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
]. 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 the existing genetic variations under environmental stress conditions [4
]. This selection can be assisted by using stress screening indices.
A number of stress screening indices described by different mathematical expressions with two to four variables have been used for identifying tolerant cultivars under stress and stress-free environments [5
]. For instance, the tolerance index (TOL) was defined as the difference between trait performances without and under stress conditions [6
]. Rosielle and Hamblin [6
] also proposed the mean productivity index (MP) as the average trait performance of a cultivar under stress and nonstress conditions. Fernandez [7
] introduced the stress tolerance index (STI) and geometric mean productivity index (GMP), where STI was the ratio of the products’ yield performance under stress and nonstress conditions and squared mean yield performance under nonstress conditions, and GMP was the square root of the product of the cultivar performance under stress and nonstress conditions. The stress susceptibility index (SSI) was used for the evaluation of trait stability and for the determination of changes in traits under different environments by utilizing the yield performance of each cultivar and mean yield performance under both conditions [8
]. The relative stress index (RSI) was introduced by Fisher and Wood [9
] in research on drought stress on spring wheat cultivars. The harmonic mean (HM) is another index that relies on trait performances. It was used in research on wheat–rye disomic addition lines and calculated as the ratio of a doubled product of cultivar yield under stress and nonstress conditions and their sum [10
]. The yield stability index (YSI) was defined as the ratio between the yield performance under and without stress [11
], while the yield index (YI) was defined as the ratio between the trait performance of certain cultivar and mean performance of all the cultivars under stress conditions [12
]. Nevertheless, simple criteria for the determination of tolerant and stabile cultivars are high values for the MP, STI, GMP, RSI, HM, YSI and YI, as well as low values for the TOL and SSI [13
A number of research papers on wheat were focused on the evaluation of low-nitrogen stress on the yield [14
]. Additionally, there have been studies on rice [16
] and rapeseed [17
] that evaluated low-nitrogen stress effects using stress screening indices. Our study focused on an analysis of nitrogen fertilization stress on grain yield and the grain protein content in winter wheat using stress screening indices. The specific objectives were: (1) to evaluate, compare and identify the most promising stress screening indices for the evaluation of cultivar nitrogen deficiency tolerance in wheat for grain yield and grain protein; (2) to evaluate the correlation between the stress screening indices and performance of the grain yield and grain protein content under high and low amounts of nitrogen and (3) to identify and select the most nitrogen deficiency-tolerant winter wheat cultivars for further breeding purposes.
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
] 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
]. 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
], and on other species like canola [30
] and maize [31
] 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
]. Interaction between TOL and grain yield under HN conditions from this study showed moderate positive correlation. Different studies on wheat [25
] 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 Table 3
and Table 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.
] 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.
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.