PONE-D-21-07853

Development of 16 novel EST-SSR markers for species identification and cross-genus amplification in sambar, sika, and red deer

PLOS ONE

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Mukesh Thakur, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

I now checked the manuscript, entitled "De novo characterization of the sambar antler velvet transcriptome and EST-SSR development for cross-genus species-identification" . I found it significant as the developed new EST-SSR markers can be applicable for population genetic studies of deer species. Based on two reviewer's comments and my own evaluation of the contents, I would recommend manuscript as "Major Revision" and would advise reviewer to estimate the genotyping errors for the markers prioritized as the significant occurrence of allele drop out, null alleles, false alleles may affect the overall explanation/outcome of the results. I suggest authors to consider the reviewer's comments and revise the manuscript accordingly.

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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: No

Reviewer #2: 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

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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: Comments:

The manuscript “Development of 16 novel EST-SSR markers for species identification and cross-genus amplification in sambar, sika, and red deer.” by Hsiao et al. developed new EST-SSR markers for genetic identification of deer species. These markers demonstrate applicability across different genera and proved suitable for identification and phylogenetic analyses across deer species. The manuscripts is well written and developed SSR markers to resolve different issue related to the deer species conservation. Although the use of the methodology and analytical approaches seem appropriate, but there are some flaws in presentation of results and it is advised to authors to incorporate suggestions:

1. Give reference in line Number 51.

2. Among the 16 SSR markers selected for the species identifications, eight markers showed lower PIC (polymorphism Information Content) value i.e. <0.5, which is not acceptable for Species, Individual and population Genetic structure Identification. Lower PIC value may reduce the discriminatory power of theses markers may reduce. Authors need to Clarify that why they are selecting these markers.

3. Authors did nice effort to screen SSR markers and demonstrated their use in the species identification but I could not see any effort to estimate the genotyping error. Genotyping error is inherent problem with SSR markers. Especially with degraded samples (like Antlers, Hair and Fecal samples). Presence of genotyping error (allele dropout and null allele) may mislead the shorting of population/species based on the allele frequency clustering. Therefore, authors need to address the Genotyping error issue for all the 16 selected markers.

4. Relatedness assessment is need to justify that the tested samples did not include only closely related individuals are not present in the data.

Reviewer #2: Reviewer comment:

In recent times, the population status of many deer species are declining with few species on the verge of extinction. Most of the published population genetic and hybridization studies on deer species have used cross species amplification microsatellites. The current study entitled "De novo characterization of the sambar antler velvet transcriptome and EST-SSR development for cross-genus species-identification" tries to fill the lacunae by designing microsatellites for Sambar and tested their efficacy in other two deer species.

The study holds significance. The authors tried to answer majority of the earlier reviewer’s comments. But still few sections are confusing and require changes to be made especially the marker designing, selection and annotation sections for better understanding for readers. Moreover, I suggest the authors to remove transcriptome and annotation part in the results and discussion sections as it has no significance in this study.

Few suggestions and questions :

Introduction: Hypothesis is not well established in the last paragraph of Introduction.

Line 80-81: Give citation for the sentence

Line 156-162: How many SSR’s were retained finally for primer designing?

Line 163-165: Elaborate the methodology a bit by including the wet lab work and any other software used in selecting 55 primers from a total of thousands of SSR’s

Line 168-169: I believe “ Information on twenty-six polymorphic markers based

on our sambar transcriptome sequence is listed in Table 1” should be moved to results. Also there is no proper justification for reporting only 26 primers out of 55 in table 1.

Line 170: Report if you have information on chromosome number in Table-1 from which the loci is picked. I believe the chromosome number is important in population genetic analysis to avoid any linkage disequilibrium.

Line 187-188: Reason for selecting 16 primers out of 55 selected and 26 in Table 1 is missing. It’s too confusing, please clarify wherever you are reducing the number of microsatellites.

Line 194: SSR sequence validation- I believe this section should go before finalizing the SSR’s for further analysis. Also mention how many microsatellites were used for validation.

Line 215-236: Though most of the above questions were answered here in these paragraphs, it’s too confusing and I believe it should be in methodology section for better understanding for readers.

Line 237: Report results of HWE and LD tests too for each species in Table 2 as they are crucial for population genetic analysis.

Line 237-238: I suggest you to merge both Tables 2 and 3 and report single table with basic diversity estimates for all three species.

Line 253-254: Overall and individual species wise PIC values were very less. Can you comment on the reliability of selected 16 markers for its use in other population genetic and hybridization studies?

Line 272: Remove asterisk marks on the FST p-values as all are significant.

Line 282-286: Move to methodology section.

Line 351: Replace ‘they’ with ‘the’.

Line 426-427: Mention population genetics and hybridization studies in the final sentence of conclusion section.

S1 Table: I suggest S1 table should be moved to methodology section as it more clear than writing part.

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Reviewer #1: No

Reviewer #2: No

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Development of 16 novel EST-SSR markers for species identification and cross-genus amplification in sambar, sika, and red deer

PONE-D-21-07853R1

Dear Sir/Madam,

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,

Mukesh Thakur, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Pls include estimates of genotyping error and relatedness as supplementary file before processing the MS for formal acceptance.

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

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

**********

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: In the revised manuscript authors responded to all the questions except questions 3 and 4. Therefore I still have a concern about these two comments given below:

Comments 1: In my previous review, I suggested for estimation of Genotyping error (allele dropout and null allele). In the response, the author stated that they checked by Program micro checker and there was no sign of error. In this regard, the author needs to add a supplementary table for the output of Microchecker and add it to the revised manuscript.

Comments 2. Authors need to estimate pairwise relatedness using Queller and Goodnight relatedness estimators as implemented in GENALEX 6.41 and the maximum likelihood method as implemented in ML-RELATE. These estimates will give a better representation of relatedness levels in the population.

Reviewer #2: The authors had made all the necessary changes to the revised manuscript as suggested by the reviewers. The revised manuscript now looks more clear and can be published.

Even though, authors have explained well in detail regarding considering the loci with lower PIC values, I still believe the loci have limitation. Moreover, table 2 can be removed as the values are based on 55 loci which are not further used in any analysis.

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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: Yellapu Srinivas