PONE-D-19-17567

Diversity in sea buckthorn (Hippophae rhamnoides L.) varieties with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Tian

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.

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Kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

The manuscript “Diversity in sea buckthorn (Hippophae rhamnoides L.) varieties with different origins based on morphological characteristics, oil traits, and microsatellite markers” has been reviewed by two reviewers. It took me a while to secure reviewers for this manuscript, hence the delay, I apologies for it. Both the reviewers have submitted their comments, and one of them has also provided a sanitized version of the manuscript which can be seen by the authors. Both the reviewers have various concerns regarding the manuscript, but more importantly both have shown concern about the plant material and data generated. Since they raised a concern on the data, it will be better that manuscript is submitted along with answers to both the reviewer’s comments.

I will like to point out some important points:

1) The use of term varieties, cultivars, subspecies and hybrids have been without much explanation. For example, what is the basis of assigning hybrid status to a particular cultivar? More clarity is required in explanation of the material. How these varieties were assigned varietal status?

2) Generation of morphological dataset is also not mentioned clearly. You have 76 varieties growing at three locations. You need to provide environmental parameters for each location. Are all 76 growing at each location? If all the varieties are not growing at same location, many of morphological traits will be influenced by environmental factors. Did you do any multilocation trials to see the influence of environment on these traits? Did you try to collect data during different years and see if the data is consistent or showing variation. A statistical analysis of such data only will generate confidence in morphological data. Even a multilocation trial of a subset will provide information on reliability of data. Please include such data.

3) The sequencing data has been published earlier and 17 of SSR are coming from that data. Only 3 new markers have been used in the present study. This undermines the amount of data presented in this MS. You have to clearly mention these facts in the MS and the abstract. In my opinion more data needs to be generated. I suggest another 25-30 SSRs should be used for analysing the diversity.

4) There is no comparison given between the varieties used in previous publication and the present one. Are you using common varities? If you are than SSR data must be same and must have been presented in previous MS already. This has not been mentioned in the MS.

I hope you will appreciate comments made by two reviewers and will appreciate the time and efforts spent by them in reviewing the manuscript. The comments are positive and are an effort to improve the quality of manuscript further.

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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: Partly

Reviewer #2: Partly

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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: 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: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

 

Reviewer #1: General comments

The present study was conducted on 78 so called “varieties” of Hippophae rhamnoides, out of which two sets belonged to two sub-species and the third one is supposedly the hybrids. The study is aimed to decipher the genetic diversity and their oil content (in “berries”) of the “varieties”. With the given results and methodology authors are attempting to generated the data on two different aspects - first assessment of agronomic traits, especially the oil content and to relate it to a reliable phenotypic marker (among the eight selected morphological traits), and genetic diversity of species by using 20 SSR markers. An attempt has been made to link these traits together, which needs to be highlighted in the Introduction pertinently in the light of earlier attempts on related/unrelated taxa.

In general the MS needs to be modified for technical reasons and usage of sources in the study. A major revision of the MS is suggested before acceptance.

Specific comments

1. I think that in such bio-prospection studies sampling strategy is very crucial. The sampling method needs to explain that how these accessions were sourced. The MS needs elaboration on -

• How many individuals of a “variety” from each site were collected?

• Are these the random collections of registered varieties from the cultivated field in the five regions OR sampled from the wild?

• It is also not clear that how the hybrids were distinguished from parents while making collections.

• Do these sites differ in climatic conditions?

• What is the link of “origin” with oil content? Did you expect that there are bound to be differences because of differences in the climatic conditions of area of collection/cultivation of the same “variety/hybrid”?

Importantly instead of the term varieties the term accessions would be appropriate, as the authors have mentioned it in Table 1. According to the definition by The International Union for the Protection of New Varieties of Plants, "a variety must be recognizable by its characteristics, recognizably different from any other variety and remain unchanged through the process of propagation".

Do these two subspecies hybridize freely in nature and such hybrids have been characterized? This needs some population analysis like by using STRUCTURE, or at least there should be a note on the characterization of hybrids (including the features), even if they are procured form some Research Institute.

2. I don’t understand the usage of term pulp/peel in the MS (also see page 15, line 251). As the entire fleshy region was separately used for extraction of oil from the "berries" (see Methods), the use of term pulp would be appropriate. One cannot expect to remove the epidermal peel especially during the mechanical homogenization process.

3. How the present study for the genetic diversity analysis of 78 cultivars is different from other previous studies? May be highlighted in the introduction. Authors may also highlight that trait: i.e. Oil yield was correlated with the “promising” accessions.

4. Although attempt has been made of possible use of MAB in future, but it has not been justified with the discussion. For example, do the authors will depend on the same plants in the cultivated lands across the region or some mapping populations will be established. In former case GPS tagging of the individuals will be required for sourcing the material on regular basis and to establish the consistency of the trait.

Materials and Methods

5. Need to mention whether hundred-berry weight, hundred-seed weight and other dimensions were taken from mature or immature berries? In Supplementary figure 1 some samples are showing immature berries e.g. sample 65, 68 etc.

6. What do the ‘Berry Shape Indices’ refer to and what are its implications on the results/oil trait/ with genetic diversity. Provide any suitable reference if possible. (Page: 8, subsection: Morphological….)

7. The usage of phrase ‘8 agronomic traits’ seems to be superfluous as these are the traits of berries itself. How the seed width is different from the seed thickness? The difference is not apparent. Table 2 and 3; as well as in text.

8. The usage of abbreviation has not been followed see table 2 and 3. Table 2 is not necessary, may be omitted or shifted to Supplementary Data. In Tables SD is not mentioned.

9. The reference is missing for the SB18-SB20 SSRs; in the text (Page 10, line 181).

Results

10. Results should be given in the format mean ± SD. Minimum and maximum can be given in supplementary tables.

11. It is not clear from the table caption and content that whether values in the Table 4 is the minimum, maximum and mean values are representing the cumulative results of 78 varieties e.g. minimum in variety… and maximum in variety…. Need to mention in the results.

12. The results of CCA are driving a correlation between phenotypic traits and oil characteristics. The authors may use the information for total oil content (pulp+seed) or oil content in pulp and seeds separately for drawing any correlation. That would possibly help as a descriptor for the potential crop in identifying the elite/superior “variety” and further can be linked to genetic diversity.

Discussion

13. Page:28, Line:449-453. The link of this part of discussion is lacking with the previous text.

14. In conclusion part authors are concluding that this information may be useful for cultivar identification but initially they started their work for the varieties. Taxonomically these two are different entities.

Some suggestion:

1. The sequence of S1 and S2 table can be reversed as per the citation in the text.

2. Page:3, Line:54. Reference 1 is incorrect. The lead author here is Bartish I.V.

3. Page:3, Line:56-57. ….flavonoids [3-7]; ….products [8-10]. Here over-citation may be avoided.

4. Page:3, Line:59. ‘Sea buckthorn oil’ instead of ‘sea buckthorn oils’

5. Page: 4, Line 74. Add a reference to the statement. The plant is able to avoid cold and is not resistant, because the leaves are shed under extreme cold condition in this plant. Even the species is not resistant to alkali too.

6. Page:4. Line:85. Use full form at first place ‘MAB’.

7. Page:5. Line:110. What was the premise of including two known elite varieties in the study? Any supportive reference(s) for the statement, and also mention the context in which these varieties are elite.

8. Page:12. Line:204-205. May be included in Material and Methods.

Reviewer #2: The publication can be accepted post minor reviews.

Some of the comments to authors have been listed below. Some changes required are highlighted in the manuscript attached.

1. The authors mention that 76 varieties were used. There is no mention of the different species they belonged to in M&M, although it has been mentioned later in the text and table. Incorporate that information in the M&M.

2. Are these 76 different varieties or just different accessions? At many places they are being referred to as ‘cultivars’ also. Please correct accordingly in the text wherever mentioned.

3. How variable are the climatic conditions of the three research institutes?

4. Line 109: ‘………provided 76 varieties’. Does this mean that all the 76 were grown at all the 3 fields? There is no clarity on this aspect in the M&M. Most quantitative traits exhibit a huge variation across environments. To study the phenotypic variations it would have been much informative if all the 76 varieties were grown together across all the three fields. Why was that not considered?

5. There is no mention of how these varieties were grown in the field, and data from how many plants were considered for the morphological and oil analysis. For eg. for hundred berry weight (HBW), berries were collected from how many different plants?

6. Line 137: For the oil extraction and FA analysis, the authors mention that ‘each sample was analyzed three times’. Why weren’t three biological replicates taken for this analysis?

7. Line 180-181: The authors have used 17 previously developed SSR markers and 3 newly developed SSR markers using RNA-Seq. What was the basis of selection of just 3 new markers from the RNA-Seq. Why weren’t more markers deployed for the genetic characterization?

8. Line 180: Please reframe the sentence. It appears that the authors have done RNA-seq to generate the 3 new SSR markers. Although, the RNA-seq had been done in previous study from where the 17 SSR were also developed (Reference 17).

9. Instead of ‘different origins’ that has been used repeatedly by authors throughout the text and tables, I suggest use the two different species and hybrid accessions.

10. Line 340: ‘All the primers’. Reframe this line. All primers did not give 59 bands. A total of 59 bands were amplified.

11. Line 341: ‘accounting for 86.44%’ . Incomplete sentence, 86.44% of what??

12. Line 372: the 3 subgroups have been referred incorrectly. They are IIa, IIb and IIc.

13. Line 421: ‘in comparison of populations’. Statement not clear. Please reframe.

14. Line 436: ‘gene sequences’. Are all the SSR markers used genic in nature?

15. Table 1: Could just be described as the ‘Accessions of sea buckthorn used for the study’

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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.

Attachments

Attachment

Submitted filename: PONE-D-19-17567_reviewer.pdf

PONE-D-19-17567R1

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Ruan,

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.

We would appreciate receiving your revised manuscript by Feb 10 2020 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,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Both the reviews for revision have been received. One of the reviewer is mostly satisfied with the response from authors but other still has certain queries. Both the reviewers have pointed out queries regarding presentation of data, especially the inconsistencies in the SSR data. One of the reviewer has provided a sanitised version of MS. The biggest problem Is the quality of written English. The MS requires substantial improvement in English quality. I suggest authors have to take help from a professional. They also have to address the inconsistencies in MS. Authors also need to answer all the questions raised about data.

[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 #1: (No Response)

Reviewer #2: (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 #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 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

**********

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

Reviewer #2: No

**********

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: Most of the queries have been answered and incorporated in text appropriately. However, some of them need quick attention.

1. In the abstract authors have mentioned they have used 23 SSR markers out of which 17 were previously reported. Did they assessed 6 new SSRs while in response/text the number of new makers tested is 3. Query:9 (revision 1)

2. The query 7 related to seed width and seed thickness is not answered suitably, though reference is given. Seed thickness can not be regarded as seed height. Seed thickness may be similar to seed width/diameter.

3. Answer/justification for query 12 need to be recheck and if so…..it must be mentioned in materials and methods of the manuscript.

Reviewer #2: General Comments:

The manuscript has a lot of issues with the language. Many such sentences have been highlighted in the document attached. Some of these appear as two half statements fused. At other places, the sentences lack clarity. The authors need to re-frame all such statements.

The Materials & Methods needs to be revised at places (please see suggestions). Most trait measures in various tables lack the unit of measurements. Please incorporate that.

Specific comments:

1. The text inconsistently mentions the deployment of 20 SSR primer pairs at certain places (line 357, 450, Header of Table 5) and at other places (Line 27, 41, 46, 197, 358, 373, 383, 451, 482) the use of 23 SSR primer pairs has been mentioned. The supplementary table (S4) gives sequence information for 23, while its header says 20 SSR primers. Table 5, gives information for 23 markers although the Header says 20. Please ensure that all these ambiguities are taken care of.

2. Abstract says 69 polymorphic bands, while in results 59 polymorphic bands are mentioned. This ambiguity also needs to be addressed.

Introduction:

1. Line 62: ‘Two important parameters in……..oil quantity are oil content’. Oil content cannot be a parameter of oil quality. So, this statement needs modification.

2. Line 78: ‘Due to small berries………………….artificial hybridization for elite accessions.’ The statement needs to be reframed.

3. Line 98: ‘The diversity analysis…………………………germplasm’. The authors appear to have have fused to incomplete sentences. This needs to be re-wrtitten.

All other such statements have been highlighted in the document attached.

Materials & Methods

1. Line 139: ‘There were three biological replicates………measurement’.

Do the authors mean that 300 berries were taken for the analysis? 100 berries from 2-5 plants/ accession is a good enough number for the analysis.

2. Line142: ‘…with over 20 measurements…for each’

This is not clear. Do the authors mean 20 berries per accession?? And how many plants did these berries belong to?

3. For the oil extraction, were the seeds and fruit pulp weighed prior to oil extraction to maintain some uniformity. This has not been mentioned in the M&M.

The oil contents in both seeds and fruit pulp as mentioned in Line 153 is expressed as percentage. Percentage of what? Seed/pulp weight? The authors need to clearly mention that in the M&M.

In the results (Line 278), the authors mention ‘….highest oil content (24.68%) based on dry weight.’ This means that the weight of the pulp/seed was considered. But, this has not been clearly mentioned either in the M&M or in the Table 3. The units for oil characteristic (min and max) have not been mentioned in the table.

4. Line 197: ‘Twenty-three polymorphic microsatellite loci (SSR) developed using RNA-Seq was evaluated and loci SB1-SB17 were previously reported’.

Please mention here the names of the SSR markers (SB1-SB23). Nowhere in the text have they been mentioned except for tables. Then it can be mentioned that SB1-17 were previously deployed (Ref. 14).

The authors need to clearly mention in the introduction itself that in a previous study, RNA seq analysis was done to generate SSR markers and these were tested on 31 accessions. The 17 SSR markers developed in that study have been utilized in the present endeavor for genetic diversity assessment of larger set of accessions. This description in the ‘introduction’ will bring more clarity in the text. This previous study and its outcomes should be mentioned clearly in the ‘Introduction’ so that its extension in the present study can be deciphered.

Results:

1. Line 246: ‘In previous mutilocation trials in Suiling (47°14′N, 127°06′E; 202 m) and Dengkou

(40°43′N, 106°30′E; 1053m, Inner Mongolia), the fruit characteristics of 11 large……’.

How many berries per accession were taken for this analysis? The data should be represented as + SD in Table S7.

2. Line 302: ‘Small variations were found in the proportion of linoleic acid in seed oil (40.44 – 42.87%). Its proportion in hybrids were slightly higher than in ssp. mongolica (42.87% vs 42.10%.....’

Are these differences significant?

3. Table 4: How is the oil content being measured? Total oil per gram weight of seeds and pulp or some other measure?

Tables and Figures

1. Table 1: Since the authors have already mentioned that 2-5 ramet plants were collected per accession. The columns indicating the number of plants taken per accession can be removed from the Table.

2. Table 3: The units for the min. and max values of the oil characteristics have not been mentioned in the table. Similarly mention the units of measurement for each of the component in Table 4.

3. Table 3 & 4: The different fatty acid names should be included in the first column. Example: Oleic (18:1), Palmitic acid (16:0) etc.

4. Table S1: This table again classifies all the lines used as ‘cultivars’. Are these accessions or cultivars? Please check.

5. Table S3 carries a different header than the one that has been listed at the end of the manuscript. Please change that.

6. Table S7: The header for this table has been titled as Table S5. Please correct. Also it mentions ‘two experimental fields’ although it has data from three places. So, please correct.

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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

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.

Attachments

Attachment

Submitted filename: PONE-D-19-17567_R1-comments.pdf

Attachment

Submitted filename: Comments.docx

PONE-D-19-17567R2

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PLOS ONE

Dear Dr. Ruan,

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:

Both the reviewers have agreed with the changes made in Manuscript. One of the reviewer has suggested some changes in the MS and has provided a sanitized version of MS, the authors are requested to include these changes in MS. I think we will not require any further reviewing and as and when MS is submitted with required changes, the MS can be accepted. 

•  

==============================

We would appreciate receiving your revised manuscript by Mar 30 2020 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,

Shailendra Goel, Ph.D.

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #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 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

**********

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

Reviewer #2: (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 #1: As most of my queries have been attended, the MS may accepted for publication provided the other reviewer has also reached a similar decision.

Reviewer #2: The authors have made the suggested changes and the manuscript should be accepted for publication after few minor changes as suggested below. These have also been highlighted in the manuscript attached.

1. Some text changes are suggested: Line 36, 77, 78, 84, 87, 362, 375, 452, 461, 462, 489

2. Line 307, 345: Lacks clarity.

3. Suggest changing Table 5 title to: ‘Genetic diversity analyses of 78 accessions of sea buckthorn germplasm using 23 SSR markers’.

4. As previously suggested to the authors, since for each of the traits multiple measurements were taken, please include +/- SD values for each trait/accession in Table S6.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-19-17567_R2-Comments.pdf

Diversity in sea buckthorn (Hippophae rhamnoides L.) accessions with different origins based on morphological characteristics, oil traits, and microsatellite markers

PONE-D-19-17567R3

Dear Dr. Ruan,

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.

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With kind regards,

Shailendra Goel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Both the reviewers are now satisfied by the changes made in the MS. Henceforth, I recommend the publication of this MS in PLOSOne. I thank you for your patience during the process and hope that authors will appreciate all the efforts from the two reviewers in improving the manuscript.

Reviewers' comments: