Genomic Regions and Candidate Genes for Seed Iron and Seed Zinc Accumulation Identified in the Soybean ‘Forrest’ by ‘Williams 82’ RIL Population

Round 1

Reviewer 1 Report

Bellaloui et al. (2023,  doi: 10.3390/plants12213709) developed a genetic map from soybean using cross between ‘Forrest’ and ‘Williams 82’ (F × W82). A total of 306 lines was used for genotyping of 306 RILs for 2075 SNP markers using Infinium SNP6K BeadChips.

In the present study, as reported in the materials and methods, the authors report construction of a genetic map which exactly contains 2075 SNP markers by genotyping 306 RILs of the same population and used the same technology. It should be clearly mentioned whether a genetic map used earlier was used or a new map was constructed. In the first case it should be explicitly mentioned in the materials and methods section citing appropriate reference, and the the authors should check whether these QTLs are co-mapped with QTLs for other traits previously reported, for example QTL for iron and zinc (by Wang et al., 2022), MO and Ni (Bellaloui et al., 2023), seed sugar (Knizia et al., 2023), isoflavone (Knizia et al., 2019). If the map is a new one, they should generate an integrated map. 

The LOD threshold value of 2.5 is low. Only QTLs above LOD threshold should be considered. 

The materials and methods section should be clearly written. Genetic mapping, QTL identification and trait phenotyping need more details. 

No comments.

Author Response

Comment

More details about the research problem are needed.

Our response to the comment

The whole manuscript was revised, and the research problem was addressed as suggested. All edits are track-changed.

Comment

Bellaloui et al. (2023,  doi: 10.3390/plants12213709) developed a genetic map from soybean using cross between ‘Forrest’ and ‘Williams 82’ (F × W82). A total of 306 lines was used for genotyping of 306 RILs for 2075 SNP markers using Infinium SNP6K BeadChips.

In the present study, as reported in the materials and methods, the authors report construction of a genetic map which exactly contains 2075 SNP markers by genotyping 306 RILs of the same population and used the same technology. It should be clearly mentioned whether a genetic map used earlier was used or a new map was constructed. In the first case it should be explicitly mentioned in the materials and methods section citing appropriate reference, and the the authors should check whether these QTLs are co-mapped with QTLs for other traits previously reported, for example QTL for iron and zinc (by Wang et al., 2022), MO and Ni (Bellaloui et al., 2023), seed sugar (Knizia et al., 2023), isoflavone (Knizia et al., 2019). If the map is a new one, they should generate an integrated map. 

The LOD threshold value of 2.5 is low. Only QTLs above LOD threshold should be considered. 

The materials and methods section should be clearly written. Genetic mapping, QTL identification and trait phenotyping need more details. 

Our response to the comment

The manuscript was revised, and the genetic map description was further detailed and appropriately referenced as the genetic map was previously reported. All is track-changed in the Material and Methods. Whenever this genetic map was used for other traits, those traits were explicitly indicated and appropriately referenced. As now, the genetic map and the traits reported in the manuscript are appropriately referenced and cited. All clarified now to avoid any confusion. 

LOD threshold of >2.5 was used.  The use of LOD threshold of >2.5 is common and used in genetic mapping published in articles published by high quality journals, including MDPI journals such as Plants,  and others such as Crop Science, Frontiers in Plant Science, and Journal of Agriculture and Food Chemistry.  So, we believe that LOD of >2.5 is reasonable as some of these values their QTL significantly contributed to the phenotypic variation of the trait as shown by R². In addition, we added a statement in Materials and Methods citing our published articles in MDPI journal such as PLANTS with references/citation [31, 32] where we used the same approach. Both citations are included in the current manuscript in Material and Methods section.

Reviewer 2 Report

This article is a good study on Seed Iron and seed Zinc using RIL populations to conduct QTL and identify related candidate genes. However, the author has many problems in writing. For example, Figure 4 does not have zinc data and needs to be carefully revised by the author. The main issues are as follows, but are not limited to them:
1. QTL naming rules refer to the article "McCouch SR, Cho YG, Yano M, Paul E, Blinstrub M, Morishima H, Kinoshita T (1997). Report on QTL nomenclature.Rice Genet Newsl 14, 11-13."
2. The names of genes in line 290-294 are inconsistent and need to be in italics.
2. Table 1 needs to be modified. You can add a column of years and change it to a three-line grid.
3. The legends in Figures 1 and 2 are inconsistent with the figures. They need to be merged and divided into a, b, c, etc.
4. Table 2. Unable to distinguish specific years
5. The specific content cannot be seen in Figure 3 and needs to be checked. It can be displayed as a bar chart or scatter chart.
6. Tables 3, 4 and 5 should have three lines.

Other minor issues, not limited to this:
1. Line 25 and 27 QTL need to be exchanged. The full name is used when it appears for the first time.
2. Line 29 5405 is changed to 5,405
3. The chromosome is Chr1, but the QTL uses 01, which is inconsistent;
4. line 74 F2:4 change to F_2:4
5. line 83 t, [9] identified two QTL, this writing method needs to be modified.
6. Line 162 some amino acids (our research is already published, for example Knizia et al. [32]). Modify this writing
7. Line 175 HNO3 is written incorrectly.
8. Line 208 delete URL
9. Line 264 61.2-61.6, need to add cM

Author Response

Comment

I feel that there are few studies on QTL positioning for iron and zinc, and more can be added.

Our response to the comment

Although we made intensive search on the topic, studies available are on Fe and Zn in leaves, stems, seed size, and immature soybean seeds. Very limited information on mature seeds. We made further search and we cited additional reference [40] that will provide the reader with additional information. All are track-changed in the Discussion section.

Comment from Reviewer 2 and Editor

This article is a good study on Seed Iron and seed Zinc using RIL populations to conduct QTL and identify related candidate genes. However, the author has many problems in writing. For example, Figure 4 does not have zinc data and needs to be carefully revised by the author. The main issues are as follows, but are not limited to them:

1. QTL naming rules refer to the article "McCouch SR, Cho YG, Yano M, Paul E, Blinstrub M, Morishima H, Kinoshita T (1997). Report on QTL nomenclature.Rice Genet Newsl 14, 11-13."

Our response to the comment

Figure 4 was revised and Figure 4 legend was corrected to include Zn.

The above reference (McCouch et al., 1997) was consulted and it was understood that the nomenclature was referred to rice QTL. We changed the type setting of fonts of all QTLs to italic. Our approach for QTL was as previously reported by Knizia et al. [31, 32] in PLANTS (MDPI journal), and Kassem [21] and Bellaloui, et al. [7], and others  in Journal of Agricultural and Food chemistry, and in Plants. These journals are of high quality and world-wide respected. We made sure that all genes names are italic now.

Comment

2. The names of genes in line 290-294 are inconsistent and need to be in italics.

Our Response to the comment

The manuscript was revised and made sure genes are in italic.

The figure 4 was revised and the data for Zn was included.

Comment and our Responses to the Comment
2. Table 1 needs to be modified. You can add a column of years and change it to a three-line grid.

Our Response to the comment: Modified and fixed as suggested.
3. The legends in Figures 1 and 2 are inconsistent with the figures. They need to be merged and divided into a, b, c, etc.

Our Response to the comment: The Figure 1 and Figure 2 were both revised and figures legend were appropriately corrected for better clarity. Putting all figures together will make the graphs crowded and decrease the resolution.
4. Table 2. Unable to distinguish specific years

Our Response to the comment: Corrected as suggested.

5. The specific content cannot be seen in Figure 3 and needs to be checked. It can be displayed as a bar chart or scatter chart.

Our Response to the comment: The content was checked and it is fine with high resolution. Please consult Word file (original) in the future and not pdf file. Also, the Legend of Figure 3 was further explained and corrected to reflect the content of the graphs. 
6. Tables 3, 4 and 5 should have three lines.

Our Response to the comment: Corrected and modified as suggested.

 

Comment

Detail comments

Other minor issues, not limited to this:
1. Line 25 and 27 QTL need to be exchanged. The full name is used when it appears for the first time.
2. Line 29 5405 is changed to 5,405
3. The chromosome is Chr1, but the QTL uses 01, which is inconsistent;
4. line 74 F2:4 change to F_2:4

Our Responses to the comment

All the above details comments from 1 to 4 were revised and addressed as suggested, except item number 1, which is maintained as is since this approach has been used before and it has been acceptable approach, for example  references/citation [31,32], and recently Bellaloui et al. (Plants, 2023, 12, 3709). Please let me know if I missed understood.

Comment and our Responses to the Comment

Comment:

5. line 83 t, [9] identified two QTL, this writing method needs to be modified.

Our Response to the comment: Revised and modified as suggested.
Comment

6. Line 162 some amino acids (our research is already published, for example Knizia et al. [32]). Modify this writing

Our Response to the comment: Modified.
Comment

7. Line 175 HNO3 is written incorrectly.

Our Response to the comment: Fixed
Comment

8. Line 208 delete URL

Our Response to the comment: Deleted
Comment

9. Line 264 61.2-61.6, need to add cM

Our Response to the comment: Added.

Reviewer 3 Report

The study presents a comprehensive study on the identification of QTLs and candidate genes related to seed iron (Fe) and zinc (Zn) accumulation in soybean. Here are some comments on the document's content and structure:

Major comments:

1. The introduction clearly states the research objectives and significance, especially QTLs related to seed Fe and Zn and their candidate genes.

2. Part of materials and methods are described in detail, facilitating replication and statistical analysis based on SAS.

3. The discussion sections effectively integrate findings with existing literature and propose future research directions.

4. The document follows a standard journal article structure.

  

In my opinion, there are still some parts in this text should be improved as follows:

1. The abstract could be more concise and highlight the most important 2-3 findings.

2. Some sentences are too long and could be broken up for better readability shown in suggestions for consideration.

3. Some figures (1-4) with poor typesetting lacks beauty.

 

Here are some examples of sentences with potential issues, along with suggested revisions for consideration:

1. Original: "The objective of the current research was to identify genetic regions (quantitative trait loci, QTL) controlling Fe and Zn accumulation in soybean seed."

   Suggested Revision: "The objective of this research was to identify genetic regions, known as quantitative trait loci (QTL), that control the accumulation of iron (Fe) and zinc (Zn) in soybean seeds."

2. Original: "A two-year experiment was conducted in two environments [(North Carolina, 2018 (NC)]; and Illinois, 2020 (IL)] with only QTL with logarithm of odds (LOD) scores ≥ 2.5 and identified by the composite interval mapping (CIM) method were reported here."

   Suggested Revision: "A two-year experiment was conducted across two environments: North Carolina in 2018 (NC) and Illinois in 2020 (IL). Only QTL with LOD scores ≥ 2.5, as identified by the composite interval mapping (CIM) method, are reported here."

3. Original: "A total of 6 QTL were identified for seed Fe; 3 QTL (qFe-01-[NC-2018]; qFe-02-[NC-2018]; and qFe-03-[NC-2018]) on Chr 1, 2, and 6, respectively, in NC, and 3 QTL (qFe-01-[IL-2020]; qFe-02-[IL-2020]; and qFe-03-[IL-2020]), respectively, on Chr 1, 2, and 12, in IL."

   Suggested Revision: "In total, 6 QTL were identified for seed iron (Fe); specifically, 3 QTL (qFe-01-[NC-2018], qFe-02-[NC-2018], and qFe-03-[NC-2018]) were located on chromosomes 1, 2, and 6, respectively, in the NC environment, and 3 QTL (qFe-01-[IL-2020], qFe-02-[IL-2020], and qFe-03-[IL-2020]) were positioned on chromosomes 1, 2, and 12, respectively, in the IL environment."

4. Original: "A significant positive correlation between seed Fe and seed Zn were observed in 2018 (R=0.42283 and P<.0001) and in 2020 (R=0.45691 and P<.0001), although the correlation was stronger in 2020."

   Suggested Revision: "A significant positive correlation between seed iron (Fe) and seed zinc (Zn) concentrations was observed in both 2018 (R=0.42283, P<.0001) and 2020 (R=0.45691, P<.0001), with a stronger correlation noted in 2020."

5. Original: "For example, it was reported that the environment and genotype × environment interaction effects influenced the phenotype of the trait in different environments (locations × years)."

   Suggested Revision: "For instance, it has been reported that both environmental factors and genotype × environment interactions can influence the phenotype of the trait across different environments and years."

 

Comments on grammar and style:

1. Overall, the English grammar is good but there are some areas for improvement.

2. Watch out for subject-verb agreement issues.

3. Some sentences lack a clear subject or main verb.

4. Inconsistent use of past tense in some sections.

5. Some figures and tables lack clear legends.

6. Ensure proper formatting of citations and references.

 

In summary, the document presents a valuable study on soybean genetics. While the content is strong, there are opportunities to improve grammar, style, and formatting for better clarity and readability. Addressing these areas would enhance the quality of the document.

 

 

 

Author Response

Comment

The study presents a comprehensive study on the identification of QTLs and candidate genes related to seed iron (Fe) and zinc (Zn) accumulation in soybean. Here are some comments on the document's content and structure:

Major comments:

1. The introduction clearly states the research objectives and significance, especially QTLs related to seed Fe and Zn and their candidate genes.
2. Part of materials and methods are described in detail, facilitating replication and statistical analysis based on SAS.
3. The discussion sections effectively integrate findings with existing literature and propose future research directions.
4. The document follows a standard journal article structure.

Our Response to the comment

We appreciate the comments of the reviewer. We addressed all of the reviewer’s comments as suggested or as appropriate. All are track-changed.

Comment and our Responses to the Detail Comments

 

Comment

In my opinion, there are still some parts in this text should be improved as follows:

1. The abstract could be more concise and highlight the most important 2-3 findings.

Our Response to the comment

Revised and addressed as suggested; all are track-changed in the text.

Comment

2. Some sentences are too long and could be broken up for better readability shown in suggestions for consideration.

Our Response to the comment

Revised and addressed as suggested; all are track changed in the text.

Comment

3. Some figures (1-4) with poor typesetting lacks beauty.

Our Response to the comment

We revised the graphs and we modified as appropriate. We think that the figures are of high resolution and pretty clear. This was addressed above as well.

Other comments and our response to comments

Here are some examples of sentences with potential issues, along with suggested revisions for consideration:

Comment

1. Original: "The objective of the current research was to identify genetic regions (quantitative trait loci, QTL) controlling Fe and Zn accumulation in soybean seed."

   Suggested Revision: "The objective of this research was to identify genetic regions, known as quantitative trait loci (QTL), that control the accumulation of iron (Fe) and zinc (Zn) in soybean seeds."

Our Response to the comment: Modified as suggested.

Comment

2. Original: "A two-year experiment was conducted in two environments [(North Carolina, 2018 (NC)]; and Illinois, 2020 (IL)] with only QTL with logarithm of odds (LOD) scores ≥ 2.5 and identified by the composite interval mapping (CIM) method were reported here."

   Suggested Revision: "A two-year experiment was conducted across two environments: North Carolina in 2018 (NC) and Illinois in 2020 (IL). Only QTL with LOD scores ≥ 2.5, as identified by the composite interval mapping (CIM) method, are reported here."

Our Response to the comment: Modified as suggested.

Comment

3. Original: "A total of 6 QTL were identified for seed Fe; 3 QTL (qFe-01-[NC-2018]; qFe-02-[NC-2018]; and qFe-03-[NC-2018]) on Chr 1, 2, and 6, respectively, in NC, and 3 QTL (qFe-01-[IL-2020]; qFe-02-[IL-2020]; and qFe-03-[IL-2020]), respectively, on Chr 1, 2, and 12, in IL."

   Suggested Revision: "In total, 6 QTL were identified for seed iron (Fe); specifically, 3 QTL (qFe-01-[NC-2018], qFe-02-[NC-2018], and qFe-03-[NC-2018]) were located on chromosomes 1, 2, and 6, respectively, in the NC environment, and 3 QTL (qFe-01-[IL-2020], qFe-02-[IL-2020], and qFe-03-[IL-2020]) were positioned on chromosomes 1, 2, and 12, respectively, in the IL environment."

Our Response to the comment: Modified as suggested.

Comment

4. Original: "A significant positive correlation between seed Fe and seed Zn were observed in 2018 (R=0.42283 and P<.0001) and in 2020 (R=0.45691 and P<.0001), although the correlation was stronger in 2020."

   Suggested Revision: "A significant positive correlation between seed iron (Fe) and seed zinc (Zn) concentrations was observed in both 2018 (R=0.42283, P<.0001) and 2020 (R=0.45691, P<.0001), with a stronger correlation noted in 2020."

Our Response to the comment: Modified as suggested.

Comment

5. Original: "For example, it was reported that the environment and genotype × environment interaction effects influenced the phenotype of the trait in different environments (locations × years)."

   Suggested Revision: "For instance, it has been reported that both environmental factors and genotype × environment interactions can influence the phenotype of the trait across different environments and years."

Our Response to the comment: Modified as suggested.

Other Comments on grammar and style:

1. Overall, the English grammar is good but there are some areas for improvement.
2. Watch out for subject-verb agreement issues.
3. Some sentences lack a clear subject or main verb.
4. Inconsistent use of past tense in some sections.
5. Some figures and tables lack clear legends.
6. Ensure proper formatting of citations and references.

Our Response to the comment

We revised the entire manuscript and corrected and addressed all the above items from 1 to 6, especially the legend of the figures. We revised them as suggested or corrected them as appropriate.

Comment 

In summary, the document presents a valuable study on soybean genetics. While the content is strong, there are opportunities to improve grammar, style, and formatting for better clarity and readability. Addressing these areas would enhance the quality of the document.

Our Response to the comment

We appreciate the thoughtful comment on our research, and we appreciate the comments of the reviewers and editors for their efforts and commitments to science and education.

 

 

Round 2

Reviewer 1 Report

See detailed comment

Introduction

The introduction section is a collection of review of literature. The authors have provided ample evidences in the introduction section that many QTLs with LOD value > 3 are reported for seed Zn and Fe content in soybean, but argued that information on QTL and candidate genes are 'almost non-existent'. It is self-contradictory.

Materials: An existing genetic map was used for QTL mapping. The same map was previously used for mapping QTLs for iron and zinc (by Wang et al., 2022). However, in the present study, the same map, the same population and the same trait was used, but QTLs were located in different positions. 

The effect of location and year on QTL detection is noteworthy.  Based on the year 2018 experiment in one location QTLs for Zn was identified on Chr. nos. 2, 3, 7, 19. On the other hand, in 2020, location was changed and the QTLs for the same trait was reported on chr. 5 and chr. 8, but not on 2,3, 7 or 19. This shows high environmental variation and fluctuation in expression of the trait, or poor phenotyping. The experiment should be repeated in each location. Since there is no genotyping involved, QTLs shoudl only be identified after observing multi-locational, multi-year data. 

Hence, the results should be reported only after observing stable phenotypic expression.   

Authors' argument that many others have published QTL with LOD score over 2.5 and below 3.0, so we should also support a LOD score of 2.5 as critical LOD is childish. Conventionally, a LOD score of 3.0 is the critical LOD as published in over hundreds of research paper, discussions and textbooks. Rather than reporting a QTL with LOD< 3.0, the authors should use more stringent experimental condition to improve the precision of QTL identification. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The author has made careful modifications, but there are still some problems; for example, the specific content cannot be seen in the pictures below, Figure 1, Figure 2, and Figure 3. It is impossible to judge the accuracy of the author's results. It is also recommended that each figure be marked with A, B, etc.

1. Line 204 2075 is changed to 2,075. The author needs to check the full text.

2. The format of references needs to be carefully checke

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf