Marigold (Tagetes erecta) MADS-Box Genes: A Systematic Analysis and Their Implications for Floral Organ Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

The authors conducted a very thorough analysis of the MADS-box gene family, genes present in the genome of eukaryotes but primarily essential for autotrophic organisms for flower induction. They identified 65 genes and localized their expression in various tissues. The data analysis was carried out meticulously, and the results are clearly presented, with noteworthy graphs. The authors need to address a few comments to improve the methodology section, particularly regarding the plant material used and the reference genome. I recommend a "major revision" to ensure the authors have sufficient time to respond thoroughly.

Introduction

Introduction section thoroughly describes the MADS box TFs domains architecture, as well their broad array of functions in flower development. Within this context, I suggest including some useful references in lines 51/52 related to their role in hypertrophic inflorescence induction (inflorescence size). https://doi.org/10.3390/agriculture11070622; https://doi.org/10.3389/fpls.2023.1198909; https://doi.org/10.3390/plants12020407; https://doi.org/10.1134/S1607672918060145

Material and Methods

Subsections must be written according to the MDPI instructions for authors in italics.

For the plant material section (subsection 2.1), references 42 and 46 do not specify the details of the material used. I highly recommend providing more specific information about the inbred line of marigold utilized in the study. Please include details such as the source or provider of the material and the method of generation, if applicable. This additional information will enhance the clarity and reproducibility of your research.

Additionally, the “growth conditions” were not thoroughly specified. I suggest providing additional details related to the growth conditions. Furthermore, I recommend creating a new subsection 2.2 dedicated solely to the sampling process, which you have precisely described, and which is a crucial part of your trial.

Subsection 2.1.  Please, specify the reference genome employed for Your analysis with its entry name. Is this one: ASM3086718v1: Tagetes erecta genome assembly ASM3086718v1 - NCBI - NLM (nih.gov) ? Furthermore, provide the database employed for retrieving the genome as material of Your trial.

For each website You have to specify the date of access.

For all the software employed specify version and the company.

In line 97 should be beneficial specifying the assessment of RNA concentration and quality. So, specify the spectrophotometer (model ecc..).

Line 216 STRING database should be written with the capital letters. Please specify the version as well the access date. Furthermore, You should  incorporate the reference suggested by STRING website for supporting the project: 10.1093/nar/gkac1000

The same for Cytoscape.

Results

Results are clearly presented. Figures 5,6 and 8 necessitate a better quality of the pictures.

Discussion

The Discussion section is very informative. However, I recommend providing a rationale for the selection of the inbred line used in your study, specifically noting that it "has one whorl of ray florets in the periphery of the capitulum," and explaining the importance of this choice in your comprehensive MADS-box analysis.

Author Response

Comments 1:

(Introduction) Introduction section thoroughly describes the MADS box TFs domains architecture, as well their broad array of functions in flower development. Within this context, I suggest including some useful references in lines 51/52 related to their role in hypertrophic inflorescence induction (inflorescence size). https://doi.org/10.3390/agriculture11070622; https://doi.org/10.3389/fpls.2023.1198909; https://doi.org/10.3390/plants12020407; https://doi.org/10.1134/S1607672918060145

Response 1:

Thank you for highlighting this important point. We agree with your suggestion and have accordingly reviewed the relevant references. We have now supplemented the role of MADS-box transcription factors in inflorescence architecture, which can be found in the revised manuscript on [page 1, line 44].

 

Comments 2: 

(Material and Methods) Subsections must be written according to the MDPI instructions for authors in italics.

Response 2:

We sincerely thank you for your careful review. We have changed the secondary subsections to italics and have also addressed other formatting issues, as per MDPI's instructions. The adjustments can be found in the revised manuscript on [page 3, line 120; page 3, line 126; page 3, line 138; page 4, line 157; page 4, line 178; page 4, line 188; page 5, line 199; page 5, line 215; page 5, line 223; page 5, line 239; page 7, line 262; page 8, line 298; page 11, line 327; page 13, line 371; page 14, line 391; page 15, line 438; page 16, line 485].

 

Comments 3: 

(Material and Methods) For the plant material section (subsection 2.1), references 42 and 46 do not specify the details of the material used. I highly recommend providing more specific information about the inbred line of marigold utilized in the study. Please include details such as the source or provider of the material and the method of generation, if applicable. This additional information will enhance the clarity and reproducibility of your research.

Response 3:

Thank you for your valuable feedback. To clarify the origin of the M525B material used in my study, I have revised the references and included a detailed description of the source and generation method of the M525B material in the manuscript. The revisions are as follows: "The marigold inbred line M525B-1, characterized by a single whorl of ray florets at the periphery of the capitulum, was developed through 10 generations of self-crossing of M525B, a male fertile type isolated from the two-type line M525AB by He" (https://doi.org/10.1186/s12870-020-02644-5, https://doi.org/10.1371/journal.pone.0150892). The adjustments can be found in the revised manuscript on [page 3, line 121-123].

 

Comments 4: 

(Material and Methods) Additionally, the “growth conditions” were not thoroughly specified. I suggest providing additional details related to the growth conditions. Furthermore, I recommend creating a new subsection 2.2 dedicated solely to the sampling process, which you have precisely described, and which is a crucial part of your trial.

Response 4:

Thank you for raising this point. First, since the growth of marigold does not have any specific requirements, we have revised the section title to "2.1 Plant Material" to avoid any potential misunderstanding for the readers. Additionally, we have added a new subsection, "2.2 RNA Preparation," to provide readers with a clearer understanding of our sample preparation process. The adjustments can be found in the revised manuscript on [page 3, line 120-137].

 

Comments 5: 

(Material and Methods) Subsection 2.1. Please, specify the reference genome employed for Your analysis with its entry name. Is this one: ASM3086718v1: Tagetes erecta genome assembly ASM3086718v1 - NCBI - NLM (nih.gov) ? Furthermore, provide the database employed for retrieving the genome as material of Your trial.

Response 5:

Thank you for pointing this out. We apologize for the oversight. We have now included the reference genome information in the manuscript.’ The genome and protein sequences of marigold were obtained from the NCBI database (Tagetes erecta genome assembly: GCA_026213115.1, https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_026213115.1/). The adjustments can be found in the revised manuscript on [page 3, line 140-141].

 

Comments 6: 

(Material and Methods) For each website You have to specify the date of access.For all the software employed specify version and the company.

Response 6:

Thank you very much for your reminder. We have now provided the version, URL, access dates, and citations for each website and software, such as "HMMER version 3.3.2 program (http://hmmer.org, accessed on December 30, 2023). The adjustments can be found in the revised manuscript on [page 3, line 139-148; page 4, line 149-156; page 4, line 157-177; page 4, line 179-183; page 4, line 189-194; page 5, line 202-205; page 5, line 216-221].

 

Comments 7: 

(Material and Methods) In line 97 should be beneficial specifying the assessment of RNA concentration and quality. So, specify the spectrophotometer (model ecc..).

Response 7:

Thank you for your reminder. We have now included the model of the spectrophotometer used for assessing RNA concentration and quality. The adjustments can be found in the revised manuscript on [page 3, line 136].

 

Comments 8: 

(Material and Methods) Line 216 STRING database should be written with the capital letters. Please specify the version as well the access date. Furthermore, You should incorporate the reference suggested by STRING website for supporting the project: 10.1093/nar/gkac1000.The same for Cytoscape.

Response 8:

Thank you very much for your reminder. We have now provided the version, URL, access dates, and citations for STRING and Cytoscape. The adjustments can be found in the revised manuscript on [page 5, line 216; page 5, line 220].

 

Comments 9: 

(Results) Results are clearly presented. Figures 5,6 and 8 necessitate a better quality of the pictures.

Response 9:

Thank you for your feedback. We have improved the quality of Figures 5, 6, 7, and 8 to ensure that the content we wish to convey is clearer to the readers. The adjustments can be found in the revised manuscript on [page 10, line 310; page 11, line 322; page 12, line 343; page 13, line 367].

 

Comments 10:

(Discussion) The Discussion section is very informative. However, I recommend providing a rationale for the selection of the inbred line used in your study, specifically noting that it "has one whorl of ray florets in the periphery of the capitulum," and explaining the importance of this choice in your comprehensive MADS-box analysis.

Response 10:

We agree with your suggestion. Therefore, we have added a rationale for the selection of the inbred line and explained the importance of this choice in the Discussion section. The revised text is as follows: "To further analyze the expression patterns of MADS-box genes in marigold's ray and disc florets, we selected the M525B-1 inbred line, which exhibits clearly differentiated florets without transitional forms, as our research material." The adjustments can be found in the revised manuscript on [page 17, line 510-513].

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewed manuscript, titled "Marigold (Tagetes erecta) MADS-box Genes: A Systematic Analysis and Their Implications for Floral Organ Development” by Liu et al. is genomic analysis of the genes that contain the highly conserved and highly relevant MADS containing genes in the aforementioned organism.  This work is primarily computational in nature, and there is extensive efforts made to identify these genes, characterize their expression profiles, and to provide an analysis of as many features as possible (intron-exon structure, conservation, expansions, etc).  Overall this work provides insight into this gene family, and the authors work provides a meaningful addition for researchers.  I have no major concerns about this manuscript, however I believe that there are a few areas that I would suggest revision prior to publication of the finished work.

 

Comments:

The introduction does not read well in the format that the authors have structured it.  There are a number of very useful elements, however the structure jumps around.  Paragraph 1 is largely focused on a structural description of the conserved domains of these genes, P2 is focused on why these are important genes, P3-4 is why this organism is relevant for study and of interest.  This reviewer would recommend altering the order (not necessarily the information present) to allow for the reader to better understand the relevance and importance of this work prior to the structural binding features of the protein, etc.

The materials and methods seem clear and are appropriate in their level of detail and clarity.  As a number of online tools were used in the analysis, however, the authors need to include very specific information to allow reproducibility.  Accession dates are lacking, as are details pertaining to specific parameters used and the rationale for the specific parameters – default settings?  Any changes to default settings?  Why were the specific parameters utilized for this work and the analysis?

The authors find a meaningful amount of gene clustering as a result of this family being duplicated throughout the evolutionary history of this organism, and other related ones. 

This reviewer has a very strong interest in genomic organization – and I have authored a number of papers that are focused on the functional genomic clustering of genes.  I am including one such reference below – and as it is one that I am an author on, there is no pressure to have these authors include this work in their manuscript, however there are many appropriate others in the literature that the authors can use.  I would like the authors elaborate of the significance of the duplications and the colocalization of genes into functional groupings.

Reference:

Cittadino, G.M.; Andrews, J.; Purewal, H.; Estanislao Acuña Avila, P.; Arnone, J.T. Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya. J. Fungi 2023, 9, 523. https://doi.org/10.3390/jof9050523

Comments on the Quality of English Language

The language is fine as written.  Minor copy editing would be beneficial, I believe that the authors can accomplish this themselves.

Author Response

Comments 1:

The introduction does not read well in the format that the authors have structured it. There are a number of very useful elements, however the structure jumps around. Paragraph 1 is largely focused on a structural description of the conserved domains of these genes, P2 is focused on why these are important genes, P3-4 is why this organism is relevant for study and of interest. This reviewer would recommend altering the order (not necessarily the information present) to allow for the reader to better understand the relevance and importance of this work prior to the structural binding features of the protein, etc.

Response 1:

Thank you very much for your valuable feedback. I have carefully reviewed the introduction section of my manuscript and have made structural adjustments. The revised structure is as follows: P1 introduces the typical capitulum of Asteraceae plants, P2-3 explains the structure and functions of MADS-box genes, P4 discusses the research progress of MADS-box genes in the complex floral patterns of Asteraceae, and P5 addresses the significance of marigold as a model for heterogamous capitulum and the importance of further research. I hope this revision makes the article clearer and easier to understand for you and other readers. The adjustments can be found in the revised manuscript on [page 1, line 31-page 3, line 118].

 

Comments 2: 

The materials and methods seem clear and are appropriate in their level of detail and clarity. As a number of online tools were used in the analysis, however, the authors need to include very specific information to allow reproducibility. Accession dates are lacking, as are details pertaining to specific parameters used and the rationale for the specific parameters – default settings? Any changes to default settings? Why were the specific parameters utilized for this work and the analysis?

Response 2:

Thank you very much for your reminder. We have now provided the version, URL, access dates, and citations for online tools. Additionally, we have specified the non-default parameters used and provided explanations for their selection. The adjustments can be found in the revised manuscript on [page 3, line 139-148; page 4, line 149-156; page 4, line 157-177; page 4, line 179-183; page 4, line 189-194; page 5, line 202-205; page 5, line 216-221].

 

Comments 3: 

The authors find a meaningful amount of gene clustering as a result of this family being duplicated throughout the evolutionary history of this organism, and other related ones. 

This reviewer has a very strong interest in genomic organization – and I have authored a number of papers that are focused on the functional genomic clustering of genes. I am including one such reference below – and as it is one that I am an author on, there is no pressure to have these authors include this work in their manuscript, however there are many appropriate others in the literature that the authors can use. I would like the authors elaborate of the significance of the duplications and the colocalization of genes into functional groupings.

Reference: Cittadino, G.M.; Andrews, J.; Purewal, H.; Estanislao Acuña Avila, P.; Arnone, J.T. Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya. J. Fungi 2023, 9, 523. https://doi.org/10.3390/jof9050523

Response 3:

Thank you very much for your valuable suggestion. After carefully reviewing the article you provided, we recognized gaps in our previous discussion. As a result, we have expanded the discussion section of our manuscript to include insights on functional genomic clustering. We hope this addition clarifies the significance of the duplications and the collinearity analysis of genes into functional groupings. The adjustments can be found in the revised manuscript on [page 10, line 312-315; page 15, line 409-417].

Author Response File: Author Response.pdf