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PONE-D-19-28312

Genetic analysis of QTLs controlling allelopathic characteristics in sorghum

PLOS ONE

Dear Dr. Shehzad,

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.

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Craig Eliot Coleman, PhD

Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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: Yes

Reviewer #2: No

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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 authors used two sorghum populations, i.e. SDRS and F2:3, to study the inhibitory effect of sorghum WSE on the seed germination and on the root length of lettuce seedlings. They used four concentrations of the WSE (25%, 50%, 75% and 100%). The authors identified several QTL in the F2:3 and in the association mapping populations. The work is designed in a proper way, however, I have the following comments;

• I couldn't find any figures attached to the manuscript that could have helped me to better understand the experimental design.

• Please, add how the heritability was calculated. Was the H2 for germ50% 0.07?

• Please, remove the last paragraph of the "Bioassay of allelopathic effect using water-soluble extract (WSE)" as it was explained again in the next paragraph "Statistical analysis".

• For QTL mapping, was threshold 2.5 chosen based on 1000 permutation or as an arbitrary threshold?

• I couldn't find any figure or data presenting the map of the F3 or the association panel. Please, provide this data.

• In such low density map, I would recommend using window size larger than 10 cM, e.g. 30 cM, to consider the following sentence correct "The adjacent QTLs identified for the same trait with non-overlapping intervals on same chromosome were considered as different QTLs". In addition, one can consider two adjacent qtl as different ones only when their effect is different, e.g. one has an effect from A and the other from B.

• Please change this title “Linkage disequilibrium (LD)-based association mapping” to “Population structure and association mapping” or add two subtitles “Population structure” and “Association mapping”

• “the P-values representing the significance of LD was measured”, how was P value calculated? What is threshold of 2.5 and strict threshold =3 ? Please explain. Have you used an arbitrary threshold or Bonferroni correction? I believe a P value of 2.5 is relatively low and will increase the false positives.

• Last paragraph of Linkage disequilibrium (LD)-based association mapping, “As genome-wide association studies results in spurious associations in particular with low number of markers.” Not clear what Authors want to say.

• In Marker localization and homology with known genes “The Map Viewer of NCBI website (http://www.ncbi.nlm.nih.gov/mapview/) was used to identify loci previously identified as linked to known genes in genome-based sequence information.” I don’t understand whether the author did that to know if their SSR were previously mapped, which could be done from previously published articles, or they did that to just know what is known. Please, clarify on that.

• In “Construction of linkage map……”, please, move the following sentence to discussion “These findings confirms the consistency and accuracy of the linkage map reported here.

• The discussion part, in general, needs to be restructured to avoid repeating sentences and contents. Below are some suggestions,

• when comparing the discrepancy in locating some SSR, e.g. SB3664 and SB2613, please give references after "in previous report".

• The paragraph that starts with "We used two approaches and two different types of populations ……………..", it reads like a review not discussion. Can be deleted or modified.

• "We used four different concentrations of WSE, ……………...." and then "This confirms...…....". It isn't clear what confirms what.

• It will be more coherent to gather all the discussion related to QTL and LD mapping in one paragraph, as now in several places I find the same conclusion, i.e. the suitability of the 2 approaches to map significant QTL ....."

• Why discussing SOR1? Please take the space needed to elaborate on why specifically the authors discussed this gene

• "A single common co-localized major QTL qtl10RL on Chr 10 between markers Xtxp270 (32.6 bp) and SB5329 (35.5 bp) was detected for inhibition of root length by WSE used in 50%, 75% and 100% concentrations in both approaches of QTL mapping". The content of this sentence was mentioned earlier in the discussion. Please, remove this sentence and all other repeated sentences.

• The author should carefully interpret the results of the "regional mapping" and the physical co-location of with known genes as the QTL interval is normally large and contain several genes, especially with this low dense map. I would rather call them candidate genes

Reviewer #2: Allelopathic characteristics of sorghum has been studied at large, however, the reports of QTL for allelopathic characteristics were not reported. In this line, the study “Genetic analysis of QTLs controlling allelopathic characteristics in sorghum” has a merit. Authors did very good job by conducting experiment in optimal way and results are also very interesting however authors need to focus on the results of the study. I propose to recommend for publication in PLOSONE with few suggestions to improve the manuscript.

Major revision

1. The phytotoxin sorgoleone has been largely associated for allelopathic characteristics of sorghum. Due importance for sorgoleone production is not given in this study. It is suggested to study the QTL for sorgoleone content and yield. Further, QTL for sorgoleone needs to be associated with other components of allelopathic characteristics sorghum.

2. Validation of expression stability of QTL is missing in this study. It is suggested validate the identified QTL multiple seasons and location would be logical.

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Reviewer #1: Yes: Mohamed El-Soda

Reviewer #2: Yes: Nagaraja Reddy

[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 to be viewed.]

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PONE-D-19-28312R1

Genetic analysis of QTLs controlling allelopathic characteristics in sorghum

PLOS ONE

Dear Dr. Shehzad,

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 26 2020 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.

Please include the following items when submitting your revised manuscript:

• 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Craig Eliot Coleman, PhD

Academic Editor

PLOS ONE

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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)

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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: Yes

**********

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

Reviewer #1: Yes

**********

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: Yes

**********

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: Yes

**********

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: Authors have partially addressed the comments as they gave answers to the queries raised during the first round but only in their letter not in the main text. Please amend the manuscript based on the suggested changes. For example;

1. Please, add how heritability was calculated, and explain why you refer to SOR1 in the discussion part

2. Please, add to your methods how the threshold was chosen for QTL and association mapping. Please add R2 and how you calculated "FDR-corrected P value" for the association mapping, that might strengthen your point of using 2 as a threshold.

3. Please, check the language throughout the manuscript as many sentences are not correct. For example, "Each trait was analyzed with empirical experiment-wise threshold values for significance (P = 0.05) from estimating 1000 permutations"

4. In the QTL part, please discuss the QTL co-location as this will support your results.

5. Please rephrase the sentence you added to introduction, “Researchers studying secondary metabolites affecting germination of parasitic weed Striga asicatica (witchweed) first discovered it (Chang et al. 1986). Retrobiosynthetic NMR analysis was ................. ,

materials and methods, "We physically localized the SSR loci that were strongly linked with the traits by BLAST searches of sequences in http://www.phytozome.net/sorghum ……… ",

and to the discussion "also reported an increase in inhibitory effects with increasing concentration of allelochemicals. After statistical analyses, we detected 75% and 100% WSE as optimum --------", as they don’t read well.

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7. PLOS authors have the option to publish the peer review history of their article (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: Mohamed El-Soda

[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 analysis of QTLs controlling allelopathic characteristics in sorghum

PONE-D-19-28312R2

Dear Dr. Shehzad,

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.

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Kind regards,

Craig Eliot Coleman, PhD

Academic Editor

PLOS ONE

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