PONE-D-21-02525

Comparative transcriptomics of leaves of five mulberry accessions and cataloguing structural and expression variants for future prospects

PLOS ONE

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Reviewers' comments:

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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?

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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: Title: Comparative transcriptomics of leaves of five mulberry accessions and cataloguing structural and expression variants for future prospects

In this manuscript the authors have sequenced the leaf tissues from five species of Morus sp. namely M. indica variety K2 and V1, M. laevigata and M. serrata. and M. notabilis. Initially they have compared the various assemblers used for transcriptome assembly. They have shown that Trinity assembler performs better than the rest of the assemblers. The authors have identified common ortholog transcripts among the five species and seen their differential expression. They have also validated some genes using qRTPCR.

Further the authors have studied the SNP variations in the five genotypes and identified several. The authors have also done the GO enrichment analysis in the five genotypes.

Here are a few comments:

1. In figure 2 the authors should include the description of the contigs used for making the heat map.

2. A comparative heat map for the GO enrichment analysis can be made for all the five genotypes.

3. What are the pathways that are differentially regulated in these five species?

4. Genes that could be playing important role in plant-insect interaction could be selected and SNP identified from those could be highlighted.

5. I could not find any SRA number in the manuscript so I presume the authors have not deposited their data in the public repositories.

Reviewer #2: The article reports interesting analyses where the authors have compared the transcriptome data of five genotypes of mulberry to find out the differentially expressed genes and variants. Here, authors have also tried to find out the impact of variants on different traits that are shown by the genotypes. This study might have larger implications in various mulberry breeding programs and would be a data source for breeding experiments. Besides the scientific significance of the manuscript or the amount of data it encompasses, the manuscript requires some reworking to ensure technical soundness, clarity and readability. My specific comments on this manuscript are as follows;

Abstract: It is not well written, unnecessary extension of several experiments only. The abstract should reflect the actual outcome in a consolidated manner. It is very difficult what the comparisons mean and what they are referring to. Name of cultivars and comparisons do not match.

Major comments:

Introduction should be arranged in a more systematic manner. Research of the several recent workers specifically on the genotypes properties should be cited at relevant places. Authors should also take care of grammatical errors. Therefore, a serious rework to improve this section is advised. Authors should also discuss potential mechanisms that might have shaped these genotypes.

Authors have taken three wild types and two cultivated varieties which show contrasting characters. Although this data is given in the text, it would be better it is given in tabular format along with some characteristic feature of cultivars chosen such as stress tolerance/sensitive. Also, total read number, unigenes, transcripts can be mentioned in the same table

Methods: Here also scope exists to give a structured outlook of this section. Language and grammatical errors should also be corrected by the authors. My specific comments about this sections are given below that must be addressed.

1. Authors have used standalone version of Annocript to annotate the assembled transcriptomes which rely on several databases. They should mention the version of these databases too along with the version of the annocript tool.

2. Authors have used Proteinortho for identification of orthologs. The standalone version of this software is based on finding the best reciprocal BLAST hits. Since similarity based searches are often debated, the authors must provide the details of this step like nature of input sequences, cutoff values, e-values, version of BLAST program etc.

3. Explain algebraic connectivity in little more detail

4. Authors have tried to validate their transcriptome by means of qPCR experiments. It is advisable to give correlation between them by using a coefficient.

5. The results obtained from Gene Ontology enrichment analyses could be extrapolated for genotype specific traits. Although authors have put a section on the same, it is not reflected in the discussion portion.

6. Will authors explain what they mean by GO enrichment analyses of ‘Unique Genes’?

7. It is of utmost importance to deposit the raw sequence files, assembly to a public database for easy accessibility.

8. in the heat map and qpcr graphs, for better understanding, give gene annotation as well, in the present version only the an identity number is given.

9. Important and interesting information is given in supplementary figures related to GO analysis. Can authors present this data in main figure(s) in a way to compare varietal differences in three main figures representing cellular component, molecular function and biological process.

In the discussion section

Discussion should be based on the results that authors have produced rather than presenting their data in this section.

Conclusion should be elaborated more.

English issues:

There are many mistakes throughout the text. Authors must ensure the homogeneity, correctness across the manuscript. A few of them are given below.

1. ... the leaf transcriptomes --> transcriptome of leaves.

2....comparison b/w genotypes like KvsV should be uniform everywhere

5. as the data ----> as it

Upregulation ----> up-regulation throughout the text

Misssense ----> miss-sense

de novo ---> de novo

transabyss ---> Transabyss throughout the text

Nomenclature of species is inconsistent, some times in italics where as some times in regular

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Reviewer #1: No

Reviewer #2: No

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Comparative transcriptomics of leaves of five mulberry accessions and cataloguing structural and expression variants for future prospects

PONE-D-21-02525R1

Dear Dr. Khurana,

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 Jain

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #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 #2: Yes

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

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 #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 #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 #2: (No Response)

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Reviewer #2: Yes: Harsh Chauhan