield and Isengrain, Tosunbey, Vulkan and BC Darija in terms of the grain protein content. of 100


Introduction
Nitrogen, as one of the essential nutrients for plant growth, development and productivity, is a substantial requirement for efficient and profitable crop production. Its deficiency may lead to changes in the gene expression, plant metabolism, growth rates and, finally, crop yield and quality reduction [1,2]. In order to avoid crop failure, N fertilizers are usually applied in large quantities. However, the excessive application of N fertilizers beyond nitrogen plant requirements has adversely affected the environment, and it has become obvious that, due to a number of various damaging effects on the environment and economic costs, the high-nitrogen fertilizer consumption has to be optimized [3].
An increased awareness of environmental protection and sustainable production raises the necessity to incorporate a selection of low-nitrogen-tolerant winter wheat cultivars for high yield and quality in the breeding process. Consequently, breeding for high-nitrogen use efficiency (NUE) has become of great importance. The most commonly used concept of breeding for NUE is to select superior germplasms for the grain yield per se by exploiting

Plant Materials and Field Experiment
The experimental material included forty-eight winter wheat cultivars released from 1936 to 2016, mostly by Croatian-HR breeding institutes (33 cultivars) and the rest by breeding programs from seven other countries (Austria-AU, France-FR, Hungary-HU, Italy-IT, Romania-RO, Russia-RUS and Turkey-TR).
Cultivars were evaluated in field trials under two N fertilization levels during two consecutive growth seasons (2016/17 and 2017/18) at Osijek (45 • 33 N, 18 • 40 E), Zagreb (45 • 48 N, 15 • 58 E) and Poreč (45 • 13 N, 13 • 35 E). These locations represent different climatic conditions and soil types in Croatia. In general, Osijek and Zagreb have continental climates, dry with an Eutric Cambisol soil type and humid with a carbonate alluvium soil type, respectively. Poreč has a Mediterranean climate and red soil.
The experiment was set up as a split-plot factorial design in three replications with two N fertilization levels as the main plots and 48 wheat cultivars as the subplots. The plot sizes were 7.56 m 2 at Osijek and Poreč and 4.95 m 2 at Zagreb. The sowing rate was 350 viable kernels m −2 in all trials and for all cultivars. In order to avoid edge effects on the experimental plots, buffer plots were sown at the beginning and at the end of the main N Agronomy 2021, 11, 1544 3 of 15 treatment plots. Basic and pre-sowing fertilizations of 74-kg N ha −1 , 80-kg P 2 O 5 ha −1 and 120-kg K 2 O ha −1 were applied by adding 100 kg ha −1 of urea (46% N) and 400 kg ha −1 of NPK (7:20:30) in order to achieve the experimental conditions as similar as possible to those conditions in wheat production in the region where nitrogen is applied before planting. The N treatment comprised two N fertilization levels, 0-kg N ha −1 (low N, LN) and 100-kg N ha −1 (high N, HN), in a form of calcium ammonium nitrate (27% N). HN-level plots were top-dressed by 50-kg N ha −1 in tillering (GS23-25 after [18]) and by 50-kg N ha −1 during the stem extension (GS33-35 [18]) growth stages.
Herbicides, insecticides and fungicides to control major weeds, insects and foliar diseases were applied in accordance with the commercial practices of wheat production in the Southeastern European region. All trials were harvested at harvest maturity (GS92 [18]) by plot combines and weighed. Grain yield values were recalculated to a 0% moisture content and kg ha −1 . Grain protein content (%) was measured using an Infratec 1241 Grain Analyzer (Foss, Denmark) from the grain samples of 0.5 kg collected after the harvest across all experimental plots.

Statistical Analysis
Combined analysis of variance (ANOVA), best linear unbiased prediction (BLUP) for grain yield and grain protein content, principal component analysis (PCA) and 3D visualization of the results were performed by R software [19]. ANOVA was performed using linear model (aov function) with fixed effects for the year, location and cultivar effects, including their interactions. The screening indices were computed, and Pearson's correlation coefficients, as well as Spearman's rank order, were estimated using iPASTIC [13], which describes the stress indices as follows: The tolerance index (TOL): where Yp is the yield of the cultivar under HN conditions (e.g., yield under normal conditions to achieve the full potential), and Ys is the yield of the cultivar under LN conditions (e.g., yield under stress conditions); The stress susceptibility index (SSI): where SI = 1− Yms Ymp (SI is the stress intensity, and Yms and Ymp are the yield means of all cultivars under stress and without it, respectively); The yield stability index (YSI): the yield index (YI): the mean productivity index (MP): the geometric mean productivity index (GMP): the stress tolerance index (STI): The combined ANOVA revealed varying effects of the tested factors on the grain yield at the high-nitrogen level (Yp), low-nitrogen level (Ys) and nine indices ( Table 1). The cultivar significantly affected the Yp, Ys, MP, GMP, HM, STI and YI values. The effect of location was significant for Yp; Ys and only three screening indices (MP, GMP and HM). Similarly, the year significantly affected the Yp; Ys and TOL, STI and YSI. The cultivar × location (G × L), cultivar × year (G × Y), location × year (L × Y) and cultivar × location × year (G × L × Y) interactions were significant for the Yp, MP, GMP, HM and STI. The G × L and L × Y interactions were significant for the TOL, while the G × L and G × Y interactions were significant for the YI. In addition, L × Y was significant for the YSI, and G × L was significant for the RSI. None of the factors significantly affected the SSI (stress screening index). df -degrees of freedom, ns-not significant, ** and ***-significant at the levels of probability of p < 0.01 and 0.001, respectively, Yp-grain yield under HN, Ys-grain yield under LN, TOL-tolerance index, MP-mean productivity index, GMP-geometric mean productivity index, HM-harmonic mean index, SSI-stress susceptibility index, STI-stress tolerance index, YI-yield index, YSI-yield stability index and RSI-relative stress index.
The combined ANOVA for the grain protein content and nine indices showed that the effects of the cultivar, location, year, G × L, G × Y, L × Y and G × L × Y were significant for the Yp, MP, GMP and HM and not significant for the SSI and RSI ( Table 2). The cultivar significantly affected the Ys, STI and YI, while the location affected the Ys, TOL, STI and YSI. The effect of the year was significant for the Ys. The STI and YI were significantly affected by the G × L, G × Y and G × L × Y interactions. The Ys was affected by the G × Y and L × Y interactions, and the TOL was affected by the L × Y interaction. df -degrees of freedom; ns-not significant; *, ** and ***-significant at the level of probability of p < 0.05, 0.01 and 0.001, respectively; Yp-grain yield under HN; Ys-grain yield under LN; TOL-tolerance index; MP-mean productivity index; GMP-geometric mean productivity index; HM-harmonic mean index; SSI-stress susceptibility index; STI-stress tolerance index; YI-yield index; YSI-yield stability index and RSI-relative stress index.

BLUP Values for Grain Yield and Grain Protein Content and Stress Tolerance Indices
The BLUP values for the grain yield under high-(Yp) and low (Ys)-nitrogen conditions and nine stress tolerance indices across the cultivars are shown in Table 3   Yp-grain yield under HN, Ys-grain yield under LN, TOL-tolerance index, MP-mean productivity index, GMP-geometric mean productivity index, HM-harmonic mean index, SSI-stress susceptibility index, STI-stress tolerance index, YI-yield index, YSI-yield stability index and RSI-relative stress index.
The BLUP values for the grain protein content under the high-and low-nitrogen conditions and nine stress tolerance indices are shown in Table 4   Yp-grain yield under HN, Ys-grain yield under LN, TOL-tolerance index, MP-mean productivity index, GMP-geometric mean productivity index, HM-harmonic mean index, SSI-stress susceptibility index, STI-stress tolerance index, YI-yield index, YSI-yield stability index and RSI-relative stress index.

Correlation Analysis of Grain Yield, Grain Protein Content and Stress Screening Indices
The correlation analysis for the grain yield and grain protein content under the low and optimal nitrogen conditions is shown in Table 5. High negative correlations were found between the grain yield and grain protein content performance for both (stress and nonstress) conditions, with r values ranging from −0.67 ** to −0.74 **. Table 5. Correlation coefficients (r) for the grain yield and grain protein content (GPC) in stress and nonstress conditions. The results of the correlation analysis for the grain yield and different stress tolerance indices revealed the variegated strengths and directions of the relationships (Figure 1). The significance of the correlations also varied (Supplementary Table S1). Very high positive correlations were found between the Ys and MP (r = 0.98 **), GMP (r = 0.98 **), HM (r = 0.99 **), STI (r = 0.98 **) and YI (r = 1.00 **), while no correlation was found between the Ys and SSI, YSI and RSI. A low, insignificant positive correlation was found between the Ys and TOL (r = 0.23). In terms of the yield performance under the high-nitrogen treatment, a very high positive correlation was found between the Yp and MP (r = 0.99 **), GMP (r = 0.98 **), HM (r = 0.98 **), STI (r = 0.98 **) and YI (r = 0.94 **). Additionally, a moderate positive significant correlation between the Yp and TOL (r = 0.55 **) and low, significant positive correlation between the Yp and SSI (r = 0.33 *) were found. A low, significant negative correlation between the Yp and YSI (r = −0.33 *) and Yp and RSI (r = −0.33 *) was observed. The TOL screening index was moderately positively correlated with the MP (r = 0.41 **), GMP (r = 40 **), HM (r = 0.39 **) and STI (r = 0.40 **) and highly with the SSI (r = 0.97 **). High negative correlations were found for both the TOL and SSI with the YSI and RSI (r = −0.97 ** to −1.00 **). The MP had a perfectly positive correlation with the GMP (r = 1.00 **), HM (r = 1.00 **) and STI (r = 1.00 **) and an almost perfect correlation with the YI (r = 0.98 **). The GMP relationships with the HM, STI and YI were the same. The STI was highly positive correlated to the YI (r = 0.98 **).
with the MP (r = 0.41 **), GMP (r = 40 **), HM (r = 0.39 **) and STI (r = 0.40 **) and highly with the SSI (r = 0.97 **). High negative correlations were found for both the TOL and SSI with the YSI and RSI (r = −0.97 ** to −1.00 **). The MP had a perfectly positive correlation with the GMP (r = 1.00 **), HM (r = 1.00 **) and STI (r = 1.00 **) and an almost perfect correlation with the YI (r = 0.98 **). The GMP relationships with the HM, STI and YI were the same. The STI was highly positive correlated to the YI (r = 0.98 **). A correlation analysis for the grain protein content and different stress tolerance indices is shown in Figure 2. The results were similar to the results of the correlation analysis for the grain yield. Very high positive correlations were found between the Yp and MP (r = 0.99 **), GMP (r = 0.99 **), HM (r = 0.99 **), STI (r = 0.95 **) and YI (r = 0.95 **), while a moderate positive correlation was found between the Yp and TOL (r = 0.58 **). No correlations were found between the Yp and the SSI, YSI and RSI. Almost identical values were calculated for the correlation coefficients between the Ys and the MP (r = 0.99 **), GMP (r = 0.99 **), HM (r = 0.99 **), STI (r = 0.98 **) and YI (r = 1.00 **). A low positive correlation was found between the Ys and TOL (r = 0.30 *) but no significant correlations between the Ys and SSI, YSI and RSI. The correlation coefficients, together with the level of significance for the grain protein content and stress screening indices, are shown in Supplementary   Figure 1. Correlation coefficients (r) and correlogram for the grain yield and stress screening indices. Ns-not significant, *, ** and ***-significant at the level of probability p < 0.05, 0.01 and 0.001, respectively.
A correlation analysis for the grain protein content and different stress tolerance indices is shown in Figure 2. The results were similar to the results of the correlation analysis for the grain yield. Very high positive correlations were found between the Yp and MP (r = 0.99 **), GMP (r = 0.99 **), HM (r = 0.99 **), STI (r = 0.95 **) and YI (r = 0.95 **), while a moderate positive correlation was found between the Yp and TOL (r = 0.58 **). No correlations were found between the Yp and the SSI, YSI and RSI. Almost identical values were calculated for the correlation coefficients between the Ys and the MP (r = 0.99 **), GMP (r = 0.99 **), HM (r = 0.99 **), STI (r = 0.98 **) and YI (r = 1.00 **). A low positive correlation was found between the Ys and TOL (r = 0.30 *) but no significant correlations between the Ys and SSI, YSI and RSI. The correlation coefficients, together with the level of significance for the grain protein content and stress screening indices, are shown in Supplementary Table S2. The TOL screening index was in a moderately positive correlation with the MP (r = 0.45 **), GMP (r = 0.44 **), HM (r = 0.43 **) and STI (r = 0.46 **) and in a high positive correlation with the SSI (r = 0.88 **). Additionally, high negative correlations were found for the TOL and SSI with the YSI and RSI (r = −0.88 ** to −1.00 **). In addition, the MP was in perfectly positive correlation with the GMP (r = 1.00 **), HM (r = 1.00 **) and STI (r = 1.00 **) and in almost perfect correlation with the YI (r = 0.99 **). The GMP relationships with the HM, STI and YI followed the trend found for the grain yield data. Additionally, the STI was also highly positive correlated to the YI (r = 0.98 **).

Figure 2.
Correlation coefficients (r) and correlogram for the grain protein content and stress screening indices. ns-not significant, *, ** and ***-significant at the level of probability p < 0.05, 0.01 and 0.001, respectively.

Identifying Nitrogen Deficiency-Tolerant Winter Wheat Cultivars
According to the MP, GMP, HM, STI and YI indices, cultivars Sofru (G40), BC Opsesija (G10) and MV-Nemere (G29) may be highlighted as the cultivars with the highest grain yield performances under stress (Ys) and nonstress (Yp) conditions (Supplementary Figure S3). Opposite to these cultivars, cultivars U-1 (G44), Bezostaja-1 (12) and BC Tena (11) had the lowest values for grain yield under both N treatments. The TOL, YSI and RSI indices indicated cultivars Katarina (G24) and Ficko (G17) as N deficiency-tolerant cultivars for the grain yield. Correlation coefficients (r) and correlogram for the grain protein content and stress screening indices. ns-not significant, *, ** and ***-significant at the level of probability p < 0.05, 0.01 and 0.001, respectively.
For the grain protein content, and according to the MP, GMP, HM, STI and YI values of the indices, U-1 (G44) had the highest value, along with the cultivar OS-Olimpija (G31), for both nitrogen conditions (Supplementary Figure S2). Cultivars Forcali (G19), Viktoria (G45) and BC Tena (G11) also revealed a high tolerance to the LN conditions for the grain protein content. The TOL, YSI and RSI indices highlighted Isengrain (G23), Tosunbey (G43), Vulkan (G46) and BC Darija (G5) as the LN-tolerant cultivars for the grain protein content.

Principal Component Analysis for Indices, Grain Yield and Grain Protein Content
A PCA biplot for the grain yield and different stress tolerance indices is shown in Figure 3. The total contribution to the first two components of variation was 99.9%. The first principal component (PC1) contributed to the variations by 64.9% and indicated a strong correlation with the YI, Ys, GMP, MP, HM, STI and Yp. The second principal component (PC2) contributed to the variations by 35%, and it had a strong correlation with the YSI, SSI, RSI and TOL.
(G45) and BC Tena (G11) also revealed a high tolerance to the LN conditions for the grain protein content. The TOL, YSI and RSI indices highlighted Isengrain (G23), Tosunbey (G43), Vulkan (G46) and BC Darija (G5) as the LN-tolerant cultivars for the grain protein content.

Principal Component Analysis for Indices, Grain Yield and Grain Protein Content
A PCA biplot for the grain yield and different stress tolerance indices is shown in Figure 3. The total contribution to the first two components of variation was 99.9%. The first principal component (PC1) contributed to the variations by 64.9% and indicated a strong correlation with the YI, Ys, GMP, MP, HM, STI and Yp. The second principal component (PC2) contributed to the variations by 35%, and it had a strong correlation with the YSI, SSI, RSI and TOL. As in the case of the PCA for the grain yield, the total contribution of the first two principal components for the grain protein content was 99.9%, while the first principal component (PC1) also contributed to the variations by 64.9% and indicated a strong correlation with the YI, Ys, GMP, HM, MP, STI and Yp (Figure 4). The second principal component (PC2) contributed to the variations by 35%, and it had a strong correlation with the YSI, RSI, SSI and TOL.

Discussion
This study was focused on evaluation of stress screening indices and analysis of low nitrogen tolerance for grain yield and grain protein content of winter wheat cultivars originated from different breeding institutions based on stress screening indices. It was con-

Discussion
This study was focused on evaluation of stress screening indices and analysis of low nitrogen tolerance for grain yield and grain protein content of winter wheat cultivars originated from different breeding institutions based on stress screening indices. It was confirmed, as in other studies [2,20,21] that the low nitrogen soil conditions significantly reduce winter wheat grain yield and grain protein content performance.
Grain yield and grain protein content were in high negative correlation independently on N conditions. That negative correlation is in agreement with other studies on wheat [22][23][24]. The result fully support previous conclusions about difficulties for breeders to simultaneously increase protein content while maintain grain yield [25].
The revealed significance of cultivar, location and year effects, and most of their interactions in this study support previously reported data on wheat grain yield and grain protein variation. A study on Croatian winter wheat [21] reported significant effects of cultivar and environment on grain yield and grain protein content, while genotype by environment interaction effect was significant only on grain yield. Other study [26] showed significant effect of genotype by environment interaction on both, grain yield and grain protein content. Our data indicated significance of all main effects as well as almost all combinations of cultivar, location and year interactions for grain yield and grain protein content, with exception of G × L interaction for grain protein content and G × L × Y interaction for grain yield and grain protein content under low nitrogen conditions. Significant variations were detected by ANOVA for investigated screening indices. In comparison to study from Zhao et al. [27], we have not observed significant location effect for STI and YSI and year effect for MP, GMP and HM. In contrast, we observed significant G × Y effect for MP. Also, we observed only significant year effect on YSI, while mentioned study observed significant effects of cultivar, location, year, G × Y and G × L. This discrepancy may be due to genetic divergence of cultivars investigated and differences in environmental conditions of experiments. TOL, YSI, MP, HM, GMP are stress screening indices that uses only grain yield and grain protein content performance of certain cultivar under stress and non-stress conditions, while SSI and RSI are using mean performance of all cultivars in both conditions besides performance of each cultivar in both conditions. YI uses only performance of cultivar under stress condition and mean performance of all cultivars under stress condition, while STI uses performance of single cultivar under stress and non-stress conditions and mean performance of all cultivars under optimal conditions. Correlation coefficient between YSI and other screening index always have same value but opposite sign from correlation coefficient between SSI and that same index. This is also confirmed in study on low nitrogen tolerance on rice [16].
This study compared several stress screening indices as tool for selection of low nitrogen tolerant winter wheat cultivars. Indices MP, GMP, HM, STI and YI showed very high positive correlation with grain yield and grain protein content under stress and non-stress conditions. Previous studies on wheat [28,29], and on other species like canola [30] and maize [31,32] confirm that trend for grain yield. According to number of studies STI, GMP and MP indices were reported as indices of choice for identification of stress tolerant wheat cultivars [33][34][35]. Interaction between TOL and grain yield under HN conditions from this study showed moderate positive correlation. Different studies on wheat [25,[36][37][38] found similar results under HN conditions. However, these studies observed significant negative correlation between yield under stressful conditions and TOL, while in our case that correlation was insignificant.
The grain yield and grain protein content of the wheat cultivars under nonstress and stress N conditions were apparently associated with the genetic structure of the cultivars. Significant differences were found between and among the cultivars, irrespective of the N conditions. Three-dimensional diagrams for the grain yield (Supplementary Figure S1) and grain protein content (Supplementary Figure S2) highlighted the cultivars with the highest values of grain yield or grain protein content, along with the highest MP, GMP, HM, STI and YI values. As such, they can be denoted as LN-tolerant cultivars.
The classification of cultivars according to their performances under stress and nonstress conditions was described in the study by Fernandez et al. [7]. Cultivars expressing uniform and high performances in both stress and nonstress conditions belong to group A; cultivars that expresses high performances only in conditions with an optimal amount of nitrogen and not under stress conditions are in group B. Cultivars that performs better in stress conditions than in nonstress conditions are in group C, and cultivars whose performances in grain yield and quality parameters are low in the stress and optimal conditions belong to group D. Ideal stress selection indices should clearly distinguish these groups. According to the results from Tables 3 and 4, none of the studied cultivars belonged to group C in terms of the yield and grain protein contents. In our study, cultivars with a STI higher than 1 were placed in group A for the grain yield and grain protein contents. These cultivars had higher performances under stress conditions than the average performances of all the cultivars under the optimal conditions. The cultivars that belonged to group D were easily identified by 3D diagram, as they had the lowest performances in both conditions and the lowest STI. The biggest challenge was to identify the cultivars in group B.
Fernandez [7] noted that SSI and TOL cannot differentiate groups A and C while MP cannot differentiate groups A and B. STI have same drawback as MP, but it is better in distinguishing A and B than GMP and MP. As it is described in study of Thiry et al. [39], SSI and TOL can distinguish stress tolerant and stress susceptible cultivars, while GMP, HM and MP relies on high performance, therefore these five indices have been divided into two groups PCI (production capacity indices STI, GMP, MP) and RCI (resilience capacity indices SSI, TOL). SSI and TOL classify cultivars only based on reduction of yield performance under stress conditions [9]. Zhao et al. [27] points out that cultivar selection based only on GMP and MP is partial and could lead to errors, because these cultivars show average performance of grain yield under different nitrogen levels. Combined use of STI with GMP and MP improves cultivar selection.
Based on results from our study we cannot unequivocally separate a unique index as superior for application in a breeding program. Thus, we suggest selection based on combination of several stress tolerance indices such as mean productivity index (MP), geometric mean productivity index (GMP), harmonic mean index (HM), stress tolerance index (STI), and yield index (YI) combination.
As suggested by Brancourt-Hulmel et al. [40] and Cormier et al. [26] breeding programs targeting to produce N-efficient cultivars for low-input environments should include testing and selection at low-input conditions to maximize selection gains for grain yield. Complementary approach pointed out by Przystalski et al. [41] suggests combining information from both organic (low input) and non-organic (high input) experiments to optimize the selection of wheat cultivars for organic farming systems and Hitz et al. [42] indicated that selection at low N is necessary to identify high NUE genotypes. Contrary, Annicchiarico et al. [43] found no clear advantage when targeting organic production of direct selection for grain yield in organic systems relative to indirect selection in conventional systems. This implies that there is no clear-cut recommendation which approach is superior and individual breeding strategies under low N environments should be adjusted according to the regional agro-ecological conditions.

Conclusions
In this study 48 winter wheat cultivars were evaluated under high-nitrogen (HN) and low-nitrogen (LN) conditions. We determined Sofru, BC Opsesija and MV-Nemere as the most tolerant cultivars for the grain yield in low-nitrogen conditions based on five selection indices (the MP, GMP, HM, STI and YI) and potentially useful as parental donors. For the same indices, in terms of the grain protein content, cultivars U-1, OS-Olimpija, Forcali, Viktoria and BC Tena were selected as the most tolerant under low-nitrogen conditions. In consideration of three other indices (the TOL, YSI and RSI), cultivars Katarina and Ficko were denoted as LN-tolerant cultivars in terms of the grain yield and cultivars Isengrain, Tosunbey, Vulkan and BC Darija in terms of the grain protein content. Our results showed that the MP, GMP, HM, STI and YI could be used in the identification of cultivars that give high yields and high-quality grain in conditions with and without a sufficient amount of nitrogen. The TOL, YSI and RSI showed weak or no correlations with the yield and grain protein content performance under stress and optimal conditions. Cultivar evaluation based on stress tolerance indices can be useful for wheat breeders, as they can track cultivar performances under stress conditions. Additionally, tolerant cultivars could be used for developing new high-yielding cultivars in stress conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11081544/s1, Figure S1. 3D diagrams for the stress screening indices and grain yield under HN (Yp) and LN (Ys) treatments of winter wheat cultivars, Figure S2. 3D diagrams for the stress screening indices and grain protein content under HN (Yp) and LN (Ys) treatments of winter wheat cultivars.