PONE-D-21-09114

Genome-wide identification of the COBRA-like gene family in rapeseed (Brassica napus L.)

PLOS ONE

Dear Dr. Liu,

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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Kun Lu, Ph.D.

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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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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

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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 manuscript, the authors conducted a genome-wide analysis of the COBRA-like gene family in rapeseed. They also performed phylogenetic analysis, cis-element, and gene expression analysis. However, I do have some concerns before its final acceptance.

1. In phylogenetic analysis COBLs, authors had done analysis using protein sequences from Brassica and A. thaliana only. They can draw a better picture/results if they use more species for phylogenetic analysis.

2. Why authors randomly selected the five genes out of 44 for Q-RT PCR analysis? In my opinion, there should be a criterion/logic for selection. Like, (a) selection based on their subclass division; phylogenetic based, (b) selection of genes based on their in-silico expression results.

3. “BnaCOBLs were involved in stem lodging resistance” based on these experiments, it overestimated results. In my opinion, some more experiments should be conducted to further functional validations of results to meet the journal standards. Functional validation of genes using transgenic approach/VIGS/CRISPAR.

4. It is also suggested that authors should improve the discussion section. They have to discuss their results and compare them with some earlier and recently published papers in more depth and clarity.

5. Please provide the Pfam ID used for COBL gene identification.

6. Detail of BnaActin gene used in this study. i.e., NCBI ID, or any other database ID should be provided.

7. How many biological replicates of each tissue were used for qRT-PCR analysis, since it is not clear from the martial and methods section. Please clear?

8. How they named COBL genes? Please provide details.

Reviewer #2: In this paper, using the method of biological information, 44 COBRA-like genes were identified in rapeseed. Then, the phylogenetic relationships, the chromosome location, the domain composition, and the putative cis element distribution of BnaCOBLs were determined. Different expression levels in stems with distinct lodging resistance suggested that there was some association between BnaCOBLs and stem lodging resistance. The logic of the article is very clear, and the scientific issues of concern are also well explained. But there are still several issues that need attention:

(1) In line 127, when determining the promoter, you should not only use the length as the standard, but should combine some basic characteristics of the promoter, such as the -10 region and the -35 region. Because the promoter may exist in the last gene, or the two genes mentioned in your article share the promoter. The sequence of the promoter directly affects the results of the subsequent analysis. For example, the speculation of gene function in line 445 may be biased.

(2) Line 117. Why you choosed 10 motifs and motif width of 8-50 amino-acid residues as this papaer's parameters. The basis should be pointed out here.

(3) In the discussion section, when the article directly quoted the content of the article results, the corresponding picture number was not marked. I think it will be easier for readers to read this article by marking the corresponding picture numbers.

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Reviewer #1: No

Reviewer #2: Yes: Guanghui Xiao

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Attachments

Attachment

Submitted filename: WHQ comments.docx

PONE-D-21-09114R1

Genome-wide identification and expression profiling of the COBRA-like genes reveal likely roles in stem strength in rapeseed (Brassica napusL.)

PLOS ONE

Dear Dr. Liu,

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 10 2021 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. 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,

Kun Lu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments (if provided):

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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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yang et al., used bioinformatics method to analyze the COBL gene family in rapeseed. The results are interesting, and may have potential guiding significance for lodging resistance improvement. The major comments are:

1. Whether the animal (like mouse or human) has COBL homeologues, if has, please introduce the gene function of COBL genes in Introduction.

2. The authors are better to give the uniform naming in B. napus, B. rapa and B. oleracea based on Fig. 1 result, and combine the Table S2 and S3 together. Otherwise the readers hard to compare the same homeologue in different species.

3. Based on the protein location predication result, the BnaCOBL10 and BnaCOBL42 were not on cell membrane, while these two genes were in the same subgroup (Fig. 2). The authors may discuss the possibility of the differentiation of gene function in Discussion.

4. The authors need to check the italics of the gene name fully (e.g. Line 37, 44, 56, 57, 79, 141, 223, 226, 229, 230 etc.)

Reviewer #4: In this manuscript, 44 COBRA-like genes were identified in rapeseed by sequence identity and conservative COBRA domain. The author characterized these genes on phylogenetic grouping, the chromosome location, the domains, and the putative cis-element distribution in promoters of BnaCOBLs. The expression profiles and the protein domains of AtCOB and AtCOBL4 subgroup imply these AtCOB subgroup members may be involved in stem development and stem breaking resistance of rapeseed, rather than members in AtCOBL4 subgroup, which are functional in crops of grass family. The logic of the article is clear, and the scientific issues of concern are also well explained. But I think authors should address following points in this manuscript before publishing in “ PLOS ONE ”:

1. The conclusion in line 79 to 80 seemed to be expanded according to the results and discusses.

2. As the topology of the phylogenetic tree (Fig 1), BnaCOBL5 and BnaCOBL44 did not belong to any subgroup orthologs from AtCOBLs. Where they evolved from? Maybe these two genes are not the members of COBL family in rapeseed.

3. The alternative splicing of subgroup AtCOBL4 presented in the Result section should be supported by figures.

4. Why the members in AtCOBL4 subgroup with incomplete protein still are expressed in stems at a certain level?

5. In line 19 in the Abstract section, whether the expansion of COBL family in B.napus was attributable to ‘whole-genome duplication’ or ‘whole-genome triplication’ as written in line 423?

6. Some syntax and formatting errors such as, in line 243, ‘The kind of gene pair was’ should revise as ‘This kind of gene pairs were’ in this context; in Table 1, the ‘p-value’ in line where the ‘Gene Name’ is ‘BnaCOBL20’, does not line up with other elements in the same line.

7. Add some correlation descriptions between the genes (such as BnaCOBL9, BnaCOBL35 and BnaCOBL24 etc.) expression change and stem strength in results.

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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 #3: No

Reviewer #4: 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.

Genome-wide identification and expression profiling of the COBRA-like  genes reveal likely roles in stem strength in rapeseed (Brassica napus L.)

PONE-D-21-09114R2

Dear Dr. Liu,

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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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,

Kun Lu, Ph.D.

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 #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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 #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: 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 #3: Yang et al. addressed all the comments, and revised the manuscript fully. The manuscript is matched the publication criteria of PLOS ONE right now.

Reviewer #4: This paper is focused on identification and expression profiling of the COBRA-like  genes reveal likely roles in stem strength in rapeseed, it is well organized and its presentation is good. I think it meets the current publication criteria of PLOS ONE.

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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 #3: No

Reviewer #4: No