Gene co-expression network analysis to identify critical modules and candidate genes of drought-resistance in wheat

Liangjie Lv; Wenying Zhang; Lijing Sun; Aiju Zhao; Yingjun Zhang; Limei Wang; Yuping Liu; Ziqian Li; Hui Li; Xiyong Chen

doi:10.1371/journal.pone.0236186

Peer Review History

Original SubmissionApril 30, 2020
27 May 2020 Decision Letter - Wujun Ma, Editor PONE-D-20-08497 Gene co-expression network analysis to identify critical modules and candidate genes of drought-resistant in wheat PLOS ONE Dear Dr. chen, 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 seek professional proofreading to the manuscript; Address all concerns raised by the two reviewer one by one; Please submit your revised manuscript by Jul 11 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, Wujun Ma Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In your Methods section, please provide additional details regarding the plant materials used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials. 3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free. Upon resubmission, please provide the following: The name of the colleague or the details of the professional service that edited your manuscript A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a supporting information file) A clean copy of the edited manuscript (uploaded as the new manuscript file) 4. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. 5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 6. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 1 and 2 in your text; if accepted, production will need this reference to link the reader to the Table. [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? 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 ******** 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: No Reviewer #2: Yes ****** 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: No ****** 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: Drought stress is an important limiting factor in wheat production. Lv et al analyzed differiential expression genes of wheat between drought stress and normal growth conditions by means of RNA-seq and gene co-expression network analyses. They detected three modules and 26 twelve hub genes that are associated with drought resistance mechanisms, and newly identified five candidant genes for drought resistance. The study is interesting. However, several main issues need be addressed, and the manuscript need to be carefully revised due to many underexpressing stentences and many wrongtypos/grammars. For examples, 1 DESCRIBE the treated samples at which growth stages, which tissues in Material, and RESULTS parts and Figure1. Figure1 should explain themeanings of C vs A, CR VS DR, ... 2 Title ,drought-resistant should be corrected to drought-resistance 3. In Abstract and result parts, in the "genes involved in the modules, such as dark turquoise, yellow and brown, were found", dark turquoise, yellow and brown should be explained by expert names, such as detailed genes/pathways. 4. In Abstract, Twelve central, greatly correlated genes in stage-specific modules were subsequently confirmed and validated at the transcription levels, including TraesCS7D01G417600.1, TraesCS5B01G565300.1, TraesCS4A01G068200.1, TraesCS2D01G033200.1, TraesCS6B01G425300.1, TraesCS7A01G499200.1, TraesCS4A01G118400.1, TraesCS2B01G415500.1, TraesCS1A01G129300.1, TraesCS2D01G326900.1, TraesCS3D01G227400.1 and TraesCS3B01G144800.1, the genes should be written with the gene name, such as PP2C, ERF1B... 5. Lines 26-27, and results, what are five of the genes newly identified for drought resistance in the study? How do you verify their functions in drought resistance? 6. The MS English writing must be carefully improved and throughtly edited by a native English speaking expert. 7. Each figure, SHOULD BE NUMBERED. Reviewer #2: Major issues 1. The English language needs professional proofreading as there are massive typos and grammar errors in the main document. 2.A short introduction referring to the current understanding of drought-resistant in wheat is suggested. 3. A total of twelve genes (TraesCS7D01G417600.1, TraesCS5B01G565300.1, TraesCS4A01G068200.1, TraesCS2D01G033200.1, TraesCS6B01G425300.1, TraesCS7A01G499200.1, TraesCS4A01G118400.1, TraesCS2B01G415500.1, TraesCS1A01G129300.1, TraesCS2D01G326900.1, TraesCS3D01G227400.1 and TraesCS3B01G144800.1) were validated at the transcription levels, why? Minor issues 1. Please remove the background in the text. For example, Line 61-70; Line79-101, et al. 2. What is the “follow-up test material”(Line105)? 3. line 110, punctuation missing in the sentence. 4. line 108-109, “During the growth of wheat (the roots, leaves, and crowns), the material was collected from 6 separate plants tissue from each material and pooled for RNA extraction” could be change into “The roots, leaves, and crowns were collected from 6 separate plants from each material and pooled for RNA extraction” 5.Line 150, 2-ΔΔCT should be 2-ΔΔCT. ****** 6. 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: Yes: Rugen Xu [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. https://doi.org/10.1371/journal.pone.0236186.r001
Revision 1
24 Jun 2020 Author Response Referee #1: We want to begin by thanking Referee #1 for writing that “Drought stress is an important limiting factor in wheat production. The study is interesting.” We also appreciated the constructive criticism and suggestion. We addressed all the points raised by the reviewer, as summarized below. 1. Describe the treated samples at which growth stages, which tissues in Material, and Figure1 should explain the meanings of C vs A, CR VS DR, ... (in results). As the reviewer suggested, in the material method section, we added a description of the growth stage and tissue of the treated samples and added the meaning interpretation of C vs A, CR vs DR… in the legend of Figure 1 on the result section. 2. Thanks to the referee’s comment, we have changed the “drought-resistant“ to “drought-resistance“ in the title. 3. In Abstract and result parts, in the "genes involved in the modules, such as dark turquoise, yellow and brown, were found", dark turquoise, yellow and brown should be explained by expert names, such as detailed genes/pathways. Thank you very much for your comments. In the abstract and results section, the module's names were described in dark turquoise, yellow and brown ... This is because when WGCNA is used for gene clustering analysis, genes that are functionally related or similar are grouped and represented by the same color. The same color module contains many genes, which cannot be enriched into a single pathway or explained by expert names. So only colors can be used to describe modules. 4. In Abstract, Twelve central, greatly correlated genes in stage-specific modules were subsequently confirmed and validated at the transcription levels, including TraesCS7D01G417600.1, TraesCS5B01G565300.1, TraesCS4A01G068200.1, TraesCS2D01G033200.1, TraesCS6B01G425300.1, TraesCS7A01G499200.1, TraesCS4A01G118400.1, TraesCS2B01G415500.1, TraesCS1A01G129300.1, TraesCS2D01G326900.1, TraesCS3D01G227400.1 and TraesCS3B01G144800.1, the genes should be written with the gene name, such as PP2C, ERF1B... As the reviewer suggested, We looked up the names of these genes and added them in parentheses. 5. Lines 26-27, and results, what are five of the genes newly identified for drought resistance in the study? How do you verify their functions in drought resistance? Referee #2 asks what are five of the genes and how to verify….. Lines 351-353, we added the specific names of these five genes to the paper. We treated wheat Jimai 418 in the seedling stage with drought (20%PEG) for a different time and extracted RNA for RT-PCR verification. It was found that the expression levels of these five genes were significantly different under drought stress, which was speculated to be related to response to drought stress. We would like to note that we investigated the five genes in the previous study and found no reported that the five specific genes were associated with drought resistance in wheat. Therefore, in this manuscript, we focused on these five genes and will carry out transgenic verification in subsequent experiments. 6. The MS English writing must be carefully improved and thoroughly edited by a native English speaking expert. As suggested by the reviewer we have invited professional scientific editing service (peerwith) and native expert (Ally Oakes) to polish the language of the article. Please see the attachment for the editing certificate. Our colleagues studying abroad have also made careful improvements to the English expressions throughout the manuscript. All changes have been revised in the manuscript with track changes. 7. Because of the referee’s comment, we have numbered the pictures in the manuscript. Referee #2: We would like to thank the referees for their thoughtful review of our manuscript. We believe that the additional changes we have made in response to the reviewer's comments have made this a significantly stronger manuscript. Below is our point-by-point response to the referee’s comments. Major issues 1. The English language needs professional proofreading as there are massive typos and grammar errors in the main document. The language of the article has been polished by professional scientific editing service (peerwith) and native expert (Ally Oakes). Please see the attachment for the editing certificate. Our colleagues studying abroad have also made careful improvements to the English expressions throughout the manuscript. All changes have been revised in the manuscript with track changes. 2. A short introduction referring to the current understanding of drought-resistant in wheat is suggested. According to the referee’s suggestion, we added a brief introduction to the current understanding of drought-resistant in wheat at the end of the first paragraph. 3. A total of twelve genes (TraesCS7D01G417600.1, TraesCS5B01G565300.1, TraesCS4A01G068200.1, TraesCS2D01G033200.1, TraesCS6B01G425300.1, TraesCS7A01G499200.1, TraesCS4A01G118400.1, TraesCS2B01G415500.1, TraesCS1A01G129300.1, TraesCS2D01G326900.1, TraesCS3D01G227400.1 and TraesCS3B01G144800.1) were validated at the transcription levels, why? According to the k-mean diagram of 9 modules determined in this paper, the drought-treated tissues and developmental stages were compared with the transcriptional results, and eight modules with consistent results were obtained for analysis. The 38 genes with the highest correlation degree were selected by using the protein interaction diagram. After consulting the gene function and referring to previous literature, we speculated that these 12 genes might be related to drought. Minor issues 1. Thanks to the referee’s comment, the wrong background have removed in the text. 2. Line105, the “follow-up test material” was changed to “test material”. 3. Line 110, to better understand we have punctuated in this sentence. 4. lines 108-109, we agree with the reviewer's comment and changed to “The samples were collected from 3 separate plants and pooled for RNA extraction.” 5. Line 150, according to the referee’s suggestion, 2-ΔΔCT was changed to 2-ΔΔCT. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0236186.r002
1 Jul 2020 Decision Letter - Wujun Ma, Editor Gene co-expression network analysis to identify critical modules and candidate genes of drought-resistant in wheat PONE-D-20-08497R1 Dear Dr. chen, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Wujun Ma Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0236186.r003

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .