Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages

Stephanie Saade; Chris Brien; Yveline Pailles; Bettina Berger; Mohammad Shahid; Joanne Russell; Robbie Waugh; Sónia Negrão; Mark Tester

doi:10.1371/journal.pone.0236037

Peer Review History

Original SubmissionFebruary 16, 2020
7 May 2020 Decision Letter - Chengdao Li, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-20-04581 Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages PLOS ONE Dear Dr Saade, 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 paper is not written in the standard format for PLOS ONE. Please make changes in your revision. Please read both reviewers' comments and address the points raised. We would appreciate receiving your revised manuscript by 8 June 2020. 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The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No 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: In the manuscript entitled “Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages”, the authors performed GWAS for salt-tolerance related traits using 377 spring barley accessions, attempting to identify genetic components of barley salinity tolerance at two stages. Definitely, the study provides useful information of major QTLs related to salt tolerance in barley. However, in the MS, there are many issues should be definite and addressed. In my opinion, in the MS, the phenotypic data of traits at the reproductive stage was not well organized. For example, how about the correlation of the yield traits under salinity field condition between the seasons 2013/2014 and 2014/2015? If the correlation of the trait is not good between the two years, it will influence the results of the GWAS based on average value of these traits. For the experiment design, the time interval (within 11 days after 200 mM salt treatment in the soil pot) may be not enough to show the difference among accessions. Is it a reason to explain why no common QTLs is detected between the two stages ? More discussion would be addressed in the part of discussion. The figure 1 is not easy to understand. How to reflect the genotypic difference of the effect of salt treatment on the traits in this figure? In the legend of figure 2, the authors did not explain marker term and marker-by- treatment term. In figure 3, there was no significant correlation between ion content and other traits. In the experiment, the fourth complete leaf was used to analyze Na and K content at the vegetative stage, why choose the fourth complete leaf for analysis? Minor comments: the notes of x-axis in the figure S2 is missing. Table S5 had the data of TGW (‘Thousand grain weight’ in short?), but this trait was not mentioned in the MS. Reviewer #2: The authors presented an interesting paper with the aims to find new genetic components of salinity tolerance in barley at the vegetative and reproductive stages. They sreened a large population of barley cultivars using the high-throughput phenotyping platform, which is novel and very important for improving the efficiency and accuracy of the labour-intensive phenotyping process. I only have two minor suggestions. 1.) The paper contains lots of data, but these were mostly presented in the Supplementary Files. One would suggest that a couple of these figures/tables should be presented in the paper. This will be useful for the readers to understand the high-tech and highly efficient phenotyping platform for screening large number of lines of crops such as barley and wheat. 2.) The paper combines the Results with Discussion, which is no an issue for many journals. It does not seem to be a standard format for PLoS One. I leave this for the Editor to sort out. ****** 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.0236037.r001
Revision 1
5 Jun 2020 Author Response We thank the reviewers for their comments, which we have replied to below. Reviewer #1: In the manuscript entitled “Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages”, the authors performed GWAS for salt-tolerance related traits using 377 spring barley accessions, attempting to identify genetic components of barley salinity tolerance at two stages. Definitely, the study provides useful information of major QTLs related to salt tolerance in barley. However, in the MS, there are many issues should be definite and addressed. - In my opinion, in the MS, the phenotypic data of traits at the reproductive stage was not well organized. For example, how about the correlation of the yield traits under salinity field condition between the seasons 2013/2014 and 2014/2015? If the correlation of the trait is not good between the two years, it will influence the results of the GWAS based on average value of these traits. Response: The data used to run the GWAS model is the spatial adjusted data for site (assuming 4 sites: 2013_control, 2013_salt, 2014_control, 2014_salt) as described in the Materials and methods section Lines 291-293 and 295-297. The correlations between 2013_control - 2014_control and 2013_salt - 2014_salt are high (a bit too high as they are inflated due to the spatial adjustment per site). Pearson correlation coefficient between years Trait Control Salt Both treatments HEA 0.8407097 0.932204 0.87892784 MAT 0.8662038 0.904021 0.89078656 RIP 0.6053287 0.704846 0.67635931 HEI 0.9999970 0.999999 0.99224917 EAR 0.9684144 0.797763 0.9669035 GPE 0.9498838 0.951435 0.92541853 DRY_WT 0.8855445 0.937100 0.90577249 YLD 0.9938078 0.995805 0.95926533 HI 0.8951714 0.967731 0.84466089 We added a sentence “, using predicted means adjusted for year and condition.” on line 304 to re-emphasize that. - For the experiment design, the time interval (within 11 days after 200 mM salt treatment in the soil pot) may be not enough to show the difference among accessions. Is it a reason to explain why no common QTLs is detected between the two stages? More discussion would be addressed in the part of discussion. Response: The ionic phase of salt response is due to the accumulation of the sodium ions in the shoot. The shoot ion-independent phase (also known as osmotic phase) happens within few hours to few days of the salt treatment [1]. The ionic phase is more extensively studied compared with the shoot ion-independent phase, where phenotyping is the bottleneck. Our aim was to use high-throughput phenotyping to study the shoot ion-independent phase (Lines 130-132). Therefore, 11 days is actually more than enough. However, we hypothesized in the manuscript (Lines 550-551, 563-564) that the plants were too young when the salt was imposed possibly making it hard to see differences among accessions. To address the reviewer’s comment and clarify that the shoot ion-independent phase of the plant response to salt stress is rapid and is manifested by growth reduction, we added the sentence “shoot ion-independent tolerance takes place in the early stage of the salt stress before sodium ions had the time to accumulate. During this rapid response of the plant to stress, growth is reduced” (Lines 96-98). - The figure 1 is not easy to understand. How to reflect the genotypic difference of the effect of salt treatment on the traits in this figure? Response: The aim of Fig 1 is to show that the salt treatment (200mM) is considered appropriate for our studies because it significantly affected all studied traits without causing major premature plant senescence. As shown in Fig 1, there is significant difference between average of trait under control compared with average of trait under salt treatment. The sentence “and natural genetic variation across the population in the magnitude of this response.” at Line 342 was not referring to Fig 1. We deleted it to avoid confusion. - In the legend of figure 2, the authors did not explain marker term and marker-by- treatment term. Response: we addressed this by explaining the terms in the legend (this is Fig 3 now). We also added the explanation of those terms to the legends of S3 and S5 Figs. - In figure 3, there was no significant correlation between ion content and other traits. In the experiment, the fourth complete leaf was used to analyze Na and K content at the vegetative stage, why choose the fourth complete leaf for analysis? Response: (This is Fig 4 now) Choosing the first or second fully expanded leaf is a standard practice in salinity stress evaluation. This ensures that the leaves collected from varying genotypes have been exposed to the same period of treatment. Traits measured at The Plant Accelerator® are growth related (absolute growth rate and relative growth rate) and leaf Na and K contents. The former correlates with the shoot ion-independent phase of the response to salt treatment while the latter correlates with the ionic phase. This explains why Na and K content did not significantly correlate with the other traits at The Plant Accelerator®. Na content usually correlates with senescence as the plant response to salt stress. As mentioned in Line 383-385, detecting the known locus on chromosome 4H for sodium and potassium contents proves that the genetic material is appropriate for this study. To address the reviewer’s comment, we added “This ensured that the collected leaf had been exposed to the same period of stress despite variations in plant growth across accessions” (Lines 199-201) to the Materials and methods section to explain that choosing the first or second fully expanded leaf is a standard practice in salinity stress evaluation. We also re-emphasized in the Discussion section that Na and K content are related to the ionic phase (Line 380). - Minor comments: the notes of x-axis in the figure S2 is missing. Response: We fixed this by adding the “chromosome” label, which was missing from the x-axis of the lower panel. - Table S5 had the data of TGW (‘Thousand grain weight’ in short?), but this trait was not mentioned in the MS. Response: Data on TGW trait was initially collected. However, the trait had very low heritability and was not reliable so we decided not to include it in the manuscript. We kept the phenotypic data (Table S2) and GWAS results (Table S3 b, field) of the TGW (an important yield component) for transparency and future reference for readers. Reviewer #2: The authors presented an interesting paper with the aims to find new genetic components of salinity tolerance in barley at the vegetative and reproductive stages. They sreened a large population of barley cultivars using the high-throughput phenotyping platform, which is novel and very important for improving the efficiency and accuracy of the labour-intensive phenotyping process. I only have two minor suggestions. 1.) The paper contains lots of data, but these were mostly presented in the Supplementary Files. One would suggest that a couple of these figures/tables should be presented in the paper. This will be useful for the readers to understand the high-tech and highly efficient phenotyping platform for screening large number of lines of crops such as barley and wheat. Response: We addressed this by: - Moving the table that compares trait (ear number per plant and grain number per ear) means between accessions by condition (control or saline) and genotype (at peak markers) from supplementary information to the main text as Table 2. - Moving the plots of smoothed absolute growth rate and relative growth rates at The Plant Accelerator® from supplementary information to the main text as Fig 1. 2.) The paper combines the Results with Discussion, which is no an issue for many journals. It does not seem to be a standard format for PLoS One. I leave this for the Editor to sort out. Response: We understand that combining the Results with Discussion is not a standard format. However, it is not uncommon. We think that combining the results and discussion makes this paper concise, especially that results from both the field and The Plant Accelerator® need to be presented and discussed. In addition, the guidelines (https://journals.plos.org/plosone/s/submission-guidelines#loc-results-discussion-conclusions) state “Results, Discussion, Conclusions. These sections may all be separate, or may be combined to create a mixed Results/Discussion section (commonly labeled “Results and Discussion”) or a mixed Discussion/Conclusions section (commonly labeled “Discussion”).” Similarly, https://journals.plos.org/plosone/s/file?id=4497/Main%20Body%20-%20ONE%20Formatting.pdf show the results and discussion section together. Therefore, we prefer to keep the Results and discussion combined. 1. Munns R, Tester M. Mechanisms of salinity tolerance. Annual Review of Plant Biology. 2008;59:651-81. doi: 10.1146/annurev.arplant.59.032607.092911. PubMed PMID: WOS:000256593200026. Attachments Attachment Submitted filename: Saade_PLOSONE_rebuttal_PONE-D-20-04581.docx https://doi.org/10.1371/journal.pone.0236037.r002
29 Jun 2020 Decision Letter - Chengdao Li, Editor Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages PONE-D-20-04581R1 Dear Dr. Saade, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Chengdao Li, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0236037.r003
Formally Accepted
6 Jul 2020 Acceptance Letter - Chengdao Li, Editor PONE-D-20-04581R1 Dissecting new genetic components of salinity tolerance in two-row spring barley at the vegetative and reproductive stages Dear Dr. Saade: I'm 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 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 plosone@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 Professor Chengdao Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0236037.r004

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