Dear Dr. Assfaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please submit your revised manuscript by May 26 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Bahram Heidari

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: This study examines the genetic diversity and population structure of 45 soybean genotypes from the IITA breeding program, utilizing both phenotypic traits and SNP markers. The authors employ multivariate statistical methods such as principal component analysis (PCA) and cluster analysis to assess the genotypic and phenotypic variation. However, there are a few suggestions that could further improve the manuscript, as outlined below:

Line 39: Replace this "Soybean [Glycine max (L.) Merrill], is the leading oil seed crop, accounting for 57% of the global oil production." With this "Soybean [Glycine max (L.) Merrill] is one of the leading oilseed crops globally, accounting for approximately 57% of global vegetable oil production."

Line 41-43: Replace this "It is cultivated in many countries and is a major source of vegetable oil and protein used in food, animal feed, and for industrial purposes." With this "It is cultivated in numerous countries and serves as a major source of vegetable oil and protein, essential in food, feed, and various industrial applications."

Line 44-45: Replace this "It was domesticated in the eleventh (11th) century BC around Northeast China and was then transported throughout Asia, the USA, Brazil, and Argentina." With this "Soybeans were domesticated around the 11th century BC in Northeast China and subsequently spread across Asia, the USA, Brazil, and Argentina."

Line 46-47: Replace this "Nigeria is the second-largest producer of soybeans in Africa, after South Africa. The first soybeans were brought into Nigeria in 1908, but the first successful cultivation was in 1937 with the Malayan variety, which was reported as commercial production in Benue State." With this "Nigeria ranks as the second-largest producer of soybeans in Africa, following South Africa. Soybeans were first introduced to Nigeria in 1908, with successful commercial cultivation beginning in 1937, using the Malayan variety in Benue State."

Line 49-50: Replace this "The crop is adapted to various environmental conditions and is primarily cultivated under rain-fed conditions." With this "The crop is adapted to diverse environmental conditions and is predominantly grown under rain-fed conditions."

Lines 55–57: How can the genetic improvement of soybeans be specifically tailored to address malnutrition in Nigeria or similar regions? Are there particular traits being targeted that would make soybeans more beneficial nutritionally?

Line 184: Have you considered including an Analysis of Variance (ANOVA) along with mean comparison for the phenotypic traits assessed before performing the multivariate statistical analyses such as PCA and clustering? This could help to highlight significant phenotypic variation among the genotypes and provide a clearer basis for the subsequent analyses.

Line 259: The discussion section could benefit from incorporating the results from the ANOVA and mean comparison analyses, especially when interpreting the genetic variability observed in the study.

Reviewer #2: 1- The results of ANOVA are needed.

2- Add the number of plants used for phenotypical measurements.

3- Add the method of cluster analysis.

4- Write the formulas used for parameters estimation to the materials and methods section.

5- Consider eigenvectors equal to or greater than 0.4 as high in the PCA analysis and reinterpret.

6- Calculation of genetic distances between cluster groups and genetoypes are required.

7- I have mentioned some suggestions in the text of the article that should be corrected.

**********

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Reviewer #1: No

Reviewer #2: Yes:  Massoud Dehdari

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-25-08250_revised1.pdf

Dear Dr. Assfaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jul 31 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Yêyinou Laura Estelle Loko

Academic Editor

PLOS ONE

Additional Editor Comments:

Reviewer 1

The authors reported on “Genetic diversity and population structure analysis of soybean [Glycine max (L.) Merrill] genotypes based on agro-morphological traits and SNP markers”, an aspect that is key to the success of every breeding program as a perquisite to successful varietal development. The authors have done well in the presentation of the manuscript sections though with minor corrections to improve the manuscript quality. My few comments for each section are below.

Abstract

L24&235: “…genotypes were 25 further assessed for SNPs-based genotypic diversity analyses”. Please include the type of marker and the quantity.

L28: take out the word “corresponding” the markers are not corresponding to the traits used for pheno-based phylogeny

L29 and 30: it is better to list a few of the desirable traits in the genotypes rather than just using a general perception.

L33: you said “considerable genetic variation” but concluded with “high genetic variation” which one should the reader accept?

Introduction

L53: I think you meant “…..high-yielding, climate, and stress-resilient…..”

Materials and Methods

L115 – 122: Experimental materials should come before experimental design and management.

L130: should be “…agro-morphological traits including plant height…”

L134 – 145: I assume you did ANOVA but chose not to report it. If no please it should be part of your results or at least in your supplementary, file but reported in your main text. ANOVA is the basis for establishing variability that is your diversity per se. you have data from three locations, readers will like to see environment, genotype, and their interaction effects prior to be interested in other results. Please include the process in handling these aspects of the analysis in the phenotypic data analysis focusing on choice of model used, justification, and how you generated the data for cluster analysis. You did cluster analysis but you kept information on the data used (BLUES or BLUP) and why?

L135: what do you mean by similarity coefficient? Did you use similarity matrix or dissimilarity matrix for clustering? In my opinion, your focus is diversity so dissimilarity matrix like, Gower and Euclidean are fine.

L143: You did PCA with two packages. I am familiar with both packages and can tell that both of them do not do PCA. Only one does the PCA while the other is for visualization. So please be specific which one was used for PCA and which was for visualization.

L149: what do you mean immature? I assume you meant fully expanded but young leaf. If so, please correct and if not please clarify.

L170 – 183: your MAF threshold seems very low (0.01), the standard is usually 0.05. Do you have a reason for bringing it down to 0.01? how did you handle markers with high heterozygosity? Did you check for call rate prior to QC and what was your threshold?

L177 and 178: You used both VCFtool and PLINK for filtering and marker diversity indices assessment. Please be clear which one is for which activity? BIC is not to estimate but to define optimum subpopulation.

Results

NB: Please include the proposed analysis in the materials and methods in the results section.

L185 – 196: You cited table 4 before fig 1. Please arrange the results in same manner.

L210: fig 2 should be placed after the text just before table 5.

L212 – 215: there is grammar issue here, please attend to it.

L220: Please subject the cluster performance to f-statistics to see whether the cluster groups are significantly different from one another for each of the traits. That way, you can identify which clusters actually possess what traits with statistical confidence.

L237: Your BIC vs cluster number graph had a sharp drop from one to two and at this point was also the lowest indicating that two was the optimal subpopulation but you said three. Could you justify? I suggest you verify the subpopulation with alternative analysis like PCA scree plot. If it happens that you consider two subpopulations then DAPC may have to change.

L249: Fig 5 seems to have been misplaced. The figure was titled phylogenetic three but was placed under population structure. If I am wrong then please provide the results for the phylogenetic analysis. This can be compared to the phylogenetic analysis from the phenotypic data to see how the genotypes grouping aligns.

Discussion

L313: The authors said “The minor allele frequency (MAF) values measure the ability of markers to discriminate among genotypes” this is new and contrary to what is known about MAF. If MAF does this, then what is function of PIC?

Conclusion

The conclusion is too long and needs to be reduced and more concise.

Other information

The figures not easily readable.

Reviewer 2

1-Add error1 and error2 as SOV in the ANOVA table

2-For yield, D50F and D95M traits, you should introduce the superior genotype at each location because the interaction has become significant.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

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

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: 1-Add error1 and error2 as SOV in the ANOVA table

2-For yield, D50F and D95M traits, you should introduce the superior genotype at each location because the interaction has become significant.

Reviewer #3: The authors reported on “Genetic diversity and population structure analysis of soybean [Glycine max (L.) Merrill] genotypes based on agro-morphological traits and SNP markers”, an aspect that is key to the success of every breeding program as a perquisite to successful varietal development. The authors have done well in the presentation of the manuscript sections though with minor corrections to improve the manuscript quality. My few comments for each section are below.

Abstract

L24&235: “…genotypes were 25 further assessed for SNPs-based genotypic diversity analyses”. Please include the type of marker and the quantity.

L28: take out the word “corresponding” the markers are not corresponding to the traits used for pheno-based phylogeny

L29 and 30: it is better to list a few of the desirable traits in the genotypes rather than just using a general perception.

L33: you said “considerable genetic variation” but concluded with “high genetic variation” which one should the reader accept?

Introduction

L53: I think you meant “…..high-yielding, climate, and stress-resilient…..”

Materials and Methods

L115 – 122: Experimental materials should come before experimental design and management.

L130: should be “…agro-morphological traits including plant height…”

L134 – 145: I assume you did ANOVA but chose not to report it. If no please it should be part of your results or at least in your supplementary, file but reported in your main text. ANOVA is the basis for establishing variability that is your diversity per se. you have data from three locations, readers will like to see environment, genotype, and their interaction effects prior to be interested in other results. Please include the process in handling these aspects of the analysis in the phenotypic data analysis focusing on choice of model used, justification, and how you generated the data for cluster analysis. You did cluster analysis but you kept information on the data used (BLUES or BLUP) and why?

L135: what do you mean by similarity coefficient? Did you use similarity matrix or dissimilarity matrix for clustering? In my opinion, your focus is diversity so dissimilarity matrix like, Gower and Euclidean are fine.

L143: You did PCA with two packages. I am familiar with both packages and can tell that both of them do not do PCA. Only one does the PCA while the other is for visualization. So please be specific which one was used for PCA and which was for visualization.

L149: what do you mean immature? I assume you meant fully expanded but young leaf. If so, please correct and if not please clarify.

L170 – 183: your MAF threshold seems very low (0.01), the standard is usually 0.05. Do you have a reason for bringing it down to 0.01? how did you handle markers with high heterozygosity? Did you check for call rate prior to QC and what was your threshold?

L177 and 178: You used both VCFtool and PLINK for filtering and marker diversity indices assessment. Please be clear which one is for which activity? BIC is not to estimate but to define optimum subpopulation.

Results

NB: Please include the proposed analysis in the materials and methods in the results section.

L185 – 196: You cited table 4 before fig 1. Please arrange the results in same manner.

L210: fig 2 should be placed after the text just before table 5.

L212 – 215: there is grammar issue here, please attend to it.

L220: Please subject the cluster performance to f-statistics to see whether the cluster groups are significantly different from one another for each of the traits. That way, you can identify which clusters actually possess what traits with statistical confidence.

L237: Your BIC vs cluster number graph had a sharp drop from one to two and at this point was also the lowest indicating that two was the optimal subpopulation but you said three. Could you justify? I suggest you verify the subpopulation with alternative analysis like PCA scree plot. If it happens that you consider two subpopulations then DAPC may have to change.

L249: Fig 5 seems to have been misplaced. The figure was titled phylogenetic three but was placed under population structure. If I am wrong then please provide the results for the phylogenetic analysis. This can be compared to the phylogenetic analysis from the phenotypic data to see how the genotypes grouping aligns.

Discussion

L313: The authors said “The minor allele frequency (MAF) values measure the ability of markers to discriminate among genotypes” this is new and contrary to what is known about MAF. If MAF does this, then what is function of PIC?

Conclusion

The conclusion is too long and needs to be reduced and more concise.

Other information

The figures not easily readable.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Nikwan Shariatipour

Reviewer #2: Yes:  Massoud Dehdari

Reviewer #3: Yes:  DR. ADEJUMOBI IDRIS ISHOLA

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-25-08250 revised2.pdf

Attachment

Submitted filename: PLOSOne Reviewer.docx

Dear Dr. Assfaw,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Yêyinou Laura Estelle Loko

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #2: 1-DF=g(r-1)=45*(3-1)=90

I think the DF for Error2 is 90. Please response to this comment.

2-For yield, D50F and D95M traits, you should introduce the superior genotype at each location because the interaction has become significant.

Reviewer #3: Table 2: what does the SNP designation in the Table represent?

Table 3: the caption should be well-placed on top of the Table and not in the first cell. In addition, add the status of each trait as separate column in the Table (qualitative or quantitative).

Line 146: linear mixed model is LMM or call it mixed linear model (MLM) not LMER (this is a function in lmerTest R-package).

Line 157: I do not think LSD can do a good mean comparison for 45 genotypes if you look thoroughly on how it calculates the critical value for comparison. Why not explore HSD that is more robust.

Line 160 – 165: Kindly include the number of traits used for the cluster analysis and how they were selected. The method is Ward.D2 and not Ward.D2 . cluster analysis is performed using based R function not dendextend package. The package is only used to plot the dendrogram.

Line 168-173: Kindly provide the reason for the PCA analysis. Was it used to select the traits used for clustering? if so, please it should come before cluster analysis.

Line 257: you cited S5 Table before S3 Table in line 261 and I think the order should be reversed since it is their first citation in the manuscript

Line 370: I think the discussion is too long and contain many redundant statements. Endeavour to reduce the discussion and be more precise.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes:  Massoud Dehdari

Reviewer #3: Yes:  Adejumobi Idris Ishola (PhD)

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Genetic diversity and population structure analysis of soybean [Glycine max (L.) Merrill] genotypes based on agro-morphological traits and SNP markers

PONE-D-25-08250R3

Dear Dr. Abebawork Tilahun Assfaw,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yêyinou Laura Estelle Loko

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #2: I think your manuscript is now ready for publication in the PLOS One journal. I wish you success in your future research.

Reviewer #3: (No Response)

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes:  Masoud Dehdari

Reviewer #3: Yes:  Idris Ishola Adejumobi (PhD)

**********