Development of Genome-Wide Unique Indel Markers for a Heat-Sensitive Genotype in Wheat (Triticum aestivum L.)
, Kunpeng Xu 1, Meng Wang 2, Zhenchuang Wang 1, Ziyang Cai 2, Ao Li 1, Anxin He 3, Xiaoming Xie 2, Lingling Chai 2, Mingjiu Liu 1, Xingqi Ou 1 and Zhongfu Ni 2
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsReview Report
The title is clear and succinct, accurately conveying the study's focus on genome-wide unique Indel markers associated with a heat-sensitive wheat genotype. The abstract effectively summarizes the study, outlining the objectives, methodology, key findings, and their significance. To enhance its impact, the abstract could place greater emphasis on the broader applications of these findings in advancing wheat breeding programs and improving heat tolerance in wheat cultivars.
The introduction establishes the context of the research by emphasizing the challenges of heat stress on wheat productivity in the face of climate change. For convenience, please incorporate study of Ahmad et al., 2024: https://pubs.acs.org/doi/full/10.1021/acsomega.3c10379.
The introduction highlights the importance of Chromosome Segment Substitution Lines (CSSLs) and emphasizes the necessity of genome-wide markers, effectively establishing the study's relevance. While the rationale for the research is clearly presented, incorporating recent findings on heat tolerance mechanisms in wheat could provide a more comprehensive context.
The methodology is thoroughly described, offering a clear and systematic explanation of the resequencing process, indel identification, and primer design, ensuring transparency and reproducibility. The authors employed robust bioinformatics and experimental validation approaches. Some technical details (e.g., thresholds for indel selection) could be elaborated for reproducibility. A comparison of the chosen methodology with alternative marker development approaches would provide added depth.
The results are presented comprehensively, including: The development of genome-wide unique primer pairs is a significant contribution. The authors achieved a high success rate in polymorphism testing (59.69% to 99.81%), indicating the reliability of their markers. Highlight practical scenarios where these markers can be applied in wheat breeding programs.
Consider statistical analysis or comparisons with existing marker systems to contextualize the efficiency.
The discussion effectively interprets the results, linking them to broader goals of wheat genetics and stress tolerance: The potential application of these markers in Marker-Assisted Selection (MAS) is well-explained. Include potential limitations, such as scalability to other wheat varieties or environmental conditions.
Discuss challenges in integrating these markers into large-scale breeding programs.
The conclusion summarizes the study's contributions concisely. The authors claim that their markers provide a solid foundation for CSSL development and quantitative trait analysis. However, a clearer statement on future directions, such as field-level validations or integration into global wheat improvement initiatives, would add value.
The references are relevant and up-to-date. However, more citations from recent high-impact studies on wheat genetics and heat tolerance could strengthen the scientific foundation.
Comments for author File: 
Comments.pdf
Review Report
The title is concise and effectively communicates the study's purpose, focusing on genome-wide unique Indel markers for a heat-sensitive wheat genotype. The abstract provides a clear summary of the objectives, methodology, results, and significance. However, it could benefit from a stronger emphasis on the broader implications of the findings for wheat breeding and heat tolerance.
The introduction establishes the context of the research by emphasizing the challenges of heat stress on wheat productivity in the face of climate change. For convenience, please incorporate study of Ahmad et al., 2024: https://pubs.acs.org/doi/full/10.1021/acsomega.3c10379. For gene perspectives, consult https://link.springer.com/article/10.1007/s12298-024-01528-4.
Introduction effectively outlines the significance of Chromosome Segment Substitution Lines (CSSLs) and the need for genome-wide markers. The rationale for the study is clear, but the introduction could integrate recent studies on heat tolerance mechanisms in wheat for a broader perspective. For example, https://link.springer.com/article/10.1007/s12298-021-01080-5.
The methodology is detailed and well-structured, describing the resequencing process, indel identification, and primer design. The authors employed robust bioinformatics and experimental validation approaches. Some technical details (e.g., thresholds for indel selection) could be elaborated for reproducibility. A comparison of the chosen methodology with alternative marker development approaches would provide added depth.
The results are presented comprehensively, including: The development of genome-wide unique primer pairs is a significant contribution. The authors achieved a high success rate in polymorphism testing (59.69% to 99.81%), indicating the reliability of their markers. Highlight practical scenarios where these markers can be applied in wheat breeding programs.
Consider statistical analysis or comparisons with existing marker systems to contextualize the efficiency.
The discussion effectively interprets the results, linking them to broader goals of wheat genetics and stress tolerance: The potential application of these markers in Marker-Assisted Selection (MAS) is well-explained. Include potential limitations, such as scalability to other wheat varieties or environmental conditions.
Discuss challenges in integrating these markers into large-scale breeding programs.
The conclusion summarizes the study's contributions concisely. The authors claim that their markers provide a solid foundation for CSSL development and quantitative trait analysis. However, a clearer statement on future directions, such as field-level validations or integration into global wheat improvement initiatives, would add value.
The references are relevant and up-to-date. However, more citations from recent high-impact studies on wheat genetics and heat tolerance could strengthen the scientific foundation.
Author Response
Comment 1: The title is clear and succinct, accurately conveying the study's focus on genome-wide unique Indel markers associated with a heat-sensitive wheat genotype. The abstract effectively summarizes the study, outlining the objectives, methodology, key findings, and their significance. To enhance its impact, the abstract could place greater emphasis on the broader applications of these findings in advancing wheat breeding programs and improving heat tolerance in wheat cultivars.
Response: Thanks for your valuable comment. We have done it (lines 27-29).
Comment 2: The introduction establishes the context of the research by emphasizing the challenges of heat stress on wheat productivity in the face of climate change. For convenience, please incorporate study of Ahmad et al., 2024: https://pubs.acs.org/doi/full/10.1021/acsomega.3c10379.
Response: Thank you very much. However, the study you mentioned (Ahmad et al., 2024) focuses on drought resistance and has no direct relationship with heat-stress tolerance. Hence, we did not incorporate this study into our manuscript.
Comment 3: The introduction highlights the importance of Chromosome Segment Substitution Lines (CSSLs) and emphasizes the necessity of genome-wide markers, effectively establishing the study's relevance. While the rationale for the research is clearly presented, incorporating recent findings on heat tolerance mechanisms in wheat could provide a more comprehensive context.
Response: Thanks for your valuable comment. We have done this (lines 49-54).
Comment 4: The methodology is thoroughly described, offering a clear and systematic explanation of the resequencing process, indel identification, and primer design, ensuring transparency and reproducibility. The authors employed robust bioinformatics and experimental validation approaches. Some technical details (e.g., thresholds for indel selection) could be elaborated for reproducibility. A comparison of the chosen methodology with alternative marker development approaches would provide added depth.
Response: Thanks for your valuable comment. The thresholds for indel selection were listed in Lines 147-148. Figure 1 listed all actions taken to develop genome-wide unique indel markers in wheat. Compared with known marker systems, a significant improvement in our chosen methodology is to check the uniqueness of primer pairs across the whole wheat genome. We have emphasized this in Lines 209-219. Since most PCR-based markers in wheat were designed two decades ago before the advent of high-quality reference genome, we are unable to compare their chosen methodologies with our study.
Comment 5: The results are presented comprehensively, including: The development of genome-wide unique primer pairs is a significant contribution. The authors achieved a high success rate in polymorphism testing (59.69% to 99.81%), indicating the reliability of their markers. Highlight practical scenarios where these markers can be applied in wheat breeding programs.
Response: Thanks for your valuable comment. We have pointed out possible applications of these indel markers in Lines 416-424.
Comment 6: Consider statistical analysis or comparisons with existing marker systems to contextualize the efficiency.
Response: Thanks for your valuable comment. Fifty-four Xbarc SSR markers were randomly selected to amplify the 16 wheat accessions, with an aim to compare the newly developed indel markers with an existing marker system. Results showed that 13.33% to 28.89% of Xbarc SSR markers were polymorphic between 15 individual wheat accessions and E6015-3S, which were much lower as compared to that in indel markers. We summarized these results in Lines 281-288 and listed the detailed information in Table S8.
Comment 7: The discussion effectively interprets the results, linking them to broader goals of wheat genetics and stress tolerance: The potential application of these markers in Marker-Assisted Selection (MAS) is well-explained. Include potential limitations, such as scalability to other wheat varieties or environmental conditions.
Response: Thanks for your valuable comment. These newly developed indels markers are highly unique and polymorphic in the studied 16 wheat accessions. Hence, they are believed to be with good scalability to other wheat varieties. We discussed this in Lines 374-383. Currently, we are performing another study using these indels to amplify 150 breeding lines to check whether potential limitations exist, and we will publish this result in another paper.
Comment 8: Discuss challenges in integrating these markers into large-scale breeding programs.
Response: Thanks for your valuable comment. We have discussed this in Lines 382-397.
Comment 9: The conclusion summarizes the study's contributions concisely. The authors claim that their markers provide a solid foundation for CSSL development and quantitative trait analysis. However, a clearer statement on future directions, such as field-level validations or integration into global wheat improvement initiatives, would add value.
Response: Thanks for your valuable comment. We summarize future directions in Lines 412-419.
Comment 10: The references are relevant and up-to-date. However, more citations from recent high-impact studies on wheat genetics and heat tolerance could strengthen the scientific foundation.
Response: Thanks for your valuable comment. We have done this. Please see citations [4], [5], [6] and [9] for details. We provided a more detailed context regarding the global impact of heat stress on wheat production in Lines 40-44 and recent genetics studies on heat-stress tolerance in Lines 49-53.
Reviewer 2 Report
Comments and Suggestions for AuthorsReview Report for AGRONOMY 3385226
Development of Genome-wide Unique Indel Markers for a Heat-Sensitive Genotype in Wheat (Triticum aestivum L.)
Necessary corrections and recommendations in the manuscript:
Row 5 (in the Authors names)
Authors wrote: ...Xingqi Ou1 and Zhongfu Ni2
Reviewer: Delete "and" between the Authors names.
Please, correct this in the Authors names.
Rows 63-66 (in Introduction)
Authors wrote: Our ongoing research is trying to develop a few series of inter-varietal CSSLs using an HS sensitive genotype E6015-3S [19] as the recipient parent and a few tolerant accessions as the donor parent, to thoroughly study the quantitative inheritance of HS tolerance
in wheat.
Reviewer: It is not clear what is the goal of Yours Investigation.
Maybe change the begining of the sentence to: The goal of our ongoing research is is trying to develop ...
Please correct this in Introduction.
Rows 69-73 (in Introduction)
Authors wrote: In this study, we developed 11,016 primer pairs targeting 5,036 indel sites, with an average density of 0.36 indels per Mbp along the whole genome. They are ideal DNA markers for inter-varietal CSSL development in wheat, owing to their genomewide uniqueness, high polymorphism, and easy-to-use characteristics.
Reviewer: From my point of view, the content of this sentences are direct Authors results of their investigation. Because of this, my recommendation is to move these sentences somewhere to Chapter "Results or Conclusions".
Please correct this in Introduction.
Rows 194-202 (in the Title of Figure 2)
Authors wrote: All content of Figure 2.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 2" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start.
Maybe use capital letters in brackets: (A), (B), (C), (D)...
Please, correct this in Figure 2.
Rows 231-236 (in the Title of Figure 3)
Authors wrote: All content of Figure 3.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 3" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start.
Maybe use capital letters in brackets: (A), (B), (C), (D)...
Please, correct this in Figure 3.
Rows 266-274 (in the Title of Figure 4)
Authors wrote: All content of Figure 4.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 4" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start.
Maybe use capital letters in brackets: (A), (B), (C), (D)...
Please, correct this in Figure 4.
Rows 298-305 (in the Title of Figure 5)
Authors wrote: All content of Figure 5.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 5" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start.
Maybe use capital letters in brackets: (A), (B), (C), (D)...
Please, correct this in Figure 5.
Rows 366-367 (the end of Chapter Discussion – beginning of Chapter Supplementary Materials)
Authors did not write: Chapter "5. Conclusions".
Reviewer: Looking evidence and arguments presented in this Manuscript, Authors presented many results of their investigation and divide them to four "Results Subchapters". Author's results highlight key findings in several areas of their investigation.
Looking this, Chapter 5.Conclusions must contain direct Authors findings or results from their Investigation.
Please write Chapter 5 Conclusions.
Rows 507-509 (In References)
Authors wrote: ... Thambugala, D.; et al. Multiple wheat ...
Reviewer: Delete "et al." From sentence
Please, correct this in this reference.
Reviewer arguments for Major Revision of the paper
- In General, the subject of this study is compatible with the purpose of the Agronomy, including
Development of Genome-wide Unique Indel Markers for a Heat-Sensitive Genotype in Wheat (Triticum aestivum L.).
- The main question addressed by the research is to trying to develop a few series of inter-varietal Chromosome segment substituted lines (CSSL) using an heat stress (HS) sensitive genotype E6015-3S as the recipient parent and a few tolerant accessions as the donor parent, to thoroughly study the quantitative inheritance of HS tolerance in wheat.
- The title of Manuscript is clear and understandable.
- Abstract is complete with short information about Manuscript.
- Authors have gives appropriate keywords.
- Manuscript is divided to four Chapters and some subchapters.
- Authors did not write: Chapter "5. Conclusions".
- I consider that the topic is interesting and original to the field, because as the Authors wrote, systematic construction of inter-varietal chromosome segment substituted lines has been infrequently studied or reported in wheat.
- In this study, Authors developed 11,016 primer pairs targeting 5,036 indel sites, with an average density of 0.36 indels per Mbp along the whole genome. This primer pairs as ideal DNA markers for inter-varietal CSSL development in wheat, owing to their genome-wide uniqueness, high polymorphism, and easy-to-use characteristics.
- Development of 11,016 genome-wide unique primer pairs targeting 5,036 indel sites represents an ideal genotyping platform for the inter-varietal CSSL construction in hexaploid wheat.
- To overcome the problem of multi-locus, special emphasis Authors placed on exploring genome-wide unique indel markers which was proved to be a highly effective way.
- Introduction have appropriate literature sources and necessary information for this Chapter. The cited literature in the Introduction provides a good insight into the research issues.
- In the last part of Introduction it is not clear what is the goal of Authors Investigation. Authors must better define the Goal of their Investigation.
- When we compare Authors investigation with other published material, chromosome segment substitution lines (CSSLs) have long been important tools in plant genome research, especially in the detection of QTLs and causal genes for crop improvement. The newly developed genome-wide unique indel markers have laid a solid foundation for the development of inter-varietal CSSLs, which are key materials in studying the quantitative inheritance of heat stress tolerance in wheat.
- Observing methodology in this Manuscript, Authors good explained in Chapter 2. Materials and Methods. This Chapter Authors have divided to six subchapters:
2.1. Plant materials
2.2. Whole-genome resequencing, sequence alignment, and variation calling
2.3. Genome-wide unique indel marker development
2.4. Uniqueness check of indel markers among sequenced wheat genomes
2.5. DNA extraction and PCR amplification
2.6. Phylogenetic analysis
- From my point of view, all subchapters are good explained and contain enough information for this Manuscript. Materials and Methods in this Manuscript are well described.
- In the end of Chapter Materials and Methods Authors specify how they performed Phylogenetic analysis. Bands on polyacrylamide gels visualized by silver staining Authors scored as present (1) or absent (0). These data Authors collected and analyzed with the numerical taxonomy multivariate analysis system. A similar matrix Authors constructed using indel data based on Dice’s coefficient, which considers only one-to-one matches between two taxa for similarity. Based on the obtained similarity matrix Authors constructed a dendrogram to determine the genetic relationships among studied wheat accessions.
- Chapter 3. Results Authors divided to four subchapters.
- Results and findings in this Manuscript are presented in five Figures and eleven S-Tables. All Figures and Tables have their allegations in the Manuscript. From my point of view all Figures are very complex but because four Figures are coloured, their readability is bigger. There is no reason for any changes.
- I have only one suggestion for the content of the titles of Figures 2 - 5. Authors use lowercase letters (a,b,c,d...) which indicate subfigures in the titles of "Figure 2 - 5". This lowercase letters (a,b,c,d...) are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. From my point of view for better readability better choice would be maybe to use capital letters in brackets: (A), (B), (C), (D)...
- Chapter "Discussion" have enough literature sources and is well written. In Discussion Authors compared their results with those of other authors.
- There is no Chapter 5.Conclusions in this manuscript. Very important Chapter of Manuscript is "5.Conclusions" and in this Manuscript is missing. Looking evidence and arguments presented in this Manuscript, Authors presented many results of their investigation and divide them to four "Results Subchapters", five Figures and eleven S-Tables. Author's results highlight key findings in several areas of their investigation. Looking this, Chapter 5.Conclusions must contain direct Authors findings or results from their Investigation. Conclusions must be consistent with the evidence and arguments presented in Results and Discussion.
- In Manuscript Authors used 60 different sources of literature. For this type of Investigation and discussion, this number of literature sources is quite enough. Most of literature sources are more recent. 20% of literature sources describe the problems on other plant species which is acceptable. From my point of view references used in this Manuscript are appropriate.
- There are also couple technical errors that must be correct, indicated in the: "Necessary corrections and recommendations in the manuscript" for Authors.
- The English used in manuscript is at a good level and understandable.
Comments for author File: Comments.pdf
Author Response
Necessary corrections and recommendations in the manuscript:
Comment 1: Row 5 (in the Authors names)
Authors wrote: ...Xingqi Ou1 and Zhongfu Ni2
Reviewer: Delete "and" between the Authors names. Please, correct this in the Authors names.
Response: Thank you for your advice. We have done it (line 5).
Comment 2: Rows 63-66 (in Introduction)
Authors wrote: Our ongoing research is trying to develop a few series of inter-varietal CSSLs using an HS sensitive genotype E6015-3S [19] as the recipient parent and a few tolerant accessions as the donor parent, to thoroughly study the quantitative inheritance of HS tolerance in wheat.
Reviewer: It is not clear what is the goal of Yours Investigation. Maybe change the begining of the sentence to: The goal of our ongoing research is is trying to develop ...Please correct this in Introduction.
Response: Thank you for your advice. We have done it (lines 75-89).
Comment 3: Rows 69-73 (in Introduction)
Authors wrote: In this study, we developed 11,016 primer pairs targeting 5,036 indel sites, with an average density of 0.36 indels per Mbp along the whole genome. They are ideal DNA markers for inter-varietal CSSL development in wheat, owing to their genomewide uniqueness, high polymorphism, and easy-to-use characteristics.
Reviewer: From my point of view, the content of this sentences are direct Authors results of their investigation. Because of this, my recommendation is to move these sentences somewhere to Chapter "Results or Conclusions". Please correct this in Introduction.
Response: Thank you for your advice. We have done it (lines 411-419).
Comment 4: Rows 194-202 (in the Title of Figure 2)
Authors wrote: All content of Figure 2.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 2" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. Maybe use capital letters in brackets: (A), (B), (C), (D)... Please, correct this in Figure 2.
Response: Thank you for your advice. We have done it (lines 230-238).
Comment 5: Rows 231-236 (in the Title of Figure 3)
Authors wrote: All content of Figure 3.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 3" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. Maybe use capital letters in brackets: (A), (B), (C), (D)... Please, correct this in Figure 3.
Response: Thank you for your advice. We have done it (lines 267-272).
Comment 6: Rows 266-274 (in the Title of Figure 4)
Authors wrote: All content of Figure 4.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 4" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. Maybe use capital letters in brackets: (A), (B), (C), (D)... Please, correct this in Figure 4.
Response: Thank you for your advice. We have done it (lines 309-317).
Comment 7: Rows 298-305 (in the Title of Figure 5)
Authors wrote: All content of Figure 5.
Reviewer: Lowercase letters (a,b,c,d...) that indicate subfigures in the title of "Figure 5" are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. Maybe use capital letters in brackets: (A), (B), (C), (D)... Please, correct this in Figure 5.
Response: Thank you for your advice. We have done it (lines 341-348).
Comment 8: Rows 366-367 (the end of Chapter Discussion – beginning of Chapter Supplementary Materials)
Authors did not write: Chapter "5. Conclusions".
Reviewer: Looking evidence and arguments presented in this Manuscript, Authors presented many results of their investigation and divide them to four "Results Subchapters". Author's results highlight key findings in several areas of their investigation. Looking this, Chapter 5.Conclusions must contain direct Authors findings or results from their Investigation. Please write Chapter 5 Conclusions.
Response: Thank you for your advice. We have done it (lines 411-419).
Comment 9: Rows 507-509 (In References)
Authors wrote: ... Thambugala, D.; et al. Multiple wheat ...
Reviewer: Delete "et al." From sentence Please, correct this in this reference.
Response: Thank you for your advice. We have done it (lines 568-570).
Reviewer arguments for Major Revision of the paper
Comment 1: - In General, the subject of this study is compatible with the purpose of the Agronomy, including Development of Genome-wide Unique Indel Markers for a Heat-Sensitive Genotype in Wheat (Triticum aestivum L.).
- The main question addressed by the research is to trying to develop a few series of inter-varietal Chromosome segment substituted lines (CSSL) using an heat stress (HS) sensitive genotype E6015-3S as the recipient parent and a few tolerant accessions as the donor parent, to thoroughly study the quantitative inheritance of HS tolerance in wheat.
- The title of Manuscript is clear and understandable.
- Abstract is complete with short information about Manuscript.
- Authors have gives appropriate keywords.
- Manuscript is divided to four Chapters and some subchapters.
- Authors did not write: Chapter "5. Conclusions".
Response: Thank you very much for your appreciation. We have added Chapter "5. Conclusions" (lines 411-419).
Comment 2: - I consider that the topic is interesting and original to the field, because as the Authors wrote, systematic construction of inter-varietal chromosome segment substituted lines has been infrequently studied or reported in wheat.
- In this study, Authors developed 11,016 primer pairs targeting 5,036 indel sites, with an average density of 0.36 indels per Mbp along the whole genome. This primer pairs as ideal DNA markers for inter-varietal CSSL development in wheat, owing to their genome-wide uniqueness, high polymorphism, and easy-to-use characteristics.
- Development of 11,016 genome-wide unique primer pairs targeting 5,036 indel sites represents an ideal genotyping platform for the inter-varietal CSSL construction in hexaploid wheat.
- To overcome the problem of multi-locus, special emphasis Authors placed on exploring genome-wide unique indel markers which was proved to be a highly effective way.
- Introduction have appropriate literature sources and necessary information for this Chapter. The cited literature in the Introduction provides a good insight into the research issues.
- In the last part of Introduction it is not clear what is the goal of Authors Investigation. Authors must better define the Goal of their Investigation.
Response: Thank you very much for your appreciation. We have reorganized related context about the goal of investigation (lines 75-89).
Comment 3: - When we compare Authors investigation with other published material, chromosome segment substitution lines (CSSLs) have long been important tools in plant genome research, especially in the detection of QTLs and causal genes for crop improvement. The newly developed genome-wide unique indel markers have laid a solid foundation for the development of inter-varietal CSSLs, which are key materials in studying the quantitative inheritance of heat stress tolerance in wheat.
- Observing methodology in this Manuscript, Authors good explained in Chapter 2. Materials and Methods. This Chapter Authors have divided to six subchapters:
2.1. Plant materials
2.2. Whole-genome resequencing, sequence alignment, and variation calling
2.3. Genome-wide unique indel marker development
2.4. Uniqueness check of indel markers among sequenced wheat genomes
2.5. DNA extraction and PCR amplification
2.6. Phylogenetic analysis
- From my point of view, all subchapters are good explained and contain enough information for this Manuscript. Materials and Methods in this Manuscript are well described.
- In the end of Chapter Materials and Methods Authors specify how they performed Phylogenetic analysis. Bands on polyacrylamide gels visualized by silver staining Authors scored as present (1) or absent (0). These data Authors collected and analyzed with the numerical taxonomy multivariate analysis system. A similar matrix Authors constructed using indel data based on Dice’s coefficient, which considers only one-to-one matches between two taxa for similarity. Based on the obtained similarity matrix Authors constructed a dendrogram to determine the genetic relationships among studied wheat accessions.
Response: Thank you very much for your appreciation.
Comment 4: - Chapter 3. Results Authors divided to four subchapters.
- Results and findings in this Manuscript are presented in five Figures and eleven S-Tables. All Figures and Tables have their allegations in the Manuscript. From my point of view all Figures are very complex but because four Figures are coloured, their readability is bigger. There is no reason for any changes.
- I have only one suggestion for the content of the titles of Figures 2 - 5. Authors use lowercase letters (a,b,c,d...) which indicate subfigures in the titles of "Figure 2 - 5". This lowercase letters (a,b,c,d...) are not readable. They look like part of sentence. You must read carefully, to find where the next subfigure start. From my point of view for better readability better choice would be maybe to use capital letters in brackets: (A), (B), (C), (D)...
Response: Thank you very much for your advice. We have done it (lines 230-238, 267-272, 309-317, 341-348).
Comment 5: - Chapter "Discussion" have enough literature sources and is well written. In Discussion Authors compared their results with those of other authors.
- There is no Chapter 5.Conclusions in this manuscript. Very important Chapter of Manuscript is "5.Conclusions" and in this Manuscript is missing. Looking evidence and arguments presented in this Manuscript, Authors presented many results of their investigation and divide them to four "Results Subchapters", five Figures and eleven S-Tables. Author's results highlight key findings in several areas of their investigation. Looking this, Chapter 5.Conclusions must contain direct Authors findings or results from their Investigation. Conclusions must be consistent with the evidence and arguments presented in Results and Discussion.
Response: Thank you very much for your advice. We have done it (lines 411-419).
Comment 6: - In Manuscript Authors used 60 different sources of literature. For this type of Investigation and discussion, this number of literature sources is quite enough. Most of literature sources are more recent. 20% of literature sources describe the problems on other plant species which is acceptable. From my point of view references used in this Manuscript are appropriate.
- There are also couple technical errors that must be correct, indicated in the: "Necessary corrections and recommendations in the manuscript" for Authors.
- The English used in manuscript is at a good level and understandable.
Response: Thank you very much for your appreciation.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Suggestions for correction are in the attached file.
Best regards,
Comments for author File: Comments.pdf
Author Response
Comment 1: include a specific example of how markers can be used in breeding programs.
Response: Thank you very much for your advice. We have done it (lines 26-30).
Comment 2: prioritize only the most significant information.
Response: Thank you very much for your advice. But according to Reviewer 1 and Reviewer 2, the abstract in our manuscript effectively summarizes the study. So, we kept this context unchanged.
Comment 3: Add a concluding sentence summarizing the future implications of the findings.
Response: Thank you very much for your advice. We have done it (lines 26-30).
Comment 4: Provide a more detailed context regarding the global impact of heat stress on wheat production, especially in developing regions.
Response: Thank you very much for your advice. We have done it (lines 40-44).
Comment 5: Clarify the specific gap in the literature this study aims to address.
Response: Thank you very much for your advice. We have done it (lines 75-85).
Comment 6: Expand on the explanation of how indel markers are more advantageous compared to other marker types
Response: Thank you very much for your advice. We have done it (lines 81-82).
Comment 7: Reduce technical jargon and provide definitions for broader audiences.
Response: Thank you very much for your advice. We have done it (lines 87-89).
Comment 8: Detail the criteria used to select wheat accessions studied and how this impacts the results.
Response: Thank you very much for your advice. We have done it (lines 112-117).
Comment 9: Specify the controls used in PCR experiments to ensure data reproducibility.
Response: Thank you very much for your advice. We have done it (lines 181-183).
Comment 10: Elaborate on the choice of NTSYS software for phylogenetic analysis.
Response: Thank you very much for your advice. We have done it (lines 188-189).
Comment 11: Include a table summarizing the main parameters used in bioinformatics analysis.
Response: Thank you very much for your advice. We have listed the main parameters used in bioinformatics analyses in Figure 1 (lines 159-161).
Comment 12: Clearly discuss the variability observed among chromosomes in terms of indel density.
Response: Thank you very much for your advice. We have clearly stated the variability observed among chromosomes in terms of indel density in the manuscript (lines 203-207).
Comment 13: Include a comparison between the obtained results and similar studies to validate the findings.
Response: Thank you very much for your advice. We have done it (lines 280-287).
Comment 14: Discuss how the results could be applied to other crops beyond wheat.
Response: Thank you very much for your advice. However, to the best of our knowledge, application of indels developed in this study in other crops beyond wheat will not be possible. So, we did not discuss this in the revised manuscript.
Comment 15: Expand on the potential economic benefits of using indel markers.
Response: Thank you very much for your advice. We have done it (lines 384-385).
Comment 16: Relate the findings to global food security challenges.
Response: Thank you very much for your advice. We have done it (lines 412-419).
Comment 17: The article provides a significant contribution to the field of wheat genetics, particularly in constructing inter-varietal chromosome substitution lines. However, adjustments in communication aspects and data presentation can greatly enhance comprehension and impact.
Response: Thank you very much for your advice. We have adjusted both the communication aspect and the data presentation as reviewers have suggested. We believe that all these changes will strengthen the results and conclusions and improve our research.
Comment 18: Reformulate to emphasize the innovative aspects of the study.
Response: Thank you very much for your advice. We have done it (lines 412-419).
Comment 19: Include a more specific suggestion on how markers can be validated in the field.
Response: Thank you very much. If I understand correctly, you are suggesting us to provide a clearer statement on future directions, such as field-level validations or integration into global wheat improvement initiatives. We summarized possible future directions in the revised manuscript (lines 412-419).
Comment 20: Eliminate repetition from the abstract.
Response: Thank you very much for your advice. But according to Reviewer 1 and Reviewer 2, the Discussion in our manuscript effectively interprets the study. So, we kept this context unchanged.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors
I would like to thanks to Authors on the acceptance of my Review Recommendations for correction of their Manuscript.
Before we close this review and give the agreement for publishing, Authors need to do one correction.
In the List of References Authors wrote:
Row 581: ...Thambugala, D.; et al. Multiple wheat...
Reviewer: ''et al.'' must be delete.
I have not any more suggestion for corrections.
The Manuscript has been sufficiently improved to warrant publication in Agronomy journal.
Author Response
Comment 1: I would like to thanks to Authors on the acceptance of my Review Recommendations for correction of their Manuscript. Before we close this review and give the agreement for publishing, Authors need to do one correction. In the List of References Authors wrote: Row 581: ...Thambugala, D.; et al. Multiple wheat...Reviewer: ''et al.'' must be delete. I have not any more suggestion for corrections. The Manuscript has been sufficiently improved to warrant publication in Agronomy journal.
Response: Thanks for your valuable comment. We have done it (line 556).
