Advancing Regional Adaptation and Nitrogen Stress Resilience Through Integrative Phenotyping of Watkins Wheat Landraces via Source–Sink Dynamics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A review of the manuscript entitled: Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation.

The manuscript addresses the timely and essential topic of agroecological adaptation of wheat landraces to nitrogen stress conditions. The authors utilize a comprehensive collection of 308 wheat accessions from the Watkins collection, from which 48 contrasting genotypes were selected for detailed study.

However, while the relevance of the problem is indisputable, certain elements exhibit redundancy concerning approaches previously employed, including methodologies for assessing stress tolerance. It would be beneficial to accentuate the methodological or conceptual innovation of this research. Furthermore, the manuscript contains numerous deficiencies and shortcomings that need to be addressed and rectified. However, the study lacks crucial components essential for classifying it as pioneering, namely genotypic data (e.g., single-nucleotide polymorphism markers, genome-wide association study) and its restriction to hydroponic conditions, which impedes its applicability to field conditions. Additionally, the study's biological analysis is incomplete, as evidenced by differences in gene expression and root system architecture. The findings of preliminary studies are also absent. Furthermore, a lack of description of the indicators analyzed. Only abbreviations are provided. The formulas for calculations were incorporated as an image and not generated in the equation editor. The evaluation of root morphological traits, particularly those crucial under nitrogen stress conditions, remains unclear. The results tables are excessively detailed. It is recommended that such data be included in the supplement, while the manuscript should focus on presenting the most significant results obtained. The figures, however, are primarily illegible and inadequately described. The descriptions of the figures must be self-explanatory. The interpretation of the results is shallow. The authors neglect to discuss the molecular mechanisms of adaptation, possible signaling pathways activated by N stress, and the potential use of landraces as donors of alleles favorable for resistance (no reference to MAS or genomic selection).

 

 

Author Response

Reviewer 1:

A review of the manuscript entitled: Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation.

The manuscript addresses the timely and essential topic of agroecological adaptation of wheat landraces to nitrogen stress conditions. The authors utilize a comprehensive collection of 308 wheat accessions from the Watkins collection, from which 48 contrasting genotypes were selected for detailed study. However, while the relevance of the problem is indisputable, certain elements exhibit redundancy concerning approaches previously employed, including methodologies for assessing stress tolerance. It would be beneficial to accentuate the methodological or conceptual innovation of this research.

Response: We sincerely thank the reviewer for their thoughtful feedback and for recognizing the relevance of our study on agroecological adaptation in wheat landraces under nitrogen stress. We appreciate the opportunity to clarify and strengthen the methodological and conceptual innovations of our work. In response to the reviewer’s valuable suggestion, we have revised the manuscript to explicitly highlight the novel aspects of our approach.

Furthermore, the manuscript contains numerous deficiencies and shortcomings that need to be addressed and rectified. However, the study lacks crucial components essential for classifying it as pioneering, namely genotypic data (e.g., single-nucleotide polymorphism markers, genome-wide association study) and its restriction to hydroponic conditions, which impedes its applicability to field conditions.

Response: We sincerely thank the reviewer for their thoughtful critique and for recognizing the importance of our study on agroecological adaptation in wheat landraces under nitrogen stress.

While our study builds on established frameworks for stress tolerance assessment, we emphasize the following novel aspects:

Holistic Integration of Phenotypic and Geographic Data: By combining high-throughput phenotyping with multivariate analyses (PCA, hierarchical clustering) and geographic mapping, we traced evolutionary trajectories of source-sink dynamics in the historically underexplored Watkins collection. This approach bridges gene-bank conservation with actionable insights for breeding, a conceptual leap beyond single-trait evaluations in prior studies.

Contrasting Genotype Selection: The deliberate focus on 48 extreme genotypes from a diverse panel of 308 landraces revealed genotype × nitrogen interaction dynamics that challenge the conventional emphasis on elite cultivars. This strategy identified novel metabolic pathways and allelic combinations linked to nitrogen resilience, offering a roadmap for low-input agroecosystems.

Multi-Index Validation: The simultaneous application of nine stress indices (TOL, STI, GMP, etc.) and their integration via 3D scatterplots and rank-based clustering provided a robust, systems-level assessment of nitrogen adaptation. This methodological rigor transcends earlier single-index approaches.

These points are now explicitly highlighted in the Introduction and Discussion sections to underscore the study’s unique contributions.

The reviewer rightly highlights the potential of genotypic data (e.g., GWAS, SNP markers) to deepen mechanistic insights. While the current study focuses on phenotyping and stress-index validation as a critical first step, we fully acknowledge the importance of genomic analyses.

Foundation for Future Work: This phenotypic screening establishes a baseline for ongoing genomic investigations. We are currently conducting genome-wide association studies (GWAS) on the Watkins panel to identify loci associated with nitrogen use efficiency (NUE) and source-sink traits. These findings will be reported in a follow-up publication.

Gene-Bank Prioritization: The identified high-performing landraces (e.g., WATDE0013, WATDE0020) are prioritized for genotyping, enabling allele mining for marker-assisted breeding.

We have added a paragraph in the Discussion to clarify this trajectory and its implications for sustainable breeding.

The controlled hydroponic system was essential for isolating genotypic responses to nitrogen stress, minimizing confounding environmental variables. However, we agree that field validation is crucial for translational impact.

Controlled-Environment Rationale: Hydroponics allowed precise manipulation of nitrogen levels and root phenotyping, critical for dissecting seedling-stage source-sink dynamics.

Additionally, the study's biological analysis is incomplete, as evidenced by differences in gene expression and root system architecture.

Response: We appreciate the reviewer’s feedback. This study primarily focuses on phenotypic analysis, specifically examining source-sink variation under stress indices, and does not include genomic analysis at this stage. However, we acknowledge the importance of integrating genomic insights, and we are currently continuing our research. Genomic selection will be incorporated in future work, and we plan to expand on this aspect in subsequent studies. Thank you for your understanding.

The findings of preliminary studies are also absent. Furthermore, a lack of description of the indicators analyzed.

Response: We appreciate the reviewer’s comment. Preliminary findings are available in our previously published research paper: https://www.nature.com/articles/s41586-024-07682-9, where we identified and sorted 48 genotypes from this panel. In the current study, we specifically focused on source-sink dynamics within these genotypes. We havel referenced this prior work in the revised manuscript to provide a more comprehensive context for our analysis.

Only abbreviations are provided. The formulas for calculations were incorporated as an image and not generated in the equation editor.

Response: Thank you for your valuable feedback. We have now included the full descriptions for all abbreviations used in the manuscript. Additionally, the formulas for calculations have been reformatted and generated using the equation editor, ensuring clarity and consistency throughout the document. We hope these revisions address the concerns raised.

The evaluation of root morphological traits, particularly those crucial under nitrogen stress conditions, remains unclear.

Response: Thank you for your comment. In this study, we focused solely on the total biomass trait and nine stress indices, and did not include root morphological traits. As such, we did not conduct specific morphological studies under nitrogen stress conditions. We recognize the importance of these traits and plan to explore them in future research.

The results tables are excessively detailed. It is recommended that such data be included in the supplement, while the manuscript should focus on presenting the most significant results obtained.

Response: Thank you for your suggestion. We have revised the manuscript by moving the detailed results to the supplementary tables, as recommended. The main manuscript now focuses on presenting the most significant findings to ensure clarity and conciseness. We hope this improves the overall presentation of the results.

The figures, however, are primarily illegible and inadequately described.

Response:We appreciate the reviewer’s feedback regarding the figures. We have revised the figures to enhance their legibility, improving both the resolution and clarity of the labels and data.

The descriptions of the figures must be self-explanatory.

Response: We have updated the figure captions to provide more detailed and clear descriptions, ensuring that the figures are better aligned with the content of the manuscript.

The interpretation of the results is shallow.

Response: Thank you for your constructive comment. We acknowledge that the interpretation of the results may have been too brief in the initial manuscript. In the revised version, we have expanded on the discussion, providing a deeper analysis of the results and their implications, particularly in the context of source-sink dynamics under stress conditions.

The authors neglect to discuss the molecular mechanisms of adaptation, possible signaling pathways activated by N stress, and the potential use of landraces as donors of alleles favorable for resistance (no reference to MAS or genomic selection).

Response: We appreciate the reviewer’s insightful comment. In the revised manuscript, we have expanded the discussion to include the molecular mechanisms of adaptation to nitrogen stress, highlighting potential signaling pathways that could be activated under such conditions. Additionally, we have addressed the potential of landraces as valuable sources of alleles for resistance to nitrogen stress and discussed how molecular marker-assisted selection (MAS) and genomic selection could be utilized to enhance breeding efforts for improved nitrogen use efficiency. We hope these additions provide a more comprehensive understanding of the implications of our findings.

Reviewer 2 Report

Comments and Suggestions for Authors

The peer-reviewed article “Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation” is devoted to the evaluation wheat Watkins collection under normal and low nitrogen regimes. The authors conducted a large-scale study, including 308 wheat genotypes, as well as various indexing assessments. However, there are significant comments and questions regarding the article.

1. Keywords. Keywords are redundant. Almost all of them duplicate phrases in the title, for example, Watkins wheat landraces; genetic diversity; nitrogen stress resilience; agroecological adaptation.
2. stress → stresses;
3. Table 1. (NH4)2SO4 → (NH₄)₂SO₄. Same for all elements.
4. Table 2. All formulas in table 2 should be significantly improved. It is recommended to print them rather than use screenshots.
5. Table 2. It's unclear what Pattern of selection means?
6. What units are used to measure Yp, Ys and other values? Why are some values reduced to hundredths and some to thousandths? Same for all index values.
7. Figure 2. It is difficult to visualize and distinguish into which group the samples belong.
8. Figure 8. It is necessary to enlarge the picture for better visualization.
9. Figure 9. What can be understood from this figure?
10. Abbreviation section does not include all abbreviations.

Author Response

Reviewer 3:

The peer-reviewed article “Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation” is devoted to the evaluation wheat Watkins collection under normal and low nitrogen regimes. The authors conducted a large-scale study, including 308 wheat genotypes, as well as various indexing assessments. However, there are significant comments and questions regarding the article.

Keywords. Keywords are redundant. Almost all of them duplicate phrases in the title, for example, Watkins wheat landraces; genetic diversity; nitrogen stress resilience; agroecological adaptation.

Response: Thank you for your observation regarding the redundancy of the keywords. We have revised the list to eliminate duplication and now include the following more specific terms: Genetic diversity, nitrogen use efficiency (NUE), climate resilience, biomass partitioning, sustainable breeding. We believe these keywords more accurately reflect the core aspects of the study.

stress → stresses;

Response: Thank you for your observation, we have corrected.

Table 1. (NH4)2SO4 → (NH₄)₂SO₄. Same for all elements.

Response: Thank you for your observation, we have corrected as subscript.

Table 2. All formulas in table 2 should be significantly improved. It is recommended to print them rather than use screenshots. 

Response: Thank you for your valuable feedback. We have now included the full descriptions for all abbreviations used in the manuscript. Additionally, the formulas for calculations have been reformatted and generated using the equation editor, ensuring clarity and consistency throughout the document. We hope these revisions address the concerns raised.

Table 2. It's unclear what Pattern of selection means?

Response: We have revised the table and removed unneccery part from it.

What units are used to measure Yp, Ys and other values? Why are some values reduced to hundredths and some to thousandths? Same for all index values.

Response: Thank you for your question. In the revised manuscript, we have clarified the units used to measure Yp (potential yield), Ys (stress yield), and the other values. Yp and Ys are expressed in grams per plant (g/plant), while the stress indices are calculated as dimensionless ratios.

Figure 2. It is difficult to visualize and distinguish into which group the samples belong.

Response: Thank you for your comment. We appreciate your feedback. The details of each group have already been presented in the Results section under the heading "Three-dimensional scatter plots illustration." We hope this provides clarity on the grouping of the samples.

Figure 8. It is necessary to enlarge the picture for better visualization.

Response: Thank you for your question. The picture has been enlarged and now clear in visualization

Figure 9. What can be understood from this figure?

Response: Thank you for your question. There is no figure 9 in our manuscript, if you are asking about figure 8, then there is ranking of 48 landraces.

Abbreviation section does not include all abbreviations.

Response: Thank you for your question. It has been deleted from manuscript as per journal format. Included only in text.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled “Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation” presents the findings from an analysis of a segment of the Watkins collection, which includes 48 landraces grown in hydroponic boxes under both normal and nitrogen-deficient conditions. This research is particularly relevant given the increasing costs of mineral fertilizers and the associated ecological challenges, highlighting the need for low-input cultivars. As genetic diversity related to nitrogen metabolism genes has diminished, this collection of landraces represents a valuable source of new alleles that could be utilized in modern wheat breeding programs. This study is the first to demonstrate the varied responses of a core collection to nitrogen deficiency, employing a range of parameters that reflect productivity and stress resilience. The authors utilized correlation and principal component analysis (PCA) to examine the relationships between these indicators and to identify the most contrasting accessions.

There are several significant concerns regarding the study:

Modern high-input cultivars should be included in the experiments as a control to validate the robustness of the methodology.

Line 136: “into 96-well hydroponic boxes” – The conditions for plant growth in hydroponic systems and in soil differ dramatically. There are significant doubts regarding how the results obtained from hydroponic experiments can be applied to field conditions. The formation and functioning of the root system in water versus solid substrates vary considerably, and different groups of genes are involved in the absorption of nutrients from water compared to colloidal soil particles. Therefore, this methodological aspect warrants further discussion. Alternatively, the authors should provide correlations—either from their original experiments or from existing literature—that demonstrate a relationship between agrochemical experiments conducted in hydroponic systems and those performed in field conditions.

The limitations of applying the results and the constraints of the approximations related to breeding should be included in the discussion.

In section 2.1,  Material Screening and Selection you stated that out of an estimated 308 landraces,  exhibiting the most pronounced phenotypic divergence were selected. and how these 308 accessions were estimated? A reference or the results of this experiment should be provided to support this claim. Consequently, the rationale behind the selection of the 48 studied accessions remains unclear.

Line 140-141 states, “four independent trials across seasonal cycles (Spring 2023, Winter 2023, Spring 2024, Winter 2024) were conducted to account for environmental variability.” What is the rationale for accounting for environmental variability if the experiment was conducted “under rigorously standardized conditions” (line 131)?

There are some other issues that should be addressed:

The introduction should be organized into distinct paragraphs. For instance, it could be structured as follows:

Line 41 “Wheat (Triticum aestivum L.)…”

Line 49 “Watkins wheat landraces…”

Line 64 “Nitrogen availability directly…”

Lin 86 “Various stress…”

Line 109: “Through this comparative analysis…”

Page 8 is difficult to read because of the overwhelming number of line names, abbreviations, and numbers. There should be a more effective way to present this data. For example, select major uncorrelated independent indices for the description, while omitting those that correlate with them.

Line 358 states, “the dataset, likely related to key variables such as Mean and GMP.” However, there appears to be an inconsistency, as lines 499-500 indicate, “Prin1 was strongly associated with stress tolerance indices such as STI and Stress Index, indicating that it primarily captures variation related to nitrogen stress tolerance”

The sentences such as “[38] highlighted GMP as…” (line 93), “[39] described STI as the ratio of the product” (line 95), “[40] introduced the SSI index…” (line 98), “[39] also emphasized the use of STI” (line 99), and “Additionally, [41] employed these” (line 100) should be rephrased. It is not advisable to begin a sentence with a numerical reference.

Line 49 – At least one sentence should be added to describe the history and origin of the Watkins collection, including when and why it was developed, as well as the reason it is named  For those unfamiliar with this collection, it would be beneficial to understand its significance.

Table 1. The indices in the molecular formulas should be in lowercase.

Line 158: “Figure 1: It represents the experimental design:” – there should be the general title of the Figure, e.g. “Figure 1: The design of the experiment for low and normal nitrogen supply treatments of wheat landraces.”

Tables 3, 4, 6, and 7 should be moved to the supplementary materials.

Table 2 – Formulas should be displayed as symbols rather than inserted as images.

The titles of Figures 4 and 6 should be expanded.

Author Response

Reviewer 4:

The manuscript titled “Genetic Diversity and Nitrogen Stress Resilience in Watkins Wheat Landraces: Bridging Gene-Bank Conservation and Agroecological Adaptation” presents the findings from an analysis of a segment of the Watkins collection, which includes 48 landraces grown in hydroponic boxes under both normal and nitrogen-deficient conditions. This research is particularly relevant given the increasing costs of mineral fertilizers and the associated ecological challenges, highlighting the need for low-input cultivars. As genetic diversity related to nitrogen metabolism genes has diminished, this collection of landraces represents a valuable source of new alleles that could be utilized in modern wheat breeding programs. This study is the first to demonstrate the varied responses of a core collection to nitrogen deficiency, employing a range of parameters that reflect productivity and stress resilience. The authors utilized correlation and principal component analysis (PCA) to examine the relationships between these indicators and to identify the most contrasting accessions.

Response: We deeply appreciate the reviewer’s insightful comments and their acknowledgment of the significance of our research on agroecological adaptation in wheat landraces under nitrogen stress. Their feedback has provided us with a valuable opportunity to refine and better articulate the methodological and conceptual advancements of our study. As suggested, we have carefully revised the manuscript to more clearly emphasize the innovative aspects of our approach.

There are several significant concerns regarding the study:

 

Modern high-input cultivars should be included in the experiments as a control to validate the robustness of the methodology.

Response: We used Fielder (a high-input cultivar) as the control. While we cannot add new cultivars to this completed study, we are currently testing three modern high-input cultivars in related experiments. We appreciate the suggestion and will consider it for future work.

Line 136: “into 96-well hydroponic boxes” – The conditions for plant growth in hydroponic systems and in soil differ dramatically. There are significant doubts regarding how the results obtained from hydroponic experiments can be applied to field conditions. The formation and functioning of the root system in water versus solid substrates vary considerably, and different groups of genes are involved in the absorption of nutrients from water compared to colloidal soil particles. Therefore, this methodological aspect warrants further discussion. Alternatively, the authors should provide correlations—either from their original experiments or from existing literature—that demonstrate a relationship between agrochemical experiments conducted in hydroponic systems and those performed in field conditions.

Response: We appreciate the reviewer's insightful comment regarding the differences between hydroponic and soil-based growth systems. The hydroponic system allowed us to precisely control nitrogen availability and accurately measure root biomass, which would be extremely difficult in soil-based systems due to challenges in root separation. The 96-well hydroponic boxes enabled us to screen a large number of wheat landraces simultaneously under standardized conditions, which is particularly valuable for genomic studies and germplasm characterization. Source-Sink Focus: As our study primarily investigated source-sink dynamics, the hydroponic system provided clear advantages for measuring these physiological parameters without soil interference. While we did not conduct parallel soil experiments in this study, our ongoing field trials with selected genotypes will help validate these hydroponic findings. We have added this discussion to the manuscript (Section 4.2) to provide better context for interpreting our results.

The limitations of applying the results and the constraints of the approximations related to breeding should be included in the discussion.

Response: We appreciate this constructive suggestion. As requested, we have now added a dedicated section in the Discussion outlining the limitations of our findings, particularly regarding their applicability to breeding programs. This includes constraints related to genetic approximations, environmental dependencies, and potential scalability challenges.

In section 2.1,  Material Screening and Selection you stated that out of an estimated 308 landraces,  exhibiting the most pronounced phenotypic divergence were selected. and how these 308 accessions were estimated? A reference or the results of this experiment should be provided to support this claim. Consequently, the rationale behind the selection of the 48 studied accessions remains unclear.

Response: We appreciate the reviewer’s comment. Preliminary findings are available in our previously published research paper: https://www.nature.com/articles/s41586-024-07682-9, where we identified and sorted 48 genotypes from this panel. In the current study, we specifically focused on source-sink dynamics within these genotypes. We havel referenced this prior work in the revised manuscript to provide a more comprehensive context for our analysis.

Line 140-141 states, “four independent trials across seasonal cycles (Spring 2023, Winter 2023, Spring 2024, Winter 2024) were conducted to account for environmental variability.” What is the rationale for accounting for environmental variability if the experiment was conducted “under rigorously standardized conditions” (line 131)?

Response: We appreciate the reviewer's thoughtful question regarding our experimental design. The rationale for conducting trials across multiple seasons despite standardized conditions is two-fold:

While we maintained strict control over growth parameters, seasonal biological variation in plant responses can still occur due to subtle environmental factors beyond our control. Multiple trials help ensure our findings reflect consistent biological responses rather than season-specific artifacts.

Repeating experiments across seasons serves as an internal validation of our protocols, confirming that results are reproducible regardless of minor variations in execution timing or environmental conditions outside the growth chambers.

There are some other issues that should be addressed:

The introduction should be organized into distinct paragraphs. For instance, it could be structured as follows:

 

Line 41 “Wheat (Triticum aestivum L.)…”

 

Line 49 “Watkins wheat landraces…”

 

Line 64 “Nitrogen availability directly…”

 

Lin 86 “Various stress…”

 

Line 109: “Through this comparative analysis…”

Response: We sincerely appreciate your constructive feedback on improving the manuscript structure. As suggested, we have now reorganized the Introduction into distinct thematic paragraphs for better clarity and logical flow. The revised structure has been followed as per suggestion of reviewer.

Page 8 is difficult to read because of the overwhelming number of line names, abbreviations, and numbers. There should be a more effective way to present this data. For example, select major uncorrelated independent indices for the description, while omitting those that correlate with them.

Response: We appreciate your feedback regarding the readability of Page 8. To improve clarity, we have revised this section by focusing on the major uncorrelated independent indices in the main description while omitting redundant correlated variables.

Line 358 states, “the dataset, likely related to key variables such as Mean and GMP.” However, there appears to be an inconsistency, as lines 499-500 indicate, “Prin1 was strongly associated with stress tolerance indices such as STI and Stress Index, indicating that it primarily captures variation related to nitrogen stress tolerance”

Response: We have revised the relevant sections.

The sentences such as “[38] highlighted GMP as…” (line 93), “[39] described STI as the ratio of the product” (line 95), “[40] introduced the SSI index…” (line 98), “[39] also emphasized the use of STI” (line 99), and “Additionally, [41] employed these” (line 100) should be rephrased. It is not advisable to begin a sentence with a numerical reference.

Response: Thanks for suggestions, we have revised this paragarph and added author name before ciation.

Line 49 – At least one sentence should be added to describe the history and origin of the Watkins collection, including when and why it was developed, as well as the reason it is named  For those unfamiliar with this collection, it would be beneficial to understand its significance.

Response: We have added a sentence: We leveraged the extensive genetic, geographic, and phenotypic variation present in the A. E. Watkins bread wheat landrace collection (referred to as 'Watkins' in this study), which includes 827 accessions gathered from 32 countries during the 1920s and 1930s [9]

Table 1. The indices in the molecular formulas should be in lowercase.

Response: We appreciate your careful reading of our manuscript. As suggested, we have revised Table 1 to ensure all indices in the molecular formulas now appear in lowercase letters. This correction has been implemented throughout the table to maintain consistency with standard chemical notation.

Line 158: “Figure 1: It represents the experimental design:” – there should be the general title of the Figure, e.g. “Figure 1: The design of the experiment for low and normal nitrogen supply treatments of wheat landraces.”

Response: We sincerely appreciate your attention to detail in reviewing our manuscript. Following your suggestion, we have revised the figure caption for Figure 1 to provide a more informative and accurate description.

Tables 3, 4, 6, and 7 should be moved to the supplementary materials.

Response: Thank you for your suggestion. We have revised the manuscript by moving the detailed results to the supplementary tables, as recommended. The main manuscript now focuses on presenting the most significant findings to ensure clarity and conciseness. We hope this improves the overall presentation of the results.

Table 2 – Formulas should be displayed as symbols rather than inserted as images.

Response: Thank you for your valuable feedback. We have now included the full descriptions for all abbreviations used in the manuscript. Additionally, the formulas for calculations have been reformatted and generated using the equation editor, ensuring clarity and consistency throughout the document. We hope these revisions address the concerns raised.

The titles of Figures 4 and 6 should be expanded.

Response: We sincerely appreciate your attention to detail in reviewing our manuscript. Following your suggestion, we have revised the figure captions for Figures 4 and 6 to provide a more informative and accurate description.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In the v2 version, the title and abstract have been modified to emphasise the "integrative phenotyping" aspect and the analysis of regional adaptation. This is to highlight conceptual novelty. However, the originality of the approach to existing studies still requires a broader justification.

The figure descriptions in v2 have been expanded; for example, the description of Figure 1 includes a detailed description of the groups (A-D). However, some figures (e.g., scatterplot matrix) are challenging to interpret without a full legend. The quality of the graphics has not been improved.

In the v2 version, sections on correlation analysis of stress indicators (3.2), PCA, and cos² analysis (3.3) have been added; however, the conclusions remain primarily descriptive and lack integration with the biological background. No attempt has been made to relate the results to possible physiological or genetic pathways.

 

Author Response

Response to Comments:

Comments: In the v2 version, the title and abstract have been modified to emphasise the "integrative phenotyping" aspect and the analysis of regional adaptation. This is to highlight conceptual novelty. However, the originality of the approach to existing studies still requires a broader justification.

Response: We appreciate the reviewer’s feedback on the revised title and abstract. In version 2, we have modified these sections to emphasize the "integrative phenotyping" approach and its application to regional adaptation analysis, aiming to highlight the conceptual novelty of our study. To further address the comment on originality, we have now added a broader justification of our approach in the introduction. Specifically, we have compared our methodology and findings with prior studies on regional adaptation and stress resilience in wheat, emphasizing the novel integration of phenotypic, physiological, and genetic data. These changes underline the study's unique contribution to the field.

Comments: The figure descriptions in v2 have been expanded; for example, the description of Figure 1 includes a detailed description of the groups (A-D). However, some figures (e.g., scatterplot matrix) are challenging to interpret without a full legend. The quality of the graphics has not been improved.

Response: Thank you for pointing out the need for enhanced figure descriptions and improved graphics quality. In version 2, we expanded the descriptions for key figures, such as Figure 1, detailing the grouping (A-D) and its relevance. To address concerns about interpretability, we have now included comprehensive legends for figures like the scatterplot matrix, ensuring all elements are clearly explained. Additionally, the graphics quality has been improved in the current version by using higher resolution formats and more consistent design elements to enhance readability.

Comments: In the v2 version, sections on correlation analysis of stress indicators (3.2), PCA, and cos² analysis (3.3) have been added; however, the conclusions remain primarily descriptive and lack integration with the biological background. No attempt has been made to relate the results to possible physiological or genetic pathways.

Response: We acknowledge the reviewer’s concern that the conclusions in version 2 were primarily descriptive and lacked integration with the biological context. In the revised version, we have incorporated a detailed on how the results from the correlation analysis (3.2) and PCA with cos² analysis (3.3) relate to physiological and genetic pathways.  This integration aims to provide a more comprehensive biological interpretation of our findings.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thanks for the significant correction to the article. The resubmitted article can be accepted for publication in the Diversity journal.

Best regards.

Author Response

Dear Reviewer

Thank you for your positive feedback and for accepting our revised manuscript for publication in Diversity. We sincerely appreciate the time and effort you and the reviewers have dedicated to evaluating our work. We are pleased to know that the corrections met the journal’s standards and look forward to the next steps in the publication process.

Reviewer 3 Report

Comments and Suggestions for Authors

All my comments and suggestions have been addressed. The manuscript has been received according to my recommendations. I wish authors good luck in their future investigation. 

Warm regards,

Reviewer 

Author Response

Dear Reviewer,

We sincerely appreciate your kind feedback and are delighted that our revised manuscript has been accepted for publication in Diversity. We are grateful for the time and careful consideration given by you and the reviewers, and we are pleased that our corrections align with the journal’s expectations.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Most of my comments on v2 of the manuscript have been addressed. However, the v3 version, despite the expansion of the statistical analysis, still does not integrate the results with knowledge of physiological or molecular mechanisms. The authors have still not discussed how differences in SSI or YSI-type indices may be due to specific morphological traits (e.g. root development, leaf area) or known pathways related to nitrogen management (nitrogen transporters, source-sink gene expression), no reference to the literature on the physiology of wheat response to nitrogen stress (e.g. nitrate reductase systems, chloroplast metabolism, etc.), no proposed hypotheses on potential adaptation mechanisms detected in landraces such as WATDE0013 and WATDE0020.
In this version of the manuscript, the conclusions are still mainly descriptive, with no biological depth to the interpretation of the data. This limits the potential impact of the work on the understanding of adaptation and can be seen as an understatement of the study's conceptual aim.
Attention should also be paid to the location of the figures and their descriptions in the text. This issue may have arisen during the file's conversion.

Author Response

Most of my comments on v2 of the manuscript have been addressed. However, the v3 version, despite the expansion of the statistical analysis, still does not integrate the results with knowledge of physiological or molecular mechanisms.

Response: We appreciate the reviewer’s insightful comment regarding the need for deeper integration of physiological or molecular mechanisms to contextualize the statistical findings. We agree that such integration would strengthen the biological relevance of our study.

To address this, we propose the following revisions: We have explicitly acknowledged in the Limitations section that while our study identifies statistical trends, further experimental validation  is needed to confirm mechanistic links.

We have suggest specific follow-up experiments that could bridge this gap in future work.

The authors have still not discussed how differences in SSI or YSI-type indices may be due to specific morphological traits (e.g. root development, leaf area) or known pathways related to nitrogen management (nitrogen transporters, source-sink gene expression), no reference to the literature on the physiology of wheat response to nitrogen stress (e.g. nitrate reductase systems, chloroplast metabolism, etc.), no proposed hypotheses on potential adaptation mechanisms detected in landraces such as WATDE0013 and WATDE0020.

Response: We thank the reviewer for highlighting the need to link our stress‐susceptibility (SSI) and yield‐stability (YSI) indices to concrete physiological and morphological mechanisms. In the revised manuscript we have made significant changes from line 461 to 474, 482 to 484, 512 to 519 and we have revised whole conclusion part from line 543 to 564.

In this version of the manuscript, the conclusions are still mainly descriptive, with no biological depth to the interpretation of the data. This limits the potential impact of the work on the understanding of adaptation and can be seen as an understatement of the study's conceptual aim.

Response: We sincerely appreciate your thoughtful feedback. In response, we have significantly enhanced the biological depth of our conclusions to align with the study's conceptual aim. Specifically, we have incorporated a more detailed interpretation of the data, focusing on the biological mechanisms underlying adaptation. This includes exploring potential causal links between observed traits and nitrogen stress resilience, as well as discussing their implications for regional adaptation. We believe these revisions provide a stronger foundation for the study's impact and better showcase its contribution to the understanding of adaptation in Watkins wheat landraces.

Attention should also be paid to the location of the figures and their descriptions in the text. This issue may have arisen during the file's conversion.

Response: Thanks for pointing. We have adjusted figures at their exact location, however during proofreading all figures will be adjusted by production office.