PONE-D-24-56047Fingerprinting and chemotyping approaches reveal a wide genetic and metabolic diversity among wild hops (Humulus lupulus L.).PLOS ONE

Dear Dr. Hehn,

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 Mar 30 2025 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: https://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,

Suman S. Thakur, Ph.D

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 information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

Additional Editor Comments:

Major Revision

[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: No

Reviewer #2: Yes

**********

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

Reviewer #1: No

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: The authors investigated the genetic and metabolic diversity of 36 varieties of hops in France and evaluated the molecular characterization using 16 SSR markers. Finally, they highlighted that WRKY 224 was molecular determinates for metabolic production. However, this study is a preliminary analysis of genetic diversity of hops, lacking in-depth analysis. The two major concerns are the sample selection and gene identification, which need more detail biological evidence. Here are some comments.

1.Introduction: This section needs to be more scientificity, shortening the history of hops and focusing on the genetic diversity and molecular marker analysis of wild varieties of hops. Since the review of molecular markers development is too general, which make it difficult to get the recent progress.

2.Materials and Methods: The samples selected in this paper looks randomly, and the reason why you choose these materials should be charify. Are these materials representing the natural distribution, just a regional distribution, or varieties widely used in production? All of these will affect the results of this study.

Line 317: How did you choose these 16 EST-SSR markers?

Line 345: This results should be analyzed further and more data should be provided in this section. For example, the original data of MRT should be provided. Why did the residual error of 0.606 represent the R2 of 39.6%? There was a statistical gap in this section. Additionally, how many genes involved in the metabolic differentations, and the reasons why you choose the two genes lacked the genetic evidence.

Line 453: Why did the authors point out the light conditions?

Reviewer #2: The present manuscript builds on the hypothesis that hop leaves may provide a means to distinguish wild hops based on their specialized metabolic profile. The selected metabolites include polyketide-derived prenylated phenolics, such as alpha and beta acids (hulupone and lupulone derivatives, respectively), which contribute to bitterness, and a prenylated flavonoid (a humol derivative), which contributes to antioxidant properties. The study is based on the metabolic and genetic examination of 36 wild hop accessions collected in the vicinity of Nancy, France, from various ecological habitats, such as forest edges, hedges, riparian zones, and field margins. The samples were grown and propagated under uniform conditions to enable a direct comparison of their chemical profiles. In addition, genomic DNA was analyzed using 16 hop-specific microsatellite marker loci to study genetic diversity and classify the accessions relative to global genetic data.

The metabolic profiles varied in intensity across twelve targeted metabolic identifiers: cohulupone, hulupinic acid, hulupone, adhulupone, xanthohumol, cohumulone, humulone + adhumulone, desoxyhumulone, postlupulone, lupulone E, colupulone, and lupulone + adlupulone. Although the authors conclude that the accessions group into three metabolic profiles, the data suggest that these profiles are distinguished by intensity rather than by compositional differences. Cluster analyses suggest that some WRKY enzymes could be responsible for this variation, potentially offering insight into metabolic regulation rather than differences in biosynthetic pathways. While this is briefly discussed in the conclusion, it could benefit from greater emphasis in the main text.

The paper also places the collected hop population into a broader context by incorporating global genetic data on hops, as described in the methods section. As expected, the accessions clustered within the European genetic group, and the authors conclude that the accessions likely originated from a single naturalization event. It would have been interesting if the authors had included hops from other parts of the world as out-groups, which might have provided additional insights into novel aspects of the metabolism of polyketide-derived prenylated phenolics in hops.

The manuscript is well prepared, and the analyses are robust and clearly presented. However, it appears that some of the 36 accessions are not included in every global comparison. It would have been beneficial to see all accessions consistently included in all phylogenetic analyses.

The discussion is highly relevant and well-aligned with the results. If anything, the term “chemical grouping” used to describe the division among accessions in Figures 1 and 2 might be reconsidered. These figures suggest variations in the intensity of highly similar metabolic profiles, with some accessions being low producers, others having elevated intensity, and one (Genotype 3) being a strong general producer of the compounds of interest. Terms such as “metabolic activity” or “intensity groupings” may be more appropriate descriptors.

**********

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:  Benedicte Riber Albrectsen

**********

[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.

Fingerprinting and chemotyping approaches reveal a wide genetic and metabolic diversity among wild hops (Humulus lupulus L.).

PONE-D-24-56047R1

Dear Dr. Hehn,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Suman S. Thakur, 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 #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 #2: Yes

**********

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

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 #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 #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 #2: I just had a few points to address in my initial review, and my comments on sample sizes have been appropriately addressed. ON line 245, I noticed that the manuscript currently uses ‘These method,’ which seems incorrect. Please revisit this phrase and adjust it to either ‘This method’ or ‘These methods,’ depending on your intended meaning.

**********

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 #2: No

**********