PONE-D-24-47853In-depth investigation of genome to refine QTL positions for spontaneous sex-reversal in XX rainbow troutPLOS ONE

Dear Dr. Phocas,

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 manuscripts represents high quality work of considerable interest to the readers of PlosOne. The concerns of the reviewers and editors center on flow and writing, not design or analyses. The main issues are the following.

1. Provide details on bioinformatic analyses and check on comments about analyses by reviewer 1.
2. Provide better flow (thread) through the manuscript.
3. Rev 2 asks for a broadened discussion of the spontaneous masculinization, in ecological context (can shift balance in discussion towards this point).
4. Related to that (rev2) the interaction of rearing temp and genetic factors, is interesting beyond students of aquaculture. Feel free to integrate these ideas into the discussion, again by removing or condensing other points in that section.
5. The text needs to be shortened (rev 3), but some things also need clarifying (rev 1, mainly to do with analyses and validation of findings).

Please submit your revised manuscript by Jan 23 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

The manuscripts represents high quality work of considerable interest to the readers of PlosOne. The concerns of the reviewers and editors center on flow and writing, not design or analyses. The main issues are the following.

1. Provide details on bioinformatic analyses and check on comments about analyses by reviewer 1.

2. Provide better flow (thread) through the manuscript.

3. Rev 2 asks for a broadened discussion of the spontaneous masculinization, in ecological context (can shift balance in discussion towards this point).

4. Related to that (rev2) the interaction of rearing temp and genetic factors, is interesting beyond students of aquaculture. Feel free to integrate these ideas into the discussion, again by removing or condensing other points in that section.

5. The text needs to be shortened (rev 3), but some things also need clarifying (rev 1, mainly to do with analyses and validation of findings).

6. Note, thank reviewers (one named)

[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

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: General comments

The present manuscript aims to replicate and refine the position of QTL previously identified for being associated with sex reversion in rainbow trout. To achieve this goal, the authors deploy a panel of methods that could be summarized in the following sequence: 1-sequencing females with progeny showing extreme (high and low) frequencies of reversed males. 2- Identifying SNPs within QTL regions and polymorphic in the females from (1). 3-Test QTL (Fisher exact test) on six population using SNPs from (2). 4- test all SNPs from (1) withing QTL for association with sex reversion using random forest and DAPC. 5- annotation of genes near candidate SNPs.

The manuscript is well written and quite didactic in the description of the background and methods. However, some important details are still missing in the method section and should be clarified. The methods employed in the manuscript are many, and as reader, I missed a logical thread thought the methods and results section. This could be achieved with a short introduction paragraph at the beginning of each section letting the reader know what type of information is expected from the analysis, and how it complements the analysis from the former section.

The word “validation” is used along the manuscript to describe different things, this has a potential to confuse the reader. There is a general aim to validate the QTLs from Fraslin et al. There is also an internal validation dataset within random forest. And finally, from my understanding you used a separate discovery and validation population for the Fisher test, however this remains to be clarified. I found it difficult to grab how the loci detected in the discovery population were matched in the validation group. The only mention of it is in Line 219, but I found it unclear.

Specific comments

Line 165-176. Here you skip many details about the sequence mapping, SNP calling and SNP filtering methods. I think that the exact parameters used in GATK, SAMtools etc should be given in the method section. In addition, the number of SNPs/indel reported here seems very high (about one for every 80bp if we consider the USDA_OmykA_1.1 genome is 2.3Gb long). This number is not completely surprising for a brut output from Mpileup, however the 21.10^6 SNP remaining after filtering (still one for every 110 bp), sounds like a very high density of good quality SNPs. I would therefore like to have a description of the parameters/ criterion that were used for filtering the SNPs as well as some basic statistics from the mapping (mean coverage ±sd).

Regarding the analysis performed with the SNPs from full sequence data (random forest & DAPC), you only use the genomic regions reported previously by Fraslin et al, however, if I understand correctly, you have sequenced the full genome of the females. You could thus compare the distribution of the test between putative QTL region and non-QTL regions. This would provide with an empirical null distribution of the test (Gini index & allele contribution to discriminant axes).

Table3: I don’t understand the reason for splitting the Omy1 QTL into two where Omy1-a stops at 67,960,401 bp and Omy1-b starts at the next base 67,960,402. I also see that each QTL has one given position (based on the Arlee reference genome), but despite the table legend, I didn’t find any comparison between these QTL positions and the QTL reported by Fraslin et al.

Line 384: This could be reformulated. How did the present results make LOC110527930 a less good candidate and tlx1, fbxw4… better ones? Fig3 is a dapc plot, I am not sure how it illustrates the point. (Figure numbers might have been mixed in the text, please check)

Line 419 ACP -->PCA

Line 419: What are “all those variants”? Is that the set of 33,731 SNPs? You use different subsets of data and analysis methods along the manuscript. It is thus a good idea to keep using precise wording and not hesitate to remind the reader which data you are currently commenting.

Reviewer #2: The article by Dehaullon and collaborators investigates the mechanisms underlying spontaneous masculinization in XX all-female populations of rainbow trout (Oncorhynchus mykiss). Despite the species having an XX/XY sex determination system, a small proportion of males or intersex individuals are consistently observed in these populations. This phenomenon, attributed to heritable minor sex-modifier genes, poses challenges for selective breeding and aquaculture practices. The authors focused on validating previously identified DNA markers associated with spontaneous masculinization in six French farmed populations, targeting QTL regions on chromosomes Omy1, Omy12, and Omy20.

The genome-based approaches and statistical analyses used by the authors have effectively identified functional candidate genes that may influence the reduction of germ cell proliferation and the repression of oogenesis in XX individuals. The methodology used is particularly novel, and the large volume of data generated is a significant strength of the study. Genes such as syndig1, tlx1, and hells on Omy1, along with khdrbs2 and csmd1 on Omy20, have been highlighted for further functional studies, including their interaction with environmental factors like rearing temperature. These findings provide valuable insights for improving sex-ratio control and producing more consistent all-female populations, which are preferred in aquaculture due to their delayed maturation and reduced susceptibility to diseases.

The work is highly interesting and generates a substantial amount of useful information.

I have a few minor comments for consideration:

• The authors highlight that spontaneous masculinization is a highly heritable trait controlled by minor sex-modifier genes, with several QTL regions previously identified. While the importance of monosex populations in aquaculture is well established, the implications of this phenomenon in wild populations require further exploration. Even if spontaneous masculinization occurs at a low frequency in nature, its potential effects on population dynamics, genetic diversity, or ecological stability are worth investigating. Addressing these aspects would not only broaden the ecological context of the study but also underline the relevance of understanding this process beyond its aquaculture applications.

• The conclusion highlights the challenge of undesirable masculinized progeny and the need to better understand the combined effects of genetic and environmental factors on spontaneous maleness in all-female stocks. Given the role of temperature in sex reversal, I suggest that future research could explore in more detail the interactions between genetics and rearing temperatures. Specifically, it would be interesting to consider how these factors might be controlled to achieve the desired all-female progeny, particularly when using free-hormone neomales. Additionally, it could be valuable to outline potential experimental approaches to assess the influence of rearing temperatures at different developmental stages. Exploring whether specific temperature thresholds or genetic combinations could minimize masculinization while optimizing production efficiency may also provide important insights. I believe that discussing these potential avenues for future research, based on the findings of this study, would help to further contextualize the results and expand their applicability to sex ratio management in aquaculture.

I trust that addressing these aspects will further strengthen the manuscript and provide additional perspectives on how to advance the field.

Reviewer #3: The proposed manuscript “In-depth investigation of genome to refine QTL positions for spontaneous sex-reversal in XX rainbow trout” significantly contributes to the study of evolutionary biology, particularly in understanding the evolution of sex determination and its pathways. The manuscript is technically sound and written in high-quality English. Impact of the study is well highlighted and the statistical analyses are appropriately chosen and used. It would be beneficial if the authors included Sanger sequencing results for selected SNPs, especially those that are 100% sex-linked. However, the manuscript is undoubtedly of high quality and meets the requirements for the Plos One journal. I believe it will also be of interest to readers of the journal. I have only minor suggestions:

1) Manuscript is very long and requires the readers’ full intention. The introduction spans more than three pages, and the discussion is more than 9 pages long. I think this extensive length may diminish the reader's interest. Some parts are explained in great detail and I suggest shortening these sections where possible.

2) Line 71: “primary sex determination”. Primary sexual differentiation sounds better to me.

3) Line 100: “follows” instead of “follow”?

4) Line 101: “testis or ovary” instead of “testis and ovary”?

5) Some abbreviations are defined more times (e.g., GSD). Some abbreviations do not have definitions (TGF-β, QTL).

6) Line 170: A period is missing behind a citation.

7) Line 612-613: If I follow this sentence well, it says that the gene gld-1 is localized in cytoplasm. It needs to be reformulated.

8) Line 628: “fertile” is duplicated

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes:  Martin Knytl

**********

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<div>PONE-D-24-47853R1In-depth investigation of genome to refine QTL positions for spontaneous sex-reversal in XX rainbow troutPLOS ONE

Dear Dr. Phocas,

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 text is quite verbose and the results often swing from past to present tense. With specks of MS thesis wording, where the sentences become chatty and winding using “looking into …” and “favorite causative gene” In particular shorten the gene talk section, from line 512 to 737. Not all of those points are of nr. 1 priority. And make sure you tone down talk of causative relationships, the associations are weak and effects sizes generally small. This rewriting effort could be led by the principal investigator, to train the student in a more succint writing style.

Minor points (examples of writing fixes, many other similar instances are found in manuscript, urge you to find them and fix). See accompanying.

Please submit your revised manuscript by Apr 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

PLOSsex2

The manuscript is greatly improved and the two reviewers were mostly content with the changes made.

The reviewers are generally happy, except with the length and lack of definitions of abbreviations.

Main point

The text is quite verbose and the results often swing from past to present tense. With specks of MS thesis wording, where the sentences become chatty and winding using “looking into …” and “favorite causative gene” In particular shorten the gene talk section, from line 512 to 737. Not all of those points are of nr. 1 priority. And make sure you tone down talk of causative relationships, the associations are weak and effects sizes generally small. This rewriting effort could be led by the principal investigator, to train the student in a more succint writing style.

Minor points (examples of writing fixes, many other similar instances are found in manuscript, urge you to find them and fix).

Line 32

Is “located” needed?

Line 36

“to precise their functional roles as” strange wording?

Line 59 and 62

Add references. Check if further citations are needed for other factual statements.

Line82

“is changed to the opposing pathway,” can you reword.

Line 120

Turn into past tense “Currently, there are” to “…have been”?

In all animals or just fish?

Line 135

Reword “is a repeatedly phenomenon observed “

Line 163-4

Fix sci. annotation of numbers “9.252 107 “

Line 209

Add space “instructions.After”

Line 214

Spell check.

Line 325

Reword “Among the latter,” and “had at least an effect”

Line 333

Use regular PLOS one style when referrring to supplements “given in S1 Supplementary Tables, S3 Table”

Line 333-35 “with indication of the level of significance in those populations, as well as the corresponding positions of SNPs on both reference genomes” this can be skipped.

Line 335

Use “two or more” rather than “at least two” Check if this wording is used elsewhere!

Line 341

In table 2 there is some ambiguity on the annotation. “Close to …” is used in some cases but in other exact distance (in kb) is used. Use one style or the other.

Line 347

Shorten “for the QTL that are given in Table 3” to “for the QTL (Table 3)” Please see if you can shorten the manuscript with finding more examples of “extra words” of this type.

Line 349

Reword “we enlarged the search of QTL in three different regions”

Line 355

Suggest title “redefined boundaries…” Also, suggest that you add columns with the “old” boundaries, instead of using footnotes. Makes the data more easily apprehended and reused.

Line 367

Spell out Random Forest (RF) here. Also, it is not clear from the text that this is a sliding window approach (can you specify the window size here?)

Line 384

Drop “we estimated”

Line 389

Verbose sentence, can you shorten? “Due to the very small size of our dataset, it is likely that we had too many variables in the full RF analysis, creating some « noise » by considering over 1,100 haplotypes together in the analysis to classify our 23 individuals”

Line 392

Shorten “Looking at the locations (Fig. 2) of the best ten RF ranked haplotypes on the 2 Mb evaluated on Omy1 (see S1 Supplementary Tables, S5 Table), they fell” to

“The best ten RF ranked haplotypes in the 2 Mb on Omy1 (Fig2, see S1 Supplementary Tables, S5 Table), fell”

Line 410

Can drop “All”

Line 413

Reword, don’t think you should have a favorite gene! “initially was our favourite causative gene”

Line 416

Reword “the first three best haplotypes to explain sex-reversal were”

Line 420

Strange wording of sentence, “unfortunately” should not be used!

Figure 2 and 3 legend. The RF colours score is not explained, is red or blue associating??

Figure 2 and 3. Related to above, maybe a unicolour scale makes more sense than a two colour scale, since this denotes a scale from 0-1? Also, the lines corresponding to each panel, Fisher primary, discorvery DAPC are not clearly explained. Why are there two lines for FP, and one for DAPC? Should the different regions be labelled A, B, C? (with OmyID as secondary headings?)

Line 430

Strange wording “of the best haplotypes to explain sex-reversal across all the”

Line 436

Reword “appeared then as the subsequent haplotypes ranked” to “appeared in the RF haplotype analyses, ranked”

Line 438

Strange wording “best-ranked”, find alternative and fix throughout manuscript!

Line 450

Reword “On the contrary, the DAPC allowed us to perfectly discriminate” to “On the contrary, the DAPC perfectly discriminated…” Many more cases where the text can be changed from first person to passive description, and extra words trimmed. Like the sentence preceeding this one.

Line 455

Indicate how many variants, “PCA of the XX variants in the” and replace “best” with some other word.

Line 459

Reword this sentence “In S1 Supplementary Tables, S10 Table are given the genotypes for the 100 best SNPs that …” this is example of “Thesis” writing, that can be shortened and made more consise.

Line 478

Replace “playing a role on” with “associating with” and state that this is “female to male” reversal!

Line 500

Shorten and reword to “was the motivation to further investigate the variants in larger genomic”

Line 505

Reword “population A although it was closely related”

Line 527

Most likely, not very likely.

Line 613

Put table 5 as a supplemental table. Is of minor importance!

Line 620

Reword “has been pointed out as one of”

Line 741

Reword “this genetic influence”

Line 749

Reword to “Indeed, the high heritability of spontaneous maleness in XX individuals [27]”

Line 751

Check verb “Improved” and drop “then” from sentence.

Line 773

Reword to “we found in several French… that variants in some chromosomes associated…”

[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

Reviewer #3: 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

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: all comments have been addressed and the manuscript was modified accordingly. I have no further request.

Reviewer #2: All suggested changes and recommendations have been incorporated into the revised version of the manuscript. The authors have addressed all reviewer comments, and the manuscript has been improved accordingly.

Reviewer #3: The proposed manuscript has been revised and improved. The authors have addressed all the recommendations and I agree with their responses. While the article remains lengthy, I understand that it is necessary to provide a comprehensive understanding of the multiple topics covered in the manuscript. However, one suggestion still needs to be considered: not all abbreviations have been defined. Please define the abbreviation ‘omy’ in both the abstract and the introduction, and ‘QTL’ in the abstract. I believe that the study has a sufficiently high impact and fits well with the scope of the PLoS One journal. I look forward to seeing the published version of the paper.

**********

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: Yes:  Alejandro S. Mechaly

Reviewer #3: Yes:  Martin Knytl

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

In-depth investigation of genome to refine QTL positions for spontaneous sex-reversal in XX rainbow trout

PONE-D-24-47853R2

Dear Dr. Phocas,

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.

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Kind regards,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

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