Development of a Flex-Seq SNP panel for raspberry (Rubus idaeus L.) and validation through linkage map construction and identification of QTL for several traits of agronomic importance to raspberry breeding

Jahn Davik; Paolo Zucchi; Matteo Buti; Linda Milne; Julie Graham; Daniel James Sargent

doi:10.1371/journal.pone.0328606

Peer Review History

Original SubmissionJuly 2, 2025
19 Aug 2025 Decision Letter - Hidenori Sassa, Editor Dear Dr. Davik, Please carefully examine the reviewers' comments, and revise the manuscript. Please submit your revised manuscript by Oct 03 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. 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. 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Thank you for stating the following financial disclosure: [The research leading to these results has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 101000747. The author is solely responsible for its content, it does not represent the opinion of the European Commission and the Commission is not responsible for any use that might be made of data appearing therein. https://research-and-innovation.ec.europa.eu/funding/funding-opportunities/funding-programmes-and-open-calls/horizon-2020_en This grant was awarded to JD, PZ, and LM. The funder did not play any role in any part of the research presented here.]. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 5. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [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? Reviewer #1: Partly 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 Reviewer #1: No Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Flex-Seq SNP panel for raspberry (Rubus idaeus L.) is an effective resource for genetic improvement in red raspberry. QTL analysis and GWAS using the SNP panel detected several significant associations for four agronomic traits, leading to the successful identification of multiple candidate genes. However, some issues need to be addressed before publication. 1. Line 102: As this is the first time “LEC” is being introduced, a brief explanation is required. 2. Lines 141-142: Please explain the genetic relationships among these four cultivars. The authors should perform a PCA using the Flex-Seq SNPs of the diversity panel to clarify the genetic positioning of the four cultivars within the panel. I think that using only four cultivars is insufficient for designing SNPs to evaluate the diversity of the panel. 3. Lines 179-180: These parameter values are different from those listed in Table 1. 4. Lines 200-203: Please explain here as well that raspberry has seven chromosomes. Why did the authors select only 25 SNPs rather than utilizing all available markers? 5. Lines 215 and 340: It is inappropriate for only this part to be italicized. Please make it consistent with the rest. 6. Line 231: Why are only all heterozygous markers in the diversity panel used? I think homozygous markers can also be utilized. 7. Line 233: The results of the GWAS conducted with BLINK have not been presented. 8. Lines 249-250: The Fig 1, S1 Fig, as well as the other figures, have low resolution and are difficult to interpret. Please provide higher-resolution versions. 9. Line 253: The supplementary files are not in the correct order. 10. Lines 272 and 284: Please explain the composition of the “481” samples. 11. Line 275: Could you please explain the meaning of the columns and rows in the S4 File? 12. Line 278: Please explain the Indels in Table 1. Additionally, were the Indels used in the analysis? 13. Lines 303-304: Could you please discuss the possible reasons for the discrepancy in marker ordering on the left side of Chromosome 2 shown in Fig 3? 14. Lines 314-321: Please explain about Ri2 and Ri3, including their A and/or B types. 15. Line 331: There are two notations: 'SO486' and 486. Please unify them. 16. Line 363: The S6 File was not provided, so I was unable to review it. Please describe in the Methods section the approach used to define the candidate genes, including details such as the range of genomic regions considered. 17. The authors have made the phenotypic data publicly available, but the marker genotype data has not been shared. Reviewer #2: Dear authors This study created a new Flexseq panel for red raspberries and confirmed its effectiveness through linkage analysis, QTL analysis, and GWAS. While this paper appears to be scientifically sound, I recommend revising it prior to publication to enhance reader understanding based on the following comments. Major comment ■1 To enable readers to visually understand the reliability and effects of each QTL, present histograms or box plots that divide populations by SNP or indel alleles for both QTL analysis and GWAS. ■2 Is it necessary to write something like an LD plot for the range of candidate genes for GWAS? Please consider not only the proximity of the peak and the gene locus, but also the linkage disequilibrium relationship between SNPs. ■3 Please show the population structure of the population used in GWAS. Please use any of the following methods: Admixture, principal component analysis, or phylogenetic tree. With the SNP information, I think you can perform these analyses. Minor comment ■4 Line 271 “150bp” is wrong It may be “150 bp” ■5 Line 273 I think it would be better to express it as VCF (variant call format) rather than “.vcf file”. ****** 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
29 Sep 2025 Author Response Response to Reviewer #1 General comment: Flex-Seq SNP panel for raspberry (Rubus idaeus L.) is an effective resource for genetic improvement in red raspberry. QTL analysis and GWAS using the SNP panel detected several significant associations for four agronomic traits, leading to the successful identification of multiple candidate genes. We thank the reviewer for their positive overall comments regarding our manuscript. Line 102: LGC Genomics is the name of a company. We have edited the text to make this clear. Lines 141–142: We appreciate the reviewer’s suggestion. The four cultivars referenced are well documented in previously published work (ref). As noted in the Methods, SNPs for the panel were derived not only from these cultivars but also from two additional mapping studies, ensuring broad representation. Because the original DNA samples were not available, we could not re-genotype them, and additional analyses such as PCA are therefore outside the scope of this study. Importantly, the effectiveness of the SNP panel is demonstrated through our QTL and GWAS results, which confirm that the set is adequate for diversity and trait mapping purposes. Lines 179–180: We thank the reviewer for spotting this error. The parameters in the table were the correct ones, and the methods have been edited accordingly. Lines 200–203: We have amended the text to explain our reasoning in the methods section. Lines 215 and 340: This has been done. Thank you for spotting the formatting error. Line 231: We have corrected to state all polymorphic markers were included. Thank you for spotting the error. Line 233: We thank the reviewer for noting this omission. The MLM GWAS results have now been added to the Results section and are provided in supplementary file S5 Fig. The associations are consistent with the results obtained with BLINK, supporting the robustness of the SNP panel, however, some of the associations are below the level of significance set for the BLINK analysis. Lines 249–250: High resolution versions were included in the submission and can be accessed when clicking on the links in the PDF. We have no control over the resolution in the final PDF built; this is done by PLOS. If you click on the links, you have access to the high-resolution versions, but we cannot increase the resolution in the submission as this is created automatically by PLOS from our high-resolution files. Line 253: This has now been corrected. Lines 272 and 284: This has now been corrected to 457, the number of genotypes studied and detailed in the methods. Line 275: The format is that of a standard .BED file, but headers have been added for clarity. Line 278: InDels were not considered as markers further in the study. This has now been explained in the text. Lines 303–304: A possible explanation for the discrepancy has been provided in the text. Lines 314–321: This has now been explained in the results section “Paris×486 Linkage map construction”. Line 331: We thank the reviewer for spotting this inconsistency. SO has been removed from its occurrences in the text. Line 363: We apologise for this omission. The S6 File is included with this revised submission. In addition, we have expanded the Methods section to describe the candidate gene identification approach. Data availability: We thank the reviewer for spotting the omission of the sentence detailing where the reads are deposited. The raw reads from the FlexSeq genotyping were deposited in the ArrayExpress repository at EMBL-EBI (www.ebi.ac.uk/arrayexpress) and will be released upon acceptance of the manuscript for publication. Response to Reviewer #2 General comment: This study created a new Flexseq panel for red raspberries and confirmed its effectiveness through linkage analysis, QTL analysis, and GWAS. While this paper appears to be scientifically sound, I recommend revising it prior to publication to enhance reader understanding. We thank the reviewer for their generally positive comments, they are greatly appreciated. Major comment Present histograms or box plots for allele effects at QTL and GWAS loci. We have now included boxplots illustrating allele effects at significant QTL and GWAS loci (Supplementary Figures Sx–Sy). These plots provide a visual representation of the effect sizes and enhance the interpretability of the results. Consider including LD plots for GWAS candidate gene regions. We thank the reviewer for this valuable suggestion. However, as this study is primarily focused on the development and demonstration of a broadly useful SNP panel rather than an in-depth dissection of specific trait loci, we feel that detailed LD analyses are beyond the scope of the present manuscript. Our intention is to show that the panel can successfully identify trait associations, and we hope that the community will apply it for deeper genetic analyses, including LD mapping. Show the population structure used in GWAS (Admixture, PCA, or phylogenetic tree). A NJ tree has been constructed and is included in the manuscript as a Supplementary File. Minor comments Line 271 - “150bp” should be “150 bp” Corrected Line 273 - say “VCF” rather than “.vcf file” Corrected Attachments Attachment Submitted filename: Rebuttal_Letter_PLOS_ONE.docx https://doi.org/10.1371/journal.pone.0328606.r002
15 Nov 2025 Decision Letter - Hidenori Sassa, Editor 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 Dec 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. 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'. 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We look forward to receiving your revised manuscript. Kind regards, Hidenori Sassa Academic Editor PLOS ONE Journal Requirements: 1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments: Please carefully examine the comment on the range of LD by Reviewer 2, and revise the manuscript. [Note: HTML markup is below. Please do not edit.] Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> 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 Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: (No Response) ****** Reviewer #1: I have confirmed that the authors have appropriately and thoroughly addressed the points that were raised. Reviewer #2: Dear authors The authors addressed most of my comments, but I have one concern. Major comment While the authors state that LD analysis is out of scope, this study includes analysis of candidate genes associated with GWAS peaks. I find the analytical workflow inappropriate in the following respects: The authors extracted 2 Mbp around the peak SNP, but the reason for this 2 Mbp range is unclear. If there is a reference that can be cited, please provide it. Generally, the correspondence between physical distance and genetic distance varies depending on chromosomal region such as whether the region is near a telomere or a centromere. In such cases, uniformly narrowing the range to a fixed 2 Mbp is inappropriate both experimentally and analytically. In this sense, if presenting information on related genes, it is crucial to properly verify indicators like LD. Furthermore, despite the primary objective being to investigate whether associations with traits can be established, please state, within the Candidate Gene Identification section, the rational justification for performing analysis on the candidate genes of SNP peaks. ****** 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 2
15 Dec 2025 Author Response Major comment While the authors state that LD analysis is out of scope, this study includes analysis of candidate genes associated with GWAS peaks. I find the analytical workflow inappropriate in the following respects: The authors extracted 2 Mbp around the peak SNP, but the reason for this 2 Mbp range is unclear. If there is a reference that can be cited, please provide it. Generally, the correspondence between physical distance and genetic distance varies depending on chromosomal region such as whether the region is near a telomere or a centromere. In such cases, uniformly narrowing the range to a fixed 2 Mbp is inappropriate both experimentally and analytically. In this sense, if presenting information on related genes, it is crucial to properly verify indicators like LD. Furthermore, despite the primary objective being to investigate whether associations with traits can be established, please state, within the Candidate Gene Identification section, the rational justification for performing analysis on the candidate genes of SNP peaks. Authors’ response: We thank the reviewer for reiterating this point. We have addressed the LD issue by creating LD decay plots around the most significantly trait-associated SNPs from the linkage and the GWAS analyses. These plots are added in S7 Fig. Analyses were restricted to ±5 Mb around the focal marker and we used r2 between pairwise markers and LOESS smoothing to summarize local LD patterns around. We set non-negligible LD as the distance on either side of the focal SNP at which the LOESS-predicted r2 fell below 0.10. The sizes of the LD certainly did vary. However, all the suggested candidate genes were located well within the proposed LD windows. Attachments Attachment Submitted filename: Rebuttal_Letter_PLOS_ONE_auresp_2.docx https://doi.org/10.1371/journal.pone.0328606.r004
28 Jan 2026 Decision Letter - Hidenori Sassa, Editor Development of a Flex-Seq SNP panel for raspberry (Rubus idaeus L.) and validation through linkage map construction and identification of QTL for several traits of agronomic importance to raspberry breeding PONE-D-25-35797R2 Dear Dr. Davik, 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. For questions related to billing, please contact billing support . 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, Hidenori Sassa Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0328606.r005
Formally Accepted
Acceptance Letter - Hidenori Sassa, Editor PONE-D-25-35797R2 PLOS One Dear Dr. Davik, 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 Dr. Hidenori Sassa Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0328606.r006

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