Review Reports - Genetic Characterization of Spring Wheat Cultivars from a High-Altitude Region in China Using a 100K Single-Nucleotide Polymorphism Panel

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Main Comments on manuscript agronomy-3335177

Significance of the study:

The authors studied spring wheat cultivars to analyze genetic characteristics using a 100K single-nucleotide polymorphism (SNP) chip panel. The information generated in this study could be used as breeding sources and helpful in maintaining food security.

Novelty of the study:

The authors used 115 diverse spring wheat cultivars from a high-altitude region of China, which could be used as breeding sources to improve yield and productivity. The novelty of the result is well presented. However, I have fears about the issues raised below.

Comments to the Authors.

Below are my comments on the manuscripts section by section.

I. Title

ü The title is informative.

II. Abstract

ü The abstract is well-written and includes a vital summary of the findings at an appropriate length.

III. Introductions

The introduction is practical, clear, and well-organized.

IV. Materials and Methods

The information regarding weather data is not available as it influences the results of QTL analysis. The data related to weather data should be included.

Did the authors use BLUPs or BLUEs that were not indicated in the method? Which one is used in the study?

The authors did not include the data for phenological traits (days to heading, maturity, and grain filling period). Figure S2 shows the correlations of the measured trait, which is not attached in the main manuscript. However, this information is essential to check whether covariate analysis is needed or not. For example, the correlations of days to heading (DH) with other traits are duly essential to see the impact of DH on the finding of significant markers. The authors reported significant correlations on line 144 with the statement, “Correlation analysis demonstrated that all trait pairs showed signiﬁcant positive correlations (P ≤ 0.05)”. These results show the importance of computing the covariate analysis with significant correlations, which can affect the results of significant SNPs. The issue of how the effects of flowering time should be duly considered when interpreting the results of any QTL analysis, particularly for grain yield and related traits.

The authors do not indicate how they used to find the candidate genes associated with significant SNPs, which is very important.

V. Results

ü Overall, the results are well written. However, the information below is needed.

Figures 1, 2, and 3 are not visible for comment and are unclear for publication.

The figure 3 is entirely not visible. Therefore, I cannot say anything about the graph, the model used, and the significant markers here. Even to say something about whether the model perfectly fits or not. A significant comment is made on the QQ plots for some traits, which show the overfitting of the models. Please redo the GWAS analysis for some traits. Are any of the significant SNPs fixed in subpopulations? What is the effect of population structure on the traits?

VI. Discussion

ü Comparatively, the discussion is well written.

VII. Conclusions

Minor improvement is needed on its conciseness.

VIII. References, figures, and tables.

The authors should improve the quality of Figures 1, 2, and 3 for better visibility.

The authors can also refer to the attached PDF.

Comments for author File: Comments.pdf

Author Response

Comments 1:The information regarding weather data is not available as it influences the results of QTL analysis. The data related to weather data should be included.

Response: Thank you for pointing this out. We agree with this comment. Therefore, We have supplemented supplemented the average temperature and average precipitation for 2023 and 2024 in the Materials and Methods section, specifically on pages 3, lines 96-98.

Comments 2: Did the authors use BLUPs or BLUEs that were not indicated in the method? Which one is used in the study?

Response: Many thanks. We employed Best Linear Unbiased Predictors (BLUPs), as detailed in the Methods section, specifically on page 3, line 127.

Comments 3: The authors did not include the data for phenological traits (days to heading, maturity, and grain filling period).

Response:Thank you for pointing this out.We recognize the significance of these data in comprehensively understanding the results of QTL analysis. However, the primary objective of this study is to explore the genetic characteristics of important traits in spring wheat varieties in the highland regions of China. Consequently, phenological traits were not included in the primary analysis of this study. In future research, we will more comprehensively consider the impact of phenological traits to ensure the comprehensiveness and accuracy of the results.

Comments 4: Figure S2 shows the correlations of the measured trait, which is not attached in the main manuscript.

Response: Many thanks.We have included the revised figure in the main manuscript. Please refer to Figure 1 in the article for details.

Comments 5: The authors do not indicate how they used to find the candidate genes associated with significant SNPs, which is very important.

Response: Thank you for pointing this out. We annotated candidate genes within approximately 150 kb upstream and downstream of the SNP loci. This aspect is further elaborated in the Methods section, specifically at line 136 on page 3.

Comments 6: Figures 1, 2, and 3 are not visible for comment and are unclear for publication.

Response:Many thanks.We have revised all the figures in the manuscript.

Comments 7: Conclusions: Minor improvement is needed on its conciseness.

Response:Many thanks.We have revised the conclusion section. as detailed on pages 10, lines 352-355.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Title: Title should be revised. It could be "Genetic Characterization of Spring Wheat Cultivars from a High-Altitude Region in China Using a 100K SNP Panel"

Abstract:

Rewrite the abstarct. It lacks clarity and language accuracy.

Introduction

Line 33-34. Rewrite the line with clarity "Disease resistance, high grain yield and high grain quality are the main wheat breeding targets, and directly impact the sustainability of agricultural practices and the security of food supplies "

Authors should improve clarity and language. Many sentences lack clarity and hard to understand.

-Introduction can be improved by adding recent and relevant GWAS studeies on wheat instead of mentioning GWAS in other crops. Uptill now a large number of GWAS studies are published in wheat.

Material and Methods

-A number of language and typo errors must be corrected.

- The seed rate @ 300kg/ha seems too high. Is this optimal seed rate in Qinghai province?

-The method of analyzing populationstructure, linkage disequilibrium and GWAs shoulb me moved under the subheading 'Statistical analysis'

Results

- Line 150-151, With reference to the sentence The 115 spring wheat cultivars were genotyped using the 100K SNP panel. A total of 108,836 reliable SNPs were obtained, which were found to be distributed across all 21 wheat chromosomes (Figure S3).'' I couldn't understand, how did you obtain 108,836 reliable SNPs using the 100,000 SNP panel? Could you please explain it?

-Overall the description of results is not satisfactory.

-Figures: All figures are of very poor quality. I couldn't read any of them.

Discussion

Please improve the discussion section by adding recent and very relevant results from wheat GWAS. Please renove the irrelevant traits that you didnot include in your study.

Conclusion

-Can be improved by concluding the population structure and LD decay in your population of 115 accessions.

Comments on the Quality of English Language

The clarity of the text needs improvement, and this can be achieved by enhancing the quality of the English language.

Author Response

Comments 1:Title should be revised. It could be "Genetic Characterization of Spring Wheat Cultivars from a High-Altitude Region in China Using a 100K SNP Panel"

Response:Many thanks. We have revised the title to "Genetic Characterization of Spring Wheat Cultivars from a High-Altitude Region in China Using a 100K SNP Panel."

Comments 2:Rewrite the abstarct. It lacks clarity and language accuracy.

Response:Agree.We have revised the ambiguous expressions in the summary section, specifically on line 18 of the first page of the article.

Comments 3:Line 33-34. Rewrite the line with clarity "Disease resistance, high grain yield and high grain quality are the main wheat breeding targets, and directly impact the sustainability of agricultural practices and the security of food supplies

Response:Thank you for pointing this out. We have made the modifications as suggested by the reviewers, specifically on lines 33-35 of page 1.

Comments 4:Authors should improve clarity and language. Many sentences lack clarity and hard to understand.

Response:Thank you for pointing this out. We have refined the language of the article to address the issues pertaining to its clarity and expression.

Comments 5:Introduction can be improved by adding recent and relevant GWAS studeies on wheat instead of mentioning GWAS in other crops. Uptill now a large number of GWAS studies are published in wheat.

Response: Many thanks. We agree with this comment. We have revised the phrase "such as wheat yield and quality [9], soybean growth cycle [10], and rice yield [11]" to "such as wheat yield and quality [9], cold tolerance [10], and disease resistance genetic loci [11]" in the article, specifically at lines 60 and 61 on the second page. Additionally, the corresponding references [10] and [11] have been modified accordingly, as detailed on lines 402 to 407 of the ninth page.

Comments 6:A number of language and typo errors must be corrected.

Response: Thank you for pointing this out. We have made the necessary revisions.

Comments 7:The seed rate @ 300kg/ha seems too high. Is this optimal seed rate in Qinghai province?

Response: Many thanks. we have revised, our seeding rate is 30 kg per 667m².

Comments 8:The method of analyzing populationstructure, linkage disequilibrium and GWAs shoulb me moved under the subheading 'Statistical analysis'

Response: Thank you for pointing this out. Since these methods are parallel in nature, statistical analysis describes the organization and analysis of certain data using statistical software. In contrast, the methods for analyzing population structure, linkage disequilibrium, and genome-wide association studies are conducted through bioinformatics software.

Comments 9: Line 150-151, With reference to the sentence The 115 spring wheat cultivars were genotyped using the 100K SNP panel. A total of 108,836 reliable SNPs were obtained, which were found to be distributed across all 21 wheat chromosomes (Figure S3).'' I couldn't understand, how did you obtain 108,836 reliable SNPs using the 100,000 SNP panel? Could you please explain it?

Response: Thank you for pointing this out. Because, the number of high-quality SNPs in our 100K SNP chip core is exactly 108,836.

Comments 10: Overall the description of results is not satisfactory.

Response: Many thanks. We greatly appreciate your valuable feedback on our work. To address this, we have conducted a comprehensive revision and supplementation of the results section, re-examined all the figures and tables, and made corresponding annotations in the text to ensure a clearer and more detailed description.

Comments 11: Figures: All figures are of very poor quality. I couldn't read any of them.

Response: Many thanks. We have revised the clarity of the figures in the article.

Comments 12: Please improve the discussion section by adding recent and very relevant results from wheat GWAS. Please renove the irrelevant traits that you didnot include in your study.

Response: Thank you for pointing this out. The issue has been revised in the discussion section, specifically on page 9, lines 325-335.

Comments 13: Can be improved by concluding the population structure and LD decay in your population of 115 accessions.

Response:Many thanks.We have revised the conclusion section, specifically on page 10, lines 353-354.

Comments 14:The clarity of the text needs improvement, and this can be achieved by enhancing the quality of the English language.

Response:Many thanks.We sincerely appreciate your valuable comments on our work. We place great emphasis on the issue of text clarity you raised and have taken steps to enhance the quality of the English language. We have thoroughly reviewed the text, conducted meticulous proofreading and polishing, ensuring that the expression is more accurate and fluent. We believe these improvements will help better convey our research content.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study is an important contribution to understanding the genetic characteristics of spring wheat cultivars in high-altitude regions. The use of a 100K SNP chip and the identification of significant loci and potential candidate genes are commendable scientific findings that hold promise for wheat breeding and genetic improvement programs.

However, I believe there are critical areas that need attention and improvement before the manuscript can be considered for publication. Below are my detailed comments:

Major Decision: Revision Required
The study does not estimate heritability for the traits analyzed. Heritability is crucial for determining the genetic versus environmental contribution to phenotypic traits. Without this information, the interpretation of genome-wide association study (GWAS) results becomes less valuable, as the genetic basis of the observed traits remains uncertain.

The manuscript does not clearly define the threshold levels for significant marker identification in the GWAS. It is important to state the p-value threshold (e.g., Bonferroni correction or other methods) used to minimize false positives. Providing these details will enhance the reproducibility and reliability of the findings.

The figures in the manuscript lack clarity and organization. Some of them appear overcrowded or difficult to interpret. Dont compressing the figures by combining related results into a single, cohesive image or panel.

While 218 significant loci and several candidate genes are reported, the lack of heritability estimates diminishes their practical breeding relevance. Establishing heritability would reinforce the value of the GWAS findings and help prioritize candidate genes for further research.

Recommendations for Improvement:

Include estimates of heritability for the traits under study.
Clearly define the threshold for significant loci in the GWAS analysis and discuss false discovery rate control methods used.
Revise the figures for clarity and organization, combining related data where possible.
Discuss the implications of the relatively short data collection period and how this might impact the findings.

Author Response

Comment 1: The study does not estimate heritability for the traits analyzed. Heritability is crucial for determining the genetic versus environmental contribution to phenotypic traits. Without this information, the interpretation of genome-wide association study (GWAS) results becomes less valuable, as the genetic basis of the observed traits remains uncertain.

Response: Many thanks. We have calculated the heritability for the phenotypic traits analyzed and supplemented the relevant data in Table 1, as detailed in the last column of Table 1 on page 3 of the article.

Comment 2: The manuscript does not clearly define the threshold levels for significant marker identification in the GWAS. It is important to state the p-value threshold (e.g., Bonferroni correction or other methods) used to minimize false positives. Providing these details will enhance the reproducibility and reliability of the findings.

Response: Thank you for pointing this out.We have made the necessary revisions, as detailed in the "Population Structure and GWAS" section of the Materials and Methods, lines 127-137 on page 3.

Comment 3: The figures in the manuscript lack clarity and organization. Some of them appear overcrowded or difficult to interpret. Dont compressing the figures by combining related results into a single, cohesive image or panel.

Response: Thank you for pointing this out. Based on the reviewers' comments, we have made corresponding adjustments to the clarity of all figures in the article.

Comment 4:While 218 significant loci and several candidate genes are reported, the lack of heritability estimates diminishes their practical breeding relevance. Establishing heritability would reinforce the value of the GWAS findings and help prioritize candidate genes for further research.

Response: Thank you for pointing this out. We have calculated the heritability for the phenotypic traits analyzed and supplemented the relevant data in Table 1, as detailed in the last column of Table 1 on page 3 of the article.