Review Reports - Transcriptome Sequencing Unveils a Novel Mechanism Underlying Breed Distinctions Between Thin- and Fat-Tailed Sheep

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep” presents a comparative transcriptomic analysis of tail adipose tissue from fat-tailed Kazakh sheep, thin-tailed Suffolk sheep, and their F2 hybrids. The study integrates mRNA, lncRNA, and miRNA profiling to explore regulatory networks associated with tail fat deposition. Overall, the topic is relevant to animal genetics and breeding, and the dataset has potential value. However, several methodological, analytical, and interpretative issues should be addressed to strengthen the rigor and impact of the study.

Below are my comments:

1) Each group contains only three individuals, which substantially limits statistical power and increases the risk of false positives or overinterpretation of DEGs. I would suggest acknowledging the limitations due to small sample size in the Discussion.

2) The study uses Kazakh ewes, Suffolk rams, and F2 individuals, which introduces a potential confounding effect of sex on gene expression, particularly in adipose tissue. As this is not sufficiently addressed, the authors could discuss the potential impact of sex differences on adipose transcriptomes and justify or statistically control for this factor if possible.

3) The reliance on FPKM for expression normalization is known to introduce biases; more current methods (e.g., TPM, DESeq2/edgeR raw count-based normalization) are preferred. Also, the use of TopHat and Cufflinks/Cuffdiff pipelines is outdated; other widely accepted tools (e.g., HISAT2/STAR and DESeq2) should be considered or at least discussed as a limitation. If reanalysis is not feasible, provide a justification for the chosen methods and discuss their limitations.

4) The manuscript lacks clarity regarding multiple testing correction methods used for DEG, lncRNA, and miRNA identification. Also, it is not always clear whether adjusted p-values were consistently applied across all analyses, especially for ncRNAs. Consider providing details on statistical thresholds, multiple-testing correction, and criteria used for defining genes and ncRNAs.

6) While regulatory relationships (lncRNA–mRNA and miRNA–mRNA) are predicted bioinformatically, no functional validation assays (e.g., knockdown/overexpression, luciferase reporter assays) are provided to confirm these interactions. An experimental validation for at least a subset of lncRNA–mRNA or miRNA–mRNA interactions is critical, or otherwise clearly state that these interactions are predictive and require future validation.

7) Many of the identified genes and pathways (e.g., FASN, ACACA, PPAR signaling, FABP family) have been repeatedly reported in previous studies on sheep tail fat deposition. The manuscript does not clearly explain how the findings add to existing transcriptomic studies. I would suggest comparing the results more explicitly with prior transcriptomic studies and clarifying what is new.

8) Some sections of the Results and Discussion are overly long and repetitive, particularly where similar pathways and genes are discussed multiple times across mRNA, lncRNA, and miRNA analyses.

Overall, the manuscript addresses an important topic. However, methodological limitations, particularly sample size, sex confounding, and lack of functional validation, currently weaken the strength of the conclusions.

Author Response

Rev1 Comments and Suggestions for Authors

Below are my comments:

Response 1): We concur with this insightful comment. Accordingly, we have incorporated a dedicated discussion on the limitations arising from the small sample size, and this revision can be located on page [10], paragraph [2], line [9-16] of the revised manuscript.

Response 2): We have already discussed this issue in discussion section. this revision can be located on page [10], paragraph [2], line [17-22] of the revised manuscript.

Response 3): We acknowledge that the original analysis methods have inherent limitations, and discussed this issue in the discussion section. this revision can be located on page [10], paragraph [2], line [23-30] of the revised manuscript.

Response 4): In this study, we consistently adopted the Benjamini-Hochberg (BH) method for multiple testing correction in the identification of differentially expressed mRNAs (DEGs), lncRNAs, and miRNAs. The core purpose of using this method is to control the False Discovery Rate (FDR). To ensure consistency across all analyses, we strictly used adjusted p-values instead of raw p-values for screening all differentially expressed transcripts. This uniform standard was applied to mRNAs, lncRNAs, and miRNAs without any exceptions. We have supplemented the full set of statistical thresholds and biological criteria used to define differentially expressed genes and ncRNAs in the revised manuscript. t this revision can be located on page [5], paragraph [4], line [22-39] of the revised manuscript.

Response 6) For the regulatory pairs that cannot be fully validated due to experimental conditions or resource constraints. We will also add a dedicated discussion section to elaborate on the limitations of bioinformatic predictions, emphasize that these interactions require further experimental validation in future studies, and propose potential research directions for such validation. this revision can be located on page [10], paragraph [2], line [31-36] of the revised manuscript.

Response 7) We have reorganized the discussion section to clearly distinguish between the existing findings and the new discoveries. this revision can be located on page [8], paragraph [1], line [13-44] of the revised manuscript.

8) Some sections of the Results and Discussion are overly long and repetitive, particularly where similar pathways and genes are discussed multiple times across mRNA, lncRNA, and miRNA analyses.

Response 8) The results and discussion sections have been reorganized and the content has been reorganized as well. this revision can be located on page [9], paragraph [1-5], line [1-46] of the revised manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have taken an unusual experimental approach and have employed transcriptomics methodologies, in order to study differences between sheep breeds.

Whilst this approach is interesting, there are issues that need to be clarified before further processing of the manuscript.

One. The use of these three breeds is not sufficiently justified. Moreover, the authors have included an Asian breed and a European breed, which means that the two breeds were rather distant genetically. The use of two Asian breeds, with more vlose genetic relationships might have been more useful.

Two. The small number of animals used for each breed creates problems. This is not adequately justified. Moreover, do not explain how they guaranteed the genetic background of the animals within each breed.

Three. The authors do not provide adequate details regarding the control materials and the control procedures employed in the study.

Four. Whilst visualization is excellent and the authors must be congratulated for the great graphs and designs, the use of tables is limited. This should be rectified, as it will provide quickly information in summary form to readers.

Five. The Discussion is lengthy and without appropriate subdivisions becomes boring to read and impedes flow of reading. The authors should divide it into subsections.

Six. The number of references is rather small for such a complex and detailed study. Personally, I expected at least 85-90 references for such a lengthy manuscript.

Overall. The authors must improve the manuscript by taking into account the above and resubmit it to continue the evaluation.

Author Response

Rev 2 Comments and Suggestions for Authors

The authors have taken an unusual experimental approach and have employed transcriptomics methodologies, in order to study differences between sheep breeds.

Whilst this approach is interesting, there are issues that need to be clarified before further processing of the manuscript.

Response One: We agree with your point of view. However, as the sequencing and analysis have already been carried out, the content of the data cannot be changed now. We have already discussed these limitations in the discussion. this revision can be located on page [10], paragraph [2], line [9-16] of the revised manuscript.

Response Two： n our discussion, we talked about the limitations of the small sample size. We tried our best to select the ones with relatively clear varieties and pedigrees for the experiment.

Three. The authors do not provide adequate details regarding the control materials and the control procedures employed in the study.

Response Three：We chose to conduct the research on the crossbreeding of Kazakh sheep and double-breed Suffolk sheep and their F2 hybrids based on clear and important breeding goals, rather than random breed comparisons. Core breeding goals: This study aims to analyze the key mechanisms regulating tail fat deposition at the molecular level, ultimately serving the purpose of cultivating a new breed that combines "multiple-birth traits", "excellent meat traits", "moderate tail fat deposition", and can adapt well to the high-altitude and harsh grazing conditions of the Xinjiang cold pastoral area. Basis for parent selection: Maternal parent (Kazakh sheep): It is a typical fat-tailed breed in Xinjiang, with extremely strong adaptability to the local high-altitude and rough feeding conditions, but its reproductive rate is relatively low. Paternal parent (double-breed Suffolk sheep): It is a new breed with multiple births and lean meat traits (derived from the classic thin-tailed Suffolk sheep), aiming to introduce its high reproductive capacity and excellent meat production performance.

Response Four： We have added some important tables, namely Table 1 to Table 5.

Five. The Discussion is lengthy and without appropriate subdivisions becomes boring to read and impedes flow of reading. The authors should divide it into subsections.

Response Five：The discussion has been divided into subsections as required.

Six. The number of references is rather small for such a complex and detailed study. Personally, I expected at least 85-90 references for such a lengthy manuscript.

Response Six：The references have been added to 72 as required.

Overall. The authors must improve the manuscript by taking into account the above and resubmit it to continue the evaluation.

Reviewer 3 Report

Comments and Suggestions for Authors

Transcriptome sequencing unveils a novel mechanism underlying breed distinctions between thin- and fat-tailed sheep

Dear Authors,

the manuscript is interesting and well-prepared. Determining the molecular mechanism of fat deposition and confirming that the Lnc RNA-524-mRNA-miRNA regulatory axis is a key pathway in tail fat formation will certainly allow for improvement of the traits responsible for this effect. However, in my opinion, it would be worthwhile in future studies to increase the number of individuals in each treatment to 10 or more, if possible. Three individuals per group could result in excessive effect of individual variability on obtained results and make it difficult to extrapolate the results to a wider range of individuals in the population. Furthermore, it's worth noting the more detailed description of the statistical analysis methods in lines 168-171 and the specification of individual components in parentheses in the Figures.

Below are suggestions and comments that may be helpful in the revision process:

Lines 9-10

Indentation of text in lines after number or number sign/hash can be adapted to this from lines 6 and 7.

Line 37

Ovis aries (binominal nomenclature, italics needed).

Lines 48, 51, 56, 58 and 70

One space required after dot before beginning of new sentence.

Lines 76-78

Early studies were mentioned in sentence, reference/s can be added.

Lines 127-129

Three animals in one treatment can give low power of a test, additionally effect of individual variability during experiment can have also on obtained results. Maybe in future experiments it will be possible to increase number of animals in treatments, even to 10 animals in each.

Lines 146-150

In description of Figures 1A-1E brackets can be used, respectively to appear in the text: (A-B), (C), (D), (E). That should distinguish them from the entire text.

Line 165

Table S1

Line 168

SPSS 22.0 software was used (name and address of producer must be also added in brackets).

Line 169

One-way ANOVA and independent sample t-test was used to compare variables between treatments. Information about checking of normality distribution data in each treatment using Shapiro-Wilk’s test and homogeneity of variance between treatments (Levene’s test before one-way ANOVA and F-test in case of t-test).

Lines 263 and 270

Table S2

Lines 290-295

The same as in lines 146-150: (A), (B), (C).

Lines 316-318

The same as in lines 146-150: (A), …, (D).

Line 322

p-value instead of P-value can be used. Three animals in treatment randomly allocated as a sample from population.

Line 342

‘… Table S10)

Lines 362 and 365

Space after Table required in case of (Table S14) and (Table S15).

Line 383

(Fig.2)

Lines 530-534

Space between Table and supplement number required in case of Table S2 – Table S10.

Lines 561-592

Explanation of abbreviations must have the same indentation. The easiest way will be to insert table with two columns and hide the borders, after that text in second column can be aligned to left side.

Lines 594-702

References section

References must be adapted to the pattern described in Instructions for Authors: https://www.mdpi.com/journal/genes/instructions

Space between references must be removed.

Italics must be added to the Journal’s name and volume number of reference.

Year of publication must be added with bold.

Doi number/link must be added to references which obtained it during the publication process.

I.e.:

Fu, L.; Shi, J.; Meng, Q.; Tang, Z.; Liu, T.; Zhang, Q.; Cheng, S. Verification of key target molecules for intramuscular fat deposition and screening of SNP sites in sheep from small-tail han sheep breed and its cross with suffolk. Int. J. Mol. Sci. 2024, 25(5), 2951. https://doi.org/10.3390/ijms25052951

Author Response

Rev3 Comments and Suggestions for Authors

Dear Authors,

Response： I completely agree with your point of view. We have already discussed the limitations of the sample in our discussion. We will increase the sample size for further research in the future. this revision can be located on page [10], paragraph [2], line [9-16] of the revised manuscript.

Below are suggestions and comments that may be helpful in the revision process:

Lines 9-10

Indentation of text in lines after number or number sign/hash can be adapted to this from lines 6 and 7.

Response: The modifications have been carried out as per the requirements.

Line 37

Ovis aries (binominal nomenclature, italics needed).

Response: The modifications have been made as per the requirements (on page [2], paragraph [1], line [1] of the revised manuscript).

Lines 48, 51, 56, 58 and 70

One space required after dot before beginning of new sentence.

Response: The modifications have been made as per the requirements (on page [2], paragraph [1], lines [9], [11], [15], and [16] of the revised manuscript).

Lines 76-78

Early studies were mentioned in sentence, reference/s can be added.

Response: References have been added. They are on page [2], paragraph [2], line [13] of the revised manuscript.

Lines 127-129

Response: The limitations of the sample have already been discussed in the discussion.

Lines 146-150

In description of Figures 1A-1E brackets can be used, respectively to appear in the text: (A-B), (C), (D), (E). That should distinguish them from the entire text.

Response: The modifications have been made as per the requirements. They are located on page [17], paragraph [2], lines [30-2] of the revised manuscript.

Line 165

TableS1

response: The modifications have been made as per the requirements. They have been carried out on page [4], paragraph [3], line [4] of the revised manuscript.

Line 168

SPSS 22.0 software was used (name and address of producer must be also added in brackets).

response: The modifications have been carried out as requested. They have been made on page [4], paragraph [3], line [7] of the revised manuscript.

Line 169

response: The modifications have been carried out as requested. They have been made on page [4], paragraph [3], lines [5-7] of the revised manuscript.

Lines 263 and 270

TableS2

response: The modifications have been made as per the requirements. They have been carried out on page [6], paragraph [1], lines [6] and [11] of the revised manuscript.

Lines 290-295

The same as in lines 146-150: (A), (B), (C).

response: The modifications have been carried out as requested. In the revised manuscript, on page [17], paragraph [2], lines [1], [2], and [5].

Lines 316-318

The same as in lines 146-150: (A), …, (D).

response: The modifications have been made as requested. They are located on page [17], paragraph [3], lines [1-4] of the revised manuscript.

Line 322

p-value instead of P-value can be used. Three animals in treatment randomly allocated as a sample from population.

response: The modifications have been made as per the requirements. They are located on page [16], paragraph [3], line [7] of the revised manuscript.

Line 342

‘… Table S10)

response: The modifications have been made as per the requirements. They are located on page [7], paragraph [2], line [13] of the revised manuscript.

Lines 362 and 365

Space after Table required in case of (Table S14) and (Table S15).

response: The modifications have been made as per the requirements. They can be found on page [9], paragraph [2], line [2] of the revised manuscript.

Line 383

(Fig.2)

response: The modifications have been made as per the requirements. They are located on page [8], paragraph [2], line [2] of the revised manuscript.

Lines 530-534

Space between Table and supplement number required in case of Table S2 – Table S10.

response: The modifications have been made as per the requirements. They have been carried out on page [16], paragraph [6], line [2] of the revised manuscript.

Lines 561-592

response: The modifications have been carried out as requested. They are located on page [11], paragraph [1], lines [1-32] of the revised manuscript.

Lines 594-702

References section

References must be adapted to the pattern described in Instructions for Authors: https://www.mdpi.com/journal/genes/instructions

Space between references must be removed.

Italics must be added to the Journal’s name and volume number of reference.

Year of publication must be added with bold.

Doi number/link must be added to references which obtained it during the publication process.

I.e.:

Fu, L.; Shi, J.; Meng, Q.; Tang, Z.; Liu, T.; Zhang, Q.; Cheng, S. Verification of key target molecules for intramuscular fat deposition and screening of SNP sites in sheep from small-tail han sheep breed and its cross with suffolk. J. Mol. Sci. 2024, 25(5), 2951. https://doi.org/10.3390/ijms25052951

response: The references have been modified as per the requirements.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript and addressed the reviewer’s comments. The principal limitations of the study, including the small sample size, potential confounding effects of sex, the use of FPKM-based normalization and older RNA-seq analysis pipelines, and the absence of functional validation of predicted regulatory interactions, are now explicitly discussed in the revised manuscript.

Although these limitations constrain the strength of statistical and mechanistic inference, they do not preclude the study from contributing descriptive and exploratory insights. Provided that the conclusions remain appropriately qualified and that predictive regulatory relationships are not presented as experimentally validated mechanisms, the revised manuscript is suitable for publication.

A minor comment: Please revise the formatting of all tables to comply with MDPI style.

Author Response

A minor comment: Please revise the formatting of all tables to comply with MDPI style.

Response：The table has been modified as per the requirements.

Reviewer 2 Report

Comments and Suggestions for Authors

All the issues were addressed in the revised manuscript. The authors have performed significant and extensive supplementary work and have included additional breeds and extra information in their raw data. That way, they added valuable data in the revised manuscript.
All these changes make this paper and excellent study of international interest, which is now ready for immediate acceptance.

Author Response

Response： We sincerely appreciate your positive evaluation and insightful comments on our revised manuscript.