PONE-D-20-14708

Comparative study on cytogenetics and transcriptome between diploid and autotetraploid rice hybrids harboring double neutral genes

PLOS ONE

Dear Dr. Shahid,

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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We look forward to receiving your revised manuscript.

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Allah Bakhsh

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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #2: Yes

Reviewer #3: 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: Yes

Reviewer #3: 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: In this paper Chen et al., 2020, compared cytological and transcriptome data between autotetraploid and diploid rice hybrid during meiosis and single microspore stages to understand the molecular mechanism of neutral genes for overcoming pollen sterility in autotetraploid rice hybrids. Using cytological analysis they identified lower chromosomal abnormalities in autotetraploid compared to other lines. The authors provide a theoretical basis for molecular mechanisms of heterosis in autotetraploid rice.

I would suggest to improve the quality of the figures and have some minor comments for the paper:

- Page 5, Line 2-3, which lines were crossed?

- Page 8, in the first paragraph, authors needs to describe the general design of experiment, which lines they crossed and which diploid and tetraploid lines they developed for analysis. Also authors needs to explain what they are showing in the figure.

- The nomenclature of hybrids used in Figure 2 is different than what is described at Page 8, line 19-21

- Page 9, Line 19-20, authors needs to describe at least some of the genes validated by qRT-PCR

- Page 10, Line 20, please explain what these results show?

- Similarly Page 12, Line 17, what you want to show by these results?

Reviewer #2: The manuscript entitled “Comparative study on cytogenetics and transcriptome between diploid and autotetraploid rice hybrids harboring double neutral genes” is focused on the role of neutral genes in diploid and autotetraploid rice. The mechanism of pollen sterility is very complex and hot issues for rice breeders. Neutral genes are great source to overcome reproductive isolation in indica-japonica hybrids. The results indicated that meiosis-specific and meiosis-related genes, and amino acids and photosynthesis metabolism-related genes contribute to higher yield and pollen fertility in autotetraploid rice hybrid. Overall, the manuscript is well written and executed but there are few issues which should be resolved before final acceptance.

Major Concern

Authors have published an article about double neural genes previously (Wu et al. 2017; Rice). What are major differences between these studies. If any data similar, should cite a reference. How about data submission to NCBI?

Minor concerns

P7L21, P8L1-5, please delete/re-write from the results section.

P8 Authors could add more cytological results, such as images about abnormal chromosome behavior in diploid and autotetraploid rice.

Figure 1 legends need to improve.

Reviewer #3: The authors try to clarify the role of double neutral genes associated with pollen fertility in autotetraploid rice hybrid; however, all results are on the way to the goal and far from the conclusions that the authors claimed in the Discussion. There are several mistakes in the manuscript that authors need to properly addressed. I have following concerns about the manuscript:

Introduction

Page 5 line 2-4: ‘we developed autotetraploid and diploid rice hybrids by crossing T449, …’ Are autotetraploid and diploid rice all crossing T449? I think this is a wrong sentence.

Methods

Page 6 line 22. gene Ontology should be Gene Ontology

Results

Page 8 line 1-7: This paragraph is almost the introduction.

Page 8 line 8-9: Please re-phrase.

Page 8 line 11: I cannot see the difference in pollen fertility between the materials in figure 1 clearly. The seed set of the materials should be present in the manuscript.

Page 8 line 15-21: How many PMCs at each stage during meiosis the authors observed? Why are there no figures of the abnormal chromosome behavior in DF1 and TF1?

Page 8 line 19: “…was lower than its parents in the metaphase I, …” I could only found T449 in the figure 2, where are the data of T431, T435 and T438 during meiosis?

Page 9 line 2: Lower pollen fertility of diploid hybrid (~70%) was found, the seed set of diploid hybrid was similar to the parents (90%). The pollen fertility of TF1 was similar to DF1, but the seed set is lower than diploid hybrid. How to explain it? What’s the connection of Double neutral genes in TF1 and DF1? What’s the purpose of this analysis? To evaluate the heterosis of autotetraploid hybrid and diploid hybrid, or ploidy analysis between TF1 and DF1, or double neutral genes analysis, it makes me very confusing.

Page 9 line 17: the table S4 should be present in the figure.

Page 9 line 18: Figure S4, the authors randomly selected 10 genes for qPCR assay, why all these genes showed up-regulation. How about the down-regulated genes?

Discussion

The discussion is not so well written. How to get the conclusion of the first subhead in the discussion? Is this only base on hybrid vigour? “But the frequency of chromosome behavior of the diploid rice hybrids carrying San and Sbn neutral genes and its parents were similar” How to get this result? Where are the statistical results of this part? I CAN’T FIND IN THE RESULTS SECTION.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[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.]

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PONE-D-20-14708R1

Comparative study on cytogenetics and transcriptome between diploid and autotetraploid rice hybrids harboring double neutral genes

PLOS ONE

Dear Dr. Shahid,

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.

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

I have received comments on your revised version of your manuscript. You are kindly requested to address some of minor comments by one of the reviewers. Overall the article seems much improved. You can kindly requested to return the manuscript within a week after the minor changes are adhered to it.

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

Please submit your revised manuscript by Oct 12 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,

Allah Bakhsh

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)

Reviewer #2: All comments have been addressed

Reviewer #3: 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

Reviewer #3: Yes

**********

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

Reviewer #1: (No Response)

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 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

Reviewer #3: 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: (No Response)

Reviewer #2: Yes

Reviewer #3: 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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The authors have revised the manuscript carefully according to reviewers’ suggestion. The authors have added new table and figures, revised sentences in the results section, and figures and figure legends. So, readers can follow the contents easily and obtain very useful information from the paper. This manuscript could be accepted for publication after being incorporated few minor suggestions as follow.

1. In Plant material: It is mentioned that “E249, and its autotetraploid rice line, T449, harboring San and Sbn”. The authors need to write the origin of these materials in detail.

2. Page 27. Bar=100um maybe Bar=100µm.

3. Please cross check all the references carefully.

4. Please be consistent with line name, E24-2x could be change as E24

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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: No

Reviewer #2: No

Reviewer #3: No

[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.

Comparative study on cytogenetics and transcriptome between diploid and autotetraploid rice hybrids harboring double neutral genes

PONE-D-20-14708R2

Dear Dr. Shahid,

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,

Allah Bakhsh

Academic Editor

PLOS ONE

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