De Novo Transcriptome Sequencing in Kiwifruit (Actinidia chinensis var. deliciosa (A Chev) Liang et Ferguson) and Development of Tissue-Specific Transcriptomic Resources

Round 1

Reviewer 1 Report

Authors have taken into account of all suggestions, and improved the manuscript sufficiently.

Author Response

Reviewer 1

Authors have taken into account of all suggestions, and improved the manuscript sufficiently.

Answer:

Thank you very much for all your suggestions and to help me to improve the manuscript.

Reviewer 2 Report

In summary, the authors have taken on board the feedback from reviewers like myself and others but improvement is still necessary. A series of additional figures have been added to the work in support of the objective stated in the paper. Some of the figures are arguably SI material rather than most-important-point-being-made to support claims made in the conclusion material. As a reader, I have understood that the outcomes of this work is to make the transcriptome, annotate it, and turn it into a resource which undoubtedly they authors did. To what end will the resource be useful in practice? Well the authors should set up a hypothesis that they can identify logical key genes from it I guess. Still not sure what hypotheses are set up in the introduction. The authors later find that a key gene is mevalonate kinase that straddles gene networks in development. I suggest the authors forshadow this clearly in the introduction. The authors do tell the reader it would be advantageous to use a transcriptomic resource in kiwifruit to identify key genes in flowering and soft fruit development, but they can set this up to read in a conventional hypothesis driven fashion. Tell the reader it is not clear what processes and genes may be important in kiwifruit. The last sentence in the introduction could be written to reflect this: '...to identify most important proteins involved in these developmental processes. Here we use our resource presented here to identify these key genes in kiwifruit development, illustrating the utility and power of this resource'. The network analyses are largely what that statement and the conclusions are based on.

Will this resource be accessible online?

Feedback to sections of the manuscript:

Results sub-heading "Refined de novo transcriptome assembly" looks good and makes sense. Maybe consider just writing out the most key data (median length, CEGMA and BUSCO scores) from Table 1 and then moving all the other data in T1 to the supplement.

Figure 2 is good.

Under the sub-heading in the Results section "Gene annotation and enrichment analysis", it is not clear to me which direction these GO term analyses are given. For instance, in shoot vs leaf in the text, is this saying that 23.1% of the top hits are for metabolic process transcripts differentially expressed higher in shoots rather than leaves? Or are these 23.1% are more highly in expressed in leaves? Figure 3 could use some customisation to fit the needs of a display item in a journal with consistent font sizes and labelling. The hyperlink blue should be changed to something readable like black. The final audience will not be clicking hyperlinks to the GO terms in the figure.

The sub-heading "Transcriptome annotation and differential expressed genes" should perhaps come ahead of the GO analysis if this is where the differential expression is actually presented. It was very confusing and honestly, I'm not sure I am understanding the logic in the presentation order. The DE analyses are what underpins the GO analyses of this vs that yes? If so then put the DE first and explain to the reader "These DE comparisons were then used to perform a series of GO analyses." Line 261 "appreciated" meaning "increased"?

Figures, particularly the network figures, need to be reviewed for clarity and consistency in font. I can read these when the document is at >200% zoom. 

Discussion: 
The authors have made the greatest progress with laying out their cases for the importance of processes and genes in the discussion based on their network analyses and other analyses. This is a pleasure to see. 

Summary:
The paper still needs work in language and keeping the figures concise and consistent. The introduction improved as well but could use a few sentences to set up/propose that the data will find interesting processes/genes. As it reads, it states developing this resource is needed and here it is, but the authors can frame their conclusions as unknowns in the introduction to demonstrate the utility of the transcriptomic resource.

Author Response

Reviewer 2

- In summary, the authors have taken on board the feedback from reviewers like myself and others but improvement is still necessary. A series of additional figures have been added to the work in support of the objective stated in the paper. Some of the figures are arguably SI material rather than most-important-point-being-made to support claims made in the conclusion material. As a reader, I have understood that the outcomes of this work is to make the transcriptome, annotate it, and turn it into a resource which undoubtedly they authors did. To what end will the resource be useful in practice? Well the authors should set up a hypothesis that they can identify logical key genes from it I guess. Still not sure what hypotheses are set up in the introduction. The authors later find that a key gene is mevalonate kinase that straddles gene networks in development. I suggest the authors for shadow this clearly in the introduction. The authors do tell the reader it would be advantageous to use a transcriptomic resource in kiwifruit to identify key genes in flowering and soft fruit development, but they can set this up to read in a conventional hypothesis driven fashion. Tell the reader it is not clear what processes and genes may be important in kiwifruit. The last sentence in the introduction could be written to reflect this: '...to identify most important proteins involved in these developmental processes. Here we use our resource presented here to identify these key genes in kiwifruit development, illustrating the utility and power of this resource'. The network analyses are largely what that statement and the conclusions are based on.

Answer:

Thank you very much for all your suggestions and comments. The manuscript has been improved. In addition, a brief comment about gibberellins and mevalonate was included in the introduction (lines 80 to 86) and the last sentence to which you refer was partially included.

- Will this resource be accessible online?

Answer:

Yes, it will. Raw data is already available. All raw reads in FASTQ format including paired-end and replicates are available from the NCBI Short Read Archive (SRA) database under bioproject number PRJNA564374 (lines 158 to 160). In addition, useful information and dissected results by plant tissue will be published with supplementary material.

 

-Feedback to sections of the manuscript:

Results sub-heading "Refined de novo transcriptome assembly" looks good and makes sense. Maybe consider just writing out the most key data (median length, CEGMA and BUSCO scores) from Table 1 and then moving all the other data in T1 to the supplement.

Answer:

I agree and thank you for your suggestion. Table 1 was modified and in the current version only total contigs, Trinity genes, average, CEGMA, and BUSCO are showed. The rest of the information was included in Table S1b of Supplementary material.

 

- Figure 2 is good.

Under the sub-heading in the Results section "Gene annotation and enrichment analysis", it is not clear to me which direction these GO term analyses are given. For instance, in shoot vs leaf in the text, is this saying that 23.1% of the top hits are for metabolic process transcripts differentially expressed higher in shoots rather than leaves? Or are these 23.1% are more highly in expressed in leaves?

Answer:

I understand the confusion, literally: “in shoot vs leaf comparisons for biological processes, the top hits GO terms were metabolic process (23.1 %; GO:0008152)...” Here we are not talking about differentially expressed genes (DEGs), we are taking into account all of the significant transcripts to each tissue comparison. This would be a way to show an overall view of each tissue comparison according to biological processes and molecular function. The detailed result of each tissue comparison is shown in Tables S2 an S3. Differential expression analysis gene by gene comes after this.

- Figure 3 could use some customisation to fit the needs of a display item in a journal with consistent font sizes and labelling. The hyperlink blue should be changed to something readable like black. The final audience will not be clicking hyperlinks to the GO terms in the figure.

Answer:

I agree and thank you for the suggestion. Figure 3 was modified and improved increasing font size and changing blue to black.

- The sub-heading "Transcriptome annotation and differential expressed genes" should perhaps come ahead of the GO analysis if this is where the differential expression is actually presented. It was very confusing and honestly, I'm not sure I am understanding the logic in the presentation order.

Answer:

Ok, I hope that after the previous explanation the doubts have been clarified. Therefore, the presentation order in the result section would be as follow:

Results

1. Refined de novo transcriptome assembly

Overall result about de assembly

2. Gene annotation and enrichment analysis

GO analysis by Panther considering leaf, flower and fruit comparisons in order to obtain an overview of biological process and molecular function involved on each tissue development.

3. Transcriptome annotation and differential expressed genes

In this sub-heading we show the functional annotation and differentially expressed genes.

4. Protein-protein interaction network

Besides, supplementary material was also reorganized.

- The DE analyses are what underpins the GO analyses of this vs that yes? If so then put the DE first and explain to the reader "These DE comparisons were then used to perform a series of GO analyses." Line 261 "appreciated" meaning "increased"?

Answer:

Thank you for your comments. DEGs mean “Differentially Expressed Genes” and this result is showed after GO analysis. GO analysis shows an overview of molecular processes and molecular function and then we analyzed the DEGs by pathway and finally we created the IPP network in order to visualize the proteins and pathways involved.

The term "appreciated" is correct, the sentence: “If we compare up and down-regulated contigs, the previous stage of each plant tissue comparison showed a lower up-regulated contigs excepting in the case of fruit at 7 vs 160 days where a higher number of up regulated contigs for fruit at 7 vs 160 days was appreciated”

 

-Figures, particularly the network figures, need to be reviewed for clarity and consistency in font. I can read these when the document is at >200% zoom.

Answer:

Thank you for your suggestion. This figure was previously improved. Pathways and protein nodes are clearly presented but I understand that the protein names are very long, however they look sharp. Anyway, the protein names are detailed in Supplementary Table 6.

-Discussion:

The authors have made the greatest progress with laying out their cases for the importance of processes and genes in the discussion based on their network analyses and other analyses. This is a pleasure to see.

Answer:

Thank you for the comments and to help me to improve the manuscript.

- Summary:

The paper still needs work in language and keeping the figures concise and consistent. The introduction improved as well but could use a few sentences to set up/propose that the data will find interesting processes/genes. As it reads, it states developing this resource is needed and here it is, but the authors can frame their conclusions as unknowns in the introduction to demonstrate the utility of the transcriptomic resource.

Answer:

Thank you very much for all the suggestions and comments. The current version of the manuscript was improved according to the reviewer’s comment including deeper corrections in the English language.

 

Reviewer 3 Report

Still manuscript focused on RNA-seq and in silico studies only. I think data should be validate by qRT-PCR to concludes something. 

Rest is fine.

Author Response

Reviewer 3

Still manuscript focused on RNA-seq and in silico studies only. I think data should be validate by qRT-PCR to concludes something.

Rest is fine.

Answer:

Thank you for your comments. I agree a qRT-PCR validation would be convenient, however, this work has been focused on in silico study in order to provide a useful transcriptomic information tool. Further studies will be focused on each specific tissue.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In the manuscript entitled “De novo transcriptome sequencing in kiwifruit [Actinidia deliciosa (A Chev) Liang et Ferguson] and development of tissue-specific transcriptomic and proteomic resources” authors performed RNA sequencing analysis of vegetative, floral and fruit growth in kiwifruit. Also performed gene annotation and enrichment analysis, DEG analysis and protein-protein interaction network. Overall, this study provides excellent information that could be used in future studies related to kiwifruit. However, some issues need to be resolved (given below) before its final acceptance.

 

1. In the title of manuscript and results, the author's emphasis on term “proteomic resources.” They performed only protein-protein interaction, so it does not seem to be the entire proteomic resource and should remove this term.
2. There is very less elucidation/explanation of the identified DEGs in various tissues in the result section. It should be provided in more detail with figures.
3. RNA-Seq data should be validated by qRT-PCR of some important DEGs.
4. Since the finding in this paper was interesting, but discussion of results needs improvements. In my opinion, they have to discuss their results and compared them with earlier and recently published papers in more depth.
5. Recently, a high-quality Actinidia chinensis and Actinidia eriantha genomes were published; these papers did not cite and utilized in the analysis of their results.

Reference:

Wu, H., Ma, T., Kang, M. et al. A high-quality Actinidia chinensis (kiwifruit) genome. Hortic Res 6, 117 (2019). https://doi.org/10.1038/s41438-019-0202-y

 

Wei Tang, Xuepeng Sun, Junyang Yue, Xiaofeng Tang, Chen Jiao, Ying Yang, Xiangli Niu, Min Miao, Danfeng Zhang, Shengxiong Huang, Wei Shi, Mingzhang Li, Congbing Fang, Zhangjun Fei, Yongsheng Liu, Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping, GigaScience, Volume 8, Issue 4, April 2019, giz027.

 

Yue, J., Liu, J., Tang, W. et al. Kiwifruit Genome Database (KGD): a comprehensive resource for kiwifruit genomics. Hortic Res 7, 117 (2020). https://doi.org/10.1038/s41438-020-0338-9

Author Response

Reviewer 1

In the manuscript entitled “De novo transcriptome sequencing in kiwifruit [Actinidia deliciosa (A Chev) Liang et Ferguson] and development of tissue-specific transcriptomic and proteomic resources” authors performed RNA sequencing analysis of vegetative, floral and fruit growth in kiwifruit. Also performed gene annotation and enrichment analysis, DEG analysis and protein-protein interaction network. Overall, this study provides excellent information that could be used in future studies related to kiwifruit. However, some issues need to be resolved (given below) before its final acceptance.

1. In the title of manuscript and results, the author's emphasis on term “proteomic resources.” They performed only protein-protein interaction, so it does not seem to be the entire proteomic resource and should remove this term.

Answer: I agree with the reviewer's comment, the term “protein resources” was deleted in the title and throughout the whole manuscript 

2. There is very less elucidation/explanation of the identified DEGs in various tissues in the result section. It should be provided in more detail with figures.

Answer: I agree with the reviewer's suggestions, new comments were added in the result section (lines 279 to 292) and a new figure was included in order to visualize the DEGs between different plant tissue comparisons (new Fig. 6).

3. RNA-Seq data should be validated by qRT-PCR of some important DEGs.

Answer: In agreement with the reviewer's opinion, I understand that would be the usual procedure, but this work covers many plant tissues and it aims to provide a useful information tool for each of the aforementioned tissues in kiwifruit. A similar approach is shown by Brian et al. (2021): “A gene expression atlas for kiwifruit (Actinidia chinensis) and network analysis of transcription factors” in BMC Plant Biology. This last study was also mentioned in the present work. In this context, further studies of qPCR validation are needed but in a new specific study.

4. Since the finding in this paper was interesting, but discussion of results needs improvements. In my opinion, they have to discuss their results and compared them with earlier and recently published papers in more depth.

Answer: I agree with the reviewer suggestions, the discussion section has been improved and new comments were added (lines 400 to 421) including 13 new references.

5. Recently, a high-quality Actinidia chinensis and Actinidia eriantha genomes were published; these papers did not cite and utilized in the analysis of their results.

Reference:

Wu, H., Ma, T., Kang, M. et al. A high-quality Actinidia chinensis (kiwifruit) genome. Hortic Res 6, 117 (2019). https://doi.org/10.1038/s41438-019-0202-y

Wei Tang, Xuepeng Sun, Junyang Yue, Xiaofeng Tang, Chen Jiao, Ying Yang, Xiangli Niu, Min Miao, Danfeng Zhang, Shengxiong Huang, Wei Shi, Mingzhang Li, Congbing Fang, Zhangjun Fei, Yongsheng Liu, Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping, GigaScience, Volume 8, Issue 4, April 2019, giz027.

Yue, J., Liu, J., Tang, W. et al. Kiwifruit Genome Database (KGD): a comprehensive resource for kiwifruit genomics. Hortic Res 7, 117 (2020). https://doi.org/10.1038/s41438-020-0338-9

Answer: These papers have been cited and duly referenced in the revised version of the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

The submitted manuscript is the valuable addition to the knowledge of Actinidia at molecular level.  Especially, the use as plant materials the different developmental stages of few kind of vegetative and generative organs may be very interesting for the readers. My question and remarks:

1) About fruits - what kind of male cultivar was used as pollinator?

2) Presented data should be discussed with the recent papers by Wu et al., 2019, as well as Tang et al., 2019, which also concern kiwifruit sequencing.

3) Don't use the term (Line 34) "Actinidia deliciosa or Actinidia chinensis deliciosa". According to the book The Kiwifruit Genome (2016; Testolin et al., as Editors), the correct and current nomenclature is: Actinidia chinensis var. deliciosa.

Author Response

Reviewer 2

The submitted manuscript is the valuable addition to the knowledge of Actinidia at molecular level.  Especially, the use as plant materials the different developmental stages of few kind of vegetative and generative organs may be very interesting for the readers. My question and remarks:

1) About fruits - what kind of male cultivar was used as pollinator?

Answer: The pollen donor was ‘Belen’ variety which has been specified in the plant material section (lines 101 to 102).

2) Presented data should be discussed with the recent papers by Wu et al., 2019, as well as Tang et al., 2019, which also concern kiwifruit sequencing.

Answer: These new references were included in the revised version of the manuscript. In addition, the manuscript was improved by adding new comments in the introduction (lines 58 to 61 and 68 to 80), result (lines 279 to 292) and discussion (lines 400 to 421) sections including 13 new references.

3) Don't use the term (Line 34) "Actinidia deliciosa or Actinidia chinensis deliciosa". According to the book The Kiwifruit Genome (2016; Testolin et al., as Editors), the correct and current nomenclature is: Actinidia chinensis var. deliciosa.

Answer: Thank you very much for your suggestion, this term was replaced throughout the whole manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript aims to address the need to understand kiwifruit -omics, namely transcriptomics, across the whole plant (except roots) in an effort to provide useful insight into this economically important plant. The collection of RNA-seq data during plant development is a powerful approach, especially if supported with phenotypic data or functional genetics studies. Unfortunately, this manuscript is strictly focused on RNA-seq and in silico studies only. The manuscript would be imporved if there were some work to identify/assign transcripts from their work to the most likely candidates in the published A. deliciosa genome for the purpose of providing additional data to help annotate a genome and to start checking their own inferences about what is important in their data. For instance, the mevalonate kinase is claimed to have increased expression/importance and possibly due to multiple genomic copies. This could be checked using reported data. The manuscript would also benefit from similar comparative work to link the author's data to what is known in different A. deliciosa datasets (or even A. arguta). 

Specific feedback:

Line 14: Not "from" China but "to" China.
Line 16: Contractions and informal language here should be written less casually. For instance, one could say: "Currently, only a small body of transcriptomic and proteomic data are available for A. deliciosa."
Botanical Latin names should be italicised.
Some sentences in the abstract are very long and meandering. 
Line 48: "Relative to other trees"? Kiwifruit grows on trees not vines?
Lines 51-63: Despite the claim by the authors of there being little published molecular work, that there are multiple genome sequences and -omics studies suggests otherwise. perhaps the authors should list these studies here but add concise about how no expression mapping of gene expression is known from commercial cultivars important to the export market.
Line 136: "Just" here is an example of the subjective and casual language the whole manuscript is written in. The extraneous words that add ambiguity or subjectivity to the results should be removed. How small does a transcript need to be to be "just" XXXbp?
Lines 184-185: it is not surprising that flowers and developing flowers would be highest in gene expression diversity. These are organs undergoing development of diverse tissues and biological functions (ex. cell cycle control during gamete development, developmental genes associated with organ patterning etc.)
Line 299: IPP? PPI I think is what is meant?
Lines 318-320: Do the authors mean to say the enzyme is a lead candidate in the developmental cycle of the plant? The citation #42 claiming multiple copies leads to higher expression (not always true if the genes subfunctionalize) is from what plant system? Could the authors use the published genomes to identify (or not identify) multiple copies of mevalonate kinase exist in Actinidia genomes?

Author Response

Reviewer 3

The manuscript aims to address the need to understand kiwifruit -omics, namely transcriptomics, across the whole plant (except roots) in an effort to provide useful insight into this economically important plant. The collection of RNA-seq data during plant development is a powerful approach, especially if supported with phenotypic data or functional genetics studies. Unfortunately, this manuscript is strictly focused on RNA-seq and in silico studies only. The manuscript would be improved if there were some work to identify/assign transcripts from their work to the most likely candidates in the published A. deliciosa genome for the purpose of providing additional data to help annotate a genome and to start checking their own inferences about what is important in their data. For instance, the mevalonate kinase is claimed to have increased expression/importance and possibly due to multiple genomic copies. This could be checked using reported data. The manuscript would also benefit from similar comparative work to link the author's data to what is known in different A. deliciosa datasets (or even A. arguta). 

Specific feedback:

Line 14: Not "from" China but "to" China.

Answer: I agree, it was modified (line 15)

Line 16: Contractions and informal language here should be written less casually. For instance, one could say: "Currently, only a small body of transcriptomic and proteomic data are available for A. deliciosa."

Answer: I agree, this sentence was revised according to the suggestions of the reviewer (line 17). In addition, the manuscript has been revised by an English spoken college.

Botanical Latin names should be italicized.

Answer: Thank you for your suggestion, all of the botanical names including those in the references are right now italicized in the revised version of the manuscript.

Some sentences in the abstract are very long and meandering. 
Line 48: "Relative to other trees"? Kiwifruit grows on trees not vines?

Answer: I agree with the comment of the reviewer, this line has been eliminated.

Lines 51-63: Despite the claim by the authors of there being little published molecular work, that there are multiple genome sequences and -omics studies suggests otherwise. perhaps the authors should list these studies here but add concise about how no expression mapping of gene expression is known from commercial cultivars important to the export market.

Answer: Thank you, this paragraph was modified according to your suggestions (lines 54 to 61)

Line 136: "Just" here is an example of the subjective and casual language the whole manuscript is written in. The extraneous words that add ambiguity or subjectivity to the results should be removed. How small does a transcript need to be to be "just" XXXbp?.

Answer: I agree with the reviewer's comment, "Just" had no sense in this context, this term has been eliminated.

Lines 184-185: it is not surprising that flowers and developing flowers would be highest in gene expression diversity. These are organs undergoing development of diverse tissues and biological functions (ex. cell cycle control during gamete development, developmental genes associated with organ patterning etc.).

Answer: I agree and thank these valuable comments to improve the final version of the manuscript.

Line 299: IPP? PPI I think is what is meant?.

Answer: Thank you, IPP was modified by PPI

Lines 318-320: Do the authors mean to say the enzyme is a lead candidate in the developmental cycle of the plant? The citation #42 claiming multiple copies leads to higher expression (not always true if the genes subfunctionalize) is from what plant system? Could the authors use the published genomes to identify (or not identify) multiple copies of mevalonate kinase exist in Actinidia genomes?

Answer: Thank you very much for your suggestion. This paragraph was modified and the reference was eliminated in order to avoid confusion. Additionally, introduction (lines 58 to 61 and 68 to 80), result (lines 279 to 292), and discussion (lines 400 to 421) sections were improved in the current version of the manuscript including 13 new references.

Author Response File: Author Response.docx