Review Reports - Transcriptome Analysis Reveals Key Genes Involved in Fruit Length Trait Formation in Pepper (<i>Capsicum annuum</i> L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “Transcriptome Analysis Reveals Key Genes Involved in Fruit Length Trait Formation in Pepper (Capsicum annuum L.)” addresses an important agronomic trait relevant to crop yield and marketability. However, several key issues need to be addressed before it can be considered for publication.

The manuscript repeatedly emphasizes the importance of yield- and quality-related traits, particularly fruit length. While this contextualizes the study, the repeated statements reduce clarity and conciseness. Consider streamlining the introduction to focus on novel insights from your work without redundancy.
There are inconsistencies in gene nomenclature throughout the manuscript (e.g., SlOFP20, CaIQD1, CaOFP20, CaTRM-like). Ensure uniform formatting according to journal guidelines, including consistent use of italics for gene symbols and proper capitalization for species-specific prefixes.
Some statements, such as “directly linked to both yield potential and aesthetic appeal,” could be clarified. Rewriting as “directly influences both yield potential and consumer appeal” would improve precision and readability.
The number of biological and technical replicates for phenotypic measurements, cytological analysis, RNA-seq, and qRT-PCR experiments is not provided. Without this information, it is difficult to assess the statistical reliability and reproducibility of the results.
The manuscript lacks details on growth conditions, including light intensity, photoperiod, temperature, humidity, and fertilizer regime. These factors are critical for reproducibility, especially when comparing developmental stages or genotypes.
The description of RNA as derived from “mixed tissue samples (0d, 15d, 30d)” is ambiguous. Pooling across developmental stages may mask stage-specific expression patterns. Clear justification for the pooling strategy or separate analysis of individual stages is recommended.
RNA quality is only assessed by agarose gel electrophoresis. Providing RIN values from a Bioanalyzer or TapeStation would strengthen confidence in RNA integrity and sequencing reliability.
The study uses TopHat 2.0 for read alignment, which is no longer considered a standard. Using modern aligners such as HISAT2 or STAR would improve mapping accuracy and data robustness.
While the study focuses on fruit length, WGCNA analysis mentions correlations with “fruit width” in some modules. Clarifying trait selection and ensuring consistency with the study focus is recommended.
The manuscript emphasizes 30 DAF for gene expression and phenotypic differences. While this is justified by maximal elongation, additional systematic analysis across more stages or replicates would strengthen conclusions regarding temporal dynamics of gene regulation.
The co-expression network analysis identifies correlations among transcription factors, hormone signaling genes, and cell wall-modifying genes. However, causative relationships are not established. Functional experiments, such as gene knockouts, overexpression, or hormone treatments, would provide stronger evidence for regulatory interactions.
Although auxin and gibberellin pathways are thoroughly analyzed, other enriched hormone pathways (e.g., brassinosteroids, ethylene, cytokinins) are only mentioned superficially. A more comprehensive exploration of these hormones could provide a fuller understanding of the hormonal regulation of fruit elongation.
The manuscript frequently references functional data from tomato and Arabidopsis. While informative, it is unclear whether homologous genes have conserved roles in pepper. Clarifying these species-specific differences and potential limitations would strengthen interpretation.
The discussion highlights functional redundancy among AUX/IAA genes, but the manuscript does not clearly distinguish which specific genes are critical in pepper. Highlighting candidate genes with potential stage-specific or genotype-specific functions would improve clarity.
MYB and ERF/AP2 families are emphasized, but downstream targets beyond XTH15-like and Expansin-A10 are not experimentally validated. Identifying or validating additional targets would provide stronger mechanistic insight into the regulatory networks controlling fruit elongation.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Through RNA-seq analysis, the study identified key genes involved in the determination of fruit length in pepper. However, some minor improvements are recommended for the manuscript to be considered for publication.

Question 1. The text presents relevant and well-structured information; however, a careful review of the formatting is recommended, particularly with regard to scientific nomenclature. Several species names are not italicized. Correcting these details is essential to ensure the accuracy and rigor expected in scientific publications.

Question 2. The second paragraph of the introduction is too long, which disrupts the flow of reading. I suggest splitting it into two or more paragraphs.

Question 3. In the topic “2.4 RNA Extraction, Library Preparation, and Sequencing”

Which sequencing platform was used? What was the sequencing coverage? What was the read length?

Question 4. Describe how the qPCR primers were designed. Additionally, list all the primers used.

Question 5. In the topic “2.8 Quantitative Real-Time PCR (qRT-PCR)”

I suggest specifying from which samples the RNA was extracted, the amount of RNA (in ng) used in the reactions, and the name of the commercial kit employed for cDNA synthesis.

Question 6. Include a reference for the 2^−∆∆Ct method.

Question 7. In Figure 7, which statistical test was used? Include this information both in the Methods section and in the figure legend."

Question 8. Specify the objective magnification used for acquiring microscopy images.