Estimation of the heritability of medicinal compound contents in Glycyrrhiza uralensis

Takahiro Tsusaka; Miki Sakurai

doi:10.1371/journal.pone.0327885

Peer Review History

Original SubmissionFebruary 27, 2025
27 May 2025 Decision Letter - Mehdi Rahimi, Editor PONE-D-25-10815Evaluation of genetic inheritance of the medicinal compound contents in Glycyrrhiza uralensisPLOS ONE Dear Dr. Tsusaka, 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 Jul 11 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. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have carried quite detailed amount of work and carried out large experimentation on the genetic inheritance of the contents of seven medicinal compounds in G. uralensis and observed high heritability for the contents of glycyrrhizin, liquiritin, liquiritin apioside, isoliquiritin, and isoliquiritin apioside. This suggests that selective breeding could be an effective strategyfor developing G. uralensis populations with high medicinal compound contents. G. uralensis cultivars with both high root yields and high medicinal compound contents could be developed and utilzed for commercial application. It is advised that kindly highlight the importance of these metabolites in the available herbal drugs or any other formulations used in their market so that in the future course of time,those newly breeding lines could be grown. Reviewer #2: This manuscript by Takahiro Tsusaka and Miki Sakurai described that they investigated the genetic inheritance (heritability) of medicinal compound contents in G. uralensis by analyzing clonal lines propagated from stolons. And they conclude that there is strong potential for selective breeding to enhance the medicinal quality of G. uralensis by targeting high-yielding genotypes with superior compound profiles and that this potential may also apply to the sustainable cultivation of other high-quality medicinal plant resources. The work is interesting. But there are still some parts need to be enhanced. 1. In the parts of plant materials and cultivation, the relationship among the materials used in the different experiments is not clear enough, especially when comparing the status of annual and biennial G. uralensis (Is the annual liquorice material a subset of the biennial? ), and could be supplemented with a designed figure or table to illustrate this issue? 2. In the parts of plant materials and cultivation, Please provide the latitude and longitude information of the experimental site. 3. Sample size varies considerably among the clonal lines, from 3 to 9, and it is recommended that the data be supplemented to a sample size of 5 or more. 4. Genetic analysis using clonal lines requires sufficient genetic distance among different lines. However, the 26 clonal lines in this study came from the same batch of seeds, and how about the genetic distance between them? So experimental evidence of how genetically distant they are from each other is requested. 5. The data for the primary root in Figure 4, ‘Correlation of primary root and adventitious root components’, were measured only once (n=1). It is recommended to add repeated measurements of primary roots or to justify the single measurement. 6. Figure 1 (compound structure) is not cited in the main text. It is recommended that it be mentioned in the methods or results section or that Figure 1 be removed from the text. ****** 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: 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". 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Revision 1
17 Jun 2025 Author Response Dear Reviewers Thank you for the thoughtful and constructive feedback you provided regarding our manuscript. We agree with you and have incorporated these suggestions throughout our paper. Detailed responses on your suggestions are as follows: Reviewer #1’s comments Reviewer #1: The authors have carried quite detailed amount of work and carried out large experimentation on the genetic inheritance of the contents of seven medicinal compounds in G. uralensis and observed high heritability for the contents of glycyrrhizin, liquiritin, liquiritin apioside, isoliquiritin, and isoliquiritin apioside. This suggests that selective breeding could be an effective strategy for developing G. uralensis populations with high medicinal compound contents. G. uralensis cultivars with both high root yields and high medicinal compound contents could be developed and utilzed for commercial application. It is advised that kindly highlight the importance of these metabolites in the available herbal drugs or any other formulations used in their market so that in the future course of time, those newly breeding lines could be grown. Response to Reviewer #1 Thank you very much for your constructive feedback on our manuscript. We fully agree with your suggestion. In response, we have added a description of the industrial importance of the medicinal compounds in lines 422–425 of the Discussion section. Reviewer #2’s comments Reviewer #2: This manuscript by Takahiro Tsusaka and Miki Sakurai described that they investigated the genetic inheritance (heritability) of medicinal compound contents in G. uralensis by analyzing clonal lines propagated from stolons. And they conclude that there is strong potential for selective breeding to enhance the medicinal quality of G. uralensis by targeting high-yielding genotypes with superior compound profiles and that this potential may also apply to the sustainable cultivation of other high-quality medicinal plant resources. The work is interesting. But there are still some parts need to be enhanced. 1. In the parts of plant materials and cultivation, the relationship among the materials used in the different experiments is not clear enough, especially when comparing the status of annual and biennial G. uralensis (Is the annual liquorice material a subset of the biennial? ), and could be supplemented with a designed figure or table to illustrate this issue? 2. In the parts of plant materials and cultivation, Please provide the latitude and longitude information of the experimental site. 3. Sample size varies considerably among the clonal lines, from 3 to 9, and it is recommended that the data be supplemented to a sample size of 5 or more. 4. Genetic analysis using clonal lines requires sufficient genetic distance among different lines. However, the 26 clonal lines in this study came from the same batch of seeds, and how about the genetic distance between them? So experimental evidence of how genetically distant they are from each other is requested. 5. The data for the primary root in Figure 4, ‘Correlation of primary root and adventitious root components’, were measured only once (n=1). It is recommended to add repeated measurements of primary roots or to justify the single measurement. 6. Figure 1 (compound structure) is not cited in the main text. It is recommended that it be mentioned in the methods or results section or that Figure 1 be removed from the text. Response to Reviewer #2 Thank you very much for your valuable and insightful comments. We have carefully considered each point and revised the manuscript accordingly. Our detailed point-by-point responses are provided below. Comment 1: In the parts of plant materials and cultivation, the relationship among the materials used in the different experiments is not clear enough, especially when comparing the status of annual and biennial G. uralensis (Is the annual liquorice material a subset of the biennial? ), and could be supplemented with a designed figure or table to illustrate this issue? Response: We have added a schematic figure illustrating the experimental design and the plant materials used in each experiment, which is included in Appendix S1. Comment 2: In the parts of plant materials and cultivation, Please provide the latitude and longitude information of the experimental site. Response: We have added the latitude and longitude information of the experimental site in lines 107–108 of the Materials and Methods section. Comment 3: Sample size varies considerably among the clonal lines, from 3 to 9, and it is recommended that the data be supplemented to a sample size of 5 or more. Response: Due to the difficulty of cultivating G. uralensis, we were unable to maintain uniform replication numbers across all clonal lines. Since the plant requires several years to grow, it is not feasible to increase the number of samples at this stage of the study. However, we recognize that a sample size of five or more is desirable to ensure adequate statistical power for variance analysis. Therefore, we re-estimated the broad-sense heritability values using only the 15 clonal lines with five or more replicates and compared the results to those obtained using all 26 lines with three or more replicates. The heritability estimates based on the 15 lines were as follows: glycyrrhizin 0.69, liquiritin 0.64, liquiritin apioside 0.86, liquiritigenin 0.29, isoliquiritin 0.66, isoliquiritin apioside 0.91, and isoliquiritigenin 0.15. These values were nearly identical to those obtained from the dataset of 26 clonal lines, indicating that the use of lines with three or more replicates still yields reliable results. Accordingly, we have clearly stated this limitation in lines 418–422 of the Discussion section and explained that, based on this consideration, we chose to proceed with the analysis using clonal lines with three or more replicates. Comment 4: Genetic analysis using clonal lines requires sufficient genetic distance among different lines. However, the 26 clonal lines in this study came from the same batch of seeds, and how about the genetic distance between them? So experimental evidence of how genetically distant they are from each other is requested. Response: Since genotyping would require cultivating additional plants, which takes several years, it is difficult to include such data in the current study. To provide some support for the genetic diversity among the clonal lines, we have added a statement in lines 115–116 of the Materials and Methods section noting that the clonal lines were randomly selected from a population of 240 individuals. In addition, we have included Appendix S2, which presents phenotypic variation in compound contents and a clustering analysis based on these traits among the 31 clonal lines used in the experiments. We plan to conduct a genotypic analysis in a subsequent study and report the results in a future publication. Comment 5: The data for the primary root in Figure 4, ‘Correlation of primary root and adventitious root components’, were measured only once (n=1). It is recommended to add repeated measurements of primary roots or to justify the single measurement. Response: Due to the biological characteristics of G. uralensis, which is highly self-incompatible and genetically heterogeneous, it is not possible to obtain genetically identical plants from seeds. As a result, only one main root sample per clonal line was available for analysis, and increasing the number of replicates was not feasible. We have acknowledged this limitation and added a statement in lines 419–420 of the Discussion section to clarify this point. Comment 6: Figure 1 (compound structure) is not cited in the main text. It is recommended that it be mentioned in the methods or results section or that Figure 1 be removed from the text. Response: Thank you for pointing this out. We have added a reference to Figure 1 in lines 175–178 of the Results section to address this issue. We look forward to hearing from you regarding our submission and would be happy to address any further questions or comments you may have. Best regards. Takahiro Tsusaka (TT), Miki Sakurai (MS) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0327885.r002
24 Jun 2025 Decision Letter - Mehdi Rahimi, Editor Evaluation of genetic inheritance of the medicinal compound contents in Glycyrrhiza uralensis PONE-D-25-10815R1 Dear Dr. Tsusaka, 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, Mehdi Rahimi, 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: The authors have adequately addressed my comments raised in a previous round of review and I think that this manuscript can be accepted for publication. ****** 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 ******** https://doi.org/10.1371/journal.pone.0327885.r003
Formally Accepted
Acceptance Letter - Mehdi Rahimi, Editor PONE-D-25-10815R1 PLOS ONE Dear Dr. Tsusaka, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@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 Associate Prof. Mehdi Rahimi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0327885.r004

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