PONE-D-22-35149De novo assembly of Iron-Heart Cunninghamia lanceolat a  transcriptome using Illumina sequencing and EST-SSR marker development for genetic diversity analysis of Chinese firPLOS ONE

Dear Dr. Xiao,

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Pramod Prasad, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments:

Substantial revisions wrt to the queries raised by both the reviewers.

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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: No

Reviewer #2: No

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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: No

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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: No

Reviewer #2: No

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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: This manuscript developed EST-SSR makers for Iron-Heart Cunninghamia lanceolat based on the transcriptome data. The results might provide some information for analyzing the genetic diversity of Chinese fir.

However, there are many problems in this manuscript.

Firstly, the whole languages were very poor, and need to further be reedited and reviewed. Generally, the writing of the manuscript was used the third person. In this paper, the whole was used the first person.

Each section of the manuscript seems mixed and unrefined. In abstract, it seemed long that makes some key results not underlined. Some sentences were repeat used in the introduction and discussion. Some sentences were long and disorderly.

In methods section, the writing of genetic diversity index was nonstandard.

Within Figure 4, the abbreviation should be noted in the title.

Discussion section, there were many sentences repeat with the results. Thus, “Unigene assembly and annotation” and “Marker discovery” could be merged.

In “Transferability of SSR markers ……”, the genetic diversity level of Iron-Heart C. lanceolate far lower than those in Duan Hongjing’s study…., “The phylogenies of the red-heart Chinese fir and Iron-Heart C. lanceolat were relatively close…..” These should be much related the limited samples that make the rationality insufficient.

Reviewer #2: This piece of work demonstrates the versatility of EST-SSR markers for phylogenetic evaluation and genetic diversity analysis of C. lanceolata. Such work should be attempted in future as such markers and the primers developed are vital resources for endemic species evaluation for conservation and beneficial needs. But the presentation and the detailing of the research here needs a lot to be desired. In fact, the research results presented is fragmented and could have been detailed. Please take care of the following points to considerably improve the chances of publication in this journal.

1. English editing of the revision is a must. At most places, clarity is missing and there are insincerities in writing.

2. Title- please edit: De novo assembly of Iron-Heart Cunninghamia lanceolata transcriptome and EST-SSR marker development for genetic diversity analysis

3. Abstract is data heavy and long. Just discuss brief results for a summative understanding.

4. Mononucleotide repeats should not be considered at all. Please focus from di- to hexa-nucleotide repeats.

5. Please include RepeatMasker based repeat analysis (additional data).

5. Please include COG-based annotation results.

6. Why PAGE and silver nitrate solution was used for determination of PCR products? Why not agarose-based determination?

7. Whether the unigenes obtained were all 'coding for ORFs'?

8. How did you differentiate between KEGG and pathway analysis?

9. Please present the pfam annotations as a Table (1902 annotations were unique)

10. Please provide figure 3A and B as a table and present the motif types as a figure.

11. Discussion is weak. Please discuss your data with previous findings.

12. BioProject??

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Reviewer #1: No

Reviewer #2: Yes: Bharat Bhusan Patnaik

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Attachments

Attachment

Submitted filename: PONE-D-22-35149_review_06.03.2023.pdf

PONE-D-22-35149R1De novo  assembly of Iron-Heart Cunninghamia lanceolata  transcriptome and EST-SSR marker development for genetic diversity analysisPLOS ONE

Dear Dr. Xiao,

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.

==============================Please make necessary changes suggested by the reviewers.==============================

Please submit your revised manuscript by Sep 30 2023 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,

Pramod Prasad, Ph.D.

Academic Editor

PLOS ONE

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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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

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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 #3: Yes

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

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 #3: (No Response)

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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 #3: Yes

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6. Review Comments to the Author

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

Reviewer #3: PONE-D-22-35149_R1.

Manuscript entitled “De novo assembly of Iron-Heart Cunninghamia lanceolata transcriptome and EST-SSR marker development for genetic diversity analysis” by Liu et al describes transcriptome sequencing and annotation of Iron-Heart C. lanceolata, mined SSRs from transcriptome and development of 15 polymorphic EST SSRs. It adds 15 novel SSR markers to the valuable species having less genomic resources. The experimental design and the approaches used in this work seem both correct for the most part. Overall, the article is informative and written well. However, in my opinion, some aspects of the manuscript need to be revised before considering this work suitable for publication.

Following are few comments based on the R1 copy

1. Line # 125-130: explains the 3 objectives. I feel the third objective is redundant and it forms the part of 2nd objective. This could be modified suitably.

2. Line #340 & 465: Word transferability is used to denote the SSR markers amplifications in accessions from different regions. But the term transferability is used for their amplification or applicability across species i.e., cross species amplifications. So, usage of this term could be avoided here.

3. Table 4: Generally, the SSRs are classified as class I (>20 bp) & ClassII (12-20 bp) based on size of repeat motifs. Which allows the users to select markers. Usually, Class I are more polymorphic. Please add few lines in the results and discussion based on this classification

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Reviewer #3: Yes: Dr. Siddanna Savadi

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De novo  assembly of Iron-Heart Cunninghamia lanceolata  transcriptome and EST-SSR marker development for genetic diversity analysis

PONE-D-22-35149R2

Dear Dr. Xiao,

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.

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Kind regards,

Pramod Prasad, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript is now suitable for publication. 

Reviewers' comments: