Genome-Wide Association Analysis of Traits Related to Nitrogen Deficiency Stress in Potato

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study evaluated 144 tetraploid potato genotypes over two years to identify traits related to nitrogen deficiency stress tolerance, using physiological and agronomic measurements. Through genome-wide association analysis, 18 significant markers were linked to traits like chlorophyll content, tuber number, leaf area, and perimeter, with distinct genomic distributions under control and stress conditions. These findings enhance understanding of genetic mechanisms for nitrogen stress tolerance, aiding marker-assisted selection in potato breeding. However, these comments will improve the manuscript

1. The introduction needs to improve, should add previously reported 2-3 studies QTLs via biparental mapping for potato, also add QTNs/SNPs identified via GWAS related to investigated traits.
2. The introduction is general and need to more specify about identified traits and concise previous QTLs/SNPs related to physiological and agronomic measurements.
3. Line# 231-245, To enhance the presentation of the correlation analysis, I recommend replacing or supplementing the table with a comprehensive figure generated using the GGally R package. This visualization would effectively display overall correlations, environment-specific correlations, data normality, and scatter plots in a single, cohesive figure, providing a clearer and more concise representation of the data compared to the current table format.
4. While the results appear reliable, I strongly recommend comparing the identified SNP markers with previously reported markers in the literature. This comparison will clarify which markers are novel and which are consistent with known associations, enhancing the study’s contribution to the understanding of nitrogen stress tolerance in potatoes.

Author Response

Dear reviewer,

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in blue in the re-submitted file.

1.The introduction needs to improve, should add previously reported 2-3 studies QTLs via biparental mapping for potato, also add QTNs/SNPs identified via GWAS related to investigated traits.

We have revised the introduction and added a new paragraph discussing two previous studies that identified QTLs in potato using biparental mapping. Additionally, we have included new citations related to QTNs/SNPs identified through GWAS for the traits investigated in our study. We believe these additions enhance the background and address your suggestion appropriately.

2. The introduction is general and need to more specify about identified traits and concise previous QTLs/SNPs related to physiological and agronomic measurements.

We have added a paragraph in the introduction specifically addressing the traits analyzed in our study. This section also includes a more concise summary of previously reported QTLs and SNPs related to physiological and agronomic measurements, as suggested.

 

3. Line# 231-245, To enhance the presentation of the correlation analysis, I recommend replacing or supplementing the table with a comprehensive figure generated using the GGally R package. This visualization would effectively display overall correlations, environment-specific correlations, data normality, and scatter plots in a single, cohesive figure, providing a clearer and more concise representation of the data compared to the current table format.

We have not employed that package in our analysis. Instead, we developed Figure 1 based on the correlation table, which we believe effectively presents the relationships in a clear and concise manner.

4. While the results appear reliable, I strongly recommend comparing the identified SNP markers with previously reported markers in the literature. This comparison will clarify which markers are novel and which are consistent with known associations, enhancing the study’s contribution to the understanding of nitrogen stress tolerance in potatoes.

We have added a paragraph to the manuscript comparing the SNP markers identified in our study with those previously reported in the literature. This comparison highlights both novel associations and markers consistent with earlier findings, thereby strengthening the contribution of our work to the understanding of nitrogen stress tolerance in potatoes

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the current manuscript, the authors conducted association analyses for 144 potato genotypes, correlating to their measured agronomic traits. In general, the study appears to have been a tremendous effort and was done meticulously; however, the presentation of the data lacks transparency and it would be essential that the authors provide extensively more information on their obtained data before the paper is accepted for publication.

 

My comments:

1. In light that a major pillar for this work being the SNP array data, it appears odd that no experimental procedure, nor detailed rendition of the data was part of the main body of the paper. Please include such content in the Materials and Methods as well as the Results sections of the paper.
2. It may be helpful to turn Table 1 into a figure to more comprehensively present the dataset. Supplementary Table S1: please provide the raw numbers.
3. Please rectify the display in Table 2.
4. Some numbers in Figure 1’s X-axis need spacing to be adjusted.

Author Response

Dear reviewer,

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in blue in the re-submitted file.

 

1. In light that a major pillar for this work being the SNP array data, it appears odd that no experimental procedure, nor detailed rendition of the data was part of the main body of the paper. Please include such content in the Materials and Methods as well as the Results sections of the paper.

 We have included the experimental procedure about the SNP array in Material and Methods and Results sections.

  2. It may be helpful to turn Table 1 into a figure to more comprehensively present the dataset. Supplementary Table S1: please provide the raw numbers.

Thank you very much for the comment, we have revised Supplementary Table S1 to include the raw data as requested. To improve clarity, we have separated the information into two distinct tables: one containing the values for the control group, and another presenting the data from the stressed plants.

 

3. Please rectify the display in Table 2.

 The display issue has been corrected. Following the suggestions made by the other reviewers, the original table 2 has been revised and transformed into a figure. It now corresponds to Figure 1, which we believe offers a clearer and more effective representation of the data.

 

4. Some numbers in Figure 1’s X-axis need spacing to be adjusted.

 We have revised the figure and adjusted the spacing of the numbers as suggested. Additionally, please note that due to other modifications in the manuscript, Figure 1 has now been renumbered and corresponds to Figure 2.

Reviewer 3 Report

Comments and Suggestions for Authors

I would like to congratulate the authors on their work. The manuscript holds great importance for potato breeding and provides valuable data in identifying SNP markers. I have made some comments on the results and discussion, which I believe could further enhance the quality of the manuscript.

Line 173: Add the factorial design of the experiment and include the statistical analysis package used in the R program.

Table 1 presents the ANOVA summary, where three factors and their interactions can be observed. However, why did the authors not check the triple interaction between YEAR × VAR × STRESS and between YEAR × STRESS?

Lines 213-215: "The results reveal significant influences of variety (VAR) and nitrogen deficiency stress (STRESS) on crucial agronomic and physiological parameters. Traits such as tuber weight and Yield are notably influenced by variety (VAR) and its interactions, like STRESS×VAR." ... If the interaction between factors is significant, I suggest the authors comment only on the interaction and not on each factor in isolation. This comment should be applied to the other variables as well.

Lines 221-223: This paragraph would fit better in the discussion section.

Table 2 presents Pearson’s correlation, but this analysis was not mentioned in the methodology nor specified.

Still in Table 2, I suggest the authors add the unit of measurement for the variables in this table and in the methodology.

I recommend adding two Pearson’s correlation tables, one for the nitrogen-deficient environment and another for the environment without nitrogen stress. This way, the authors can assess the behavior of the traits in each environment.

Lines 229-230: "These results suggest that, even under N deficiency, plants with better photosynthetic development accumulate more reserves." ... This part of the text is already a discussion; I suggest relocating it to the discussion section.

Lines 251-253: This part described by the authors constitutes a discussion.

Lines 261-264: I suggest the authors move this paragraph to the methods section.

Lines 324-325: This paragraph is too short; I suggest either merging it with another paragraph or expanding it further.

In reference 19, the year 2024 appears twice

Author Response

Dear reviewer,

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in blue in the re-submitted file.

 

Line 173: Add the factorial design of the experiment and include the statistical analysis package used in the R program.

We confirm that the description of the factorial design of the experiment, as well as the statistical analysis package used in R, have already been included in that section.

Table 1 presents the ANOVA summary, where three factors and their interactions can be observed. However, why did the authors not check the triple interaction between YEAR × VAR × STRESS and between YEAR × STRESS?

We have modified de Table 1 including the interactions between YEAR × VAR × STRESS and between YEAR × STRESS

 

Lines 213-215: "The results reveal significant influences of variety (VAR) and nitrogen deficiency stress (STRESS) on crucial agronomic and physiological parameters. Traits such as tuber weight and Yield are notably influenced by variety (VAR) and its interactions, like STRESS×VAR." ... If the interaction between factors is significant, I suggest the authors comment only on the interaction and not on each factor in isolation. This comment should be applied to the other variables as well.

Thank you for your suggestion regarding the interpretation of interactions versus individual factors. After careful consideration, this comment has been removed in the final version of the manuscript. We appreciate the feedback and have addressed the interaction effects appropriately in the revised analysis.

Lines 221-223: This paragraph would fit better in the discussion section.

We agree with your suggestion, and it has already been relocated to the Discussion section in the revised version of the manuscript.

Table 2 presents Pearson’s correlation, but this analysis was not mentioned in the methodology nor specified.

We confirm that the Pearson’s correlation analysis has now been clearly described and specified in the Materials and Methods section of the revised manuscript.

Still in Table 2, I suggest the authors add the unit of measurement for the variables in this table and in the methodology.

We have now included the units of measurement for all relevant variables both in the Materials and Methods section and in the table legends where applicable.

I recommend adding two Pearson’s correlation tables, one for the nitrogen-deficient environment and another for the environment without nitrogen stress. This way, the authors can assess the behavior of the traits in each environment.

We have included the correlation table for the control (non-stressed), as recommended. Moreover, following feedback from another reviewer, we have presented this information in the form of a figure (Figure 1), which offers a more intuitive and visually accessible overview of the trait relationships.

Lines 229-230: "These results suggest that, even under N deficiency, plants with better photosynthetic development accumulate more reserves." ... This part of the text is already a discussion; I suggest relocating it to the discussion section.

We agree that the statement is more appropriate for the Discussion section, and it has already been relocated accordingly in the revised version of the manuscript.

Lines 251-253: This part described by the authors constitutes a discussion.

This section has already been relocated to the Discussion in the revised manuscript.

Lines 261-264: I suggest the authors move this paragraph to the methods section.

The paragraph has already been moved to the Methods section in the revised version of the manuscript.

Lines 324-325: This paragraph is too short; I suggest either merging it with another paragraph or expanding it further.

We have added a new paragraph to expand the discussion and provide greater depth to the topic addressed.

In reference 19, the year 2024 appears twice

We confirm that the duplication of the year in Reference 19 has already been corrected in the revised version of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have revised the manuscript appropriately and addressed all concerns. I recommended it for publication.
Minor comment: Figure 1 is well prepared, but it it more better to add significance with values like “*” for significance.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have significantly improved the quality of the manuscript and this reviewer deems it qualified for acceptance upon meeting the editorial office’s criteria.