Identification and Characterization of CCD Gene Family in Rose (Rosa chinensis Jacq. ‘Old Blush’) and Gene Co-Expression Network in Biosynthesis of Flower Scent
Round 1
Reviewer 1 Report
Authors have made a good attempt in writing the manuscript. Overall, presentation of the manuscript is very clear and good for readers. A few suggestions for improvement as follows:
Title: italicise Rosa chinensis
Abstract: why the abstract content was partitioned? Should be in a paragraph only
Line 93-101: This sentence is too long and can be confusing. Author should revise and separate into more understandable sentences.
Line 117: Remove full stop after Rosaceae
Line 118-119: Tbtools or TBtools. Please clarify. This software/program should be cited in reference too
Line 123-125: Please provide citations for CLustalW and Iqtree
Line 183: Figure 1 is blurry. Please provide a better picture with a higher resolution
Line 190: Figure 3 is also blurry
Line 253: Figure 4A? where is A in figure 4?
Author Response
Abstract: why the abstract content was partitioned? Should be in a paragraph only
Answer: It has been modified according to your comments. Remove the break sign to make the abstract a paragraph.
Line 93-101: This sentence is too long and can be confusing. Author should revise and separate into more understandable sentences.
Answer: It has been modified according to your comments. To break a long sentence into short sentences, see lines 94 and 96. Thank you for your valuable advice.
Line 117: Remove full stop after Rosaceae
Answer: It has been modified according to your comments. Remove the dot.
Line 118-119: Tbtools or TBtools. Please clarify. This software/program should be cited in reference too
Answer: It should be TBtools, Tbtools has been changed to TBtools in line 118, and references have been added. Thank you for your valuable advice.
Line 123-125: Please provide citations for CLustalW and Iqtree
Answer: I have added references, please refer to lines 122 and 124.
Line 183: Figure 1 is blurry. Please provide a better picture with a higher resolution
Answer: It has been changed to a higher resolution picture, and provided the full text picture of the original image compression package, uploaded to the journal submission system.
Line 190: Figure 3 is also blurry
Answer: It has been changed to a higher resolution picture, and provided the full text picture of the original image compression package, uploaded to the journal submission system.
Line 253: Figure 4A? where is A in figure 4?
Answer: Sorry, the old figure 4 was not labeled A, B (The left side was A, and the right side was B). Two figures were added before this one, so the old Figure 4 became the new Figure 6. The new figure 6 was labeled A, B. Please see line 274. Thank you for your careful review.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The authors have done a study related to identification and characterization of CCD family members in Rose. Also they have analyzed gene co-expression network associated with biosynthesis of flower scent. The results are useful. However, the current version should be improved. My comments:
- Line 107: I suggest using it: "Twenty CCD family member sequences"
- "ZmVP14" , provide the complete name" Z. mays VP14"
- Line 125: which method was there used? Probably ML. Add to text.
- Lines 123-128: Add to text the references of used tools/databases.
- Lines 174-175: gene family name should not be written in italic format. Please check the whole text.
- Lines 229-235: These results are related which figure or table?
- Line 306: gene should be provided in italic format.
- Results are repeated in Discussion section. Discussion needs to improve. Key results should be interpreted.
- I suggest adding this sentence to line 359: Besides, CCD showed diverse expression, indicating that modifications including mutations have occurred in function regions, regulatory regions and coding sequence site, of duplicated members, affecting the expression as well as function (Faraji et al, 2021, Heidari et al, 2021).
References:
Faraji et al, 2021: https://doi.org/10.3390/plants10122597
Heidari et al, 2021: https://doi.org/10.3390/agronomy11081651
Author Response
Reviewer 2
Line 107: I suggest using it: "Twenty CCD family member sequences"
"ZmVP14", provide the complete name" Z. mays VP14"
Answer: It has been modified as you suggested, thank you for your valuable advice.
Line 125: which method was there used? Probably ML. Add to text.
Answer: As you mentioned, the ML method was used for tree building, which has been supplemented in line 126 of the manuscript.
Lines 123-128: Add to text the references of used tools/databases.
Answer: According to your suggestion, the database of sequence download—NCBI was added, as shown in line 124 of the manuscript.
Lines 174-175: gene family name should not be written in italic format. Please check the whole text.
Answer: Gene family name has been changed to a non-italic format in the whole text. Thank you for your valuable advice.
Lines 229-235: These results are related which figure or table?
Answer: This paragraph was a simple description of CCDs and RNA-seq data. It did not involve analysis results, so there were no figures or tables.
Line 306: gene should be provided in italic format.
Answer: NUDX has been changed to italics according to your suggestion, thank you for your valuable advice.
Results are repeated in Discussion section. Discussion needs to improve. Key results should be interpreted.
Answer: The parts of the discussion that were duplicated in the results have been removed and the discussion has been improved according to your modifications. Thank you for your valuable advice.
I suggest adding this sentence to line 359: Besides, CCD showed diverse expression, indicating that modifications including mutations have occurred in function regions, regulatory regions and coding sequence site, of duplicated members, affecting the expression as well as function (Faraji et al, 2021, Heidari et al, 2021).
References:
Faraji et al, 2021: https://doi.org/10.3390/plants10122597
Heidari et al, 2021: https://doi.org/10.3390/agronomy11081651
Answer: The following sentence has been added to line 411 as per your comment: Besides, CCDs showed diverse expression, indicating that modifications including mutations have occurred in function regions, regulatory regions and coding sequence site, of duplicated members, affecting the expression as well as function [38,39].
Author Response File: Author Response.docx
Reviewer 3 Report
Please see the attachment
Comments for author File: 
Comments.pdf
Author Response
- Although they presented the expression patterns of 15 CCD family members at different flowering stages shown in Table 3 using in silico analysis, it is difficult to understand the data and so they should be changed it to graph. Also, they should do qRT-PCR experiments to confirm in silico data and compare them.
Answer: The expression trend data of the 15 RcCCDs shown in Table 3 at different flowering stages were plotted as a heat map. 10 of the 15 RcCCDs were differentially expressed at the EF, SF, LF stages, so a heat map of the 10 genes was plotted for Figure 4.
Among the 15 RcCCDs, 8 genes were detected at EF, SF and LF stages by qRT-PCR. Among them, 6 genes were detected in EF, SF and LF simultaneously, and their expression trends were the same as the RNA-seq, which proved that the transcriptome data were accurate and reliable. The explanation was added in line 252 of the manuscript, and Figure 5 was added in line 261. In Figure 5, the transcriptome expression and qRT-PCR relative expression of the CCD family members at different flowering stages (as shown in Table 3) were shown simultaneously.
- They described GC-MS analysis in Materials and methods. However, I could not find this result in the Results part. Please show this data.
Answer: The GC-MS section of the materials and methods described the method for determining and calculating detection amounts of volatile compounds used in WGCNA. These works were not done by me, but were the results of a preliminary laboratory study.
It was my mistake to confuse you by including this part of the method in the materials and methods. I have deleted the GC-MS analysis in the materials and methods in the new revised manuscript.
- They used WGCNA analysis to show correlation between co-expression module of 15CCD family members and volatile compounds produced in rose. Because these data were very dependent on computational analysis, they should do GC-MS analysis to confirm this analysis.
Answer: The detection amounts of the 18 volatile compounds used in WGCNA were obtained by GC-MS, and they were used as phenotypic data for WGCNA along with expression data from RNA-seq to obtain modules related to these volatile compounds using the WGCNA package.
- Although they concluded that 6 modules were highly correlated with volatile compounds, the description in the Results part was very ambiguous. Please explain the correlation between 6 modules and volatile compounds’ biosynthesis in more detail in the Results part.
Answer: I apologized for the confusion caused by my ambiguous description, and I have revised some of the results as detailed in the text, lines 305, 308, 315, 328, 345. Thank you for your valuable advice.
My minor concerns are as below.
- What is the Figure 4A?
Answer: Sorry, the old figure 4 was not labeled A, B. Two figures were added before this one, so the old Figure4 became the new Figure 6 The new figure 6 was labeled A, B. Please see line 274.Thank you for your careful review.
- Please include detailed explanation in Figures 4, 5, and 6
Answer: Figures 4, 5, and 6 were updated to Figures 6, 7, and 8 after adding new figures. Detailed descriptions have been added in the figure notes of Figures 6 (line 275), 7 (line 291), and 8 (line 300), thank you for your careful review.
Author Response File: Author Response.docx
Reviewer 4 Report
Dear authors,
before I give my final evaluation with all comments I need some answers to questions which arose during I read your manuscript. These quesitions are not in regard of the description of the CCD genes, which needs thorough revision. Moreover, I was wondering a bit on the part of WGCNA: In Shi et al. 2022 you analyzed the same compounds and the same set of RNAseq data as it seems. Nevertheless, you come to different conclusions when it comes to module-trait relationships (Fig. 6 this manuscript, Suppl. Fig. 2 Shi et al.). Can you answer to this contradiction?
Additionally, one gets the impression in the manuscript that the transcriptome and GC-MS data are completely new what seems to be not the case. Is this true or not?
Furthermore, you used volatile data from tianmideng from all three stages whereas for the other three cultivars just SF data were used. Can you tell me the reason and is it really applicable to compare it like this?
Best regards
Author Response
Reviewer 4
- before I give my final evaluation with all comments I need some answers to questions which arose during I read your manuscript. These quesitions are not in regard of the description of the CCD genes, which needs thorough revision. Moreover, I was wondering a bit on the part of WGCNA: In Shi et al. 2022 you analyzed the same compounds and the same set of RNAseq data as it seems. Nevertheless, you come to different conclusions when it comes to module-trait relationships (Fig. 6 this manuscript, Suppl. Fig. 2 Shi et al.). Can you answer to this contradiction?
Answer: I used the same data as Shi et al. 2022 in this paper.
Data introduction:
A total of 18 sets of RNA-seq data were obtained for the three stages of R. hybrida 'tianmidemeng' and the SF stages of the other three cultivars (R. hybrida 'Elle', R. hybrida 'First blush' and R. hybrida 'Chingge'). Among them, R. hybrida 'tianmidemeng' was a strand-specific RNA library, and the other three cultivars used a strand-nonspecific common RNA library.
Shi et al. 2022 identified lncRNAs using the RNA-seq of the strand-specific RNA library of R. hybrida 'tianmidemeng', and later screened the RNA-seq of the common RNA library of the other three cultivars to obtain lncRNAs. lncRNAs from the four cultivars were used for WGCNA to obtain module-trait relationships.
I performed the strand-specific RNA library RNA-seq of R. hybrida 'tianmidemeng' together with the common RNA library RNA-seq of the other three cultivars for reference transcriptome analysis. mRNA was used for differential expression analysis, and the differential genes used for WGCNA were also mRNAs non lncRNAs, so the results were different.
- Additionally, one gets the impression in the manuscript that the transcriptome and GC-MS data are completely new what seems to be not the case. Is this true or not?
Answer: The transcriptome and GC-MS in the manuscript were not entirely new and were from a previous study by the group, see reference (Shi, et al., 2022). I describe the NCBI accession number of the transcriptome data in the manuscript at line 132 (NCBI database, BioProject PRJNA667625, SraAcc SRR12779319, SRR12779320, SRR12779321, SRR12779322, SRR12779323, SRR12779324, SRR12779325, SRR12779326, SRR12779327). I apologized for the confusion caused by my unclear presentation. I have added a reference to the transcriptome data in line 134 [26] (Shi, et al., 2022) and also added notes and a reference to the GC-MS data in line 154 [26] (Shi, et al., 2022).
The differential expression analysis of the transcriptome data was handled by myself, so I have provided a methodological description in the materials and methods. However, GC-MS was not determined by me, and I directly used the results of predecessors, so the determination of aromatic compounds was not described in materials and methods.
- Furthermore, you used volatile data from tianmideng from all three stages whereas for the other three cultivars just SF data were used. Can you tell me the reason and is it really applicable to compare it like this?
Answer: The GC-MS and RNA-seq data of R. hybrida ‘tianmidemeng’ at three stages (EF, SF, LF) were used to resolve the pattern of changes in the accumulation of volatile compounds during flower opening and the expression of related genes. The results showed that R. hybrida ‘tianmidemeng’ had the highest accumulation of volatile compounds in the SF stage, see reference (Shi, et al., 2022) FIGURE 2.
The other three cultivars, R. hybrida 'Elle', R. hybrida 'First blush' and R. hybrida 'Chingge ', which have different degrees of scent, were measured only at the SF stage to carry out the analysis of differences in volatile compounds and related genes among the rose varieties with different degrees of scent. This was part of the results of the research group's previous study, not the main content of this paper, and it was not publicly available for now, so it was not specifically stated in this paper.
These 18 sets of transcriptome data about 3 stages and 4 cultivars were determined for volatile compounds by GC-MS, and the expression data and phenotypic data matched and met the requirements of WGCNA, while WGCNA required more than 15 sets of RNA-seq, so these data were selected for the analysis in this paper.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
In the new version, the authors have improved the manuscript.
Author Response
Thank you for your careful review.
Reviewer 3 Report
No answer given.
Author Response
In the last comment reply (round 1), I answered all the questions, pasted the text in the web page, and uploaded the Riviewer.docx file. Answers of your comments came after string “Reviewer3”, so it was possible that I put all the reviewers' answers in one word file, which made it inconvenient for you to read. I have created a separate file for the Reviewer3 responses in this round and uploaded it.
Author Response File: Author Response.docx
Reviewer 4 Report
Dear authors,
thank you for the thorough information to my questions. Please increase the font size in Figs. 1, 3 and 8. It´s not possible to recognize what´s written in these figures without zooming in the PDF.
Author Response
I have increased the font size of Figure 1, 3, 8 as you suggested, so that the text could be seen clearly without special enlargement. At the same time, I re-uploaded the compressed package of the modified image. Thank you for your careful review.
