Uncovering anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja) using RNA-seq

Luis Miguel Riveros-Loaiza; Nicolás Benhur-Cardona; Liliana Lopez-Kleine; Johana Carolina Soto-Sedano; Andrés Mauricio Pinzón; Teresa Mosquera-Vásquez; Federico Roda

doi:10.1371/journal.pone.0273982

Peer Review History

Original SubmissionJune 9, 2022
18 Jul 2022 Decision Letter - Pankaj Bhardwaj, Editor PONE-D-22-16485The contribution of gene expression to anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja)PLOS ONE Dear Dr. Roda, 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 Sep 01 2022 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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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly 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: Yes 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: Yes Reviewer #2: Yes ****** 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: Differential expression between potatoes with different colorations were analyzed and genes related to the flavonoid-anthocyanin biosynthetic pathway were identified by authors. It’s important for the genetic understanding of anthocyanin pigmentation in potato. The manuscript, however, should be revised with more concise words. Other Comments for Manuscript: 1. Line 69-80, It would be better to be concise. 2. Line 85-86, “RGCCA” instead of “Regularized and Sparse Gener-alized Canonical Correlation Analysis (RGCCA)”. 4. Line 94, “shown in shown in the top left”, twice? In Fig 1.tiff, Smaller font of the name of the potato accessions would be better. 5. Line 141, Significant level for p-values is 0.01 or 0.05? 6. Line 227, In Fig 5, it would be better to scale in the X axis for each chromosome individually, instead of in an accumulated scale for all 12 chromosomes. 7. Line 344, “were likely responsible for” instead of “where likely responsible for”? 8. Line 375-397, The two sections, “Selection of material to be sequenced” and “RNA extraction and sequencing”, could be merged. Anyway, the sequencing methods could be described in a brief way. 9. Line 383, 37 (even 94) accessions are not a large number of accessions, considering the sequencing cost. How the statistical analyses support “one transcriptome per accession”? 10. The genetic backgrounds of 37 landraces are different, even though all these landraces belong to Phureja group. Did the difference interfere the Differential Expression Analyses? It should be discussed in the manuscript. 11. The quality of the written English is not satisfying. There are spelling and vocabulary mistakes needing corrections in the manuscript. Such as “an-thocyanin” in abstract. References in the text need to be enclose in brackets, such as line 378. 12. There are two names of the MYB gene (Soltu. DM.10G020850), StAN1 and StAN2. 13.The differential genes analyzed were not verified by any experiments, such as qPCR and dual luciferase reporter assay. 14. The WRKY13 gene in this manuscript has been reported and should be included in the discussion. StWRKY13 promotes anthocyanin biosynthesis in potato (Solanum tuberosum) tubers. Functional Plant Biology, 2022, 49(1):102-114 14. “our results are summarized in Fig 4” in line 167 should be deleted. Reviewer #2: The manuscript “The contribution of gene expression to anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja)” is well presented. This can be accepted after addressing minor comments as below: Title: Modify it, something like: ‘Uncover anthocyanin diversity in potato landraces applying RNA-seq.......approaches’ Abstract: Line 26, modify it, or mention first and second crop. Since there are several other vegetables rich in antioxidants like tomato etc. Introduction: Line 40-41, modify, not good sentence. Modify from L40 to 64, in a story format. Its a bit scattered. Results: L89-90: Besides two, mention the correlation between phylogeny and anthocyanin production as well. L102: (ie spots) ?? Correct ‘i.e.’ spots Check for typo and grammatical errors in throughout the manuscript. ****** 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: Jagesh Kumar Tiwari ******** [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.0273982.r001
Revision 1
12 Aug 2022 Author Response Detailed responses to reviewer # 1 comments: Other Comments for Manuscript: 1. Line 69-80, It would be better to be concise. We made this section more concise. 2. Line 85-86, “RGCCA” instead of “Regularized and Sparse Gener-alized Canonical Correlation Analysis (RGCCA)”. We thank the reviewer for the correction and implemented the suggested change (line 83-85). 4. Line 94, “shown in shown in the top left”, twice? We thank the reviewer for identified the duplication in the text and corrected it (line 90). In Fig 1.tiff, Smaller font of the name of the potato accessions would be better. We thank the suggestion. Unfortunately, we consider that if the font was further reduced it would not be visible. 5. Line 141, Significant level for p-values is 0.01 or 0.05? The significant level for p-value is 0,1. thank you for noticing this. We have fixed this error in the manuscript (line 121). 6. Line 227, In Fig 5, it would be better to scale in the X axis for each chromosome individually, instead of in an accumulated scale for all 12 chromosomes. We appreciate the suggestion but respectfully disagree because we think that the suggested change would add complexity to Fig 5. Therefore, we did not implement this change. 7. Line 344, “were likely responsible for” instead of “where likely responsible for”? We made the change and apologize for the error (line 342). 8. Line 375-397, The two sections, “Selection of material to be sequenced” and “RNA extraction and sequencing”, could be merged. Anyway, the sequencing methods could be described in a brief way. We implemented the suggested change. 9. Line 383, 37 (even 94) accessions are not a large number of accessions, considering the sequencing cost. How the statistical analyses support “one transcriptome per accession”? We are aware that it would have been ideal to have replicates per genotype and a larger number of sampled accession but we the statistical tests that we perform are robust to these caveats and allow us to address our research questions. Specifically, we conducted differential expression analyses using different accessions of the same coloration as biological replicates (at least 9 replicates per category). Additionally, we conducted correlation and co-expression analyses, which do not require replication per accession and work appropriately with 37 data points. We believe that this is a correct strategy to identify genes that show similar patterns across different accessions with the similar anthocyanin content, which is the goal of our study. Our goal is not to characterize or compare specific accessions and therefore we do not require replication per accession. We also believe that 37 accessions is a sufficiently big population to study the diversity of gene expression and anthocyanin content. Finally, as shown in the manuscript, most of the genes identified in our analyses have previous anthocyanin annotations, which confirms that or strategy is appropriate to identify anthocyanin genes. We clarify this in the methods section (line 389-394). 10. The genetic backgrounds of 37 landraces are different, even though all these landraces belong to Phureja group. Did the difference interfere the Differential Expression Analyses? It should be discussed in the manuscript. When we selected accessions with similar coloration patterns, we selected representative accessions in the different phylogenetic groups (Supplementary Figures 1 and 3). In this way we minimized the effect of population structure in our analyses. This was clarified in the corrected version of the manuscript (line 374-378). 11. The quality of the written English is not satisfying. There are spelling and vocabulary mistakes needing corrections in the manuscript. Such as “an-thocyanin” in abstract. References in the text need to be enclosed in brackets, such as line 378. Following the reviewer suggestion, we improved readability and corrected typos and other errors. We apologize for the typos and language errors present in the previous version of the manuscript. In this version of the manuscript, we carefully proofread the text and corrected all the errors highlighted by the editor. 12. There are two names of the MYB gene (Soltu. DM.10G020850), StAN1 and StAN2. The code: Soltu. DM.10G020850 is just for StAN2 not for StAN1. We corrected this error. 13.The differential genes analyzed were not verified by any experiments, such as qPCR and dual luciferase reporter assay. The goal of this study was to identify genes that showed population-level correlations between expression and anthocyanin content. Most of the candidate genes identified have already been associated to anthocyanin production in previous studies of potato cultivars. In the future we plan to validate some of the identified candidates with transgenesis and qPCR however we believe it is beyond the scope of this manuscript. 14. The WRKY13 gene in this manuscript has been reported and should be included in the discussion. StWRKY13 promotes anthocyanin biosynthesis in potato (Solanum tuberosum) tubers. Functional Plant Biology, 2022, 49(1):102-114 We appreciate this suggestion and included the suggested reference (line 609-610). 15. “our results are summarized in Fig 4” in line 167 should be deleted. We were grateful for this recommendation and we removed this section from the paragraph (line 169). Detailed responses to reviewer # 2 comments: Reviewer #2: The manuscript “The contribution of gene expression to anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja)” is well presented. This can be accepted after addressing minor comments as below: Title: Modify it, something like: ‘Uncover anthocyanin diversity in potato landraces applying RNA-seq.......approaches’ The title was changed to “Uncovering anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja) using RNA-seq”. Abstract: Line 26, modify it, or mention first and second crop. Since there are several other vegetables rich in antioxidants like tomato etc. The three crops that provide the highest proportion of antioxidants in the human diet are maize, tomato and potato. We have made the respective change in the manuscript. Introduction: Line 40-41, modify, not good sentence. Modify from L40 to 64, in a story format. Its a bit scattered. We have made the requested change and those paragraphs of the manuscript have been re-written (line 39-63). Results: L89-90: Besides two, mention the correlation between phylogeny and anthocyanin production as well. We are grateful for this recommendation and made the suggested correction (line 88-89). L102: (ie spots) ?? Correct ‘i.e.’ spots We were grateful for this recommendation and we made this correction (line 101). Check for typo and grammatical errors in throughout the manuscript. Following the reviewer suggestion, we improved readability and corrected typos and other errors. We apologize for the typos and language errors present in the previous version of the manuscript. In this version of the manuscript, we carefully proofread the text and corrected all the errors highlighted by the editor. Attachments Attachment Submitted filename: Response_to_Reviewers_PONE-D-22-16485.docx https://doi.org/10.1371/journal.pone.0273982.r002
19 Aug 2022 Decision Letter - Pankaj Bhardwaj, Editor Uncovering anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja) using RNA-seq PONE-D-22-16485R1 Dear Dr. Roda, 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, Pankaj Bhardwaj, 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 #1: All comments have been addressed Reviewer #2: 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: The The author answered all the comments. I think this vision is appropriate for publication. I have no more comment. Reviewer #2: Maybe accepted for publication. Authors have addressed all the comments. Hence the manuscript may be accepted. ****** 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: Yes: Jagesh Kumar Tiwari ******** https://doi.org/10.1371/journal.pone.0273982.r003
Formally Accepted
30 Aug 2022 Acceptance Letter - Pankaj Bhardwaj, Editor PONE-D-22-16485R1 Uncovering anthocyanin diversity in potato landraces (Solanum tuberosum L. Phureja) using RNA-seq Dear Dr. Roda: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Pankaj Bhardwaj Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273982.r004

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