Review Reports

S/NO

SECTION/ LINES/PAGES

COMMENTS

1.     1

Abstract

The abstract, being a standalone section, does not clearly state the knowledge gap addressed by the authors. Please include a statement in line 17 to explicitly highlight this gap.

2.     2

Introduction

The study objective presented in Lines 113–114—"However, the genetic mechanisms underlying growth traits remain poorly understood,"—along with the aim stated in the abstract (line 27), which refers to identifying candidate genes associated with growth in C. fuscus through the integration of QTL mapping and RNA-seq analysis of DEGs between two extreme body size groups, is currently unclear and somewhat confusing. The authors are advised to revise and harmonize these statements to present a clear, consistent study objective throughout the manuscript.

3.      

Materials and Methods

The authors need to address the following points to enhance the clarity and rigor of the methodology:

1. Selection Criteria: How were the experimental catfish selected for this analysis? Please specify the criteria or parameters used to categorize them for inclusion in the study.
2. Growth Conditions: Were all selected catfish subjected to the same rearing conditions, including factors such as water quality, temperature, stocking density, and diet? This information is essential to ensure that observed growth differences are genetically, rather than environmentally, driven.
3. Sample Size: How many big-sized and small-sized catfish were selected for the analysis? Providing the exact number of individuals in each group will improve the transparency and reproducibility of the study.

4.      

Line 138

Which version of SPSS software was used

5.      

Line 143

The authors state that “At 6 months of age, 254 individuals were randomly selected for body weight measurement,” which appears inconsistent with the figure of 200 subjects mentioned in the abstract (line 33). This discrepancy raises an important question: How many catfish were used in the study? The authors need to clarify the total number of subjects involved and reconcile this inconsistency to ensure accuracy and transparency in the study’s design and reporting.

6.      

Line 144

Line 144 states that “from these, the 9 largest and 9 smallest individuals were chosen to form 3 big-sized groups (B1, B2, B3) and 3 small-sized groups (S1, S2, S3),” but the basis for this classification is unclear. The authors should clarify how the largest and smallest individuals were determined—for example, was it purely based on body weight, length, or another growth metric? Additionally, they should specify whether any statistical thresholds or percentile cut-offs were applied. This clarification is necessary to ensure that the grouping criteria are transparent and scientifically sound.

7.      

Line 172

The authors should briefly indicate the RNA extraction method used in the study. For example, if a specific kit or protocol (such as TRIzol reagent, column-based kits, or a phenol-chloroform method) was employed, it should be clearly stated, including any modifications made to the standard protocol. This information is essential for reproducibility and assessment of RNA quality and integrity.

8.      

Transcriptome Library Creation and Raw Data Processing

The manuscript does not provide details on how sequencing was performed. The authors should clearly state:

1. The sequencing method used (e.g., sequencing-by-synthesis or another approach).
2. The sequencing platform employed (e.g., Illumina NovaSeq, HiSeq, or another).
3. How raw reads were processed—including any steps for quality control, such as trimming adapters, removing low-quality reads, and assessing read quality (e.g., using tools like FastQC or Trimmomatic).

Including these details is crucial for ensuring reproducibility and assessing the reliability of the RNA-seq data.

9.      

RESULTS

The authors should consider moving Figure I on page 6 to the supplementary material, as it primarily presents quality check results rather than a core output of the study. While important for validating the data used in subsequent analyses, it does not directly contribute to the main findings and would be more appropriately placed as supporting information.

10.  

All Figures

The authors are advised to use high-resolution versions of all figures, particularly Figures 2, 3, and 4, to ensure clarity and readability. The current resolution may hinder the reader's ability to interpret critical details, such as gene labels, axis titles, and data points. Enhancing figure quality will significantly improve the presentation and overall comprehension of the study's results.

Discussion

11.  

Lines 341-349

The authors have not adequately cited previous related studies. To strengthen the scientific context and demonstrate how their work builds upon or differs from existing research, they should enhance the literature review by incorporating and discussing relevant similar studies. This includes citing previous work on growth trait genetics, QTL mapping, and RNA-seq analysis in C. fuscus or other closely related fish species. Doing so will not only provide a more comprehensive background but also highlight the novelty and significance of the current investigation.

12.  

Lines 439-441

The statement, “Our findings suggest that these genes generally promote embryonic development, cell differentiation, and proliferation, consistent with observations from enrichment anal- which showed that many genes are enriched in biological process-related pathways,” may imply that the results were not experimentally validated. To maintain scientific transparency and credibility, the authors should explicitly acknowledge this as a limitation of the study. Clearly stating that the findings are based on bioinformatics predictions without functional validation will help set appropriate expectations and guide future research directions.