Pan-Genome Analysis of TRM Gene Family and Their Expression Pattern under Abiotic and Biotic Stresses in Cucumber

Comments 1: The manuscript is interesting and presents novel approaches. However, reference to Pangenomics in Introduction is scarce. In Discussion, implications on plant breeding are briefly mentioned; authors should present this fact in Introduction, together with a broad framework of Pangenomics. Then, Conclusions would be better supported.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have expanded the discussion of pangenomics in the Introduction to provide a broader context. Additionally, we have included relevant references and discussed the implications for plant breeding more thoroughly. This change can be found on pages 2-3 and pages 15-16 of the revised manuscript.

Comments 1: The authors have apparently screened the putative cucumber TRM gene family members based on the sequence homology to the Arabidopsis TRMs. Have the authors checked whether any annotated domain/s present (or conserved) among the members? While the MEME analysis gives conserved motifs among the assessed sequence, the annotated domain (if any) could be conserved relatively broadly.

Response 1: Thank you for your insightful comment. We deeply appreciate your suggestion to investigate the presence of annotated domains within the putative cucumber TRM gene family members. Unfortunately, our analysis did not identify any annotated domains among the TRM gene family members in our study. This could be due to the specificity of the cucumber genome or the potential divergence of these genes from their Arabidopsis counterparts. This would be an important direction for future research.

Thank you once again for your valuable feedback, which will guide our future studies.

Comments 2: The authors have used the SRA database derived expression data. Since the current study is basically secondary data-based, it is essential to include the state of plant/accession, experiment/treatment conditions, tissue/organ, etc. used in each of the assessments. Without such information, the assessment and conclusions made in the study will lack any reference point to base the arguments. The authors are kindly suggested to make necessary changes.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have added the relevant details to the Materials and Methods section on page 4. Additionally, some of this information is also included in the figure captions on page 10 for Figure 3 and on page 12 for Figure 5.

We appreciate your suggestion, which has helped improve the clarity and completeness of our manuscript.

Comments 3: [Supplementary] The study may have benefitted greatly had the authors tested at least some of the TRM member expression pattern in selected accessions. The confirmation of the pan-genome derived assessment by simple PCR would be very supportive to the study as well (since some species lack some TRM members and some TRM members of the same clade are of much different sizes). Authors are suggested to do so, if viable.

Response 3: Thank you for your insightful comment. We acknowledge that testing the expression patterns of TRM members in selected accessions could have significantly benefited the study. However, we did not include this in our manuscript as previous studies have already tested the TRM member expression patterns in accession 9930. To support our study further, we have added an analysis of the expression patterns of some TRM members at different stages of fruit development and in different fruit shapes. These updates can be found on pages 9-10 of the revised manuscript.

Regarding the confirmation of the pan-genome derived assessment by simple PCR, we do not have all 12 cucumber germplasm resources mentioned in the article to perform these experiments. This is an area of interest for our future research.

Thank you once again for your valuable feedback.

Comments 4: Section 2.1: Please changes the writing structures like those in 2.2-2.5.

Comments 1: Abbreviations are written in full the first time they appear in the text, and then followed by a parenthesis in which the abbreviation is written.

Response 1: Thank you for pointing this out. We agree with your suggestion. We have revised the manuscript to ensure that all abbreviations are written out in full the first time they appear in the text, followed by the abbreviation in parentheses. These revisions are reflected on page 1.

Comments 2: Please modify the writing of the gene by replacing ‘CsTRM’ with ‘CsTRM’. Please modify the writing of other genes in MS.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have made several revisions. However, CsTRM was not replaced with 'CsTRM' when referring to the protein in the revised manuscript.

Comments 3: Please change “Keywords: pan-genome; cucumber; TRM; fruit shape; abiotic stress response; biotic stress response” to “Keywords: cucumber; pan-genome; TRM gene; fruit shape; abiotic stress response; biotic stress response”

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made adjustments to the Keywords section on page 1.

Comments 4: The manuscript is mostly based on bioinformatics analysis, without any experimental content. I strongly recommend the author to at least add content on real-time fluorescence quantitative PCR.

Response 4: Thank you for your insightful comment. In response to your suggestion, we have added experimental content involving real-time fluorescence quantitative PCR. Specifically, we have included an analysis of the expression patterns of some CsTRM members using ovaries collected from cucumber plants 4 days before anthesis (4 DBA) and on the day of anthesis (0 DAA) from both the long fruit line CSSL2-7 and the round fruit line RNS7. Additionally, we analyzed the expression patterns of some CsTRM members in cotyledons of cucumber seedlings inoculated with gray mold at 0 hours, 6 hours, 24 hours, and 72 hours.

These updates can be found in Section 3.4 "Expression Profiles of CsTRM Genes in the Fruit" on pages 9-10, and in Section 3.5 "Expression Profiles of CsTRM Genes under Abiotic and Biotic Stresses" on pages 12-13 of the revised manuscript. The corresponding methods have been added to Section 2, "Materials and Methods," specifically in 2.6 "RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis" on page 4.

Thank you again for your valuable feedback.

Comments 5: Please do experiments to verify the expression of cucumber TRM gene family in various biotic and abiotic stresses.

Response 5: Thank you for your valuable feedback and suggestion to include experimental validation of the CsTRM gene family expression under various biotic and abiotic stresses. While we understand the importance of examining the CsTRM gene family under multiple stress conditions, our current study focused specifically on a particular biotic stress gray mold due to the constraints of time and resources. we analyzed the expression patterns of some CsTRM members in cotyledons of cucumber seedlings inoculated with gray mold at 0 hours, 6 hours, 24 hours, and 72 hours. The results were consistent with the RNA-seq data, confirming the reliability of the datasets and contributing to the understanding of CsTRM gene function. These updates can be found in Section 3.5 "Expression Profiles of CsTRM Genes under Abiotic and Biotic Stresses" on pages 12-13 of the revised manuscript.

We acknowledge the need for further exploration under additional stress conditions, which will be a priority for our future work. We hope the results presented in this manuscript will still provide valuable information and justify the publication of our current findings.

Thank you for your consideration.

Comments 1: The authors are kindly suggested to include the statistical significance test of the recently included expression data.

Response 1: Thank you for your valuable suggestion. We have now included the statistical significance tests for the recently added expression data. The relevant sections in the manuscript have been updated accordingly, and the changes can be found on pages 9 and 12.

Comments 1: Please add annotations in Table 1-2 to explain the meaning of abbreviations in the header.

Response 1: Thank you for your valuable feedback. We have added annotations to Tables 1 and 2 on pages 5 and 6 to clarify the abbreviations in the headers. Specifically, "PI" refers to the cucumber accession PI183967. Other accession do not have abbreviations.

Comments 2: Please add real-time fluorescence quantitative PCR analysis of CsTRMs gene under various biotic and abiotic stress conditions.