Molecular Regulation of Fruit Quality Traits in Citrus: RNA-Seq-Based Meta-Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article conducted a comprehensive meta-analysis of the fruit transcriptomes of six citrus species and re-analyzed the RNA-Seq data, aiming to identify genes commonly expressed both between and within species, candidate genes related to fruit traits, and genes overlapping with known quantitative trait loci for citrus fruit. It further analyzed the genes and modules in the fruit biology-related network. The data presented were abundant, the writing was fluent, and the scientific and technical presentation was relatively standardized. Nonetheless, the following aspects require further improvement.

1. L[100 - 101]: The description of the experimental materials still needs to be further refined. It is suggested that a brief biological overview of the six species selected for the study - sweet orange (C. sinensis), mandarin (C. reticulata), pomelo (C. maxima), clementine (C. clementina), kumquat (C. japonica), and papeda (C. papeda) - be provided in the introduction, including their typical fruit morphological characteristics, and clarifying their representative status in the systematic evolution and artificial domestication history of the Citrus genus. This will enhance the comprehensibility of the research background, especially for readers without a background in Citrus taxonomy. Additionally, it is recommended that standardized fruit phenotype images of the above six species be provided in the main text or supplementary materials to visually present their morphological differences. Also, note that there was ambiguity in the " No. of samples" column in Table 1, as it was not clear whether this number refers to the number of sequencing samples for each biological replicate or the number of different cultivars from the same species (a total of 24 cultivars). Please clearly define and explain this in the "Materials and Methods" section.
2. L[127 - 129]: The description of the Gene Ontology analysis method is not detailed enough, and the KEGG Analysis method is missing. Please complete it.
3. L[180 - 201]: The PCA plot is not labeled or titled. Please complete it.
4. L[238 - 308]: The connection between Table 3 and the text parts of sections 3.2.1 to 3.2.6 was not tight enough. The genes mentioned in the text did not appear in Table 3, and the genes in Table 3 were not fully analyzed in the text. It was necessary to strengthen the connection between Table 3 and the text parts of sections 3.2.1 to 3.2.6. In addition, the data in Table 3 needed to be verified. For example, the text mentioned that "CCD4 (LOC102621234)" was up-regulated in 3.2.5 Citrus japonica and 3.2.6 Citrus clementina, but the "observed expression pattern" in Table 3 was "C. reticulata → sinensis". Please verify the data in Table 3 to improve the rigor of the data.
5. L[309 - 330]: The text states that "biological process associated with DEGs was grouped in 38 terms ", but only 17 GO terms were shown under BP in Fig 4. It was also mentioned that "molecular functional process associated with DEGs was grouped into 37 terms ", yet only 23 GO terms were presented under MF in Fig 4. Only "cellular component associated with 5 DEGs was grouped into 5 terms " were completely consistent with the 5 GO terms under CC in Fig 4. Please improve the data analysis methods and results in this part to ensure that the data in the text and the chart are consistent.
6. L[394 - 395]：“WGCNA analysis identified 17 distinct modules from 16253 genes, size ranging from 2710 to 112 genes (Fig 6a).”However, only 16 models were shown in Fig 6a. Please correct Fig 6a.

Author Response

Respected editor and reviewer,

We sincerely thanking you for your valuable time, passion and critical reviewing for the improvement of our manuscript. We have addressed all your suggestions and the corrected text have been marked in blue colour

Reviewer’s comment 1:  L [100 - 101]: The description of the experimental materials still needs to

be further refined. It is suggested that a brief biological overview of the six species selected for the study - sweet orange (C. sinensis), mandarin (C. reticulata), pomelo (C. maxima), clementine (C. clementina), kumquat (C. japonica), and papeda (C. papeda) - be provided in the introduction, including their typical fruit morphological characteristics, and clarifying their representative status in the systematic evolution and artificial domestication history of the Citrus genus. This will enhance the comprehensibility of the research background, especially for readers without a background in Citrus taxonomy. Additionally, it is recommended that standardized fruit phenotype images of the above six species be provided in the main text or supplementary materials to visually present their morphological differences. Also, note that there was ambiguity in the " No. of samples" column in Table 1, as it was not clear whether this number refers to the number of sequencing samples for each biological replicate or the number of different cultivars from the same species (a total of 24 cultivars). Please clearly define and explain this in the "Materials and Methods" section.

Authors response:  Thank you so much for your comments. We have added the required texts related to systematic evolution (line 46- 49). We have also added the images carrying typical fruit morphological characteristics, included as Figure 1. “No.of samples” represents total number of raw fastq files taken from each sample in particular fruit stage, visible through line 113-115.

 

Reviewer’s comment 2: L [127 - 129]: The description of the Gene Ontology analysis method is not detailed enough, and the KEGG Analysis method is missing. Please complete it.

Authors response: Thank you. As suggested, detailed description of Gene Ontology analysis                               method has been provided (line 138-141)., also added Kegg analysis method (line 144-148).

 

Reviewer’s comment 3: L [180 - 201]: The PCA plot is not labeled or titled. Please complete it.

Authors response: Thank you, we have now provided the details about PCA plot (line 216-217).

 

Reviewer’s comment 4: L [238 - 308]: The connection between Table 3 and the text parts of sections 3.2.1 to 3.2.6 was not tight enough. The genes mentioned in the text did not appear in Table 3, and the genes in Table 3 were not fully analyzed in the text. It was necessary to strengthen the connection between Table 3 and the text parts of sections 3.2.1 to 3.2.6. In addition, the data in Table 3 needed to be verified. For example, the text mentioned that "CCD4 (LOC102621234)" was up-regulated in 3.2.5 Citrus japonica and 3.2.6 Citrus clementina, but the "observed expression pattern" in Table 3 was "C. reticulata → sinensis". Please verify the data in Table 3 to improve the rigor of the data.

Authors response:  Thank you. We appreciate your critical observation regarding the inconsistency between the text (Sections 3.2.1–3.2.6) and Table 3. We acknowledge that the connection between the genes discussed in the text and those presented in Table 3 was not sufficiently addressed in the original manuscript. Table 3 was intended to provide representative examples of gene expression variation across different Citrus species, highlighting how        differential expression patterns may contribute to phenotypic differences in fruit traits. While, many genes were generally upregulated across species, the table emphasized differences in the magnitude and direction of expression changes between species. The apparent mismatch arose because the text focus on species-specific functional interpretations, whereas Table 3 presented comparative expression trends across multiple species. Regarding the specific example of CCD4(LOC102621234), we have re-examined the data and verified that the expression trends reported in Table 3 are correct. All values have been cross-checked with Supplementary File 2 as

Well 9(line 318).

 

Reviewer’s comment 5: L [309 - 330]: The text states that "biological process associated with DEGs was grouped in 38 terms ", but only 17 GO terms were shown under BP in Fig 4. It was also mentioned that "molecular functional process associated with DEGs was grouped into 37 terms ", yet only 23 GO terms were presented under MF in Fig 4. Only "cellular component associated with 5 DEGs was grouped into 5 terms " were completely consistent with the 5 GO terms under CC in Fig 4. Please improve the data analysis methods and results in this part to ensure that the data in the text and the chart are consistent.

Authors response: We appreciate the reviewer’s careful observation; we have corrected the “molecular functional process associated with DEGs was grouped into 38 terms” (line 346). The completed details were displayed in the figure. The revised figure added to the manuscript as Figure 5a

 

Reviewer’s comment 6: L [394 - 395] ： “WGCNA analysis identified 17 distinct modules from

16253 genes, size ranging from 2710 to 112 genes (Fig 6a).” However, only 16 models were shown in Fig 6a. Please correct Fig 6a.

Authors response: As suggested, the Fig 6a is corrected now revised as Fig 7a, all 17 modules are displayed in the plot. Thank you for the suggestion.

We are once again thanking for your valuable suggesions for the improvement of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

All comments are attached to the manuscript.

Comments for author File: Comments.pdf

Author Response

Respected editor and reviewer,

We sincerely thanking you for your time, passion and critical reviewing for the improvement of our manuscript. We have addressed all your suggestions and the corrected text have been marked in green colour

Reviewer’s comment 1: Keywords should be listed in alphabetical order.  Trait? The keyword “Trait” is too broad to be useful and should be either specified or removed.

Authors response: Appreciate your suggestion. Now, the keywords are arranged in alphabetical order and word ‘Trait’ is removed. (Line 35)

Reviewer’s comment 2: The term ‘Citrus sinensis’ should be italicized throughout the manuscript.

Authors response:  As per suggestion, the term citrus sinensis is italicized throughout the manuscript.

 

Reviewer’s comment 3: The abbreviation "GCF" has been defined at its first occurrence in the manuscript

Authors response: As per suggestion the “GCF” is expanded to Genome Consortium Format (Line 120)

 

Reviewer’s comment 4: GO?

Authors response: As per suggestion, the term GO is now expanded to Gene Ontology (Line 129).

 

Reviewer’s comment 5: Please clarify the meaning of the terms logFC and padj.

Authors response: As suggested, the terms logFC and padj are now explained (Line 138-141). Thank you.

 

Reviewer’s comment 6: 32-34

Authors response: 32,33,34 has been changed to 31-33 (Line 152)

 

Reviewer’s comment 7: The sentence “Length of the chromosomes was taken from pseudochromosomes for the study [3]” is unclear. The authors should rephrase it for clarity and readability.

Authors response:  As per suggestion, the sentence for length of chromosomes has been changed (Line 154-155). Thank you.

 

Reviewer’s comment 8: The authors should provide the full form of the abbreviation WGCNA (Weighted Gene Co-expression Network Analysis) when it first appears in the text.

Authors response:  As suggested, the full form of abbreviation WGCNA is added as a list of abbreviations at the last (Line 159). Thank you.

 

Reviewer’s comment 9: Some references appear to be incorrectly placed in the text. The authors should ensure that citations are consistently positioned either at the beginning or at the end of the corresponding sentence

Authors response: The required changes have been made for reference citation positions.

 

Reviewer’s comment 10:  ?

Authors response: As per suggestion, [4] was changed to [3] (Line 186). Thank you for the suggestion

 

Reviewer’s comment 11: The title of this figure should be adjusted

Authors response:  As per reviewer’s suggestion, the title of this figure (PCA plot) is adjusted (Line 215-216)

 

Reviewer’s comment 12: Please ensure that all gene names are presented in italics throughout the text. (Table 3.)

Authors response: All the gene names are italicized in the table3 and text.

 

Reviewer’s comment 13: Please improve the clarity and resolution of this figure (Figure 4.)

Authors response: The clarity and resolution of the figure is improved and size is also increased to improve readability. Now it is placed as Figure 5. Thank you for the suggestion.

 

Reviewer’s comment 14: Please complete the figure title, as some text seems to be missing.

Authors response: The figure title is completed as per suggestion. Figure 5 is now Figure 6. (Line 429-431) thank you .

 

Reviewer’s comment 15: The table should be adjusted (Table 4.)

Authors response: The table is now adjusted, as per suggestion.

 

Reviewer’s comment 16: p should be italicized.

Authors response: p is italicized as suggested (Line 454).

 

Reviewer’s comment 17: Please improve the clarity and resolution of this figure (Figure 7.)

Authors response:  We have now improved the clarity and resolution of the Figure.  Figure 7 is now Figure 8.Thank you for the suggestion.

 

Reviewer’s comment 18: Please ensure that the entire reference list is reformatted according to journal requirements, including style, punctuation, and consistency (e.g., journal names, scientific names of species, etc.).

Authors response:  Thanks for the suggestion.  All references were reformatted as per MDPI style.

 

Authors sincerely thank reviewers for taking so much of time and making critical observations. We sincerely hope that these changes would be considered sufficient to take these revisions into next phase of peer-review process.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully revised and improved the manuscript point by point in accordance with the reviewers' comments, and the quality of the manuscript has been significantly enhanced. Finally, it is recommended that the authors carefully check and correct any language and grammatical errors throughout the text. After these revisions are completed, I believe the manuscript can be accepted.

Author Response

Respected reviewer,

We are highly thankful for your critical reviewing and time for improving our manuscript. We have checked the english and made necessary grammatical and typographical corrections in the manuscript. The corrections are highlighted in track change mode.

Thanking you

Reviewer 2 Report

Comments and Suggestions for Authors

The implementation of the reviewers’ suggestions and the authors’ responses to the raised questions have significantly improved the quality of the manuscript. However, further refinements are still required regarding the presentation of Figures 5a and 8, as well as Table 4, particularly in terms of quality, clarity, and the overall organization of the data and axis legends.

     

Author Response

Respected reviewer,

We are once again thanking you for your time, passion and critically reviewing our manuscript for improvement and clarity. As suggested, the quality of figure 5a and 8 were improved. The table 4 was reorganized for better clarity and readability. 

Thanking you.