Dear Dr. Hao-Xun Chang, Academic Editor, PLOS ONE:

Please receive our revised manuscript entitled “Genomic Regions and Candidate Genes Associated with Seed Nitrogen, Phosphorus, and Sulfur Accumulation Identified in the Soybean ‘Forrest’ by ‘Williams 82’ RIL Population” by Bellaloui et al.

We revised the manuscript and addressed all the reviewers’ comments as instructed, on one-by-one basis, and all are track-changes. We also addressed the comments noted in the attached file (form Reviewer 1) either as suggested or as appropriate. Our responses are below. We stated the reviewer’s comments and responded to them, as shown below. Enormous efforts and time were devoted in revising this manuscript, and we believe the reviewers’ comments improved the quality of the manuscript. We would like to note that “ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Thank you and we appreciate your service,

Reviewers comments and Authors’ Responses

Journal Requirements:

Authors' response

All journal requirements were addressed.

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Authors’ response

We revised the entire manuscript, and fixed the files’ names for tables, fugues, files, and so on according to the journal requirements.

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://www.mdpi.com/2037-0164/15/2/35

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Authors’ response

We revied the manuscript and made sure there are no similarities, unless the similarities are scientific terms or technical names. In addition, that published manuscript dealt with Fe and Zn, but the current manuscript deals with N, S, and P.

3. Thank you for stating the following financial disclosure:

[This research was funded by the U.S. Department of Agriculture, Agricultural Research Service Project 6066-21220-016-000D, SIUC, UM, and FSU.].

Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We include the appropriate statement in the manuscript as instructed “

Authors’ response

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Authors’ response

We included it in the cover letter as well, as below:

“ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

N/A

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Authors’ response

Thank you for the comment.

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Authors’ response

Thank you for the comment.

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Authors’ response

Thank you for the comment.

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Authors’ response

We revised the manuscript and made sure the presentation and English writing is sound.

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The trait seed P content has near zero heritability meaning no genotypic variation for the trait. Then all the analysis will be wrong. How to justify your study on this trait. There is some English corrections in the manuscript which needs to attended. There is markings done in the manuscript which needs to be attended. Some discussion is excessively written which needs to addressed before the final acceptance of the research paper.

Our response

We appreciate the reviewer’s important observation regarding the low broad-sense heritability (H² ≈ 0) for seed phosphorus (P) content. Indeed, low heritability suggests that the phenotypic variation is primarily governed by environmental effects and/or strong genotype-by-environment (G×E) interactions, which complicates genetic dissection. However, we respectfully argue that this does not invalidate the analysis, for the several below reasons (we further explained the below concepts in the Discussion section for the benefit for the reader, and all are track-changed).

1. Biological and Agricultural Relevance

Seed P content remains a biologically and nutritionally important trait in soybean, especially due to its connection with seed vigor, germination, and nutritional quality. Even when heritability is low, understanding the genetic basis of this trait is critical for identifying genotypes or genomic regions responsive to environmental P variability.

2. Precedent in Literature

Low heritability for P-related traits in soybean has been reported previously, especially under single-environment studies or limited replication. For instance, Li et al. (2005) and Zhang et al. (2016) both reported variable P heritability depending on tissue type, environment, and developmental stage. These studies still identified meaningful QTL by using multi-location or repeated trials; similar to our approach.

3. Detection of Stable QTL

Despite low overall heritability, we were able to identify several statistically significant QTL for seed P content in both environments (see Table 3), with LOD values ranging from 2.5 to 9.4 and phenotypic variance explained (R²) up to 12.17%. This suggests that specific loci still contribute consistently across environments, even if the overall trait expression is environmentally labile.

4. Justification Through G×E

The low heritability itself is an important genetic insight. It indicates a potential for strong environmental modulation or G×E interactions, which could be exploited through genotype-specific agronomic strategies or P management practices. Moreover, inclusion of this trait helps distinguish which QTL are environment-stable versus those that are more plastic.

References used above are below, and they are included in the manuscript.

Li, Y.D., Wang, Y.J., Tong, Y.P., Gao, J.G., Zhang, J.S., & Chen, S.Y. (2005). QTL mapping of phosphorus deficiency tolerance in soybean (Glycine max L. Merr.). Euphytica, 142, 137–142. https://doi.org/10.1007/s10681-005-1047-2.

Zhang, D., Li, H., Wang, J., Zhang, H., Hu, Z., Chu, S., Lv, H., & Yu, D. (2016). High-density genetic mapping identifies new major loci for tolerance to low-phosphorus stress in soybean. Frontiers in Plant Science, 7, 372. https://doi.org/10.3389/fpls.2016.00372.

Reviewer #2: The manuscript aims to identify new genomic regions associated with seed nitrogen, phosphorus, and sulfur accumulation with the Forrest by Williams 82 RIL mapping population. Several references were cited in the manuscript without the background information in the materials and methods, it would be beneficial if more in-depth information was provided throughout the article. While the manuscript is insightful, it requires major revision before it can be published. Some examples are listed below.

Authors response:

We revised the entire manuscript for better comprehension and further in-depth information, and we paid attention to the cited references in Materials and methods for further details and backgrounds. All edits are track-changes.

Introduction:

A good start for the introduction, more background information should be provided. For example, the introduction should briefly discuss the articles referenced, it would be best to directly reference the authors (Line 81 for example, which author; similarly in Line 97, directly citing the author would make the article easier to follow along) Several editorial recommendations include:

Authors response:

The introduction was revised and adjusted as needed for better clarification. Citations were directly added for better clarification. We worry that adding more information will be in conflict with the suggestion of Reviewer 1 as Reviewer 1 indicated that some discussion is excessively written.

Line 66 Soybean missing scientific name

Line 85 full name of the essential nutrients

Line 88 missing , in 6366

Line 97 F6:8

Line 98 N87-984-16 x TN93-99 => × (cross)

Line 98 Chrs 2, 7, 8, 14, 15, 18, suggest including linkage group after the chromosome numbers

Line 99 others (who?)

QTL first appears in article -> Quantitative trait loci

Line 101 “seven” QTL

Line 102 phosphorus => P

Line 103 F5:7

Authors response:

The above suggested comments were addressed as suggested. We included the linkage group in all tables.

Materials and Methods

Reviewer’s comment

Are there any reasons why this mapping population was selected for the study? Forrest was initially released for soybean cyst nematode resistance whereas William 82 was released for Phytophthora root rot resistance. The information in Line 136 to 141 should be included on the top along with the parental line. It would be best for the authors to provide more background information for the readers to follow along.

Authors response:

We moved the information to the top along with the parental lines, as suggested. We included detailed about Forrest and Williams 82 in Materials and methods; we included a Figure (Fig 1) and supplementary information/file (S1_File) about the background of the parent and crosses for the benefit of the reader.

Reviewer’s comment

The cultivars in the study should include the PI number and provide citation

Line 127 The parental lines should include details of the parental lines such as the year that it was released, which research station developed the cultivar, agronomic traits such as flower color, growth habit, seed composition, maturity groups etc.

Authors response

We included a figure (Fig 1) and supplementary information/file (S1_File) about the background of the parent. We also detailed their background in the Materials and methods ( we included their PI, their crosses, Fig 1; S1_File) and more for the benefit of the reader. All are included in the text and all are track-changed.

Reviewer’s comment

Were the experiments conducted on university research farms? More details are needed.

Authors response

Thank you for the suggestion. Yes, the experiments were conducted on university research farms, as described in K (2021) [27]. We have updated the manuscript with more details and to clarify the experimental sites, including affiliations and research station details, to ensure full transparency and reproducibility. All new edits are track-changed.

Reviewer’s comment

Experimental design and replications are not discussed

Authors’ response

We thank the reviewer for pointing this out. The experimental design and replication details were originally described in Knizia et al. (2021), but we further included in the manuscript further details for clarity and completeness. We have revised the Materials and methods section to include a full description of the field design, replication, and environmental factors impacting data collection.

Reviewers’ response

Line 139 What type of amino acids?

Authors’ response

Amino acids included threonine, serine, proline, glycine, alanine, cysteine, valine, methionine, phenylalanine, lysine, tryptophan. We included them in the text, and all are track-changed.

Reviewer’s comments

The manuscript references many literatures but does not provide sufficient details for the readers to follow along. For example, it does not reference the BARCSoySNP6k Chip in the texts.

Inconsistency regarding the units throughout the article, some have space while others do not

Line 182 provide link to SoyBase

Authors’ response

-Thank you for the comment. We adjusted the text and provided further information and a citation and reference, below, for better clarity for the reader.

Soybean BARCSoySNP6K: An assay for soybean genetics and breeding research. The Plant Journal (2020) 104, 800–811 doi: 10.1111/tpj.14960.

-We provided the link

Reviewer’s comment

Statistical Analysis

Has the author compared different QTL mapping methods such as CIM vs Multiple QTL Mapping?

Authors’ response

No, we did not, and our reasons for this are below, and also some clarification was added to the Material and methods:

We appreciate the reviewer’s question regarding the QTL mapping approach. In this study, we used Composite Interval Mapping (CIM) implemented in WinQTL Cartographer 2.5, which is a widely accepted and robust method for detecting QTL with moderate to high power and resolution.

We acknowledge that alternative methods such as Multiple QTL Mapping (MQM) or Inclusive Composite Interval Mapping (ICIM) may offer complementary insights, particularly in separating closely linked QTL or

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