High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double Digest Restriction-Site Associated DNA Sequencing (ddRAD-Seq)
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1. In the article “High Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)”, the authors studied the genetic mechanism of coloration of cotyledons of faba bean, which is of great importance for the development of its production. Cotyledon color is one of the important indices for identifying faba bean variety purity and measuring processing quality.
2. The topic is original and relevant for this field of science. It eliminates certain gaps in this area. The obtained data, together with more detailed information, can be used to create new varieties of faba bean.
The original conclusion for this work is that green cotyledon was controlled by one pair of recessive nuclear genes.
3. In this study, using the screened SNP markers, a high-density linkage map was constructed with a coverage length of 1476.95 cM and an average map distance of 0.96 cM. The green cotyledon trait was located using WinQTL Cart, and a vfGC candidate interval explaining 34.30 to 49.40% of the phenotypic variation was identified at LG02 (101.952 cM to 115.493 cM) and at LOD = 16.0, corresponding to chr1L 1077051302 bp to 1636400339 bp ( 559.35 Mb). No research has been conducted on this topic by other authors. The knowledge gained lays the scientific foundation for the fine localization of genes regulating green cotyledon and the development of molecular linked markers in faba bean.
4. With regard to improving the methodology, the authors should consider such issues.
For the experiment, only one variety of faba bean with yellow cotyledons and one with green cotyledons were taken. However, these traits can also be controlled by other genes. When changing varieties, the result may change. Therefore, it was advisable to take other varieties for experiments that are not genetically related to the studied varieties. The criterion χ2 should be denoted by a Greek letter, not a Latin one.
5. The conclusions provide a brief and concentrated summary of the results. However, due to the large number of candidate genes in the interval of this study, further in-depth research is needed to reveal the mechanism of green cotyledon formation in faba bean.
6. On lines 97-101, the authors report that the Qingcan variety 16 is characterized by yellow cotyledons, and the Qingcan variety 17 has green cotyledons. However, in Figure 1, Qingcan 17 seeds are both green and brown.
The results showed that all 32 plants of the F1 population were phenotypically consistent with Qingcan 16, that is, with yellow seeds. However, in table 1, F1 has green seeds.
7. References to literary sources are quite appropriate.
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer:
|
Thank you very much for taking the time to review this manuscript entitled “High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)” (ID: agronomy-3393952). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.
Comments and Suggestions for Authors
|
|
Comments 1: In the article “High Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)”, the authors studied the genetic mechanism of coloration of cotyledons of faba bean, which is of great importance for the development of its production. Cotyledon color is one of the important indices for identifying faba bean variety purity and measuring processing quality.
Response 1: Thank you very much for your recognition of our work.
Comments 2: The topic is original and relevant for this field of science. It eliminates certain gaps in this area. The obtained data, together with more detailed information, can be used to create new varieties of faba bean. The original conclusion for this work is that green cotyledon was controlled by one pair of recessive nuclear genes.
Response 2: Thank you again for your positive comments on our manuscript.
Comments 3: In this study, using the screened SNP markers, a high-density linkage map was constructed with a coverage length of 1476.95 cM and an average map distance of 0.96 cM. The green cotyledon trait was located using WinQTL Cart, and a vfGC candidate interval explaining 34.30 to 49.40% of the phenotypic variation was identified at LG02 (101.952 cM to 115.493 cM) and at LOD = 16.0, corresponding to chr1L 1077051302 bp to 1636400339 bp ( 559.35 Mb). No research has been conducted on this topic by other authors. The knowledge gained lays the scientific foundation for the fine localization of genes regulating green cotyledon and the development of molecular linked markers in faba bean.
Response 3: We sincerely appreciate your positive comments on our manuscript.
Comments 4: With regard to improving the methodology, the authors should consider such issues. For the experiment, only one variety of faba bean with yellow cotyledons and one with green cotyledons were taken. However, these traits can also be controlled by other genes. When changing varieties, the result may change. Therefore, it was advisable to take other varieties for experiments that are not genetically related to the studied varieties. The criterion χ2 should be denoted by a Greek letter, not a Latin one.
Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the χ2 in the manuscript.
Comments 5: The conclusions provide a brief and concentrated summary of the results. However, due to the large number of candidate genes in the interval of this study, further in-depth research is needed to reveal the mechanism of green cotyledon formation in faba bean.
Response 5: We strongly agree with the point you have made. However, when we applied for the project in 2018, there was no reference genome available for faba bean. Currently, we do not have the funding to conduct further in-depth research. In the future, as long as we have sufficient funding, we will continue this research. We hope for your understanding.
Comments 6: On lines 97-101, the authors report that the Qingcan variety 16 is characterized by yellow cotyledons, and the Qingcan variety 17 has green cotyledons. However, in Figure 1, Qingcan 17 seeds are both green and brown. The results showed that all 32 plants of the F1 population were phenotypically consistent with Qingcan 16, that is, with yellow seeds. However, in table 1, F1 has green seeds.
Response 6: We thank the reviewer for pointing our careless mistakes. We have changed the [F1 has green seeds] to [F1 has yellow seeds] in the revised manuscript. In Figure 1, the seeds outside the circle include the seed coat, while the seeds inside the circle have had the seed coat removed.
Comments 7: References to literary sources are quite appropriate.
Response 7: We greatly appreciate your acknowledgment of our work.
Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.
Kind regards, Hongyan Zhang E-mail: 2024990008@qhu.edu.cn
Corresponding authors : Yujiao Liu E-mail address: 1996990028@qhu.edu.cn |
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript presents valuable findings that contribute to the understanding of cotyledon color genetics in faba beans. However, by addressing these points, the authors can significantly improve the clarity, rigor, and impact of the MS.
1. The introduction provides a good overview of the importance of cotyledon color in faba beans. However, it could benefit from a more detailed discussion on the significance of chlorophyll degradation and its implications for plant health and agricultural practices. This would help set a stronger foundation for the study's relevance.
2. Consider including more recent studies related to cotyledon color genetics in faba beans and/or other legumes. This would enhance the manuscript's credibility and show how it fits into the current research landscape.
3. Clearly state the specific objectives of the study at the end of the introduction. This will guide the reader on what to expect in the subsequent sections.
4. Ensure that terminology is used consistently throughout the manuscript. For example, if "cotyledon color" is used in one section, it should be referred to in the same way throughout the MS.
5. The ddRAD-Seq protocol is described, but it would be helpful to include more details about the sample preparation and quality control measures taken before sequencing. This will improve reproducibility and understanding of the methods used.
6. Provide a clearer explanation of the statistical methods used for QTL analysis. Including details about software and parameters used would enhance the rigor of the methodology.
7. The manuscript would benefit from a more comprehensive description of the ddRAD-Seq library construction process. Including specific steps, such as the concentrations of reagents used and the rationale behind for example; the choice of enzymes, would enhance reproducibility and understanding for readers unfamiliar with the technique.
8. The manuscript mentions the use of LOD values and explains variance rates, but providing more context on the statistical methods used to derive these values would enhance the rigor of the results. Including confidence intervals or effect sizes could also provide a clearer picture of the significance of the findings.
9. Ensure that the results are presented in a logical order, correlating directly with the research objectives stated in the introduction. This will help maintain a clear narrative throughout the manuscript.
10. The discussion could be expanded to include the broader implications of the findings for faba bean breeding programs. Discussing how the identified QTLs can be utilized in practical breeding strategies would provide valuable insights for practitioners/growers and so on in the field.
11. Suggesting specific future research directions based on the findings would enhance the manuscript. For example, proposing experiments to validate the function of candidate genes identified in the QTL region could be a valuable addition.
12. A proofreading of the manuscript is recommended to correct any grammatical errors and improve overall readability.
Author Response
Dear Reviewer:
|
Thank you very much for taking the time to review this manuscript entitled “High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)” (ID: agronomy-3393952). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.
Comments and Suggestions for Authors
|
|
Comments 1: The introduction provides a good overview of the importance of cotyledon color in faba beans. However, it could benefit from a more detailed discussion on the significance of chlorophyll degradation and its implications for plant health and agricultural practices. This would help set a stronger foundation for the study's relevance.
Response 1: We gratefully appreciate for your valuable suggestions. We agree you’re your suggestions. Therefore, we have added some contents [Meanwhile, chlorophyll degradation is closely related to crop production. Delayed chlorophyll degradation in crops increases photosynthetic capacity at late stages，leading to the increase of crop production].
Comments 2: Consider including more recent studies related to cotyledon color genetics in faba beans and/or other legumes. This would enhance the manuscript's credibility and show how it fits into the current research landscape. Response 2: Thanks for your valuable suggestions. We have already added relevant contents in the introduction section.
Comments 3: Clearly state the specific objectives of the study at the end of the introduction. This will guide the reader on what to expect in the subsequent sections.
Response 3: We sincerely appreciate your valuable suggestions. We have added some contents about specific objectives of the study.
Comments 4: Ensure that terminology is used consistently throughout the manuscript. For example, if "cotyledon color" is used in one section, it should be referred to in the same way throughout the MS.
Response 4: Thank you for pointing this out. We have carefully reviewed the MS and made the necessary corrections.
Comments 5: The ddRAD-Seq protocol is described, but it would be helpful to include more details about the sample preparation and quality control measures taken before sequencing. This will improve reproducibility and understanding of the methods used.
Response 5: We strongly agree with the point you have made. We have added the sample preparation and quality control measures taken before sequencing in [2.2. The ddRAD-seq library construction and high-throughput sequencing].
Comments 6: Provide a clearer explanation of the statistical methods used for QTL analysis. Including details about software and parameters used would enhance the rigor of the methodology.
Response 6: We thank the reviewer for pointing this out. We have added details about software and parameters in [2.4. QTL detection for cotyledon color and candidate genes annotation].
Comments 7: The manuscript would benefit from a more comprehensive description of the ddRAD-Seq library construction process. Including specific steps, such as the concentrations of reagents used and the rationale behind for example; the choice of enzymes, would enhance reproducibility and understanding for readers unfamiliar with the technique.
Response 7: We thank the reviewer for pointing this out. We have added some contexts in [1 Introduction].
Comments 8: The manuscript mentions the use of LOD values and explains variance rates, but providing more context on the statistical methods used to derive these values would enhance the rigor of the results. Including confidence intervals or effect sizes could also provide a clearer picture of the significance of the findings.
Response 8: We thank the reviewer for pointing this out. We have added some contexts in [2.4. QTL detection for cotyledon color and candidate genes annotation and 3.4. QTL mapping for cotyledon color of faba bean].
Comments 9: Ensure that the results are presented in a logical order, correlating directly with the research objectives stated in the introduction. This will help maintain a clear narrative throughout the manuscript.
Response 9: We thank the reviewer for pointing this out. We have checked the logic of the paper.
Comments 10: The discussion could be expanded to include the broader implications of the findings for faba bean breeding programs. Discussing how the identified QTLs can be utilized in practical breeding strategies would provide valuable insights for practitioners/growers and so on in the field.
Response 10: We thank the reviewer for pointing this out. We have added some contexts in [4.2. Identification of QTL for green cotyledon trait in faba bean].
Comments 11: Suggesting specific future research directions based on the findings would enhance the manuscript. For example, proposing experiments to validate the function of candidate genes identified in the QTL region could be a valuable addition.
Response 11: We strongly agree with the point you have made. We have added some contexts in [4.2. Identification of QTL for green cotyledon trait in faba bean].
Comments 12: A proofreading of the manuscript is recommended to correct any grammatical errors and improve overall readability.
Response 12: Thank you very much for your suggestions. We have already proofread the manuscript in its entirety.
Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.
Kind regards, Hongyan Zhang E-mail: 2024990008@qhu.edu.cn
Corresponding authors : Yujiao Liu E-mail address: 1996990028@qhu.edu.cn |
Reviewer 3 Report
Comments and Suggestions for AuthorsAuthors tried to localize a gene responsible for the green color of faba beans. By generating up to 200 offsprings authors localized potential region containing 2000 genes.
The major concern is that the paper is somehow unfinished; the final region contains lots of genes, but does not contain probable SGR-like gene. Second hit in table 5 can be a candidate, but the matched sequence is rather short for a normal gene.
What is missing in the paper is “the gene cloning of the green cotyledon color in faba bean.“ - when authors could confirm their results by experiment, then it will be a great result and the paper would be acceptable. In the current from it has no results that can be evaluated. “High-Density Genetic Map Construction” can not be used elsewhere, only in your lab.
Additionally the English must be checked. Some sentences and paragraphs are hard to understand/ overall there are many two technical details of sequencing, but some key points of analysis are not covered.
O is the observed ratio and E is the expected ratio. - ratio of what?
the segregation ratio was 1:1
Not clear how SNP were detected.
Section 3.2 too much details, in contrast no detail on SNP, also it is more important.
LOD value - ?
To enhance the reliability of the results, we added 100 kb to each - if you have the genome, why not add only till the neighboring gene?
Comments on the Quality of English Language
The results showed that VfSGR was also anchored 291
into Chr1L 1076951302 ~ 1636500339, belonging to the DNA sequence between Vfa- 292
ba.Hedin2.R1.1g384160 (chr1L: 1196229405..1196230597) and Vfaba.Hedin2.R1.1g384240 293
(chr1L: 1196898240..1196900680), but not anchored to the specific gene sequence(Table 5). 2
-
Not clear at all.
Author Response
Dear Reviewer:
|
Thank you very much for taking the time to review this manuscript entitled “High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)” (ID: agronomy-3393952). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.
Comments and Suggestions for Authors
|
|
Comments 1: Authors tried to localize a gene responsible for the green color of faba beans. By generating up to 200 offsprings authors localized potential region containing 2000 genes. The major concern is that the paper is somehow unfinished; the final region contains lots of genes, but does not contain probable SGR-like gene. Second hit in table 5 can be a candidate, but the matched sequence is rather short for a normal gene.
Response 1: Thank you for your comments on our manuscript. Based on the high-density genetic linkage map, we have identified a QTL cluster explaining up to 49.40% of phenotypic variance. Subsequently, we compared the QTL cluster with the reference genome of “Hedin/2”, but unfortunately, no SGR or similar genes were found. Finally, we compared the vfSGR sequences published by Chen et al. with the reference genome of “Hedin/2”, we identified an mRNA named jg86978.t1 (chr1L:1196354403..1196355697 (- strand)), but still no SGR or similar genes were identified. Therefore, we speculate that the reference genome of “Hedin/2” published by Jayakodi et al. contains a certain number of gaps, leading to issues with gene structure annotation. In the future, once we obtain project support, we will carry out the work on the complete reference genome of faba bean.
Comments 2: What is missing in the paper is “the gene cloning of the green cotyledon color in faba bean.“ - when authors could confirm their results by experiment, then it will be a great result and the paper would be acceptable. In the current from it has no results that can be evaluated. “High-Density Genetic Map Construction” can not be used elsewhere, only in your lab.
Response 2: We strongly agree with the point you have made. However, when we applied for the project in 2018, there was no reference genome available for faba bean. Currently, we do not have the funding to conduct further in-depth research. In the future, as long as we have sufficient funding, we will continue this research. We hope for your understanding.
Comments 3: Additionally the English must be checked. Some sentences and paragraphs are hard to understand/ overall there are many two technical details of sequencing, but some key points of analysis are not covered.
Response 3: We sincerely appreciate your suggestions on our manuscript. We have carefully checked the sentences in the manuscript and corrected the erroneous expressions. The key points of analysis in [2.3. Development of polymorphic ddRAD markers and construction of high-density genetic map “The raw reads obtained from sequencing were identified to obtain the raw reads of each sample, and the reads were used for tag development and genotyping using Stacks, and the specific analysis process was as follows: 1. ustacks for each sample tag development, with the parameter: -m 3 -M 3 -d -R; 2. cstacks for inter-sample tag clustering and constructing a catalog, with parameters: -n 4; 3. sstacks for eachl sample comparison against catalog to detect SNPs in the samples with the parameter: default. For subsequent genetic linkage analysis, SNPs were filtered for completeness and depth with parameters: --max-missing 0.5 --min-mean DP 5. SNP markers were genotyped according to the genotypes of parent plants, and aa x bb type markers were screened for genetic linkage map construction.”].
Comments 4: O is the observed ratio and E is the expected ratio. - ratio of what? the segregation ratio was 1:1
Response 4: Thank you for pointing this out. We have changed [ratio] to [value] in the revised manuscript.
Comments 5: Not clear how SNP were detected. Section 3.2 too much details, in contrast no detail on SNP, also it is more important. LOD value - ? To enhance the reliability of the results, we added 100 kb to each - if you have the genome, why not add only till the neighboring gene?
Response 5: Thank you for pointing this out. The SNP detected and details on SNP in [2.3. Development of polymorphic ddRAD markers and construction of high-density genetic map “The raw reads obtained from sequencing were identified to obtain the raw reads of each sample, and the reads were used for tag development and genotyping using Stacks, and the specific analysis process was as follows: 1. ustacks for each sample tag development, with the parameter: -m 3 -M 3 -d -R; 2. cstacks for inter-sample tag clustering and constructing a catalog, with parameters: -n 4; 3. sstacks for eachl sample comparison against catalog to detect SNPs in the samples with the parameter: default. For subsequent genetic linkage analysis, SNPs were filtered for completeness and depth with parameters: --max-missing 0.5 --min-mean DP 5. SNP markers were genotyped according to the genotypes of parent plants, and aa x bb type markers were screened for genetic linkage map construction.”]. The LOD value in[3.4. QTL mapping for cotyledon color of faba bean“The CIM of WinQTL Cart software was used to identify the QTL of cotyledon color trait in faba bean, and QTL segments with LOD values ≥3.0 were selected (Table S4). When a QTL for a trait was detected, if multiple QTL clusters were detected at the same or similar locations, they were called QTL clusters, named 'QTL-cluster + sequence number '. A total of four QTL clusters for cotyledon color were detected, which were distributed in LG02 and LG04, with LOD values ranging from 3.16 to 26.30, and explained variance rate ranging from 7.90% to 49.40% (Table 3, Figure 3). Among them, QTL-cluster1 located on LG02 contained the most significant SNPs (79) and QTL-cluster3 located on LG04 contained the least significant SNPs (3). Since the green cotyledon trait of faba bean was controlled by a single gene, the QTL-cluster1 was initially targeted by considering LOD, explained variance rate and number of significant SNPs.”].
In this study, a high-density linkage map was constructed for the first time using the ddRAD-seq technique, and a QTL cluster vfGC was localized at LG02 that could explain 34.30% to 49.40% of the variance contribution in green cotyledon traits, and corresponding to Chr1L 1076951302 to 1636500339 (559.55 Mb). We speculate that there might be SGR or similar genes in Chr1L 1076951302 to 1636500339 (559.55 Mb). To enhance the reliability of the results, we added 100 kb to each of the flanking sequences.
Comments on the Quality of English Language
Comments 1: The results showed that VfSGR was also anchored 291 into Chr1L 1076951302 ~ 1636500339, belonging to the DNA sequence between Vfa- 292 ba.Hedin2.R1.1g384160 (chr1L: 1196229405..1196230597) and Vfaba.Hedin2.R1.1g384240 293 (chr1L: 1196898240..1196900680), but not anchored to the specific gene sequence(Table 5). Not clear at all.
Response 1: We gratefully appreciate for your valuable suggestions. We agree you’re your suggestions. Therefore, we have revised [According to the results, VfSGR belonged to a segment of chr1L, located between Vfaba.Hedin2.R1.1g384160 (chr1L: 1196229405...1196230597) and Vfaba.Hedin2.R1.1g384240 (chr1L: 1196898240...1196900680), yet it has not been anchored to a specific gene sequence on the "Hedin/2" genome (Table 5)] in the manuscript.
Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.
Kind regards, Hongyan Zhang E-mail: 2024990008@qhu.edu.cn
Corresponding authors : Yujiao Liu E-mail address: 1996990028@qhu.edu.cn |
Reviewer 4 Report
Comments and Suggestions for AuthorsThe research offers a significant contribution by developing a high-density genetic linkage map and identifying QTLs associated with cotyledon color in faba bean (Vicia faba L.). This work is original because it employs ddRAD-Seq, a robust genomic tool, to tackle a well-defined agricultural trait. The study enhances understanding of the genetic mechanisms controlling cotyledon color, which is vital for improving faba bean breeding strategies. However, the originality is partly reduced by the fact that similar mapping studies have been done for other traits in Vicia faba, and no functional genes directly tied to the green cotyledon trait were identified in this study.
The study is novel in its application of ddRAD-Seq to construct a high-density genetic linkage map for faba bean, achieving a smaller average distance between markers than previously reported studies. The detection of a QTL cluster explaining up to 49.40% of phenotypic variance further supports the novelty of this research. However, the absence of concrete identification of functional candidate genes, such as SGR or similar genes, limits the study’s immediate applicability in molecular breeding programs. Future studies could build on this foundation to pinpoint the exact genes involved.
The manuscript is generally clear and logically structured. Key results are presented effectively with supporting figures and tables that provide insights into genetic mapping and QTL analysis. Yet, some areas require attention: Overuse of technical jargon without definitions may alienate non-specialist readers. For example, terms like "ddRAD-Seq," "QTL," and "LOD" should be fully explained upon first use. While informative, the figures could benefit from more descriptive captions. For example, linking figure content explicitly to the manuscript's text would improve clarity. Finally, the discussion lacks sufficient emphasis on how these findings compare to previous studies, reducing the context's depth.
The article is well-written and adheres to scientific writing standards. However, a few areas could be improved: Consistency in tense usage is needed; some sentences switch between past and present tense unnecessarily. Some sentences, e.g., "to a certain extent constrains its basic molecular genetic research," are verbose and could be more concise. Long sentences with multiple clauses make some sections harder to follow. Breaking them into shorter, more focused sentences would enhance readability. Eliminate redundancy. For example, in the conclusion, "which demonstrated the reliability of our results in this study" can be trimmed to "demonstrating the reliability of our results."
As final recommendations I would suggest enhancing the Discussion: relate findings to prior research on genetic mapping and QTL analysis in legumes. Highlight the unique contributions of this study. You could also address limitations: Clearly state the limitations, such as the lack of functional gene identification, to provide transparency and to guide future research. Improve accessibility by providing definitions for technical terms and ensuring consistent terminology throughout the manuscript.
Author Response
Dear Reviewer:
|
Thank you very much for taking the time to review this manuscript entitled “High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq)” (ID: agronomy-3393952). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.
Comments and Suggestions for Authors
|
|
Comments 1: The research offers a significant contribution by developing a high-density genetic linkage map and identifying QTLs associated with cotyledon color in faba bean (Vicia faba L.). This work is original because it employs ddRAD-Seq, a robust genomic tool, to tackle a well-defined agricultural trait. The study enhances understanding of the genetic mechanisms controlling cotyledon color, which is vital for improving faba bean breeding strategies. However, the originality is partly reduced by the fact that similar mapping studies have been done for other traits in Vicia faba, and no functional genes directly tied to the green cotyledon trait were identified in this study.
Response 1: We strongly agree with the point you have made. However, when we applied for the project in 2018, there was no reference genome available for faba bean. Currently, we do not have the funding to conduct further in-depth research. In the future, as long as we have sufficient funding, we will continue this research. We hope for your understanding.
Comments 2: The study is novel in its application of ddRAD-Seq to construct a high-density genetic linkage map for faba bean, achieving a smaller average distance between markers than previously reported studies. The detection of a QTL cluster explaining up to 49.40% of phenotypic variance further supports the novelty of this research. However, the absence of concrete identification of functional candidate genes, such as SGR or similar genes, limits the study’s immediate applicability in molecular breeding programs. Future studies could build on this foundation to pinpoint the exact genes involved.
Response 2: Thank you again for your comments on our manuscript. Based on the high-density genetic linkage map, we have identified a QTL cluster explaining up to 49.40% of phenotypic variance. Subsequently, we compared the QTL cluster with the reference genome of “Hedin/2”, but unfortunately, no SGR or similar genes were found. Finally, we compared the vfSGR sequences published by Chen et al. with the reference genome of “Hedin/2”, we identified an mRNA named jg86978.t1 (chr1L:1196354403..1196355697 (- strand)), but still no SGR or similar genes were identified. Therefore, we speculate that the reference genome of “Hedin/2” published by Jayakodi et al. contains a certain number of gaps, leading to issues with gene structure annotation. In the future, once we obtain project support, we will carry out the work on the complete reference genome of faba bean.
Comments 3: The manuscript is generally clear and logically structured. Key results are presented effectively with supporting figures and tables that provide insights into genetic mapping and QTL analysis. Yet, some areas require attention: Overuse of technical jargon without definitions may alienate non-specialist readers. For example, terms like "ddRAD-Seq," "QTL," and "LOD" should be fully explained upon first use. While informative, the figures could benefit from more descriptive captions. For example, linking figure content explicitly to the manuscript's text would improve clarity. Finally, the discussion lacks sufficient emphasis on how these findings compare to previous studies, reducing the context's depth.
Response 3: Thanks for your suggestions. Terms like "ddRAD-Seq," "QTL," and "LOD" were fully explained in the revised manuscript. We also identified some issues with the figures and tables in the manuscript, and we have revised the manuscript's text. In addition, we have also added some contents to the discussion section to increase the depth of our article.
Comments 4: The article is well-written and adheres to scientific writing standards. However, a few areas could be improved: Consistency in tense usage is needed; some sentences switch between past and present tense unnecessarily. Some sentences, e.g., "to a certain extent constrains its basic molecular genetic research," are verbose and could be more concise. Long sentences with multiple clauses make some sections harder to follow. Breaking them into shorter, more focused sentences would enhance readability. Eliminate redundancy. For example, in the conclusion, "which demonstrated the reliability of our results in this study" can be trimmed to "demonstrating the reliability of our results."
Response 4: Thanks for your suggestions. We have changed [which demonstrated the reliability of our results in this study] to [demonstrating the reliability of our results] in the revised manuscript. At the same time, we have read through the entire manuscript again to avoid similar problems.
Comments 5: As final recommendations I would suggest enhancing the Discussion: relate findings to prior research on genetic mapping and QTL analysis in legumes. Highlight the unique contributions of this study. You could also address limitations: Clearly state the limitations, such as the lack of functional gene identification, to provide transparency and to guide future research. Improve accessibility by providing definitions for technical terms and ensuring consistent terminology throughout the manuscript.
Response 5: Thanks for your suggestions. We have added some contents to the discussion section to increase the depth of our article.
Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.
Kind regards, Hongyan Zhang E-mail: 2024990008@qhu.edu.cn
Corresponding authors : Yujiao Liu E-mail address: 1996990028@qhu.edu.cn |
