PONE-D-24-24053Glycine soja, PI424025, is a valuable genetic resource to improve soybean seed-protein content and compositionPLOS ONE

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Additional Editor Comments:

It is obvious that QTL mapping will identify several QTLs for the studied trait. However, pinpointing the strong QTLs with major effect on phenotype will help the breeders to transfer beneficial traits in breeding lines. So, authors need to pinpoint major QTLs with strong effect on phenotype. Additionally, soybean have the well annotated genome. Please do candidate gene analysis from the major QTLs regions.

Though it is good work, authors failed to discuss the obtained results appropriately. Therefore, results section needs to be improved.

Introduction and Discussions are the key sections for MS as they will give brief background about the subject/study and validation for your results, respectively. Improve these sections by citing the most recent references. Such as:

• Singh, G., Gudi, S., Amandeep, Upadhyay, P., Shekhawat, P. K., Nayak, G., ... & Ayoubi, H. (2022). Unlocking the hidden variation from wild repository for accelerating genetic gain in legumes. Frontiers in plant science, 13, 1035878.

• Gudi, S., Saini, D. K., Singh, G., Halladakeri, P., Shamshad, M., Tanin, M. J., ... & Sharma, A. (2021). Unravelling consensus genomic regions associated with quality traits in wheat (Triticum aestivum L.) using meta-analysis of quantitative trait loci. bioRxiv, 2021-11.

In material and method “Phenotype data Analysis” section come before “Molecular marker and QTL analysis”

Change title from “conclusion” to “discussion”

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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 study attempts to identify sources for superior seed-protein content and composition by generating mapping populations and then unravelling the genetic basis of traits related to elemental composition in seeds. The purpose of the study is important as improving soybean protein content and composition could amount to better nutritional and market value.

Major comments:

While the superior line identified may be possessing higher protein content and desirable alleles for protein content and composition, the study has issues with experimental plan and presentation. Most of the paper focuses on the elemental composition of C, N, S their ratios as proxies for protein content and composition.

1. Writing is not up to the mark – fails to mention key aspects such as why these traits were chosen to be studied in detail are missing and also there are no adequate and appropriate references to support the sentences in the paper at most of the places.

2. Highly Incoherent presentation of the findings and lack of depth in analysing and discussing how the findings in the study correlate with the findings from previous studies.

3. An elaboration on why sulfur is important for protein quality can be added in the introduction. Additionally, a few lines on why carbon partitioning and allocation during maturity stages is important for protein content should be mentioned here for clearer understanding of the readers.

4. In the introduction, the penultimate paragraph says that representation of QTL by the said markers is ambiguous. The physical position of the markers and the POWR1 locus can be obtained and checked for colocalization. This sentence can be changed in the manuscript.

5. The authors have not adequately mentioned or explained in this paper why NC Raleigh was chosen as a parent for crossing and generation of mapping population with PI424025B.

6. The authors must use the full form of abbreviations the first time they use it in the paper and use the abbreviated form during the subsequent uses.

7. What was the area of the plots used for the experiment?

8. As it has been mentioned that enough seeds were not available for protein estimation using NIR, the authors need to mention the quantity of seeds required for protein and oil measurement using NIR, and what quantity was actually used (for one location).

9. “Molecular marker and QTL analysis” section in Materials and methods can be merged with “Linkage map construction” part for more coherence.

10. More insights could be obtained by analysing the phenotype data in more detail. Descriptive stats, ANOVA and biplots to understand the GxE interactions across the 5 locations can be done. Also, assessing some agronomic traits would be more informative.

11. In the “Linkage map construction” section in Materials and methods, I suggest the authors to check the collinearity of the linkage map with the physical map using the physical positions of the 6K panel.

12. Since protein was not estimated at all the locations but N was, is there a formula to convert the amount of N to %protein across all locations?

13. Reference missing for the sentence - “The ratio of N to C is often reported which gave a better correlation of (0.78) with seed protein measured by NIR.”

14. The reduction in carbon source towards the terminal stages of the plant may contribute to lower protein content due to competition for carbon skeleton with oil. How does the finding in your study correlate with previous studies? Since NC Raleigh has high C content, how does that fit here since it has low protein content? Interpretations of your results must be done thoroughly by consulting previous similar studies.

15. The GmSWEET39 gene is 388KB upstream of the left marker of the QTL (SoyBase, Glyma2.0). It may be better not to consider that the QTLs on chr15 and GmSWEET39 are colocalized as mentioned in the paper. Claims must be thoroughly checked for accuracy before presenting.

16. Candidate gene analyses could be performed on the QTLs obtained to see what kind of genes present/possible molecular mechanisms are.

17. Protein content and oil have not been estimated in all but one of the locations used in the study. It is unclear as to how the C, N, S content translate to protein and oil content. Correlation alone cannot be used as a metric for using the C, N, S compositions as proxy for protein content and oil performance across different locations. Overall, the study lacks detail in discussion and analyses. Due to these reasons, the study does not have adequate merits for publication currently.

Reviewer #2: In this study authors have developed a Recombinant inbred line (RIL) population using Glycine soja as one of the parent. Phenotyping of the RILs was done for seed protein, carbon, nitrogen and sulphur content. Linkage map was developed using SNP data and new QTLs for high protein, carbon, nitrogen and sulphur content were identified were. Overall, this manuscript provides new information regarding the seed composition traits in soybean. Following points may be considered:

1. Additional information regarding candidate genes and QTLs may be provided.

2. Application of the outcome may be discussed in more detail.

3. Recently published articles related to the field may be cited and briefly discussed.

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Reviewer #1: No

Reviewer #2: No

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Glycine soja, PI424025, is a valuable genetic resource to improve soybean seed-protein content and composition

PONE-D-24-24053R1

Dear Dr. Taliercio,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Santosh Gudi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. 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: (No Response)

Reviewer #2: Yes

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

Reviewer #1: (No Response)

Reviewer #2: Yes

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4. 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: (No Response)

Reviewer #2: Yes

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5. 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: (No Response)

Reviewer #2: Yes

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6. 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: (No Response)

Reviewer #2: The revised manuscript is of high scientific quality and provides important information in this field.

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Reviewer #1: No

Reviewer #2: No

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