Comprehensive Analysis of Wild Rice Mitochondrial Genomes Reveals Structural Variation, Repeat Dynamics, and the Evolution of orf182

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Comparative Analysis of orf182 Widespread in Chinese Wild Rice Species for Hybrid Rice Breeding” by Long et al. presents a genomic analysis of mitochondrial genomes in wild rice species. The findings are timely and significant for exploring new genetic resources relevant to hybrid rice breeding. To strengthen the manuscript, the following specific revisions are recommended:

1, The manuscript requires language polishing to improve clarity and precision. For instance, the term “widespread” in the title is vague. Additionally, the first sentence of the Abstract claims that food security challenges have been "addressed", which should be rephrased for greater accuracy.

2, The current title does not fully capture the scope of the study, as the analysis of orf182 is primarily confined to the first result. Subsequent sections detail the mitochondrial genome, repeat sequence variation, structural variation, and a pan-mitochondrial genome. The authors should consider refining the title to better encompass all major findings.

3, There are inconsistencies in the figure descriptions. The number of samples depicted in Figure 1A conflicts with the description in Line 100. Furthermore, in Figure 1B (Line 474), the second sample seems to be labeled as GX51/MXS(BC5).

4, In Figure 2A-B (Line 491), the y-axis is missing a label, which should be provided.

5, Figure 5A-C lacks necessary legends to explain its content.

6, Figure S3 inconsistently uses both R and R² values. Additionally, an equal sign is missing for "Class III" in the annotation.

7, The size of Figure S7 should be enhanced to allow for clearer interpretation.

8, A clarification is needed regarding the copy number of cox1. Line 140 states it is replicated in nearly all wild rice mitogenomes, yet it is not listed among those with multiple copies in the JX2 mitogenome (Lines 122-123).

9, The conclusions drawn in Lines 169-170 and 187-188 require supporting citations.

10, The overall visual presentation of the figures, particularly Figure 2, requires improvement to meet publication standards.

Author Response

1, The manuscript requires language polishing to improve clarity and precision. For instance, the term “widespread” in the title is vague. Additionally, the first sentence of the Abstract claims that food security challenges have been "addressed", which should be rephrased for greater accuracy. Response 1:Thanks for your suggestions. We had revised the title and abstract it in line 11-13. The revised sentences were as following:The widespread adoption of hybrid rice has played a pivotal role in ensuring food security in China. However, the heavy reliance on wild-abortive (WA) cytoplasmic male sterility (CMS) systems raises potential biosafety concerns 2, The current title does not fully capture the scope of the study, as the analysis of orf182 is primarily confined to the first result. Subsequent sections detail the mitochondrial genome, repeat sequence variation, structural variation, and a pan-mitochondrial genome. The authors should consider refining the title to better encompass all major findings. Response 2: We had revised the title into “Comprehensive Analysis of Wild Rice Mitochondrial Genomes Reveals Structural Variation, Repeat Dynamics, and the Evolution of orf182”. 3, There are inconsistencies in the figure descriptions. The number of samples depicted in Figure 1A conflicts with the description in Line 100. Furthermore, in Figure 1B (Line 474), the second sample seems to be labeled as GX51/MXS(BC5). Response 3: Thank you for pointing this out. We had used 18 markers instead of 18 sample. Sorry for this mistake, and we have revised the Figure 1B. 4, In Figure 2A-B (Line 491), the y-axis is missing a label, which should be provided. Response 4: Thanks for your comment, We had added the y-axis label in this revision. 5, Figure 5A-C lacks necessary legends to explain its content. Response 5:Thank you for pointing this out. We had rewritten the legends of Figure 5A-C as following: A. Distribution of annotated protein-coding genes across individual accessions, showing the composition of core (red), dispensable (green), and private (blue) gene sets in each accession. X-axis mean the gene number, y-axis indicate the wild rice samples.. B. UpSet plot showing the intersection and distribution of gene families across accessions. Vertical bars represent the number of gene families shared among specific combinations of accessions, while the connected dots below indicate the corresponding intersection sets. Horizontal bars (left) denote the total number of genes in each accession..C. Pan-genome and core-genome size dynamics with increasing sample size. The pan-genome (teal) shows a continuous increase as additional genomes are incorporated, whereas the core genome (red) gradually decreases, indicating progressive reduction in shared genes across all accessions. 6, Figure S3 inconsistently uses both R and R² values. Additionally, an equal sign is missing for "Class III" in the annotation. Response 6: Thanks for your carefully review. We had updated it. 7, The size of Figure S7 should be enhanced to allow for clearer interpretation. Response 7: We had changed the Figure S7 in this revision.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

How were the 76 accessions shown to be sterile.  The parental line can not be sterile.  This cross is not in the m/s.

None of your supplemental figures have legends.  I think the labels for Fig S1 is wrong as well.

What are the core set of 37 genes from the 11 genomes?

Figure 3A has no units?  Also the A and B look like A is only class 1 and 3 and B 2 and 4.

In figure 5A are the differences in the core genes due to replication of the genes?  It is not clear how you can have different numbers of core genes.

Figure 6 has a typo in the Sorghum Sequence label

You need to define private genes, do you mean unique?

In section 4.3 what are the Illumina (typo) PacBio and Nanopore methods.  These need references.

Your assembly methods need to be more clear.  It is not clear what was used as a scaffold and than have added depth using Illumina to correct for sequencing errors.

Same for section 4.7 need far more detail, this reads as results not methods.

Suppl Fig S5 seem to have the same color for the insertion, deletion, translocation and inversion.

Author Response

How were the 76 accessions shown to be sterile.  The parental line can not be sterile.  This cross is not in the m/s.

Response 1:Thanks for you pointing this out. Building upon our previous work published in The Plant Journal (Xie et al., 2018), this study reports the identification of a novel cytoplasmic male sterility (CMS) gene, orf182, which confers a completely male-sterile phenotype and defines a new CMS type. To further investigate its diversity and potential applications, we screened global wild rice accessions for the presence of this sterility gene, followed by orf182 variation analysis and mitochondrial genome sequencing. In addition, we initiated new breeding combinations aiming to develop novel CMS-based resources for hybrid rice breeding. Hence, we had revised the sentence as following: Among these, 76 accessions (23.75%) exhibiting the D1-type cytoplasmic male sterility (CMS) gene orf182 were selected for subsequent genetic and genomic analyses

Xie H, Peng X, Qian M, Cai Y, Ding X, Chen Q, Cai Q, Zhu Y, Yan L, Cai Y. The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice. Plant J. 2018 Jun 7.

None of your supplemental figures have legends.  I think the labels for Fig S1 is wrong as well.

Response 2: We have add the legends for the supplement figures in this revision. We also have revised the labels in Fig S1.

What are the core set of 37 genes from the 11 genomes?

Response 3: Thanks for you pointing out this. The core set indicate the the mitochondrial gene shared with cultivated rice NIP reference. The detail including atp4, atp8, rps7, cox3, nad7, nad5, rps3, rps2, rps12, rps16, nad4, nad3, nad2, cox2, nad1, rps19, rpl2, cob, nad4L, orfX, rps4, orf182, atp9, atp6, ccmcB, atp1, nad9, rps1, matR, ccmC, ccmFn, rps13, ccmFc, rpl5, cox1 and nad6, which listed in Figure S2.

Figure 3A has no units?  Also the A and B look like A is only class 1 and 3 and B 2 and 4.

In figure 5A are the differences in the core genes due to replication of the genes?  It is not clear how you can have different numbers of core genes.

Response 4: Thanks for you pointing out this. We had revised it the figure 3. Actually, we had built a pan- mitochondrial genome by using the protein sequences. This distinction gives rise to two forms of pan-genome analysis: one based on gene families and another based on individual genes. Our analysis reveals that for each sample, the composition of core gene families remains constant; however, the total count of core genes differs, a finding illustrated in Figure 5A. The variant number of core genes were due to the gene copy number variations.

Figure 6 has a typo in the Sorghum Sequence label

Response 5:Thanks for your careful review. We had corrected it.

You need to define private genes, do you mean unique?

Response 6: Thanks for your constructive suggestion.private gene indicated it only presented in one sample. We have revised it in this revision.

In section 4.3 what are the Illumina (typo) PacBio and Nanopore methods.  These need references.

Response 7: Thanks for your kindly reminder. We had added the references in this revision.

Your assembly methods need to be more clear.  It is not clear what was used as a scaffold and than have added depth using Illumina to correct for sequencing errors.

Response 8: Thanks for your point this out. We had rewritten the methods.

Same for section 4.7 need far more detail, this reads as results not methods.

Response 9: Thanks you for your kindly suggestion. We had rewritten it.

 

Suppl Fig S5 seem to have the same color for the insertion, deletion, translocation and inversion.

Response 10: Thanks for kindly reminder. We have corrected the colors in this revision.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, 

There is a few recommendation for your article:

1. I recommend reducing the information in the final paragraph of the Introduction. It is important to focus strictly on the aim and objectives of the study. Please consider removing detailed descriptions of the methodology and any references to results. This will improve clarity and ensure better alignment with what follows.
2. Line 331 and 333: What does "the high-quality reads were assembled into sequences (bp)....." mean exactly? What are you referring to more specifically? I recommend rephrasing this and providing a more detailed description of this step for a clearer understanding of the methodology. Also, please clarify the statement "…redundant sequences were manually removed to obtain the final mitochondrial genome…”
3. line 343: Also,  the sentence "…redundant sequences were manually removed…"’—what exactly does ‘manually’ mean in this context?   This step is insufficiently detailed to ensure reproducibility.”
4. The versions of all software used and described in subsections 4.5–4.9 are not specified and Is toooo many software used... Some of them .
5. Line 350: "repeatively" or respectively ?
6. Line 356: "The mitogenome of Nipponbare was characterized by Notsu [29] were used for comparison" — why was this reference used?
7. Line 358: "Collinear regions were identified using the nucmer module with an identity threshold of 95% under the many-to-many alignment mode",  - many- to- many is not an explicative mode!! and why 95%???
8. Line 362: missing space "11wild rice"
9. Line 377: "....using mummer combined with SYRI software based on the NIP reference.." what mean "mummer" and also NIP reference???
10. The Materials and Methods section lacks sufficient detail and clarity to ensure reproducibility. Key parameters, software settings, and filtering criteria are either missing or insufficiently described.
11. Line 95-98 and other: In the Results section, a large part of the information presented in Materials and Methods is repeated. Here, you should focus strictly on the results obtained from applying the described methods and analysis tools, like "...“aiming to clarify the evolutionary relationships…” and other
12. You collected 320 accessions, of which 76 were selected for analysis, only 18 were used for phylogenetic reconstruction, and only 11 for sequencing—without explaining why this selection was made. What were the statistical, biological, or maybe financial criteria behind this selection?
13. Line 111: "....phylogenetic tree constructed by 20 MSTs..." what mean MSTs??
14. “mitogenome" and "mitochondrial genome" are used inconsistently; also "orf182/nad6" vs "orf182-nad6”.
15. Did you use bootstrap support in the phylogenetic analysis?
16. Please move subchapter 2.4 (‘2.4. Identified the Structural…’) before Figure 4
17. Lines 205–207: you mention "Whole-genome alignment using Mauve further characterized large-scale structural rearrangements, revealing 17 locally collinear blocks (LCBs) with variable ordering across accessions (Figure S6)." How are all 17 locally collinear blocks represented in Figure S6? Only segments labeled with uppercase letters from A to N are shown, which accounts for 15 letters—I assume these were intended to denote the LCBs?”
18. Line 216-217: "The gene accumulation curve (Figure 5C) indicated an open pan-genome, suggesting that new genes are likely to be identified as more mitochondrial genomes from rice are sequenced." what did you mean?
19. Line 224: "„GX34… implying a more stable evolutionary history and possible adaptation to a narrower ecological niche” - a simple speculation....
20. Line 231: What exactly  "private" and "variable" gene families mean in this context? From this sentence: "followed by private (30.77%) and variable (25%) gene families”. This results are in figure 5? Please refer to all graphic from figure 5 
21. Is there a possible confusion between ORFs and genes when interpreting all these results? How can you tell whether it is a gene or an ORF? Because ORFs, specially chimeric ORFs, are more presents in mitochondrial genomes then functional genes
22. Where are the conclusions of the manuscript?
23. The text is dense and sometimes repetitive, with many sentences that are too long and overloaded with concepts and unsupported speculation, that are not fully supported by the presented data.

Author Response

1. Reviewer 3:I recommend reducing the information in the final paragraph of the Introduction. It is important to focus strictly on the aim and objectives of the study. Please consider removing detailed descriptions of the methodology and any references to results. This will improve clarity and ensure better alignment with what follows.

Response 1: Thank you for you constructive suggestion. We had reducing the information in the last paragraph of the introduction as your recommendation.

2. Line 331 and 333: What does "the high-quality reads were assembled into sequences (bp)....." mean exactly? What are you referring to more specifically? I recommend rephrasing this and providing a more detailed description of this step for a clearer understanding of the methodology. Also, please clarify the statement "…redundant sequences were manually removed to obtain the final mitochondrial genome…”

Response 2: Thanks for your comment. We have rewritten the method of 4.4 in this revision. Raw sequencing reads were trimmed using Trimmomatic (version 0.35) to remove low-quality bases (quality score < 20) and adapter sequences, retaining reads with a minimum length of 50 bp. High-quality reads were assembled de novo using two complementary assemblers — SOAPdenovo2 v2.04 (http://soap.genomics.org.cn/) and Celera Assembler v8.0 — with k-mer sizes optimized for each dataset. Scaffolds were constructed using SSPACE.

3. line 343: Also,  the sentence "…redundant sequences were manually removed…"’—what exactly does ‘manually’ mean in this context?   This step is insufficiently detailed to ensure reproducibility.”

Response 3: Thanks for your comment. We had rewritten the method of assembly. We agree that the description of “manual removal of redundant sequences” was insufficiently detailed and may affect reproducibility.

In this study, “manual removal” refers to a curation step performed after the initial assembly, in which redundant contigs or overlapping regions were identified based on sequence similarity and alignment. Specifically, all candidate sequences were aligned using BLASTN, and regions showing >95% sequence identity and substantial overlap (≥80% of their length) were considered redundant. Among overlapping sequences, the shorter or lower-quality contig was removed, while the longer and more complete sequence was retained.

In addition, ambiguous regions were further inspected using genome alignment visualization tools to ensure consistency and continuity of the mitochondrial genome structure.

 

4. The versions of all software used and described in subsections 4.5–4.9 are not specified and Is toooo many software used... Some of them .

Response 4. Thanks for your comment. We had rewritten the methods in this revision.

5. Line 350: "repeatively" or respectively ?

Response 5: Thanks for carefully review. We had corrected it in this revision.

 

6. Line 356: "The mitogenome of Nipponbare was characterized by Notsu [29] were used for comparison" — why was this reference used?

Response 6:We thank the reviewer for the insightful question regarding the choice of Nipponbare as the reference genome. The O. sativa cv. Nipponbare mitochondrial genome was selected as the reference for several reasons. First, it remains the most comprehensively annotated and highest-quality mitochondrial genome in the Oryza genus, serving as the foundational reference for rice comparative genomics. Second, since Nipponbare represents a typical fertile (non-CMS) line, it provides an essential baseline for identifying CMS-associated structural variations (such as the chimeric gene orf182) in wild rice. Third, using a universal reference like Nipponbare ensures the reproducibility and comparability of our results with existing databases and published literature. While we acknowledge that wild rice-specific references could offer alternative perspectives, employing Nipponbare as an anchor minimizes analytical bias and provides a clear evolutionary coordinate for the structural rearrangements observed in our study.

7. Line 358: "Collinear regions were identified using the nucmer module with an identity threshold of 95% under the many-to-many alignment mode",  - many- to- many is not an explicative mode!! and why 95%???

Response 7: Thanks for your insightful comment. We agree that the term "many-to-many" can be ambiguous without context. In the context of the MUMmer suite’s nucmer module, the "many-to-many" mode refers to an alignment configuration that allows for the detection of all possible homologous relationships between multiple query and reference sequences, including complex rearrangements, duplications, and translocations. Unlike a "one-to-one" mode (which forces a single best match) or a "one-to-many" mode, the many-to-many approach is specifically designed to identify all collinear segments even when they are not uniquely orthologous. This is particularly important in comparative genomics for detecting duplicated regions or multi-copy genes that have been retained in collinear blocks. We will clarify this in the revised text to specify that "the many-to-many mode was employed to comprehensively capture all potential collinear relationships, including those involving duplicated segments."

The 95% threshold was chosen to balance sensitivity and specificity. Given that we are comparing the sample species in rice, which are evolutionarily close, this threshold allows us to reliably distinguish true orthologous collinear blocks from paralogous or repetitive elements while being stringent enough to exclude spurious noise.

 

8. Line 362: missing space "11wild rice"

Response 8: Thanks for your careful review. We had revised it in this revision.

9. Line 377: "....using mummer combined with SYRI software based on the NIP reference.." what mean "mummer" and also NIP reference???

Response 9: Thanks for your constructive suggestions. We had deleted the 4.8 section.

10. The Materials and Methods section lacks sufficient detail and clarity to ensure reproducibility. Key parameters, software settings, and filtering criteria are either missing or insufficiently described.

Response 10: Thanks for you suggestion. We had rewritten the methods section 4.4 to 4.9 in this revision.

11. Line 95-98 and other: In the Results section, a large part of the information presented in Materials and Methods is repeated. Here, you should focus strictly on the results obtained from applying the described methods and analysis tools, like "...“aiming to clarify the evolutionary relationships…” and other

Response 11: Thanks for your constructive suggestions. We have rewritten the results 1 as following: To further elucidate the evolutionary origin and distribution of this gene, we collected 320 common wild rice accessions from three provinces in China (Guangdong, Jiangxi, and Guangxi), of which 76 (23.75%) carried the D1-type CMS gene orf182 and were retained for subsequent analyses. Phylogenetic reconstruction using 18 highly polymorphic markers resolved the evolutionary relationships within the D1-type wild rice group into distinct clades (Figure 1, Tables S2, S3). Eleven representative accessions spanning nine phylogenetic clades were selected for mitochondrial genome assembly; comparative analysis of these high-quality assemblies revealed extensive structural variation and recombination events, and highlighted the potential functional role of orf182 in CMS. Based on three O. rufipogon accessions carrying orf182 from geographically and phylogenetically distinct backgrounds, male sterile lines of Mingxiangsi were successfully obtained after only five backcross generations (Figure 1B), demonstrating that the D1-type CMS system can be efficiently transferred into diverse genetic backgrounds.

12. You collected 320 accessions, of which 76 were selected for analysis, only 18 were used for phylogenetic reconstruction, and only 11 for sequencing—without explaining why this selection was made. What were the statistical, biological, or maybe financial criteria behind this selection?

Response 12: Thanks for your comment. We apologize for the writing error; the “18 materials” mentioned in the text actually refers to “18 markers”. This has been corrected in the revised manuscript. Taking into the genetic diversity and financial together, We selected 11 sample represent the 76 wild rice harbored the orf182 gene confer to CMS for sequencing and further analysis. The rewritten sentence as response 11.

13. Line 111: "....phylogenetic tree constructed by 20 MSTs..." what mean MSTs??

Response 13:Thanks for your carefully review. This is a type mistake. It is MSSs instead of MSTs. MSSs means mitotype-specific sequences. The detail information can be seen in the reference (Xie et al., 2018)

Xie H, Peng X, Qian M, Cai Y, Ding X, Chen Q, Cai Q, Zhu Y, Yan L, Cai Y. The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice. Plant J. 2018 Jun 7

14. “mitogenome" and "mitochondrial genome" are used inconsistently; also "orf182/nad6" vs "orf182-nad6”.

Response 14: Thanks for your carefully review. We uniformly designated it as “mitochondrial genome” and “orf182-nad6”

15. Did you use bootstrap support in the phylogenetic analysis?

Response 15: Thanks for your carefully review. We had used 1000 bootstrap to constructed the phylogenetic tree.

16. Please move subchapter 2.4 (‘2.4. Identified the Structural…’) before Figure 4

Response 16: Thanks for your suggestion. We had revised it as your recommendation.

17. Lines 205–207: you mention "Whole-genome alignment using Mauve further characterized large-scale structural rearrangements, revealing 17 locally collinear blocks (LCBs) with variable ordering across accessions (Figure S6)." How are all 17 locally collinear blocks represented in Figure S6? Only segments labeled with uppercase letters from A to N are shown, which accounts for 15 letters—I assume these were intended to denote the LCBs?”

Response 17: Thank you for your careful observation. The reviewer is correct that only 15 blocks are labeled with uppercase letters A-N in Figure S6. The reason for this discrepancy is that among the 17 locally collinear blocks (LCBs) identified, 15 were visualized as continuous, well-defined colinear regions and were therefore labeled with letters A-N (15 letters in total) in the figure.

The remaining two LCBs (LCB 16 and LCB 17) are not displayed as colored blocks in the figure but are instead represented only by syntenic lines connecting genomes. This is because these two regions are highly rearranged or fragmented across the aligned genomes, preventing them from being rendered as complete colored blocks under Mauve's default visualization settings. However, their collinear relationships are still reflected by the connecting lines in the figure. Thus, the total number of LCBs is 15 (labeled blocks) + 2 (represented only by lines) = 17.

To improve clarity, we will add the following explanation to the figure legend in the revised manuscript:

"Note that 15 of the 17 LCBs are displayed as colored blocks labeled A-N, while the remaining two LCBs are represented solely by syntenic lines due to their highly rearranged or fragmented nature across genomes."

We appreciate the reviewer's attention to detail, which has helped us improve the presentation of our data.

 

18. Line 216-217: "The gene accumulation curve (Figure 5C) indicated an open pan-genome, suggesting that new genes are likely to be identified as more mitochondrial genomes from rice are sequenced." what did you mean?

Response 18:We thank the reviewer for this comment. Our intention was to interpret the shape of the gene accumulation curve in Figure 5C. A curve that does not plateau as genome number increases is characteristic of an open pan-genome, meaning that the total gene repertoire is not yet fully represented. In the context of wild rice mitochondrial genomes, which can harbor variable CMS-related genes (such as orf182), this suggests that future sequencing efforts may identify additional lineage-specific genes or ORFs.

19. Line 224: "„GX34… implying a more stable evolutionary history and possible adaptation to a narrower ecological niche” - a simple speculation....

Response 19: Thank the reviewer for this critical comment. We agree that the original wording was overly speculative and not directly supported by our genomic data.

In the revised manuscript, we have removed the speculation regarding ecological adaptation and narrowed the interpretation to what the data can actually support. Specifically, we now simply note that GX34 exhibits fewer structural rearrangements compared to other accessions, which may indicate a relatively stable genomic architecture.

We appreciate the reviewer's insistence on precision and have revised the text accordingly.

20. Line 231: What exactly  "private" and "variable" gene families mean in this context? From this sentence: "followed by private (30.77%) and variable (25%) gene families”. This results are in figure 5? Please refer to all graphic from figure 5 

Response 20:Thanks for your comments. We had revised in the method section 4.9 in this revision.  

21. Is there a possible confusion between ORFs and genes when interpreting all these results? How can you tell whether it is a gene or an ORF? Because ORFs, specially chimeric ORFs, are more presents in mitochondrial genomes then functional genes.

Response 21:Thank you for raising this important point. We agree that mitochondrial genomes, especially in plants, often contain numerous ORFs, including lineage-specific and chimeric ORFs, which may lead to potential ambiguity if not clearly distinguished from functional genes.

In this study, we strictly distinguished annotated genes from ORFs based on the following criteria:

Reference-based annotation: Only sequences that matched previously annotated mitochondrial genes in well-characterized rice mitochondrial genomes (Nipponbare) or conserved plant mitochondrial genes were classified as “genes” These include protein-coding genes, rRNAs, and tRNAs with established functional annotation.

Functional annotation support: Gene identification was supported by homology searches against public databases ( NCBI NR, Pfam), and only sequences with clear functional annotation and conserved domains were retained as genes.

ORF filtering: Putative ORFs, including chimeric or lineage-specific ORFs lacking functional annotation or homologous support, were not included in the gene set used for pan-genome analysis. These ORFs were analyzed separately (or excluded, if applicable) to avoid inflating gene counts.

Consistency across samples: The pan-genome analysis was conducted using a curated set of orthologous genes to ensure comparability across accessions and to minimize the impact of spurious ORFs.

Therefore, the “genes” reported in our study refer specifically to conserved and functionally supported mitochondrial genes, rather than all predicted ORFs.

22. Where are the conclusions of the manuscript?

Response 22: The abstract is the conclusion.

23. The text is dense and sometimes repetitive, with many sentences that are too long and overloaded with concepts and unsupported speculation, that are not fully supported by the presented data.

Response 23:Thank you for this constructive comment. We appreciate the reviewer’s observation regarding the clarity and conciseness of the manuscript.

In this revision, we have thoroughly revised the manuscript to improve readability and reduce redundancy. Long and complex sentences have been shortened or restructured, and repetitive descriptions have been removed. We have also carefully reviewed all interpretations and removed or revised statements that were overly speculative or not sufficiently supported by the data.

In particular, the Results and Methods sections have been streamlined to ensure clearer presentation of key findings and a more direct linkage between data and conclusions.

We believe these revisions have substantially improved the clarity, rigor, and overall quality of the manuscript.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, 

Below are some recommendation to improve your manuscript:

Line 101, 393, : All scientific name should be in written in italics and also the gene name orf182 (in some paragraph is not)

Line 189: "mitochondrial .." should be in uppercase letters

Lines 407-410: the information is duplicated. This paragraph is from early version of the manuscript.

 

Author Response

Comments：Line 101, 393, : All scientific name should be in written in italics and also the gene name orf182 (in some paragraph is not)

A: Thanks for your constructive suggestions. We had revised it throughout the all manuscript. All revisions were highlighted in red.

Line 189: "mitochondrial .." should be in uppercase letters

A: Thanks for your comments. We had corrected it as “Mitochondrial” in line 193.

 

Lines 407-410: the information is duplicated. This paragraph is from early version of the manuscript.

A: Thanks for your constructive suggestions. We had deleted it in line 418-423 in the previous manuscript.

Author Response File: Author Response.pdf