Weighted Gene Co-Expression Network Analysis Reveals Key Pathways and Hub Genes Associated with Successful Grafting in Pecan (Carya illinoinensis)

Round 1

Reviewer 1 Report

I have reviewed the manuscript by Mo et al. entitled "Weighted gene co-expression network analysis identifies key pathways and hub genes associated with successful grafting in pecan," which describes oxidative detoxification and hormone signaling as the probable key pathways for the successful grafting of pecan. They further identified 48 hub genes identified here might be the key ones that led to the divergence of graft survival rates among different pecan cultivars.

The manuscript is nicely written and presents detailed analyses. There are some improvements which must be considered before making the final decision. 

1- In the introduction section, the authors have used most of the old references/reviews of literature. It is suggested to update the review of the literature and present literature from the last 5 years to present the current state of knowledge. Authors can use old literature only if there is no other option and mention there that it is an old finding

2- Present hypothesis and objective clearly 

3- In conclusion, instead of summarising the results, present 1-2 key findings, novelty and propoects 

Author Response

I have reviewed the manuscript by Mo et al. entitled "Weighted gene co-expression network analysis identifies key pathways and hub genes associated with successful grafting in pecan," which describes oxidative detoxification and hormone signaling as the probable key pathways for the successful grafting of pecan. They further identified 48 hub genes identified here might be the key ones that led to the divergence of graft survival rates among different pecan cultivars.

The manuscript is nicely written and presents detailed analyses. There are some improvements which must be considered before making the final decision. 

Response: Thanks very much for your comments.

 

1- In the introduction section, the authors have used most of the old references/reviews of literature. It is suggested to update the review of the literature and present literature from the last 5 years to present the current state of knowledge. Authors can use old literature only if there is no other option and mention there that it is an old finding.

Response: Thanks very much for your constructive comments. Indeed, there are some old references in our previous version, we have replaced some of them with the last 5 years` literatures.

 

2- Present hypothesis and objective clearly.

 

Response: Thanks very much for your thoughtful comments. In our latest version, we provide our objective clearly in the introduction part (Page 2, Line 77-81).

 

3- In conclusion, instead of summarizing the results, present 1-2 key findings, novelty and prospects.

 

Response: Thanks very much for your advice. We only present our key findings and the related prospects in the revised manuscript.

 

Reviewer 2 Report

Dear Editor-in-Chief of forests journal (Genetics and Molecular Biology section)

Hi and have a good day

I reviewed the article entitled” Weighted gene co-expression network analysis identifies key pathways and hub genes associated with successful grafting in pecan” written by Zhenghai  et al. The introduction, material and methods, results, and discussion show the investigation was achieved in a good manner and this research has some novelty. However, I think major corrections are necessary to improve the quality of this article for publication in the forests journal. So I suggest revision for this article in the result, discussion, and another part of MS as follows:

 

Title of article

Please bring the Latin name of the pecan into brackets.

 

Abstract part:

Application of the present study in plant breeding was brought at the end of the abstract in one or two lines.

Introduction part:

After line 42, please refer to common or specific genes involved in grafting healing esp. in pecan and in another tree in three or four lines.  

Material and methods part:

-In material and methods only used DEseq2 package for obtaining DIGs. Why the authors did not use another package like limma and so on to compare the result of DEGs and select the better DIGs?

- Please bring the R package WGCNA commands that were used in this MS as supplementary files.

-In this part, the modification could be done corresponding with the correction of the results.

Results part

-One Venn diagram was drawn between two cultivars, time treatments, and finally, the common and different DIGs were reviewed.

-In figure 2b: The spot of numbers determines on the picture.

-In figure 4b: what does it mean the eigengene expression? Is it expression value?

-In figure 5: line 242 green model must be the tan model. Please check again.

-Table 1: Please bring this table to the list of supplementary tables.

-Please bring annotation of hub genes (such as gene accession, gene symbol, gene function, and so on) that are listed in Figure 6 in one table.

- Is it possible for authors to bring real-time PCR analysis into MS for some of the hub genes detected in this research to confirm of obtained results?

-co expression network for hub genes of the tan and green model was drawn and the hub gene was detected again and interpreted in the result.

-Promoter variants of the hub gene are traditional. For improving article quality, please investigate or extract transcription factors of hub genes and bring the information of them in the results and discussion part.

Discussion part

-In this part, most of the discussion was achieved on oxidative stress and hormones. Please discuss grafting healing, callus formation, and so on in one paragraph.

-Lines 346-359 are not related to the result of this MS. please delete these lines and instead of them, explain the hub genes you obtained in the result.

- The conclusion part is like the abstract, please rewrite it again (different from the abstract) and also give suggestions for future works.

 

Best wishes

 

 

 

   

 

 

 

 

 

 

 

 

 

 

Author Response

I reviewed the article entitled” Weighted gene co-expression network analysis identifies key pathways and hub genes associated with successful grafting in pecan” written by Zhenghai et al. The introduction, material and methods, results, and discussion show the investigation was achieved in a good manner and this research has some novelty. However, I think major corrections are necessary to improve the quality of this article for publication in the forests journal. So, I suggest revision for this article in the result, discussion, and another part of MS.

 

Response: Thanks very much for your comments.

 

Title of article

Please bring the Latin name of the pecan into brackets.

 

Response: Thanks very much for your comments. We have provided it in the latest version.

 

Abstract part:

Application of the present study in plant breeding was brought at the end of the abstract in one or two lines.

 

Response: Thanks very much for your constructive comments. The future application of our results is added in the revised manuscript.

 

Introduction part:

After line 42, please refer to common or specific genes involved in grafting healing esp. in pecan and in another tree in three or four lines. 

 

Response: Thanks very much for your advice. We have re-organized the related sentences (Page 1, line 39-44).

 

 

Material and methods part:

-In material and methods only used DEseq2 package for obtaining DEGs. Why the authors did not use another package like limma and so on to compare the result of DEGs and select the better DEGs?

 

Response: Thanks very much for your insightful comments. As DESeq performs the best with the least number of false positives among the commonly used programs (edgeR, DESeq, and limma; PloS one, 2014, 9(8): e103207), we used this program for DEG detection. If we use more than one program for detecting DEGs and select the overlapping DEGs for analysis, the number of genes subjected to WGCNA will be decreased and some findings might be lost thereby. Based on these concerns, we only used DESeq for DEG analysis.

- Please bring the R package WGCNA commands that were used in this MS as supplementary files.

 

Response: Thanks very much for your comment. We have provided it as supplementary Table S1 in the revised version.

 

Results part

-One Venn diagram was drawn between two cultivars, time treatments, and finally, the common and different DEGs were reviewed.

 

Response: Thanks very much for your comment. Since graft-responsive DEGs is our main concern and the controls (expression values at 0 day after grafting) for the two cultivars were different, we divide the Venn diagram into two subfigures and the different and common DEGs are also visualized by the two Venn diagrams.

 

-In Figure 2b: The spot of numbers determines on the picture.

 

Response: Thanks very much for your comment. To make the two subfigures in Figure 2 be consistent, we also add spots in Figure 2b. The caption concerning the numbers adjacent the spots is supplied accordingly in the new manuscript.

 

-In figure 4b: what does it mean the eigengene expression? Is it expression value?

 

Response: Thanks for your comment. Eigengene (ME) is the first principal components of a given module and represents the general expression of module genes (Section 2.4, Page 3, line 142-143). In our previous manuscript, ‘Eigengene expression’ provided in Figure 4b is not appropriate, and we replace it with ‘Eigengene value’ in the revised manuscript.

 

-In Figure 5: line 242 green model must be the tan model. Please check again.

 

Response: Thanks very much for pointing out our mistake. We have revised it accordingly and we are very sorry for the mistake.

 

-Table 1: Please bring this table to the list of supplementary tables.

 

Response: Thanks very much for your advice. We have provided it as supplementary Table S5.

 

-Please bring annotation of hub genes (such as gene accession, gene symbol, gene function, and so on) that are listed in Figure 6 in one table.

 

Response: Thanks very much for your comment. The information of hub genes in both tan and green modules were listed in Table S4 in the revised version.

- Is it possible for authors to bring real-time PCR analysis into MS for some of the hub genes detected in this research to confirm of obtained results?

 

Response: Thanks very much for your comment. As RNA-seq has become a precise way for gene expression detection, we chose not to perform an additional qPCR experiment.

 

-co expression network for hub genes of the tan and green model was drawn and the hub gene was detected again and interpreted in the result.

 

Response: Thanks very much for your comment. We draw the co-expression network, as it can present the connectivity of a hub gene clearly. Expression of hub genes is presented to indicate their possible difference between the two cultivars.

 

-Promoter variants of the hub gene are traditional. For improving article quality, please investigate or extract transcription factors of hub genes and bring the information of them in the results and discussion part.

 

Response: Thanks very much for your constructive comment. Transcription factor can directly regulate the expression of genes. In terms of this, it is more important than the other hub genes. In the revised version, we extract the transcription factors in the result section (Page 9, line 279-283) and discussion them in the discussion part (Page 11, line 378-381).

 

Discussion part

-In this part, most of the discussion was achieved on oxidative stress and hormones. Please discuss grafting healing, callus formation, and so on in one paragraph.

 

Response: Thanks very much for your insightful comment. In the latest version, we use one paragraph to indicate that cell proliferation and vascular bundle development were not the key pathways leading to a low grafting survival rate of pecan (Page 11, line 356-363).

 

-Lines 346-359 are not related to the result of this MS. please delete these lines and instead of them, explain the hub genes you obtained in the result.

 

Response: Thanks very much for your comment. We have deleted the relevant text and briefly discuss the underlying functions of several hub genes in pecan grafting.

 

- The conclusion part is like the abstract, please rewrite it again (different from the abstract) and also give suggestions for future works.

 

Response: Thanks very much for your comment. In the conclusion section of our revised manuscript, we only present our key findings and future application of our work.

Round 2

Reviewer 2 Report

Dear Editor in Chief of Forests Journals

Hi and have a good day;

I studied the revised article and most of the items was corrected by the authors. In my opinion, this article is acceptable for publication in forest journal in this form. However, Supplementary table 2 was repeated many times in lines 182-187. and also figure 2 and 4 repeated twice into MS.

best wishes