Genetic Analysis of the Special Peel Color Segregation Ratio Coregulated by Anthocyanin and Chlorophyll Pathway Genes in Eggplant

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

After careful review of the manuscript, the methodology requires improvement, and certain results should be relocated to the methodology section for better clarity and structure.

L61. SmANS should be in italics

L96-172. The font type change since this line. (please review this). .

L145. Please include the sonication conditions used.

L148. HiSeq… what is the model of the Illumina HiSeq?

L154-161. The authors state that in 2014 this was conducted and it was mapped to SME_r2.5 genome. Currently, there is a website of the eggplant genome https://eggplant.kazusa.or.jp/. And several genomes were published after 2014. I suggest that the authors should perform this against the most recent available genome.

161. The authors are interested in color, however the authors did not explain where the SNPs were identified ? Authors should include more information about this.

L173. The authors explain that molecular markers were exploited. However, authors should explain in detail how this was performed

L175-L176. Please include a table with the sequence and the characteristics of the primers designed. Furthermore, what are the PCR conditions?? time? Tm? Several information is missing. Please include all this.

L177. SNPs and InDel are not the same. How do the authors design Indel Markers?? please include information of the bioinformatic tools and explain how it was do it.

L177-180. Why some markers were analyzed by acrilamide gels and this under agarose gels?? Moreover, authors need to explain the PCR conditions and the equip of the PCR was performed.

L191. Authors stated that “the constructs were transformed”. However, what was the plasmid?? map of the plasmid? Sequence?? Please include more information.

 

L276. Density tic map??? Please review

 

The authors need to improve the method section, explain more about the bioinformatic analysis in order to evaluate if the results found are correct. It is hard to analyze without more information in the method section.

 

L350. I suggest that table 6 should be in method section and include the size of the fragment amplified.

Table 6. The marker InDel-APRR2 with the ddPPgv1gv1 marks “no amplication”.. any suggestion of why this happened

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

In this study, the authors sought to elucidate the genetic basis and interaction mechanisms underlying unusual eggplant peel color segregation by investigating how genes controlling anthocyanin and chlorophyll biosynthesis jointly determine fruit color. Specifically, they aimed to identify, map, and characterize the roles of the D and P genes (anthocyanin-related) and the Gv1/Gf gene (chlorophyll-related), clarify their recessive epistatic and additive interactions, and link genetic variation to pigment accumulation and phenotypic outcomes in segregating populations, while also developing molecular markers to support genetic analysis and breeding for diverse eggplant peel colors.

Title.
The current title is accurate in identifying the principal genes and the two types of interactions under investigation, but it slightly overstates the level of mechanistic insight provided by the study. By asserting that the loci “jointly regulate” eggplant peel color, the title implies a degree of molecular or biochemical causality that is not directly demonstrated. While the study convincingly establishes genetic interaction and perfect marker linkage, it does not present functional evidence—such as pathway crosstalk, gene expression analyses, or biochemical assays—showing how these genes act together at the mechanistic level. A more restrained title would better align with the strength and scope of the data. For instance, a formulation such as “Genetic Mapping and Interaction Analysis of D, P, and Gv1 Loci Underlying Eggplant Peel Color Segregation” would accurately reflect the study’s genetic focus without implying untested mechanistic conclusions.

Abstract.
The abstract provides a generally accurate overview of the study but overstates its conclusions and omits key limitations that are important for proper interpretation. It characterizes the work as offering “valuable insights into the genetic mechanisms” underlying peel color, which is true insofar as genetic models and interactions are concerned, but it fails to clarify that no biochemical or molecular mechanisms were directly investigated. There is no acknowledgment that the characterization of gene roles relied primarily on sequencing and genetic analysis rather than functional validation, such as pigment profiling, expression analysis, or complementation tests. In addition, some phrasing is imprecise or awkward—statements such as “the objective of this research offers” should be replaced with direct descriptions of what the research did and found. Finally, while references to mutations “inhibiting” pigment synthesis are consistent with prior literature, the abstract does not make clear that the present study itself does not experimentally confirm causality for the specific alleles examined. Overall, the abstract effectively summarizes the study’s claims but inherits a broader tendency to present genetic associations and models as fully elucidated mechanisms.

Keywords.
The keyword section is serviceable but overly broad and misses opportunities to enhance the paper’s discoverability. Terms such as “eggplant,” “peel color,” and “epistasis and additive interactions” accurately describe the general topic but do not reflect the methodological or molecular specificity of the work. Incorporating more precise keywords—such as “bulk segregant analysis (BSA),” “molecular markers,” “SmMYB1,” “SmAPRR2,” or “chlorophyll biosynthesis”—would improve indexing and better signal the study’s relevance to researchers working in plant genetics, pigmentation, and genomic mapping.

Introduction.
The introduction is well written and provides a strong rationale for the study, but it slightly overpromises in its description of anticipated outcomes. In particular, statements suggesting that the work will “elucidate gene interactions within different pigment biosynthesis pathways” imply a level of pathway-level or mechanistic resolution that is not fully achieved. The study succeeds in defining genetic interactions and inheritance patterns with clarity and rigor, yet it offers limited insight into how these interactions operate within or between specific biochemical pathways.

Overall Assessment.

Taken together, the front matter is effective in conveying the importance of the research and in framing its objectives within the broader context of eggplant pigmentation genetics. However, it consistently sets expectations for mechanistic depth that are not fully met by the methods and results that follow. This disconnect reflects not a flaw in the motivation or framing of the study, but a mismatch between the strength of the genetic evidence presented and the more expansive mechanistic language used to describe its implications.

Methods.
The methods as described are generally sufficient for the study’s core technical aims—namely, the genetic mapping of the D, P, and Gf loci and the development of linked molecular markers—but they fall short of supporting the broader, more mechanistic objectives articulated in the introduction. While the experimental design enables locus identification and preliminary interaction inference at the genetic level, it is not equipped to rigorously address questions of epistasis, joint determination of peel color, or the biochemical linkage between allelic variation and pigment accumulation. Reproducibility is achieved in part through the use of standard techniques and referenced pipelines, but it is undermined by critical omissions in experimental detail and, more importantly, by structural gaps in population design and phenotypic assessment.

Plant Materials.

The choice and description of plant materials represent a major methodological shortcoming relative to the study’s stated goals. Although the parental lines are clearly phenotyped and their sources are identified, the populations employed are inherently inadequate for analyzing epistatic and additive interactions between the anthocyanin (D/P) and chlorophyll (Gv1/Gf) pathways. Each population segregates for only a single trait, and no population is described that segregates simultaneously for both anthocyanin and chlorophyll variation. Such a population is a prerequisite for any meaningful analysis of genetic interaction between these pathways, and its absence constitutes a fundamental disconnect between the experimental design and a central objective of the study. From a reproducibility standpoint, the lack of accession numbers or seed bank identifiers further limits the ability of other researchers to obtain identical germplasm, despite otherwise clear phenotypic descriptions.

Detection of Anthocyanin and Chlorophyll Content.

The pigment detection methods are technically standard but fundamentally misaligned with the study’s objectives. The stated aim is to link genetic variation to pigment accumulation in segregating populations, yet anthocyanin and chlorophyll contents are measured only in the parental lines and an F₁ hybrid. No biochemical measurements are performed in the F₂ populations where segregation of alleles occurs. This omission entirely severs the intended connection between genotype and biochemical phenotype, precluding any assessment of how different allelic combinations influence pigment levels. Although the use of commercial assay kits and spectrophotometric equations yields quantitative data, their limited application renders them insufficient—and effectively irrelevant—for addressing the study’s central genetic questions.

Phenotypic Evaluation of the E4957 F₂ Population.

Phenotypic evaluation of the E4957 F₂ population is minimally adequate for testing simple Mendelian segregation, as it allows for the classification and counting of phenotypic classes. However, the scoring system employed (3, 2, 1, 0) is subjective and non-quantitative, which sharply limits its utility for understanding how multiple genes jointly determine fruit color. The reliance on qualitative descriptors such as “purple-brown” or “purple-red” introduces observer bias and reduces reproducibility, particularly in the absence of standardized color charts, calibrated imaging, or controlled lighting conditions. Given the study’s emphasis on genetic interactions and color determination, objective colorimetric measurements—such as CIELab* values—across a population segregating for both traits would have been essential.

Genomic DNA Extraction and DNA Pool Construction.

The procedures for genomic DNA extraction and bulk construction are adequate for bulk segregant analysis. The methods described are standard, appropriate for high-throughput sequencing, and sufficient to support the downstream mapping strategy. No major technical deficiencies are evident at this stage.

SLAF-seq Combined with BSA for Mapping the D, P, and Gf Genes.

The SLAF-seq combined with the BSA approach is appropriate and effective for the primary goal of locus mapping. The use of two different reference genomes for different genes is unconventional and may complicate cross-locus comparisons, but it does not invalidate the approach. The sequencing workflow and bioinformatic analyses rely on widely used tools, including GATK and SnpEff, and are supported by appropriate citations. Reproducibility is moderate to high, although it would be improved by the inclusion of explicit software parameters, quality thresholds, and SNP filtering criteria.

Development of Molecular Markers.

The development of molecular markers is methodologically sound and aligned with the study’s mapping objectives. However, reproducibility is compromised by the omission of primer sequences from the methods section. While the primer design software is identified, the absence of the actual sequences represents a critical gap that limits independent validation and reuse of the markers.

Cloning of SmMYB1, SmANS, and SmAPRR2-Like Genes.

The cloning of candidate genes constitutes a logical extension of the mapping results but remains superficial in execution. Sequencing alleles from the two parental lines successfully identifies polymorphisms and supports candidate gene nomination, yet it does not amount to functional characterization. No expression analyses, complementation assays, or loss- or gain-of-function studies are performed, despite language suggesting that the roles of these genes were “characterized.” As such, the methods support descriptive genetic inference rather than mechanistic insight.

Overall Assessment.

In sum, the methods are technically competent and largely reproducible for a narrowly defined study focused on mapping the D, P, and Gf loci and developing linked molecular markers. However, they are incomplete and poorly aligned with the broader, more ambitious objectives of elucidating gene interactions and mechanistic determinants of peel color. The principal limitations arise not from execution, but from experimental design choices that preclude direct testing of the study’s central conceptual claims.

Results.
The results section is highly effective in accomplishing the study’s primary genetic objectives, particularly the mapping of the D, P, and Gf loci and the validation of tightly linked molecular markers. The genetic evidence supporting locus localization is strong, internally consistent, and well presented. However, the results are notably less complete with respect to the study’s broader mechanistic and interaction-focused aims. Assertions regarding gene interactions and the biochemical basis of peel color variation rest largely on segregation ratios and candidate gene sequencing, rather than on direct functional assays or biochemical measurements in segregating populations. As a consequence, the results convincingly establish genetic associations and inheritance patterns but remain inferential with respect to mechanism.

Phenotype and Anthocyanin and Chlorophyll Concentrations.

The analysis of phenotypes and pigment concentrations is only partially adequate in relation to the study’s stated goals. While anthocyanin and chlorophyll levels are measured in the parental lines and the F₁, allowing confirmation of expected differences between these genotypes, the absence of pigment quantification in the F₂ population represents a critical gap. Despite claims of a “direct correlation” between genotype and pigment accumulation, no such relationship is demonstrated in the segregating population that forms the core of the genetic analysis. Without biochemical data from the 237 F₂ individuals, the intended link between allelic variation and pigment levels cannot be substantiated, and conclusions regarding pigment accumulation remain extrapolative rather than demonstrated.

Unusual Peel Color Segregation Ratios.

The analysis of peel color segregation ratios is adequate and persuasive in demonstrating non-Mendelian patterns consistent with genetic interaction. The observed ratios provide strong support for epistatic and additive effects among the loci under study, effectively reinforcing the proposed genetic model. Nevertheless, these findings remain confined to the level of genetic inference. The results do not include corresponding measurements of anthocyanin or chlorophyll content for each phenotypic or genotypic class, leaving the biochemical consequences of these interactions uncharacterized.

Genetic Localization of the D, P, and Gf Loci.

The genetic localization of the D, P, and Gf loci is fully adequate and represents one of the strongest components of the study. The mapping strategy is robust, the intervals are clearly defined, and the results are well supported by sequencing and marker data. These sections successfully fulfill the stated mapping objectives and provide a solid foundation for downstream genetic analysis.

Development of Molecular Markers for Peel Color.

The development of molecular markers linked to peel color traits is likewise fully adequate. The markers are shown to be tightly associated with the target loci and are validated across relevant populations, underscoring their utility for both genetic analysis and potential breeding applications. This aspect of the results is conclusive and well aligned with the study’s applied objectives.

Validation of Genotypes in the E4957 F₂ Population.

Genotype validation in the E4957 F₂ population is thorough and convincing. The correspondence between marker genotypes and phenotypic classes provides strong confirmation of linkage and supports the proposed genetic model. Within the scope of genetic validation, these results are both adequate and conclusive.

Cloning and Sequence Analysis of Candidate Genes.

The cloning and sequence analysis of candidate genes are adequate for identifying putative causative mutations but fall short of the stated objective of functional characterization. The identification of sequence lesions consistent with known loss- or gain-of-function alleles strengthens the genetic interpretation of the mapping results. However, this section does not extend beyond descriptive sequence analysis. No data are presented on gene expression, protein function, or regulatory relationships among the candidates, nor is there evidence addressing how these genes interact within pigment biosynthesis pathways. Given that the objective was to “characterize the roles” of these genes, the results provide only a preliminary genetic description rather than a substantive functional or mechanistic characterization.

Discussion.
The discussion section (Sections 4.1–4.4) is partially adequate in that it effectively situates the study within both classical and contemporary genetic literature and offers a coherent synthesis of the proposed genetic interaction model among the D, P, and Gv1/Gf loci. The narrative successfully integrates historical segregation analyses with modern mapping results, reinforcing the plausibility of the genetic framework. However, this strength is undermined by a notable lack of critical self-evaluation. The discussion does not acknowledge several major methodological and interpretive limitations that materially affect the strength of the study’s conclusions, and it repeatedly overextends genetic inference into claims of biochemical mechanism that are not directly supported by the data.

A central omission is the complete absence of discussion regarding the lack of pigment measurements in the segregating F₂ population. Because anthocyanin and chlorophyll contents were not quantified in the individuals used for genetic analysis, the study cannot biochemically validate its claims of additive effects or “joint determination” of peel color. This limitation is fundamental, yet it is neither acknowledged nor addressed. Similarly, the discussion does not concede that the “characterization” of gene roles is restricted to the identification of sequence polymorphisms in candidate genes, without any functional validation through expression analysis, complementation, or loss-of-function approaches. By omitting these points, the discussion presents the study as more comprehensive than its methods and results justify.

The discussion also frequently conflates strong genetic evidence with a proven biochemical mechanism. Statements asserting that Gv1 (Gf) exerts an additive effect on D and P to determine peel color depth are framed as mechanistic conclusions, despite being based solely on visual phenotypic scoring and segregation ratios. Without quantitative pigment data or objective color measurements distinguishing genotypes such as D_P_Gv1_ and D_P_gv1gv1, the biochemical nature of this “additivity” remains hypothetical. This conflation is further evident in the presentation of the proposed biochemical pathway model. Although Figure 7 is, by necessity, a speculative schema derived from genetic data and candidate gene identities, the discussion treats it as an explanatory endpoint rather than as a testable hypothesis, without explicitly noting that no data on pathway flux, gene expression, or protein interaction were generated to support it.

An additional weakness is the uncritical treatment of the mapping results for Gf. The discussion does not address the fact that BSA-seq failed to produce a clear mapping peak for this locus, necessitating a lowered threshold and resulting in a large candidate interval of approximately 10.55 Mb. This methodological compromise is an important caveat that bears directly on the confidence with which Gf is equated to Gv1, yet it is not discussed. The absence of such caveats contributes to an overall tone that prioritizes defending the proposed model over critically evaluating the strength and limits of the evidence.

Conclusion.
The conclusion largely recapitulates the study’s principal genetic findings and, at that level, is broadly supported by the data. However, it reiterates several overreaching statements that project genetic models onto biochemical mechanisms without direct experimental support. Assertions that the combination of anthocyanin and chlorophyll determines the purple-brown peel color are logically consistent with existing knowledge but were not directly demonstrated within the study’s experimental framework, particularly in the absence of comparative pigment or colorimetric data from segregating populations. Similarly, the description of gene additive interactions as Gv1 “promoting chlorophyll accumulation and deepening anthocyanin coloration” relies on qualitative visual assessment rather than quantitative biochemical or colorimetric evidence.

The conclusion also treats the equivalence of Gf and Gv1 as effectively established, when it is more accurately a parsimonious and well-supported hypothesis. While map overlap and phenotypic correlations strongly suggest that these loci correspond to the same gene, definitive proof—such as co-segregation of flesh and peel color with the same mutation in a single population or functional validation of the allele—has not been provided. Finally, language suggesting that pigment biosynthesis pathways are “restored” or that pathway “superposition” occurs introduces speculative mechanistic interpretations. The data demonstrate genetic complementation and segregation patterns, not the restoration or direct interaction of biochemical pathways.

Overall Assessment of Discussion and Conclusion.

In the context of previous work, the discussion succeeds in positioning the study as a meaningful advance in eggplant genetics, offering a clear and testable genetic model for the interaction of the D, P, and Gv1 loci and delivering valuable molecular markers for future research and breeding. However, the presentation of limitations is inadequate and represents a significant weakness. The discussion reads largely as an affirmation of the proposed model rather than a balanced evaluation of what the data do and do not demonstrate. As a result, while the core genetic conclusions are strongly supported, the broader mechanistic interpretations adopted in the discussion and conclusion are only partially justified.

To improve rigor and transparency, the discussion would benefit from a dedicated subsection explicitly addressing limitations, particularly the absence of pigment biochemistry in the F₂ population and the preliminary nature of the functional gene “characterization.” The conclusion should be tempered to emphasize that the study provides a robust genetic framework and practical tools that lay the groundwork for future biochemical and functional analyses, rather than claiming to have fully elucidated the mechanistic basis of peel color determination.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please see my comments!

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors performed the changes requested. The Article has been improved and can be accepted.

 

Author Response

Thank you for reviewing.

Reviewer 2 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

As noted in my previous reviewer comments, the authors aimed to elucidate the genetic basis and inheritance mechanisms underlying unusual eggplant peel color segregation by mapping and analyzing the interactions among the D, P, and Gv1 (Gf) loci in a cross between green- and white-peel inbred lines. Specifically, they aimed to determine how epistatic interactions between the anthocyanin-related genes D (SmMYB1) and P (SmANS), in conjunction with the additive chlorophyll-related gene Gv1 (SmAPRR2-Like), contribute to explaining the observed F2 segregation ratios and phenotypic variation in peel color. Additionally, they aimed to identify candidate gene mutations responsible for pigment differences and to develop molecular markers for genotype–phenotype association, thereby providing insights into the coordinated regulation of anthocyanin and chlorophyll biosynthesis and facilitating marker-assisted breeding for diverse eggplant peel colors.

1. Overall assessment

This study addresses an interesting and biologically meaningful question: the genetic interaction between anthocyanin- and chlorophyll-related loci underlying unusual peel color segregation in eggplant. The segregation ratio (27:9:21:7), candidate gene identification, and marker development represent potentially valuable contributions to eggplant genetics and breeding.

However, several conclusions are currently overstated relative to the evidence presented. In particular:

• The identity of Gf and Gv1
• Definitive parental genotypes
• The additive genetic model
• Marker utility for breeding beyond the mapping population

Additionally, important methodological details and data transparency elements are missing, limiting reproducibility.

The manuscript would benefit from a major revision, primarily to:

1. Moderate interpretative claims,
2. Clarify inference versus proof,
3. Improve methodological transparency,
4. Strengthen logical consistency and terminology usage.

2. Title

Comment

The title is generally appropriate but could be improved for precision and impact.

Major issue

• The phrase “Underlying Eggplant Peel Color Segregation” is somewhat vague. Consider specifying:
• “in an F2 population”
• or referencing the unusual segregation pattern.

Minor issue

• Terminology inconsistency between Gf and Gv1 should be resolved and standardized in the title.

3. Abstract

The abstract is structured and informative, but several statements require refinement.

3.1 Segregation model description

Major comment

The phrase:

“two recessive epistatic genes and one additive gene”

is potentially misleading and insufficiently justified. The evidence supports interaction consistent with reciprocal recessive epistasis between D and P, but the additive effect of Gf/Gv1 is inferred rather than rigorously demonstrated via quantitative genetic analysis.

Recommendation: Rephrase more cautiously, e.g.:

• “consistent with reciprocal recessive epistasis between D and P, with Gv1 showing a modifying/additive interaction.”

3.2 Gf corresponding to Gv1

Major comment

The statement:

“Gf… corresponded to Gv1”

is too definitive given the mapping resolution and lack of functional validation.

Recommendation: Replace with:

• “mapped near the Gv1 locus”
• or “is likely associated with Gv1”
• unless direct allelism testing was performed.

3.3 Marker utility

Minor comment

The abstract implies general breeding applicability. However, markers were validated only within the E4957 F2 population.

Recommendation: Temper claims to reflect the validation scope.

3.4 Logical clarity

The pigment description is mostly coherent, but ensure consistency when describing chlorophyll presence in white vs. green lines.

4. Keywords

Comment

The keywords are relevant but overly general.

Recommendation

Consider adding more specific terms, such as:

• anthocyanin
• chlorophyll
• SmMYB1
• SmANS
• APRR2-Like
• segregation ratio
• epistatic interaction

This will improve discoverability and indexing.

5. Introduction

The Introduction provides adequate background but requires sharpening and clarification.

5.1 Novelty framing

Major comment

The novelty of the study is not emphasized strongly enough.

The Introduction should more clearly articulate:

• Why previous D–P or D–P–Y models are insufficient.
• Why a 27:9:21:7 segregation pattern is biologically significant.
• Why interaction between anthocyanin and chlorophyll loci represents a conceptual advance.

5.2 Premature conclusions

Major comment

The Introduction currently presents conclusions (e.g., interaction model outcomes) before results are shown.

Recommendation: Move interpretive statements to Results or Discussion.

5.3 Gf vs Gv1 Terminology

Major comment

There is ambiguity regarding:

• Whether Gf and Gv1 are identical loci,
• Allelic variants,
• Or closely linked but distinct genes.

This must be clarified clearly and consistently.

5.4 Redundancy

Some descriptions of pigment biosynthesis and gene identity are repetitive. Condensation would improve readability.

6. Materials and Methods

This section requires strengthening for reproducibility.

6.1 Phenotyping

Major issue

Peel color classification appears subjective.

• Were color categories scored visually only?
• Were objective measures used (e.g., colorimeter, Lab* values)?
• Were evaluators blinded?

Additionally:

• F2 pigment quantification data are missing.

Recommendation:

• Provide detailed phenotyping protocol.
• Include quantitative pigment data for F2 individuals, if available.
• Describe replication and scoring criteria.

6.2 Mapping methodology

Major issue

Important methodological details are missing:

• SNP filtering criteria
• Coverage depth thresholds
• Missing data handling
• LOD thresholds
• Mapping software parameters

These are essential for reproducibility.

6.3 Primer information

Major issue

Primer sequences for developed markers (InDel22522, InDel5531, InDel-APRR2) are not fully reported.

These must be included in:

• Supplementary tables.

6.4 Statistical analysis

Clarify:

• How segregation ratios were tested (Chi-square? Exact test?)
• Degrees of freedom
• Significance thresholds

7. Results

The results are promising but require more cautious interpretation.

7.1 Segregation model interpretation

Major comment

The 27:9:21:7 ratio fits a three-locus interaction model. However:

• Alternative genetic models should be briefly discussed.
• The additive characterization of Gf requires stronger statistical support.

7.2 Gf mapping strength

Major comment

The Gf signal appears weaker than D and P.

Avoid overstating locus identity unless:

• Fine mapping
• Allelism testing
• Or functional validation supports it.

7.3 Parental genotypes

Major comment

Parental genotypes are inferred based on candidate mutations but not functionally validated.

The manuscript should clearly state these are inferred genotypes, not experimentally confirmed ones.

7.4 Sequence variation interpretation

Candidate gene mutations are compelling. However:

• Functional validation (expression, complementation, enzymatic activity) is lacking.
• Statements should reflect association, not confirmed causation.

7.5 Marker concordance

The 100% genotype–phenotype concordance is impressive but only demonstrated in a single F2 population.

Avoid generalizing to broader germplasm without validation.

8. Discussion

The Discussion is conceptually strong but requires moderation of claims.

8.1 Overstatement of genetic identity

Statements implying definitive identification of:

• Gf = Gv1
• Causal mutation confirmation

should be softened unless directly proven.

8.2 Additive vs. Modifying Effect

Clarify whether Gv1:

• Shows true additive inheritance,
• Or acts as a modifying factor conditional on anthocyanin presence.

8.3 Limitations (currently underdeveloped)

A dedicated paragraph acknowledging limitations is needed, including:

• No functional validation
• Mapping resolution constraints
• Single-population validation
• Lack of quantitative pigment data in F2
• Potential environmental influence

This will significantly strengthen credibility.

8.4 Broader implications

Breeding applications should be framed as:

• Promising but requiring multi-population validation.

9. Figures and Tables

Major issues

• Ensure all referenced tables/figures are present.
• Provide raw genotype–phenotype counts.
• Provide complete marker details.

10. Writing and presentation

Minor issues

• Reduce redundancy in the Introduction.
• Ensure consistent gene naming (D vs SmMYB1; Gf vs Gv1).
• Standardize terminology.
• Moderate definitive language (e.g., “corresponded to”, “confirmed”).

11. Summary of required revisions

Major revisions needed

1. Moderate overstatements (Gf–Gv1 identity, additive claim, breeding utility).
2. Clarify inference vs. proof.
3. Provide complete mapping and SNP filtering parameters.
4. Include primer sequences and marker details.
5. Strengthen phenotyping description.
6. Add an explicit limitations section.

Minor revisions

1. Improve novelty framing in the Introduction.
2. Expand keywords.
3. Clarify terminology consistency.
4. Refine title precision.

Final recommendation

The study contains valuable genetic findings and a compelling segregation model. However, revisions are necessary to:

• Improve methodological transparency,
• Align claims with evidence,
• Enhance logical clarity,
• Increase scientific rigor.

With appropriate revision, the manuscript has strong potential for publication and contribution to eggplant genetics and breeding research.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please see my comments!

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

I appreciate the authors’ effort to revise the manuscript and respond to the previous review. The revision improves several aspects of the study, particularly the moderation of causal claims and the clarification of some methodological details. The study addresses an interesting question regarding the interaction between anthocyanin- and chlorophyll-related loci affecting eggplant peel color, and the genetic mapping work has potential value for understanding pigmentation genetics in eggplant.

However, several reviewer concerns were only partially addressed, and some revisions remain incomplete or insufficiently reflected in the revised text. In particular, the abstract wording, novelty framing in the introduction, clarification of genetic interaction terminology, and methodological transparency still require improvement. In addition, some inconsistencies and minor language errors remain.

To help further strengthen the manuscript, I provide section-by-section comments below.

1. Title

Assessment

The revised title:

“Genetic Mapping and Interaction Analysis of D, P, and Gv1 Loci Controlling Eggplant Peel Color”

is clearer and more precise than the original version. The terminology has been improved and the main genetic loci are now explicitly stated.

Remaining suggestions

While the revised title is acceptable, the authors may still consider whether it could more clearly reflect the distinctive genetic finding of the study, namely the unusual segregation pattern and the interaction between anthocyanin and chlorophyll pathways.

For example, referencing the segregation pattern or interaction between pigment pathways could further strengthen the title’s specificity.

This is optional and does not require major modification if journal title-length constraints apply.

2. Abstract

Assessment

The authors indicate that the abstract has been revised to moderate several statements. However, the revised abstract still contains wording that may overstate the level of genetic inference.

For example:

“genetic model controlled by recessive epistasis D and P, and modification/additive interaction Gv1”

The evidence presented supports epistasis between D and P, but the role of Gv1 appears to be inferred from phenotypic segregation patterns rather than formally demonstrated through quantitative genetic analysis.

Similarly, the description:

“the additive interaction gene Gf”

remains ambiguous and may imply a level of mechanistic confirmation that is not fully demonstrated.

Recommendations

Please revise the abstract to:

1. Use more cautious language when describing the role of Gv1/Gf.
   For example:
• “consistent with reciprocal recessive epistasis between D and P, with Gv1 likely acting as a modifying factor.”
2. Avoid language implying confirmed additive inheritance unless supported by formal quantitative genetic analysis.
3. Ensure that the relationship between Gf and Gv1 is described cautiously (e.g., “mapped near the previously reported Gv1 locus” or “likely associated with Gv1”).
4. Carefully proofread the abstract to correct remaining typographical errors (e.g., “chlorphyll” → “chlorophyll”).

Addressing these points will improve both scientific precision and clarity.

3. Keywords

The revised keywords are generally relevant; however, several issues remain.

Points for revision

1. The spelling of “chlorphyll” should be corrected to “chlorophyll.”
2. The current list does not fully incorporate some of the reviewer’s suggestions (e.g., segregation ratio, genetic mapping, or pigmentation pathways), which may improve indexing and discoverability.
3. The explanation that the journal limits the number of keywords to five should be checked against the current list, as the provided keywords appear to exceed this number.

Please revise the keyword list accordingly to ensure accuracy and compliance with journal guidelines.

4. Introduction

The introduction provides useful background on eggplant pigmentation genetics but still requires further refinement.

4.1 Novelty framing

The novelty of the study should be articulated more clearly. In particular:

• Why previously reported models (e.g., D–P or D–P–Y interactions) are insufficient to explain the observed phenotype.
• Why the 27:9:21:7 segregation ratio observed in this study is biologically noteworthy.
• How the interaction between anthocyanin and chlorophyll pathways represents a conceptual advance beyond previous studies.

Currently, these points are implied but not strongly emphasized.

4.2 Structure and clarity

Some sections of the introduction remain overly descriptive or repetitive, particularly in the discussion of pigment biosynthesis pathways. Condensing these sections would improve readability and allow greater focus on the specific research question addressed by this study.

4.3 Terminology consistency

The relationship between Gf and Gv1 should be clearly explained early in the introduction.

At present it remains unclear whether the authors consider:

• Gf and Gv1 to be identical loci,
• allelic variants, or
• closely linked but distinct genes.

Given the current evidence, the manuscript should consistently refer to this relationship as inferred or likely associated, rather than definitively identical.

5. Materials and Methods

The authors have added some clarification regarding phenotyping and mapping approaches, which is appreciated. However, additional details are still needed to ensure full reproducibility.

5.1 Phenotyping protocol

The explanation that peel colors are visually distinguishable is reasonable. However, the methods section should still clearly describe:

• The exact phenotyping criteria used to classify individuals into the four categories.
• Whether phenotyping was performed by one or multiple evaluators.
• Whether replicate observations were made.

Even when phenotypes are visually distinct, explicit documentation of the scoring protocol improves reproducibility.

5.2 Pigment quantification

Quantification of anthocyanin and chlorophyll only in parental lines and F1 plants is acceptable for confirming pigment associations. However, the manuscript should explicitly acknowledge that quantitative pigment data were not collected for F2 individuals, which represents a limitation when interpreting phenotype–genotype relationships.

5.3 Mapping methodology

The description of the mapping approach should clearly include:

• SNP filtering criteria
• Coverage depth thresholds
• Handling of missing data
• Criteria used for identifying candidate regions

Although sequencing and analysis were performed by a service provider, the analytical parameters must still be documented in the manuscript to ensure methodological transparency.

6. Results

The mapping and candidate gene analyses are interesting and represent a valuable component of the study. The authors have appropriately moderated several statements regarding causality.

However, the manuscript should ensure that:

1. Parental genotypes are described as inferred, not experimentally confirmed.
2. Candidate mutations in SmMYB1, SmANS, and SmAPRR2-like are presented as associations consistent with the observed phenotypes, rather than confirmed causal variants.

In addition, if the authors state that markers have been applied in additional eggplant populations, the corresponding data should either be included in the manuscript or the statement should be removed.

7. Discussion

The discussion provides a useful interpretation of the genetic results but still requires refinement.

7.1 Genetic interaction interpretation

The explanation of the role of Gv1 remains somewhat unclear. The manuscript should explicitly clarify whether:

• Gv1 exhibits true additive inheritance, or
• acts as a modifying factor influencing the phenotypic outcome of D and P interactions.

If the latter interpretation is more appropriate, this should be clearly stated.

7.2 Moderation of claims

The discussion appropriately softens statements implying that Gf and Gv1 are definitively identical loci. This cautious wording should be maintained throughout the manuscript.

Similarly, candidate gene mutations should be discussed as likely associations rather than confirmed causal variants, given the absence of functional validation.

7.3 Study limitations

The manuscript would benefit from a clear paragraph summarizing the main limitations, including:

• absence of functional validation experiments
• mapping resolution constraints
• marker validation limited to a single F2 population
• absence of quantitative pigment measurements in F2 individuals
• potential environmental effects on pigment accumulation

Explicit acknowledgment of these limitations would strengthen the scientific transparency and credibility of the study.

7.4 Breeding implications

The discussion of marker-assisted breeding applications should remain cautious, as marker performance has only been evaluated within a single population.

It would be appropriate to state that these markers show potential utility, but additional validation across diverse germplasm is required.

8. Figures and Tables

The addition of genotype–phenotype counts and marker details is appreciated and improves transparency.

Please ensure that:

• All figures and tables are clearly referenced in the text.
• Marker sequences, primer details, and expected amplicon sizes are fully reported.

9. Writing and Language

The manuscript has improved in clarity but still contains several minor grammatical and typographical errors.

Examples include:

• spelling errors such as “chlorphyll”
• occasional awkward sentence structures
• inconsistent terminology.

A careful final language edit is recommended to improve readability and ensure consistent gene nomenclature throughout the manuscript.

 

Overall Recommendation

The manuscript addresses an interesting question regarding the genetic interaction between anthocyanin and chlorophyll pathways influencing eggplant peel color, and the mapping of the D, P, and Gv1-associated loci represents a valuable contribution.

The revision has improved several aspects of the manuscript, particularly the moderation of causal claims. However, several reviewer concerns remain partially addressed, particularly regarding:

• clarity of genetic interaction interpretation
• methodological transparency
• novelty framing
• explicit acknowledgment of study limitations.

Addressing the points outlined above would substantially strengthen the manuscript’s scientific clarity, rigor, and impact.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

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Author Response

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Author Response File: Author Response.pdf