Effect of Tm-2a, Sw-5 and Ty-1 Gene Introduction on the Agronomic Performance and Metabolic Profile of Traditional Muchamiel-Type Tomato Varieties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents a scientifically significant and practically valuable approach to preserving traditional tomato varieties while enhancing virus resistance.By introgressing Tm-2a, Sw-5, and Ty-1 genes in a heterozygous state into Muchamiel-type tomatoes, the authors successfully reconcile two critical agricultural priorities: maintaining genetic diversity and mitigating viral yield losses.The experimental design is robust, featuring a two-year field trial with comprehensive metabolic profiling and rigorous statistical validation.The work advances tomato breeding paradigms and provides a transferable model for other crops.

Suggested Modifications:

1. It is best to describe the disease resistance performance of hybrid varieties in the field or laboratory.Genotypic data alone are insufficient.
2. Address how Ty-1 reduces organic acids.
3. Add statistical significance markers to Figures 2–5 for clarity.

Author Response

Comments 1. It is best to describe the disease resistance performance of hybrid varieties in the field or laboratory. Genotypic data alone are insufficient.

Response 1. Resistance assays were indeed performed under controlled laboratory conditions using agroinoculation (TYLCV) and mechanical inoculation (ToMV and TSWV), and all hybrids exhibited resistance consistent with the expected genotypes (L123-126). These results were initially omitted, as they are part of a separate manuscript currently in preparation. However, we fully agree that it is essential for the reader to know that the resistance has been experimentally confirmed. Moreover, the efficacy of the resistance genes was demonstrated in previous studies referenced in this manuscript, where the breeding lines were developed and selected through backcrossing based on their resistant phenotype and genotype.

 

Comments 2. Address how Ty-1 reduces organic acids.

Response 2. This point has been addressed in the revised manuscript by referring to the genomic context of the Ty-1 introgression and its potential impact on acidity (L504-507).

 

Comments 3. Add statistical significance markers to Figures 2–5 for clarity.

Response 3. Statistical significance is already detailed in the corresponding table (Table 3 and 4), as the interaction between two factors (variety and year) makes it difficult to represent clearly in the figures. Additionally, we noticed a compatibility issue that caused the histogram bars to disappear in the PDF version due to a conflict between Word-to-PDF converter. This issue has now been resolved in the revised version.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a study on the introduction of virus-resistance genes (*Tm-2a*, *Sw-5*, and *Ty-1*) into traditional Muchamiel-type tomato varieties. The research evaluates agronomic performance, fruit morphology, and metabolic profiles, demonstrating that heterozygous hybrids maintain desirable traits while conferring resistance. The study is well-designed, with robust methodology and clear results. However, some areas require clarification or improvement to enhance readability and scientific rigor.

Major Comments

1. Specify how environmental conditions (temperature, radiation) were controlled or monitored across years.
2. Confirm whether data met ANOVA assumptions (normality, homogeneity of variance). Mention corrections (e.g., transformations) if applied.
3. Highlight practical implications of yield differences (e.g., 12% reduction in UMH1200×MC2 in 2023). Is this economically significant?
4. Simplify tables/figures (e.g., merge Supplementary Tables S1–S3 into one concise table). Use subheadings to group related metabolites (e.g., "Flavor-Associated Compounds").
5. Address why *Ty-1* homozygosity reduced yield in prior studies but not here. Is it due to genetic background or environmental factors?
6. Elaborate on the proposed role in virus resistance. Cite mechanistic studies linking flavonoids to pathogen defense.

Minor comments:

1. Define abbreviations at first use (e.g., "NILs" in Introduction).
2. Replace "organoleptic" with "sensory" for broader readability.
3. Figure 2: Label axes clearly (e.g., "Glucose (mg/g FW)").
4. Table 2: Use consistent significant digits (e.g., "4.42 ± 0.08" vs. "3.22 ± 0.05").
5. Ensure all citations are formatted uniformly (e.g., "et al." in italics). Update citations where possible (e.g., FAO data from 2025 seems premature; verify).

Author Response

Major Comments

Comments 1. Specify how environmental conditions (temperature, radiation) were controlled or monitored across years.

Response 1. Information on how environmental conditions were monitored has been added to the Materials and Methods section, including details on the weather station used and the specific sensors for temperature and radiation (L151-156).

 

Comments 2. Confirm whether data met ANOVA assumptions (normality, homogeneity of variance). Mention corrections (e.g., transformations) if applied.

Response 2. We confirm that the assumptions for ANOVA were verified before the analysis: normality was assessed using the Shapiro-Wilk test, and homoscedasticity using the Levene median test. We acknowledge that this information was omitted in the initial version of the manuscript, and we have now included it in the revised version (L235-237).

 

Comments 3. Highlight practical implications of yield differences (e.g., 12% reduction in UMH1200×MC2 in 2023). Is this economically significant?

Response 3. We have addressed this point by adding a sentence discussing the potential economic relevance of the 12% yield reduction in UMH1200×MC2 in 2023 (L585-588). Additionally, we have added a sentence in the conclusion to highlight the economic significance of the yield increase associated with hybrid vigour, as this may enhance the competitiveness of these hybrids in the market (L610). We believe that these additions help to better emphasize the practical implications of yield fluctuations and improvements throughout the manuscript.

 

Comments 4. Simplify tables/figures (e.g., merge Supplementary Tables S1–S3 into one concise table). Use subheadings to group related metabolites (e.g., "Flavor-Associated Compounds").

Response 4. We appreciate the suggestion. However, we have chosen to keep Supplementary Tables S1–S3 separate, as each table corresponds to the description of distinct chromatographic profiles and contains different sets of metabolites. Additionally, these tables are referenced in different sections of the manuscript, and merging them would reduce clarity and hinder their specific contextual interpretation.

 

Comments 5. Address why Ty-1 homozygosity reduced yield in prior studies but not here. Is it due to genetic background or environmental factors?

Response 5. It is plausible that agro-environmental conditions can differentially affect genotypes, potentially influencing the expression of traits such as yield. However, in the present study, which included two consecutive years, no yield differences were observed between lines with and without Ty-1, suggesting that environmental conditions did not amplify any potential negative effect of the gene. Therefore, as discussed in lines 474-476, the absence of yield reduction is more likely attributable to differences in genetic background in these lines, a hypothesis supported by previous studies.

 

Comments 6. Elaborate on the proposed role in virus resistance. Cite mechanistic studies linking flavonoids to pathogen defense.

Response 6. The mechanistic role of flavonoids in virus resistance has now been clarified in the revised manuscript (L536-538). We have expanded the explanation of how increased flavonoid synthesis may modulate ROS accumulation during viral infection, enabling an effective defense response while minimizing oxidative damage. We believe this revision improves the clarity of the proposed mechanism.

 

Minor comments:

Comments 7. Define abbreviations at first use (e.g., "NILs" in Introduction).

Response 7. We have reviewed the use of abbreviations throughout the manuscript, and all abbreviations, including "NILs," are now defined upon first use and used consistently thereafter.

 

Comments 8. Replace "organoleptic" with "sensory" for broader readability.

Response 8. While we agree that "organoleptic" may not be the most appropriate term, we considered that "sensory" could be misleading, as no formal sensory analysis was conducted in this study. To improve clarity and accuracy, we have replaced "organoleptic" with "flavor-related," a term commonly used in the literature to refer to quality attributes associated with primary metabolites such as sugars and organic acids.

 

Comments 9. Figure 2: Label axes clearly (e.g., "Glucose (mg/g FW)").

Response 9. The y-axis label in Figure 2 has been updated to include the unit “FW” (fresh weight), and similar corrections have been applied consistently to all other relevant figures to ensure clarity and uniformity

 

Comments 10. Table 2: Use consistent significant digits (e.g., "4.42 ± 0.08" vs. "3.22 ± 0.05").

Response 10. The inconsistency was due to the use of both commas and periods as decimal separators in Table 5. This has now been corrected, and all values are presented with consistent decimal notation throughout the tables.

 

Comments 11. Ensure all citations are formatted uniformly (e.g., "et al." in italics). Update citations where possible (e.g., FAO data from 2025 seems premature; verify).

Response 11. We have revised all in-text citations to ensure consistent formatting, including the correct use of et al. in italics. Regarding the FAO data, we have clarified in the main text that the reported values refer to 2023 statistics, although the source was accessed in 2025. We hope this resolves any confusion.

Reviewer 3 Report

Comments and Suggestions for Authors

Congratulations on a rather well and nice article. Initially, I thought I won’t find anything to remark and will recommend publishing immediately. But something started to bug me and I think it needs to be addressed.

I am sure that most likely everything is ok with your hybrids and they are resistant to the intended/mentioned viruses. But the thing is… you do not have any proof. 1) You did not do inoculation tests to show how they react to virus. 2) You have not sequenced the genome fragments having these resistance genes, you do not know if your hybrids have a correct and working copies of these genes. At best, you talk about increased production of metabolites that previously were shown to be involved in the resistance to said viruses. But still, this is science. So, either give us proof and evidence that your hybrids are actually more resistant, or, more realistically, change some phrasing to more careful tone.

 

Line 2: The title is too bold “virus resistance introduction” (which you have not proven). But your formulated aim (or the very first sentence of abstract) is more accurate – “introduction of concrete genes, that supposedly confers resistance”. I recommend changing the title to reflect the truth more.

Maybe: “Effect of ToMV, TSWV, and TYLCV Resistance genes on the Agronomic Performance and Metabolic Profile of Traditional Muchamiel Type Varieties”. Or something similar. Maybe: Effect of Tm-2a, Sw-5, and Ty-1 gene introduction on the Agronomic Performance and Metabolic Profile of Traditional Muchamiel Type Varieties.

 

Line 585: “confers resistance” – you cannot claim this without hard evidence. If you say that. Then I can say that your hybrids are not resistant to said viruses. Prove me wrong… Or change phrasing.

Because current reality is like this: “This study demonstrates that the introduction of virus resistance genes Tm-2a, Sw-5 and Ty-1 into traditional Muchamiel varieties in a heterozygous state does not significantly compromise yield or fruit quality, while potentially may provide substantial resistance to ToMV, TSWV, and TYLCV respectively. The actual level of resistance though remains to be tested.”

 

Also
Line 30: “FAO” – use full name, Food and Agriculture Organization is not universally known.

 

Fix the three above things and I think you are good…

 

As another advice for the future, you could have done a slightly better job of introducing the viruses. As a casual reader, from your introduction, I have no idea how serious of a problem (in Spain) these viruses are. I was a plant virologist, and ill be the first one to tell that there are really crazy things like smoking old school tobacco cigars and then rubbing plant with same fingers and introducing the TMV into them. Or I saw in commercial greenhouses where tomatoes are grown in lines, close together, and people walking in narrow aisles between plant rows, spread viruses simply by rubbing their shoulders from plant to plant. But at the same time I can say that whenever we grow tomatoes outdoors, the viruses are barely noticeable and the fungi and mites cause all the problems. Out of your three viruses, ToMV does not even have the insect vector and is spread only mechanically or via infected seeds. And whiteflies and trips are quite small insects, susceptible to higher winds. I have no idea how common they are in southeastern coast of Spain. A few sentences describing these viruses (and their impact on tomatoes in your area) better could have made your “problem” and the “need of your work” more loud, more valuable.

Comments for author File: Comments.pdf

Author Response

Comments 1. I am sure that most likely everything is ok with your hybrids and they are resistant to the intended/mentioned viruses. But the thing is… you do not have any proof. 1) You did not do inoculation tests to show how they react to virus. 2) You have not sequenced the genome fragments having these resistance genes, you do not know if your hybrids have a correct and working copies of these genes. At best, you talk about increased production of metabolites that previously were shown to be involved in the resistance to said viruses. But still, this is science. So, either give us proof and evidence that your hybrids are actually more resistant, or, more realistically, change some phrasing to more careful tone. Line 2: The title is too bold “virus resistance introduction” (which you have not proven). But your formulated aim (or the very first sentence of abstract) is more accurate – “introduction of concrete genes, that supposedly confers resistance”. I recommend changing the title to reflect the truth more. Maybe: “Effect of ToMV, TSWV, and TYLCV Resistance genes on the Agronomic Performance and Metabolic Profile of Traditional Muchamiel Type Varieties”. Or something similar. Maybe: Effect of Tm-2a, Sw-5, and Ty-1 gene introduction on the Agronomic Performance and Metabolic Profile of Traditional Muchamiel Type Varieties. Line 585: “confers resistance” – you cannot claim this without hard evidence. If you say that. Then I can say that your hybrids are not resistant to said viruses. Prove me wrong… Or change phrasing. Because current reality is like this: “This study demonstrates that the introduction of virus resistance genes Tm-2a, Sw-5 and Ty-1 into traditional Muchamiel varieties in a heterozygous state does not significantly compromise yield or fruit quality, while potentially may provide substantial resistance to ToMV, TSWV, and TYLCV respectively. The actual level of resistance though remains to be tested.”

Response 1. We thank the reviewer for the comment and acknowledge the importance of providing experimental evidence of virus resistance. Resistance assays were performed under controlled conditions using agroinoculation (TYLCV) and mechanical inoculation (ToMV and TSWV), confirming the expected resistant phenotypes. These results are part of a separate manuscript in preparation and were therefore not included here. In response to the reviewers’ suggestions, we have clarified in the manuscript that resistance was experimentally confirmed (L123-126), and we also refer to previous studies where the breeding lines were validated through both genotypic and phenotypic selection. Based on this, we believe that the terminology used in the manuscript—both in the title and in statements such as line 585—is appropriate, as the resistance has been functionally confirmed, even if not detailed in this article. We hope that this clarification helps to address the concern and reinforces confidence in the findings presented.

Comments 2. Line 30: “FAO” – use full name, Food and Agriculture Organization is not universally known.

Response 2. Thank you for the suggestion. The acronym has been expanded to "Food and Agriculture Organization (FAO)" at its first mention in the text. 

Comments 3. As another advice for the future, you could have done a slightly better job of introducing the viruses. As a casual reader, from your introduction, I have no idea how serious of a problem (in Spain) these viruses are. I was a plant virologist, and ill be the first one to tell that there are really crazy things like smoking old school tobacco cigars and then rubbing plant with same fingers and introducing the TMV into them. Or I saw in commercial greenhouses where tomatoes are grown in lines, close together, and people walking in narrow aisles between plant rows, spread viruses simply by rubbing their shoulders from plant to plant. But at the same time I can say that whenever we grow tomatoes outdoors, the viruses are barely noticeable and the fungi and mites cause all the problems. Out of your three viruses, ToMV does not even have the insect vector and is spread only mechanically or via infected seeds. And whiteflies and trips are quite small insects, susceptible to higher winds. I have no idea how common they are in southeastern coast of Spain. A few sentences describing these viruses (and their impact on tomatoes in your area) better could have made your “problem” and the “need of your work” more loud, more valuable.

Response 3. Thank you for the suggestion. We have revised the introduction to better contextualize the impact of these viruses in south-eastern Spain, highlighting the limitations of vector control and the role of mechanical transmission, particularly under greenhouse conditions (L51-55).