Review Reports

Round 1

Reviewer 1 Report

Manuscript entitled "Evaluation of Stress Screening Indices for Nitrogen Deficiency Tolerance in Wheat" is fairly well written, however should be improved in a number of cases.

Introduction

1. Authors try to explain importance of identification of superior and breeding for improved N use efficiency. However, there are some claims, that should be rephrased.
2. Authors state that until recently N requirements were met by abundant use of mineral fertilizers. It is not clear if the authors are trying to suggest that recently this is not the case anymore and that N requirements are met differently (which is wrong) or that abundant use of N is not used anymore (which is in best case debatable). ? I suggest rephrasing.
3. Drought stress is not related to the study. I would suggest to delete.

 

Materials and Methods

1. It is not clear to me what a buffer plot is and how it was placed in the trial. Can you please add the explanation?
2. Sowing rate 350 seeds/m2 - do you mean 350 viable seeds or the actual number of seeds that were sown?
3. It is not clear to me why would you add 74 kg/N at the start of the trial (in autumn?)? For example, you added a total of 100 kg/ha N in the vegetation period. Why you added a 3/4 of total N dose at the start in autumn should be explained.
4. Did you adequately check the assumption required for the ANOVA? In my experiences multi year and multi location field trials can have a significant effect on those assumptions, requiring additional steps to correctly analyze the data. As nothing is mentioned in the text, please recheck that everything if in order with your data and add statements to reassure the readers that analysis are valid.

Results

1. Authors are kindly suggested to reconsider if Tables 3 and 4 are necessary in the manuscript body or should they be added in supplement section? Reconsider if more readers would be interested in comparing identification potential of different indices (as proposed multi-indices approach was better in your case) compared to presenting the data of the mean response of all genotypes?
2. In the tables there is a clear effect of 74 kg/ha N that was added at the start of the trials. As observed, LN plots had enough N available for the wheat to produce cca. 85-95% yields (my estimate from the data) of HN plots. Again, authors are encouraged to explain their approach and also reconsider if LN designation applies in this case. 
3. Authors should add explanation on the relationship of grain yield and protein content.

Discussion

1. As i can see from your results, never cultivars performed significantly better in both LN and HN environments compared to older cultivars (for example SOFRU vs BEZOSTAJA). There is a general opinion that older cultivars, that were breeded and used in the era of low fertility or low N era (before the general use of mineral N fertilizers) are more appropriate for low N conditions. Your results suggests, that this is at least speculative, however again your environment wasnt a true LN. Authors are encouraged to add a paragraph addressing this topic. It could be of general interest to the wheat growers (maybe organic??) to know the cultivars that could yield more in low N environment or have better baking properties, etc. Please consider this also in your discussion and conclusions.

Author Response

Dear Reviewer 1,

thank you very much for your careful and thorough reading of this manuscript and for thoughtful comments and constructive suggestions which help to improve the quality of this manuscript. We hope that the changes we have made are in accordance with your suggestions. Our point-by-point response follows.

Point 1. Introduction

Point 1.1. Authors try to explain importance of identification of superior and breeding for improved N use efficiency. However, there are some claims, that should be rephrased.

Response 1.1. We agree that explanation on importance of N use efficiency improvement may be upgraded. We kindly ask you to let us know what sentences we should rephrase. We will be grateful for your additional comment.

Point 1.2. Authors state that until recently N requirements were met by abundant use of mineral fertilizers. It is not clear if the authors are trying to suggest that recently this is not the case anymore and that N requirements are met differently (which is wrong) or that abundant use of N is not used anymore (which is in best case debatable). ? I suggest rephrasing.

Response 1.2. Thank you for pointing this out. The sentence is indeed unambiguous. Therefore, we have replaced it. The text now reads: In order to avoid crop failure, N fertilizer are usually applied in large quantity. However, excessive application of N fertilizers beyond nitrogen plant requirements has adversely affected environment and it became obvious that due to a number of various damaging effects on environment and economic costs, high nitrogen fertilizer consumption has to be optimized. (lines 34-35)

Point 1.3. Drought stress is not related to the study. I would suggest to delete.

Response 1.3. As you suggested, we have deleted drought stress and added two references on low nitrogen stress on wheat and two references about screening low nitrogen tolerant genotypes with stress indices on rice and rapeseed. The sentence now reads: A number of research papers on wheat were focused on evaluation of low-nitrogen stress on yield [14,15]. Also, there are studies on rice [16] and rapeseed [17] that evaluates low-nitrogen stress effect using stress screening indices. (lines 68-70)

Point 2. Materials and Methods

Point 2.1. It is not clear to me what a buffer plot is and how it was placed in the trial. Can you please add the explanation?

Response 2.1. The suggested explanation has been added. The sentence now reads: In order to avoid edge effects on experimental plots buffer plots were sown at the beginning and at the end of the main N treatment plots. (lines 93-95)

Point 2.2. Sowing rate 350 seeds/m2 - do you mean 350 viable seeds or the actual number of seeds that were sown?

Response 2.2. The suggested explanation has been added. The sentence now reads: Sowing rate was 350 viable kernels m^-2 in all trials and for all cultivars. (lines 92-93)

Point 2.3. It is not clear to me why would you add 74 kg/N at the start of the trial (in autumn?)? For example, you added a total of 100 kg/ha N in the vegetation period. Why you added a 3/4 of total N dose at the start in autumn should be explained.

Response 2.3. Definitely, the nitrogen deficiency stress would be more pronounced without applied nitrogen before the sowing. The main motivation  was to mimick the experimental conditions as similar as possible to those conditons in wheat production in the region where nitrogen is applied before planting, usually about ½ to ¾ of totally applied nitrogen. As you suggested, we have added the explanation. The sentence now reads: Basic and pre-sowing fertilizations of 74 kg N ha^-1, 80 kg P₂O₅ ha^-1 and 120 kg K₂O ha^-1 were applied by adding 100 kg ha^-1 of urea (46% N) and 400 kg ha^-1 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. (lines 95-98) 

Point 2.4. Did you adequately check the assumption required for the ANOVA? In my experiences multi year and multi location field trials can have a significant effect on those assumptions, requiring additional steps to correctly analyze the data. As nothing is mentioned in the text, please recheck that everything if in order with your data and add statements to reassure the readers that analysis are valid.

Response 2.4. Yes, to further clarify methodological approach we have added a following sentence in MM section : ANOVA was performed using linear model (aov function) with fixed effects for year, location and cultivar effects including their interactions. (lines 120-122)

Point 3. Results

Point 3.1. Authors are kindly suggested to reconsider if Tables 3 and 4 are necessary in the manuscript body or should they be added in supplement section? Reconsider if more readers would be interested in comparing identification potential of different indices (as proposed multi-indices approach was better in your case) compared to presenting the data of the mean response of all genotypes?

Response 3.1. You have raised an important point here. We are aware that tables are quite big and contain a lot of data. However, the idea was to show BLUP yield and grain protein content values of each cultivar under two N conditions and indices values for each of the genotype in order to indicate how single-stress index approach may not be accurate enough for particular cultivar screening. In addition, the data presentation in form of cultivar mean response may be found in a number of the published studies focussed on stress screening indices, so we had followed the commonly used type of data.   

Point 3.2. In the tables there is a clear effect of 74 kg/ha N that was added at the start of the trials. As observed, LN plots had enough N available for the wheat to produce cca. 85-95% yields (my estimate from the data) of HN plots. Again, authors are encouraged to explain their approach and also reconsider if LN designation applies in this case. 

Response 3.2. Similarly to the answer on Point 2.3. in MM section, as further explanation can serve the fact that in similar papers that explore the nature of NUE in wheat, roughly speaking half of the authors also applied nitrogen before planting so in this manner we followed the usual methodological practice applied in other similar studies.

Point 3.3. Authors should add explanation on the relationship of grain yield and protein content.

Response 3.3. We have added the suggested content to the manuscript as the Table 5. in which is showed high negative correlation between yield performance and grain protein content performance under HN and LN conditions together with short description of results in lines 203-209 and short explanation with few references in Discussion (lines 325-328).

Point 4. Discussion

Point 4.1.  As i can see from your results, never cultivars performed significantly better in both LN and HN environments compared to older cultivars (for example SOFRU vs BEZOSTAJA). There is a general opinion that older cultivars, that were breeded and used in the era of low fertility or low N era (before the general use of mineral N fertilizers) are more appropriate for low N conditions. Your results suggests, that this is at least speculative, however again your environment wasnt a true LN. Authors are encouraged to add a paragraph addressing this topic. It could be of general interest to the wheat growers (maybe organic??) to know the cultivars that could yield more in low N environment or have better baking properties, etc. Please consider this also in your discussion and conclusions.

Response 4.1. As you suggested, we have added the following content to the section: As suggested by Brancourt-Hulmel et al. (42) and Cormier et al. (28) 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. (43) 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. (44) indicated that selection at low N is necessary to identify high NUE genotypes. Contrary, Annicchiarico et al. (45) 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 not 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. (lines 378-390)

Reviewer 2 Report

In this work, Marko et al. studied evaluation N deficiency tolerance of wheat. This paper is very interesting and useful for further researchers who will work for wheat breeding or agronomy.

Actually, the authors just screened out several wheat genotypes. Therefore, the title should be: Screnning of wheat genotypes for nitrogen deficienccy tolerance.

Otherwise, the authors have used several indices for the evaluation of tolerance and this paper is well written. But I would appreciate if the figure quality would be better. Some of the figures are not visible and texts are too small.

In Fig. 1 and 2, please add significance level on the circles. This is more important than correlation coefficient values.

Also, the parameters should be on both axis to understand the relations well.

I think Figures 3 and 4 can be converted to Tables and present as supplementary files.

In the results, when describe the values, please include significance level after the values. e.g. (r=0.98**).

Discussion needs improvement. Please explain the relationship among the parameters. Please cite recent papers.

In such experiment, cluster analysis is preferred to proved a better idea for plant breeder.

Author Response

Dear Reviewer 2,

thank you very much for your time and thoughtful comments and constructive suggestions which help to improve the quality of this manuscript. We hope that the changes we have made are in accordance with your suggestions. Our point-by point response follows.

Point 1. In this work, Marko et al. studied evaluation N deficiency tolerance of wheat. This paper is very interesting and useful for further researchers who will work for wheat breeding or agronomy.

Response 1. Thank you for the kind and positive feedback!

Point 2. Actually, the authors just screened out several wheat genotypes. Therefore, the title should be: Screnning of wheat genotypes for nitrogen deficienccy tolerance.

Response 2. Thank you for pointing this out. We agree with this comment. Therefore, we have changed the title to Screening of wheat genotypes for nitrogen deficiency tolerance using stress screening indices.

Point 3. Otherwise, the authors have used several indices for the evaluation of tolerance and this paper is well written. But I would appreciate if the figure quality would be better. Some of the figures are not visible and texts are too small.

Response 3. Agree. The figures have been revised as suggested by your points 4-6.

Point 4. In Fig. 1 and 2, please add significance level on the circles. This is more important than correlation coefficient values.

Response 4. During submission we have uploaded Supplementary Tables 1 and 2 with significance level of correlation coefficient values. However, as you suggested, we have added the significance level on the circles. Due to iPASTIC toolkit limits we have changed the Fig.1 and Fig. 2 heatmaps by Fig. 1 and Fig. 2 R correlograms and modified the figures titles.

Point 5. Also, the parameters should be on both axis to understand the relations well.

Response 5. As suggested, the parameters have been added to both axis.

Point 6. I think Figures 3 and 4 can be converted to Tables and present as supplementary files.

Response 6. Although we agree that Figures 3 and 4 can be converted to Tables, we have used 3D visualisation to create simpler and clearer picture of differences among genotypes as well as differences among indices. However, we have deleted Figures 3 and 4 from the main body of manuscript and created Supplementary Figure 3 and Supplementary Figure 4, where the size of figures and text is increased as you suggested.

Point 7. In the results, when describe the values, please include significance level after the values. e.g. (r=0.98**).

Response 7. As suggested, we have included the significance levels after the r values throughout the Result section.

Point 8. Discussion needs improvement. Please explain the relationship among the parameters. Please cite recent papers.

Response 8. As suggested, we have added the following paragraph about relationships among indices: 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].  

Point 9. In such experiment, cluster analysis is preferred to proved a better idea for plant breeder.

Reponse 9. Here we did not opt for cluster analysis as the material included in the study represents old and new cultivars, so the primary idea of the paper was not directed toward more detailed comparison among cultivars for grain yield, grain protein content and nitrogen use efficiency. The focus of this paper was directed more in methodological sense toward identifying the most reliable stress tolerance indices in identifying the superior cultivars under limiting nitrogen conditions. But, definitely with material undergoing selection under LN or HN cluster analysis would be helpful in quick visual identification of superior genotypes.