Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits

Sandip K. Bera; Jignesh H. Kamdar; Swati V. Kasundra; Sahil V. Patel; Mital D. Jasani; A. K. Maurya; P. Dash; Ajay B. Chandrashekar; Kirti Rani; N. Manivannan; Pasupuleti Janila; Manish K. Pandey; R. P. Vasanthi; K. L. Dobariya; T. Radhakrishnan; Rajeev K. Varshney

doi:10.1371/journal.pone.0226252

Peer Review History

Original SubmissionJuly 5, 2019
27 Aug 2019 Decision Letter - Manoj Prasad, Editor PONE-D-19-15336 Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits PLOS ONE Dear Dr. Bera, 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. ============================== ACADEMIC EDITOR: You will see that both the reviewers made critical recommendations, but an important consideration is whether the study describes a technically sound research and made significant advances in the relevant area. However, as reviewers pointed out, I think that this manuscript needs additional works. Therefore, I encourage the authors to perform the revision as per the reviewer’s suggestions and if these were meticulously performed, then I am sure that the MS could be reconsidered on a later date. ============================== We would appreciate receiving your revised manuscript by Oct 11 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Manoj Prasad, PhD Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 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: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 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: No ****** 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: This manuscript was well written but it may not fit well to this journal. I gave this recommendation for three clear reasons: 1. The authors tried to describe a whole process for development of high oil and high oleate cultivars. The experiment was not specifically designed for any genetic studies or chemical analysis. 2. In the title, steady expression of high oleic acid and its effect on seed germination along with other seedling traits did not match the manuscript content. The evaluation of these traits was the selection outcome and did not necessary related to high oleic acid. 3. Even before making the cross, authors did not genotype parent ICGV06100 for FAD2 genotype. On Figure 2a and b, it was so confused. The well images were so different, but they scored them into the same genotype (for example, on Figure 2a well 6 and 7 to Aa; Figure 2b well 5, 6, and 7 images are the same but scored to different genotype Bb, BB, Bb). In addition, Figure 1a, only 4 samples but the authors mentioned 1 to 5. Figure 1b image was not clear. Furthermore, in the introduction (lines79-81), the authors mistook gadoleic acid (C20:1) as saturated fatty acid. Reviewer #2: Results: Table 1 results are not included in the results section Line 274 to 282 is this data represented in one of the figures or tables? line 291 what is the p-values for Figure 3? Line 294 p-value?, Line 296 25% protein contents, should state "with no significant differences??"" and cite the p-value associated Line 298 p-value line 305 pod yield, p-value???? Line 311 shelling percentage in table 1 data, what are the stats and p-value?? Line 314 what are the p-values for the significant interaction effects?? Line 321 where is this data table??? Line 323 p-value is needed?? Line 328 p-value is needed to support statement line 351 mention significant differences if any her in detail and in table 3, state p-value Line 361 to 362 please re-word for clarity is confusing as written Table 1, table footnotes are needed, define importance of superscripts statistically, what are the p-values table 2 define in table footnote how yield mean sq was determined, describe in footnotes the 3 locations, define PC1, etc, briefly state in footnotes stats used Table 3. table footnotes, table should in p-values, also, briefly state the methods used, stats used, so that reader can more clearly understand the data Figure 1 and Figure 2 are never discussed in the results section. also if these figures are to be used they need to include figure legends with details of the methods and quantity of the DNA starting materials for amplification Figure 3, Figure 4, Figure 6. and Figure 7, figure legend is needed, p-values needed with standard error bards, bar graphs using the current colors is difficult to read, try black and white and different patterns, % of what?? % of total fatty acid, x-axis values?? Figure 5???? not sure if this data adds to the strength of the manuscript?? Figure 8 same as previous comments of Figure 3, 4, also what are the harvest times and from what three states, figure legend needed Figure 9. this figure is never discussed in the manuscript. either include and discuss in the results/discussion or remove. not sure if it adds to the strength of the manuscript. General Line 71 and "the" rest Line 73 and the "remaining on-third" Line 74 low caloric (corrected spelling) line 105, remove "in a while" line 295 space after and line 297 space after and ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226252.r001
Revision 1
18 Sep 2019 Author Response Reviewer 1 Comment 1: The authors tried to describe a whole process for development of high oil and high oleate cultivars. The experiment was not specifically designed for any genetic studies or chemical analysis Reply 1: We accept the comment partially. Main aim of the experiment was to develop the introgression lines for high oleic traits which we have been developed successfully. Once we developed the introgression lines then only we aimed for genetics studies and analysis of biochemical parameters and effect of high oleic acid on seed traits which are in sequence one after another. Hope This properly justify our experimentation. Comment 2: In the title, steady expression of high oleic acid and its effect on seed germination along with other seedling traits did not match the manuscript content. The evaluation of these traits was the selection outcome and did not necessary related to high oleic acid. Reply 2: We beg to differ from the comment of reviewer. The title of the manuscript clearly describe three components of studies conducted by us. Component 1: Steady expression of high oleic acid in peanut Component 2: (high oleic peanut) bred by marker-assisted backcrossing Component 3: (High oleic acid ) its effect on seed germination along with other seedling traits Hence, our manuscript exclusively describe the above three components and their results. Hence, the title clearly match the manuscript. Comment 3: Point-1: Even before making the cross, authors did not genotype parent ICGV 06100 for FAD2 genotype. Point-2: On Figure 2a and b, it was so confused. The well images were so different, but they scored them into the same genotype (for example, on Figure 2a well 6 and 7 to Aa; Figure 2b well 5, 6, and 7 images are the same but scored to different genotype Bb, BB, Bb). In addition, Figure 1a, only 4 samples but the authors mentioned 1 to 5. Figure 1b image was not clear. Point 3: Furthermore, in the introduction (lines 79-81), the authors mistook gadoleic acid (C20:1) as saturated fatty acid. Reply 3: We beg to differ from the comment of reviewer. Point-1: Genotyping of ICGV 06100 was very much included in the gel picture (Figure 2a and 2b). In the figure 2a and 2b lane marked as P1 depicts the recurrent parent-1 which is ICGV 06100. However, we regret to mention here that labelling in the figure 2a and 2b was not proper which we have corrected subsequently. Point-2: We wholly accept the comments of reviewer for mismatching the labelling and quality of Fig. 1a, 1b 2a and 2b. Fig (1a, 1b, 2a and 2b) have been replaced by clear gel pictures with proper and corrected labelling. Also scoring details of these gel pictures have been added in Materials and methods (DNA extraction and marker genotyping subheading) to make the scoring clear. Point 3: Necessary correction has been made in the manuscript. Reviewer 2 Comments 1: Table 1 results are not included in the results section Reply 1: We accept the comment of reviewer. We unknowingly made mistake to mention table 1 in the manuscript which we have included under subheading “Recurrent parent genome recovery and linkage drag” of result section. Comment 2: Line 274 to 282 is this data represented in one of the figures or tables? Reply 2: We accept the comment of reviewer. We have made necessary correction and mentioned “Table 1” in the “Recurrent parent genome recovery and linkage drag” of result section Comment 3: Line 291 what is the p-values for Figure 3? Reply 3: We accept the comment of reviewer. Necessary correction has been included as P value at 5 % level of significance (added in footnote of Fig.3) Comment 4: Line 294 p-value?, Line 296 25% protein contents, should state "with no significant differences??"" and cite the p-value associated Reply 4: We accept the comment of reviewer. Necessary correction (one sentence of no significant difference as depicted in figure 4 has been added in manuscript). P value at 5 % level of significance (added in footnote of Fig.4) Comment 5: Line 298 p-value Reply 5: We accept the comment of reviewer. p-value is at 5 % level of significance (added in footnote in Fig.3) Comment 6: line 305 pod yield, p-value???? Reply 6: We accept the comment of reviewer. p-value at 5 % level of significance added in the table 1. Comment 7: Line 311 shelling percentage in table 1 data, what are the stats and p-value?? Reply 7: We accept the comment of reviewer. We have used Duncan’s Multiple Range Test (DMRT) for test of significance at 5%. Comment 8: Line 314 what are the p-values for the significant interaction effects?? Reply 8: We accept the comment of reviewer. Significance at 5% included in table 2. Comment 9: Line 321 where is this data table??? Line 323 p-value is needed?? Line 328 p-value is needed to support statement. Reply 9: We accept the comment of reviewer. Results described in the results section is completely based on figure 6 and figure 7. We depicted the data in bar diagram only not mentioned in a fresh table. The numerical values of oil content, Oleic, Linoleic and palmitic acids between states were more or less similar. Hence neither we compared it statistically nor added as a separate table. Hence, no p-value is mentioned. Comments 10: Line 351 mention significant differences if any her in detail and in table 3, state p-value Reply 10: We accept the comment of reviewer. P-vale at 5% included in the Table 3. Comment 11: Line 361 to 362 please re-word for clarity is confusing as written Reply 11: We accept the comment of reviewer. We have edited the sentence to make to make meaning clear Comment 12: Table 1, table footnotes are needed, define importance of superscripts statistically, what are the p-values Reply 12: We accept the comment of reviewer. We have made necessary correction in the Table 1. Comment 13: Table 2 define in table footnote how yield mean sq was determined, describe in footnotes the 3 locations, define PC1, etc, briefly state in footnotes stats used Reply 13: We accept the comment of reviewer. We have made necessary correction in the Table 2. Name of three locations were mentioned in the write up, hence avoid in the table to avoid clumsiness. Comment 14: Table 3. table footnotes, table should in p-values, also, briefly state the methods used, stats used, so that reader can more clearly understand the data Reply 14: We accept the comment of reviewer. Footnotes added in the table 3. Methodology and stats used have been mentioned in the material methods section. Comment 15: Figure 1 and Figure 2 are never discussed in the results section. also if these figures are to be used they need to include figure legends with details of the methods and quantity of the DNA starting materials for amplification Reply 15: We accept the comment of reviewer. Results of Figure 2 was already discussed in the manuscript. However, results of Figure 1 was missing. We have added the results of Figure 1(with legends) in Materials and methods (DNA extraction and marker genotyping), methods of genotyping has also been added and references have been added. Comment 16: Figure 3, Figure 4, Figure 6. and Figure 7, figure legend is needed, p-values needed with standard error bards, bar graphs using the current colors is difficult to read, try black and white and different patterns, % of what?? % of total fatty acid, x-axis values?? Reply 16: We accept the comment of reviewer. We have modified all the graphs accordingly. X- axis is self-explanatory (indicates % of total fat) Comment 17: Figure 5???? not sure if this data adds to the strength of the manuscript?? Reply 17: We accept the comment of reviewer. Yes, Fig. 5 is needed as it indicates stability and high yield of NRCGCS-ILs over locations by GGE biplot analysis and to identify such genotype was ours second objective. Comment 18: Figure 8 same as previous comments of Figure 3, 4, also what are the harvest times and from what three states, figure legend needed Reply 18: We accept the comment of reviewer. Figure 8 is essential, as it shows the passport data of new identified line NRCGCS-587 (COMPARED WITH RECURRENT PARENT) One sentence on harvest time i.e., Harvest on maturity of crop (110-115 days after sowing) has been added in Yield evaluation section of MABC-ILs in results section. Comment 19: Figure 9. this figure is never discussed in the manuscript. either include and discuss in the results/discussion or remove. not sure if it adds to the strength of the manuscript. Reply 19: We accept the comment of reviewer. Figure 9 has now been added in materials and methods part (seed and seedling traits), figure is a part of explanation of methodology followed to grow the seedlings to screen for important morphological traits to characterize the genotype. Comment 20: General comments Line 71 and "the" rest Line 73 and the "remaining on-third" Line 74 low caloric (corrected spelling) line 105, remove "in a while" line 295 space after and line 297 space after and Reply 20: We accept the comments of reviewer. We have incorporated all the corrections highlighted by the reviewer. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0226252.r002
5 Nov 2019 Decision Letter - Manoj Prasad, Editor PONE-D-19-15336R1 Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits PLOS ONE Dear Dr. Bera, 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. ============================== Thank you for submitting your revised manuscript. You will see that one of the reviewers make critical recommendations and it appears that additional work is needed as indicated by the reviewers. If these were meticulously performed, then I am sure that the MS could be reconsidered on a later date. ============================== We would appreciate receiving your revised manuscript by Dec 20 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Manoj Prasad, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 Reviewer #3: All comments have been addressed Reviewer #4: (No Response) ******** 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 Reviewer #3: Yes Reviewer #4: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: (No Response) Reviewer #3: The authors have responded to all questions raised by the previous reviewer except the question about the title. They need to change the title because it does not reflects the contents of manuscript.The title should mention the main achievement which is the development of high oleic content line. The title might read something like this "Development of stable lines with high oleic content through marker assisted introgression of fatty acid desaturate mutant alleles and its effect on seed germination". All other questions raised by previous reviewer has been answered and have improved the quality of manuscript significantly. Reviewer #4: I feel that the title may be modified. Currently, the title seems to indicate that variations in germination frequencies and other seedling traits are primarily due to high oleic acid, which may not be the case due to various reasons including genomic reconstitution as elaborated below. It would be advisable to list these as separate features/studies of the high oleic lines. In the abstract, the authors talk about resistance to biotic stresses but there is no mention of this data in the manuscript. This portion towards the end should be deleted. Line 66: "in around" I have the following four important queries which the authors need to clarify: 1. Lines 191-195: Aren't four SSRs per linkage group too less for estimating recurrent genome reconstitution? 2. Why was backcrossing done only till BC3? Would it be sufficient to reconstitute a significant portion of the recurrent parent genome in the absence of background selection. Since background selection was not done, in conjunction to point 1 above, there could be sufficient portions of the non-recurrent parent genome remaining in the resulting selected line which could be responsible for other variations observed in seedling traits. Also, this would raise questions about the stability of the generated line. 3. Was stable inheritance of the high oleic phenotype tested using successive generations of harvested seed? This is not clearly given in the text. How was it done? Which generations of selfed progeny were used? 4. How is passport data a good approach to test for genomic reconstitution? I am not in agreement with this approach. Line 226: Shouldn't it be "111-115"? Line 260: Any references for this formula? Line 284: Should be "codified" or "named" or "termed" instead of "decoded" Line 290: (RPG) in brackets Line 292: What does "nine were amplified" imply? Did they harbor the SunOleic95R genome segments? Is there any information available on the genetic/physical distance of these 10 SSRs? Line 368-369: "However, the..........the groups" is not clear. Rephrase. Figure 1 legend: Please correct for language ****** 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 Reviewer #3: No Reviewer #4: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226252.r003
Revision 2
12 Nov 2019 Author Response Reviewer #3: Comments: The authors have responded to all questions raised by the previous reviewer except the question about the title. They need to change the title because it does not reflects the contents of manuscript. The title should mention the main achievement which is the development of high oleic content line. The title might read something like this "Development of stable lines with high oleic content through marker assisted introgression of fatty acid desaturate mutant alleles and its effect on seed germination". All other questions raised by previous reviewer has been answered and have improved the quality of manuscript significantly. Reply: We beg to differ from the comment made by the reviewer #3. The reviewer #3 mentioned that the authors had not responded to the question about the change of title which is absolutely incorrect. During the first review reviewer #1 offered the same comment in his comment no.-2. In reply we submitted justification for not making any changes in the title of our manuscript (Can be referred earlier review). We still stand with our justification this time also and do not see any point for making changes in the current title of our manuscript. However, final decision lies with the editor. Reviewer #4: Comment 1: I feel that the title may be modified. Currently, the title seems to indicate that variations in germination frequencies and other seedling traits are primarily due to high oleic acid, which may not be the case due to various reasons including genomic reconstitution as elaborated below. It would be advisable to list these as separate features/studies of the high oleic lines. Reply: We beg to differ from the comment of the reviewer. The title of the manuscript clearly describe three components of the studies conducted by us.Component 1: Steady expression of high oleic acid in peanut; Component 2: Breeding of high oleic peanut by marker-assisted backcrossing; Component 3: Effect of high oleic acid on seed germination along with other seedling traits. Hence, our manuscript clearly describe the above three components and their results. Hence, the title clearly match the manuscript. Comment 2: In the abstract, the authors talk about resistance to biotic stresses but there is no mention of this data in the manuscript. This portion towards the end should be deleted Reply: The data on resistance to biotic stresses (Rust and Late Leaf Spot diseases) of NRCGCS-587 and ICGV 06100 (recurrent parent) have been mentioned in supplementary Table–2 (S2). It may be treated as additional information. Hence, deleting the statement on resistance to biotic stresses from the text may not be required. Comment 3: Line 66: "in around" Reply: Accepted, Necessary correction has been made in the manuscript. Comment 4: Lines 191-195: Aren't four SSRs per linkage group too less for estimating recurrent genome reconstitution? Reply: It is always better to use SSRs as many as possible for estimating recurrent genome reconstitution. However, we selected four anchored SSRs from each linkage group (altogether 80 SSRs) which are genetically mapped in the peanut consensus map. Out of which 79 SSRs gave similar expression both in recurrent parent and introgression line. It gives a fair indication about the reconstitution of the recurrent parent genome in introgression line. Furthermore, we used three cycles of backcrossing which essentially (mathematically) recovers 93.75% of the recurrent parent genome. Both these approaches are supplementary to each of above two approaches and confirm recurrent parent genome reconstitution. Comment 5: Why was backcrossing done only till BC3? Would it be sufficient to reconstitute a significant portion of the recurrent parent genome in the absence of background selection. Since background selection was not done, in conjunction to point 1 above, there could be sufficient portions of the non-recurrent parent genome remaining in the resulting selected line which could be responsible for other variations observed in seedling traits. Also, this would raise questions about the stability of the generated line. Reply: Theoretically, three cycle of backcrossing supposed to recover 93.75% recurrent parent genome. We attempted three cycles of backcrossing which helped us to recover 93.75% recurrent parent genome. In such case taking background selection additionally would not yield any more information. However, one can do any number of additional work to strengthen the result. Yes, there is a chance of having 6.25% of non-recurrent parent genome in the introgression line and literally, this 6.25% of non-recurrent parent genome may throw little or no variations among the introgression lines. Comment 6: 3. Was stable inheritance of the high oleic phenotype tested using successive generations of harvested seed? This is not clearly given in the text. How was it done? Which generations of selfed progeny were used? Reply: Yes, we have tested the high oleic phenotype in F3, F4 and F5 generations and confirmed. It is clearly mentioned in the text line no. 300 (for the year 2014), 308 (for the year 2015) and 337 (for the year 2016 in multiplication). High oleic trait was phenotyped through fatty acid analysis of kernels in Gas chromatography (Details methodology have been described in Materials and Methods section). Selfed progenies of F3, F4 and F5 generations. comment 7: How is passport data a good approach to test for genomic reconstitution? I am not in agreement with this approach. Reply: Passport data of a genotype/verities of a particular crop is an important information which is used in DUS (Distinctness, Uniformity and Stability) testing. It helps to distinguish/identify one genotype from others. If two genotypes are similar in majority of the passport traits except one or two major trait(s), the genotypes can also be refereed as near isogenic line (NIL). Here, recurrent parent and introgression line are similar in majority of their passport traits except the high oleic content which we have introgressed into the introgression line. Theoretically, 3 cycle of backcrossing supposed to recover 93.75% genome of recurrent parent. Thus introgression line, NRCGCS-587 has got more than 90% genome constitution of recurrent parent (ICGV 06100), which has been reflected in the passport data of introgression line and recurrent parent. Comment 8: Line 226: Shouldn't it be "111-115"? Reply: Accepted, Necessary correction has been made in the manuscript. Comment 9: Line 260: Any references for this formula? Reply: Accepted, Necessary correction has been made in the manuscript. Comment 10: Line 284: Should be "codified" or "named" or "termed" instead of "decoded" Reply: Accepted, Necessary correction has been made in the manuscript. Comment 11: Line 290: (RPG) in brackets Reply:Accepted, Necessary correction has been made in the manuscript. comment 12: Line 292: What does "nine were amplified" imply? Did they harbor the SunOleic95R genome segments? Is there any information available on the genetic/physical distance of these 10 SSRs? Reply: Accepted, Necessary correction has been made in the manuscript. Yes, there is always possibilities, possible explanation is mentioned in the text. Yes, Necessary information is already available in the supplementary table 1 (S1). Comment 13: Line 368-369: "However, the..........the groups" is not clear. Rephrase. Reply: Accepted, Necessary correction has been made in the manuscript. Comment 14: Figure 1 legend: Please correct for language Reply: Accepted, Necessary correction has been made in the manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0226252.r004
25 Nov 2019 Decision Letter - Manoj Prasad, Editor Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits PONE-D-19-15336R2 Dear Dr. Bera, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Manoj Prasad, PhD 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 #3: All comments have been addressed Reviewer #4: 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 #3: Yes Reviewer #4: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: Yes Reviewer #4: (No Response) ****** 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 #3: Yes Reviewer #4: (No Response) ****** 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 #3: Yes Reviewer #4: (No Response) ****** 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 #3: I am surprised at stubborn attitude of authors. Three different reviewers have pointed out discrepancy in manuscript content and the title. The reasons have been cited and alternate titles have been suggested. Reviewers have been constructive in reviewing the manuscript but authors are reiterating the same comment again and again. They have not even considered what reviewers have been pointing out. Under these circumstances, I have no other comments to make and leave it to wisdom of editor to decide what will be right. I will not like to take up this manuscript for reviewing anymore. Reviewer #4: (No Response) ****** 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 #3: No Reviewer #4: No https://doi.org/10.1371/journal.pone.0226252.r005
Formally Accepted
5 Dec 2019 Acceptance Letter - Manoj Prasad, Editor PONE-D-19-15336R2 Steady expression of high oleic acid in peanut bred by marker-assisted backcrossing for fatty acid desaturase mutant alleles and its effect on seed germination along with other seedling traits Dear Dr. Bera: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Manoj Prasad Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226252.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .