Identification and validation of intergeneric hybrids between Saccharum officinarum and Erianthus rockii using molecular and cytogenetic tools

Gang Wang; Jianqiang Wang; Wei Zhang; Huanying Xu; Qinggan Liang; Yuanxia Qin; Qingdan Wu; Jiantao Wu; Cheng Fu; Feng Zhou; Yuxing An; Qinnan Wang; Hailong Chang

doi:10.1371/journal.pone.0324128

Peer Review History

Original SubmissionDecember 27, 2024
27 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0324128.r001
2 Mar 2025 Decision Letter - Rongchun Han, Editor -->PONE-D-24-60083-->-->Identification and validation of intergeneric hybrids between Saccharum officinarum and Erianthus rockii using molecular and cytogenetic tools-->-->PLOS ONE Dear Dr. Chang, 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. The editor ask the authors to respond only to the comments raised by Reviewer 1. Please submit your revised manuscript by Apr 16 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 . 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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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: The authors express their sincere gratitude to Professor Zuhu Deng from Fujian Agriculture and Forestry University for his invaluable assistance in conducting the GISH assay. This research was funded by GDAS’ Project of Science and Technology Development (2022GDASZH-2022010102), the GDAS’s Project of Technical Innovation and Incubation Service Platform Construction (2021GDASYL 20210301001) and was supported by Provincial scientific research institutions stability support sub-project in 2020 "Breeding and construction of healthy seedling propagation system of new sugarcane varieties (lines)". We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: The author(s) received no specific funding for this work. 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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: 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: 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: Wang and colleagues proposed a combination of molecular techniques, namely tetra-primer Amplification Refractory Mutation System (ARMS)-PCR, High-Resolution Melting (HRM) curve analysis and Genomic In situ Hybridization (GISH) techniques, in validating true hybrids between Saccharum officinarum and Erianthus rockii. SNPs in the full-length ITS were identified and screened for ARMS and HRM verification. The methods are logical, and the results are valid. The authors paid efforts in improving the experimental design to raise the efficiency and convenience in hybrid identification using each molecular tool. Meanwhile, they include the data from traditional method as morphological identification in this study, making the ground of using molecular tools more sounded. The research outcomes will largely contribute to the breeding programme of sugarcanes. I have only a one major comment regarding to the study design. The three methods applied in this study have been used in other studies separately as cited (Cai et al. 2005, Aitken et al. 2007, Nair et al. 1999, Devarumath et al. 2012; Lu et al. 2012 and Lin et al. 2013). Although the authors optimized ARMS and HRM for simplified setup and shortened analysis time, I did not see the solid needs to apply all three methods as an integral. To me, the electrophoretogram of figure 5 is already evident for assessing the validity of hybrids. Could the authors explain and elaborate more why the three methods should be applied collectively? Is there any limitation of each method? Also, the GISH technique reveals the composition and the structure of chromosomes and in this case perfectly showed the n+n compositions of true hybrids. However, aneuploid is common in plants which are hybridized or mutated. Did the author considered the aneuploid issues when using this cytogenetic tool? In addition, a few minor comments the authors should settle. 1) The authority of Saccharum officinarum should be Linnaeus. Is that "Badila" is a cultivar or a line? If so, the cultivar name should be enclosed in a pair of single quotation marks i.e. 'Badila', following the International Code of Nomenclature for Cultivated Plants (ICNCP). Also, please do so for "Yunnan 15-7" if this situation is applied. 2) In line 281, the titles of Fig 6b ("the normalized fluorescence curves") and Fig 6c ("the difference plot melt curve") are not consistent with the one in Figure 6 ("Normalized Melting Curves" and "Normalized and Temp-Shifted Difference Curves"). Please be consistent for easy comprehension. 3) In Table 1, the primer names (R-D2 5'-3' RI-Dian4 5'-3') are not identical with those (FI-Er, RI-So) in the main text. Please check and revise. Reviewer #2: The study provides significant insights into sugarcane breeding by utilizing molecular and cytogenetic tools to confirm intergeneric hybrids between Saccharum officinarum and Erianthus rockii. The integration of tetra-primer ARMS-PCR, HRM curve analysis, and GISH ensures precise identification of true hybrids, addressing a critical challenge in distant hybridization. The findings highlight a distinct chromosome inheritance pattern and demonstrate the potential of E. rockii for enhancing genetic diversity in sugarcane. However, the reduced yield and sugar content in hybrids suggest the need for further backcrossing to improve agronomic traits. The study effectively establishes a foundation for utilizing E. rockii traits in modern sugarcane breeding and underscores the efficiency of nrDNA-ITS barcoding and molecular-assisted hybrid identification techniques. ****** -->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". 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.0324128.r002
Revision 1
4 Apr 2025 Author Response Response to Reviewer 1 Comments Major comment: I have only a one major comment regarding to the study design. The three methods applied in this study have been used in other studies separately as cited (Cai et al. 2005, Aitken et al. 2007, Nair et al. 1999, Devarumath et al. 2012; Lu et al. 2012 and Lin et al. 2013). Although the authors optimized ARMS and HRM for simplified setup and shortened analysis time, I did not see the solid needs to apply all three methods as an integral. To me, the electrophoretogram of figure 5 is already evident for assessing the validity of hybrids. Could the authors explain and elaborate more why the three methods should be applied collectively? Is there any limitation of each method? Also, the GISH technique reveals the composition and the structure of chromosomes and in this case perfectly showed the n+n compositions of true hybrids. However, aneuploid is common in plants which are hybridized or mutated. Did the author considered the aneuploid issues when using this cytogenetic tool? Response: Thank you for your comments. In response to your three queries, I will address them individually. 1. In general, I agree with your observation that the authenticity of hybrids can be verified solely through ARMS-PCR using the developed molecular marker. However, for any newly developed molecular marker, its reliability should be validated through additional methods. Once the reliability of a newly developed molecular marker has been confirmed through multiple methods, it becomes redundant to apply all three methods simultaneously in every case. Only ARMS-PCR, based on the developed molecular markers, is required for hybrid authenticity verification. 2. All three techniques have limitations. ARMS-PCR requires high specificity for primers and amplification conditions, necessitating numerous pre-experiments to identify optimal conditions. HRM equipment and reagents are relatively costly. The GISH experimental process is complex and not suitable for large-scale identification. 3. S. officinarum is autopolyploid with 2n = 8x = 80, while E. rockii is diploid with 2n = 30. The number of gametic chromosomes produced during meiosis is typically euploidy halved, so the chromosome number of F1 hybrids typically conforms to the n + n genetic pattern. However, when F1 hybrids are crossed further, they may produce gametes with aneuploidy halving the number of chromosomes, and the chromosome numbers of their offspring will exhibit deviations from the n + n pattern. Minor comments 1: The authority of Saccharum officinarum should be Linnaeus. Is that "Badila" is a cultivar or a line? If so, the cultivar name should be enclosed in a pair of single quotation marks i.e. 'Badila', following the International Code of Nomenclature for Cultivated Plants (ICNCP). Also, please do so for "Yunnan 15-7" if this situation is applied. Response: Agree. “Badila” is a cultivar, and “Yunnan 15-7” is a wild material found in the fields of Yunnan, China. We have, accordingly, enclosed all “Badila” and “Yunnan 15-7” in the manuscript with a pair of single quotation marks. The changes can be found in the revised manuscript lines 111-119, 158, 200-201, 225, 229, 239, 269, 298, 307-317 and 335. Minor comments 2: In line 281, the titles of Fig 6b ("the normalized fluorescence curves") and Fig 6c ("the difference plot melt curve") are not consistent with the one in Figure 6 ("Normalized Melting Curves" and "Normalized and Temp-Shifted Difference Curves"). Please be consistent for easy comprehension. Response: Agree. We have, accordingly, modified the content in the HRM assay identification of true and false F1 hybrids of the Results to emphasize this point. Line 281 to 282 in the revised manuscript this change can be found. Minor comments 3: In Table 1, the primer names (R-D2 5'-3' RI-Dian4 5'-3') are not identical with those (FI-Er, RI-So) in the main text. Please check and revise. Response: Agree. We have, accordingly, modified the primer names in Table 1 to emphasize this point. Table 1 in the revised manuscript this change can be found. ________________________________________ Response to Academic Editor Comments Comment 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 Response: Agree. We have, accordingly, changed the font size of headings at all levels to emphasize this point. In the revised manuscript this change can be found. Comment 2: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: Agree. We have, accordingly, added the following funding statement content in the ‘Funding Information’ section to emphasize this point. The following text is the funding statement: This research was funded by GDAS’ Project of Science and Technology Development (2022GDASZH-2022010102), the GDAS’s Project of Technical Innovation and Incubation Service Platform Construction (2021GDASYL 20210301001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Comment 3: Thank you for stating the following in the Acknowledgments Section of your manuscript: The authors express their sincere gratitude to Professor Zuhu Deng from Fujian Agriculture and Forestry University for his invaluable assistance in conducting the GISH assay. This research was funded by GDAS’ Project of Science and Technology Development (2022GDASZH-2022010102), the GDAS’s Project of Technical Innovation and Incubation Service Platform Construction (2021GDASYL 20210301001) and was supported by Provincial scientific research institutions stability support sub-project in 2020 "Breeding and construction of healthy seedling propagation system of new sugarcane varieties (lines)". We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: The author(s) received no specific funding for this work. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: Agree. We have, accordingly, removed funding-related text in the Acknowledgments section to emphasize this point. Line 400 to 402 in the revised manuscript this change can be found. Comment 4: We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. Response: Agree. We have, accordingly, presented raw data of Table 2 as a supporting information S1 File to emphasize this point. Line 613 to 614 in the revised manuscript this change can be found. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0324128.r003
22 Apr 2025 Decision Letter - Rongchun Han, Editor Identification and validation of intergeneric hybrids between Saccharum officinarum and Erianthus rockii using molecular and cytogenetic tools PONE-D-24-60083R1 Dear Dr. Chang, 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, Rongchun Han 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 ******** -->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 ****** -->3. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: 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 ****** -->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 ****** -->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 authors have addressed my comments adequately. Although I still feel that it is a bit overdoing by combining all three approaches together. I feel the work is technically sound and hence I recommend its 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 #1: No ******** https://doi.org/10.1371/journal.pone.0324128.r004
Formally Accepted
Acceptance Letter - Rongchun Han, Editor PONE-D-24-60083R1 PLOS ONE Dear Dr. Chang, 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. 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 Prof Dr Rongchun Han Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0324128.r005

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