Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The article is a serious study, interesting for science, but there are a number of comments and questions about it.  1. The RNA-seq compares two families after 15 days of drought (No.1 vs No.5), but it is not obvious from the description whether there is an intra-family control (0 d) for the transcriptome and how the "genotype/family" effect is separated from the "stress" effect. Currently, conclusions about "dragged-induced" for a number of genes are based on inter-family differences, which is methodically weaker without control/time series for each genotype (see the statement of experience and what RNA-seq did on 15 d, sections 4.1 and 4.5).    2. For physiological indicators, t-tests and 3 biological repeats (Figure 1, Figure 2) are indicated, but the design includes many time points (0 d, 8 d, 15 d, dehydrated), two families and several indicators. Pairwise t-tests alone are not enough without adjusting for multiple comparisons and without the "family ×time" model (for example, ANOVA/linear model). This affects the validity of the stated differences between No.1 and No.5 for all time points.   3. DEGs are defined as adj p < 0.05 and log2(fold change) > 0 (section 2.4 and 4.7). In this form, the criterion ">0" means that formally "significant" down-regulated genes do not pass the threshold (there should be |log2FC|> threshold, or separately for up/down). At the same time, the down-regulated number (4882) is further reported, which logically conflicts with the definition. It is necessary to clarify: which threshold was used according to the log2FC module, how up/down was calculated, and why this particular threshold was chosen.    4. The methods simultaneously state that the reference genome of Z. jujuba and the alignment of Hisat2 were used (4.5), and then what the de novo assembly Trinity did, followed by Corset and "unigenes as reference sequences" (4.6). These are two different pipelines (reference-based vs de novo). In its current form, it is unclear what exactly was the base for calculating expression and DESeq (reference genes or unigenes), how the multicapping problem was solved, and why FPKM was chosen (usually counts is used for DESeq). This is critical for reproducibility.  5. The GEO number GSE180449 is specified in the Data Availability section. We need to clarify whether this is really a data set specifically for this work (type/conditions/samples), and add a direct link: which sample IDs correspond to No.1_1..No.1_3 and No.5_1..NO.5_3 (you have these designations in 4.1). Without this, verification and reanalysis are difficult

Author Response

Responses to Reviewer #1

The article is a serious study, interesting for science, but there are a number of comments and questions about it. 

1. The RNA-seq compares two families after 15 days of drought (No.1 vs No.5), but it is not obvious from the description whether there is an intra-family control (0 d) for the transcriptome and how the "genotype/family" effect is separated from the "stress" effect. Currently, conclusions about "dragged-induced" for a number of genes are based on inter-family differences, which is methodically weaker without control/time series for each genotype (see the statement of experience and what RNA-seq did on 15 d, sections 4.1 and 4.5).

Responses: Thank you for your professional suggestions on the construction of this manuscript. As you mentioned, the screening of some drought-related genes is currently conducted through inter-family selection. In our preliminary experiments, we collected germplasm resources from different regions and evaluated their drought resistance, from which the genetic materials used in this study were ultimately obtained. This clone exhibits excellent traits such as drought tolerance, cold resistance, and high yield. However, during subsequent drought experiments on families of this clone, obvious phenotypic divergence was observed between No.1 and No.5—especially after 15 days of drought stress. Nevertheless, these two families still showed significantly stronger drought resistance than the other clones. Therefore, these two families were selected as the main research materials for mining drought‑resistant genes in Z. jujuba var. spinosa.

On the other hand, since both families originated from the same clone, we did not use 0‑day samples as intra‑family controls at the initial sampling stage. We will address this limitation in subsequent gene function validation experiments. Thank you again for your valuable comments and suggestions.

 

2. For physiological indicators, t-tests and 3 biological repeats (Figure 1, Figure 2) are indicated, but the design includes many time points (0 d, 8 d, 15 d, dehydrated), two families and several indicators. Pairwise t-tests alone are not enough without adjusting for multiple comparisons and without the "family ×time" model (for example, ANOVA/linear model). This affects the validity of the stated differences between No.1 and No.5 for all time points.

Responses: Thank you very much for your suggestions and guidance on this article. We have used one/two-way ANOVA instead of Student’s t-tests in the determination of physiological indices and photosynthetic parameters, and revised Figure 1 and Figure 2 accordingly (P5, L137-144; P7, L159-166).

 

3. DEGs are defined as adj p < 0.05 and log2(fold change) > 0 (section 2.4 and 4.7). In this form, the criterion ">0" means that formally "significant" down-regulated genes do not pass the threshold (there should be |log2FC|> threshold, or separately for up/down). At the same time, the down-regulated number (4882) is further reported, which logically conflicts with the definition. It is necessary to clarify: which threshold was used according to the log2FC module, how up/down was calculated, and why this particular threshold was chosen. P25, L315-332; P38-39, L512-538

Responses: Thank you sincerely for your careful reading. Based on your suggestions, we have revised the screening criteria for DEGs and adjusted the numbers of up-regulated and down-regulated genes in the main text (P18, L496) and Figure 3 (P8, L190). The criteria |log2(fold change)| ≥ 1 and FDR < 0.05 were used to screen DEGs. The two wild jujube families included 3,238 up-regulated and 2,675 down-regulated DEGs.

 

4. The methods simultaneously state that the reference genome of Z. jujuba and the alignment of Hisat2 were used (4.5), and then what the de novo assembly Trinity did, followed by Corset and "unigenes as reference sequences" (4.6). These are two different pipelines (reference-based vs de novo). In its current form, it is unclear what exactly was the base for calculating expression and DESeq (reference genes or unigenes), how the multicapping problem was solved, and why FPKM was chosen (usually counts is used for DESeq). This is critical for reproducibility.

Responses: Thank you very much for your careful reading and insights on this article. We apologise for the errors and inaccuracies in our writing. We have removed the relevant content concerning Trinity and other related matters, and provided an explanation as to why the FPKM method was selected (P17-18, L458-491).

 

5. The GEO number GSE180449 is specified in the Data Availability section. We need to clarify whether this is really a data set specifically for this work (type/conditions/samples), and add a direct link: which sample IDs correspond to No.1_1..No.1_3 and No.5_1..NO.5_3 (you have these designations in 4.1). Without this, verification and reanalysis are difficult

Responses: Thank you for your suggestion. We have included a direct link in the text along with the corresponding ID number for each sample (P16, L429-432; P20, L577). Thank you again for your careful reading and revision comments on this manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

After carefully reviewing the manuscript, several aspects require clarification and improvement, particularly in the methodology section, regarding both the bioinformatic analyses and the statistical procedures. Clearer descriptions and additional details are necessary to ensure methodological transparency and reproducibility.

Abstract

Please modify the abstract. I suggest including a brief explanation of the methodology and then the results

L22. Please include the full name of polymerase chain reaction.

 

Introduction

I suggest to include some of the pathways that the drought stress activates. This will improve the manuscript.

 

Results

L122-123. Please review the legend of the figure “of of under”.

L148-152. These paragraph should be in method section, not in result section.

L161. After all the bioinformatic analysis, do the authors perform an assembly and annotation?? If this was performed, no results are found. The following paragraph is about the DEG.

Please improve this information.

Discussion

L383. Please include some of the flavonoids of interesting of your follow-up research.

Material and methods

L411. Please review in the whole manuscript the units abbreviation (hours, minutes, etc). Hours should be “h”…

L440-L447 The methodology presents ambiguity. The authors indicate that HISAT2 was used to map reads to the reference genome, while Trinity was applied for de novo transcriptome assembly. However, the rationale for combining both approaches is not clearly explained.

The authors should clarify:

1. Whether Trinity was used for transcript discovery and annotation, while HISAT2 was used exclusively for expression quantification; and

2. How both analyses were integrated within the overall workflow.

L456. The method is DESeq2… authors should include the reference…

L475. Authors need to explain the reference gene, the calibrator sample and explain the conditions of the qRT-PCR (tm, time, phases), etc.

I suggest including the Table s9 in the main body of the manuscript.

L476-477. Authors should create a specific section about the Statistical analysis. Moreover, authros need to include the experimental design, the statistical analysis and the name of the test used. Please include all this information in a specific section after the qRT-PCR.

Conclusions

The conclusions seems a summary of the results.

L512- 529. I think these lines should be deleted.

Author Response

Responses to Reviewer #2

After carefully reviewing the manuscript, several aspects require clarification and improvement, particularly in the methodology section, regarding both the bioinformatic analyses and the statistical procedures. Clearer descriptions and additional details are necessary to ensure methodological transparency and reproducibility.

1. Abstract

Please modify the abstract. I suggest including a brief explanation of the methodology and then the results

Responses: Thank you very much for your careful reading and insights on this article. Based on your suggestion, we have modified the abstract and added descriptions of the experimental methods (P1, L14-23).

 

2. L22. Please include the full name of polymerase chain reaction.

Responses: Thank you for your suggestion. We have provided the full name of polymerase chain reaction (P1, L27).

 

3. Introduction

I suggest to include some of the pathways that the drought stress activates. This will improve the manuscript.

Responses: Thank you for your professional suggestions on the construction of this manuscript. We have revised the introduction and added descriptions related to drought stress pathways (P2, L76-78; P3, L84-92).

 

4. Results

L122-123. Please review the legend of the figure “of of under”.

Responses: Thank you for your suggestion. We have revised the legend of Figure 1 (P5, L140-143).

 

5. L148-152. These paragraph should be in method section, not in result section.

Responses: Thank you very much for your suggestion. We have moved these paragraphs to the Materials and Methods section (P7, L170-172; P17, L463-466).

 

6. L161. After all the bioinformatic analysis, do the authors perform an assembly and annotation?? If this was performed, no results are found. The following paragraph is about the DEG. Please improve this information.

Responses: Thank you for reading our article carefully. The assembly and annotation of the RNA-Seq were performed by a biotechnology company, so we have removed this section from Materials and Methods and revised the description of transcriptome data analysis accordingly (P17-18, L458-491).

 

7. Discussion

L383. Please include some of the flavonoids of interesting of your follow-up research.

Responses: Thank you for your suggestion. We have added flavonoids as the focus of our future research (P16, L408-410).

 

8. Material and methods

L411. Please review in the whole manuscript the units abbreviation (hours, minutes, etc). Hours should be “h”…

Responses: Thank you for carefully reviewing this article. We have thoroughly checked the units abbreviation formatting of the Materials and Methods section.

 

9. L440-L447 The methodology presents ambiguity. The authors indicate that HISAT2 was used to map reads to the reference genome, while Trinity was applied for de novo transcriptome assembly. However, the rationale for combining both approaches is not clearly explained.

The authors should clarify: Whether Trinity was used for transcript discovery and annotation, while HISAT2 was used exclusively for expression quantification; and How both analyses were integrated within the overall workflow.

Responses: Thank you for your professional suggestions on the construction of this manuscript. We apologise for the errors and inaccuracies in our writing. We have removed the relevant content concerning Trinity and other related matters, and revised the transcriptome data analysis (P17-18, L470-491).

 

10. L456. The method is DESeq2… authors should include the reference…

Responses: Thank you for your suggestion. We have added the references at the corresponding positions (P18, L494).

 

11. L475. Authors need to explain the reference gene, the calibrator sample and explain the conditions of the qRT-PCR (tm, time, phases), etc.

Responses: Thank you very much for your suggestions and guidance on this article. Based on your suggestion, we have revised the Reverse Transcriptase–Quantitative PCR (RT-qPCR) section (P18, L507-511, L515-518).

 

12. I suggest including the Table s9 in the main body of the manuscript.

Responses: Thank you for your suggestion. We have moved Table S9 into the main body of the manuscript and renamed it Table 1 (P18, L522).

 

13. L476-477. Authors should create a specific section about the Statistical analysis. Moreover, authros need to include the experimental design, the statistical analysis and the name of the test used. Please include all this information in a specific section after the qRT-PCR.

Responses: Thank you very much for your suggestions and guidance on this article. According to your suggestions, we have created a new section on statistical analysis (P19, L524-528).

 

14. Conclusions

The conclusions seems a summary of the results.

Responses: Thank you sincerely for providing revision suggestions for this article. The conclusions have been revised (P20, L531-550).

 

15. L512- 529. I think these lines should be deleted.

Responses: Thank you for your suggestion. We have deleted the relevant sentences. Thank you again for carefully reading this article (P20, L573-574).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for the changes made and the significant improvement in the quality of the article.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors performed the changes requested. The article can be accepted.