Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Corydalis ophiocarpa (Papaveraceae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors in “Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Corydalis ophiocarpa (Papaveraceae)” successfully reconstructed the first mitogenome of Corydalis ophiocarpa. They used a hybrid method of both Oxford Nanopore long reads and Illumina short reads technologies to generate a circular mitogenome with a length of 600 kb. It has 63 genes: 40 PCGs, 20 tRNAs, and 3 rRNAs. Also, they discovered 775 C-to-U RNA editing sites across the 40 PCGs. Interestingly, they identified 16 mt plastid DNA fragments in the mitogenome.

The paper is well-written and provides detailed results, specifically with 11 supplementary tables plus one in the main manuscript, and about 4 high-resolution figures.

However, I have a major concern that should be addressed:
Besides the fact that the raw data Oxford Nanopore long reads and Illumina short reads are not deposited, the assembled mitochondrial genome sequence itself is not deposited, nor are any accession numbers provided, even if the authors stated in the “Data Availability Statement” that all data are within the manuscript and additional files. Therefore, the authors must deposit the complete mitogenome sequence and provide the official accession number so that the community can benefit from this valuable data and verify the results.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript dedicated to the assembly, annotation, and comparative analysis of the mitochondrial genome of the medicinal plant Corydalis ophiocarpa. For the first time, the mitochondrial genome of C. ophiocarpa (600,064 bp in size) has been completely assembled and annotated. The authors discovered that the genome can exist not only as a "main ring" but also as two smaller subgenomic forms, providing repeating sequences (a 6,383 bp repeat) that mediate recombination. A detailed analysis of predicted RNA editing sites (775 C-to-U sites) was conducted. It was shown that 95.4% of the changes are nonsynonymous and result in the substitution of hydrophobic amino acids, which is characteristic of membrane protein complexes. This article is based on scientific novelty, as the mitogenome of this species is described for the first time.

Notes:

1. The authors predict 775 C-to-U editing sites, of which 739 are nonsynonymous. The “Discussion” section mentions that ~65% of predictions were confirmed in the related species C. saxicola. It would be desirable to use several key sites (for example, for the nad4 or nad7 genes, where the most changes are predicted) by direct sequencing of RNA transcripts. This enhancement increases the confidence in the conclusions. If this is not possible within the scope of this article, it should be more clearly highlighted in the “Conclusion” section as a mandatory future study.
2. Figure 1 and Supplementary Figure S1 (chloroplast genome): the mitochondrial and the chloroplast genome maps appear informative, but the fonts are very small. They are difficult to read in print or when zoomed in on-screen. Please increase the font size or select a more detailed image.
3. The text contains minor typos (e.g., "C. ophioCARa" in Section 3.3). A final proofreading is recommended.

Author Response

Comment 1: The authors predict 775 C-to-U editing sites, of which 739 are nonsynonymous. The “Discussion” section mentions that ~65% of predictions were confirmed in the related species C. saxicola. It would be desirable to use several key sites (for example, for the nad4 or nad7 genes, where the most changes are predicted) by direct sequencing of RNA transcripts. This enhancement increases the confidence in the conclusions. If this is not possible within the scope of this article, it should be more clearly highlighted in the “Conclusion” section as a mandatory future study.

Response 1: We appreciate your constructive suggestion. We concur that experimental validation of key RNA editing sites, such as those in nad4 and nad7, through RT-PCR followed by Sanger sequencing, would significantly enhance the conclusions. However, conducting these experiments necessitates primer design, PCR optimization, product purification, and Sanger sequencing, which typically takes 3-4 weeks. Given the brief revision period of 10 days, we are unable to complete these experiments at this time. To address your concern, we have explicitly indicated in the Conclusions section (Lines 491-492) that experimental validation of the predicted RNA editing sites is a critical priority for future research. We trust this revision meets your approval.

Comment 2: Figure 1 and Supplementary Figure S1 (chloroplast genome): the mitochondrial and the chloroplast genome maps appear informative, but the fonts are very small. They are difficult to read in print or when zoomed in on-screen. Please increase the font size or select a more detailed image.

Response 2: We appreciate your suggestion. We have substituted Figure 1 and the chloroplast genome map (initially Supplementary Figure S1, now renumbered as Supplementary Figure S3 due to additional revisions) with images of higher‑resolution and enlarged font sizes to enhance readability in both print and digital formats. The updated Figure 1 is located at lines 200-201 in the revised manuscript. We are confident that the current figures now comply with the journal's quality standards.

Comment 3: The text contains minor typos (e.g., "C. ophioCARa" in Section 3.3). A final proofreading is recommended.

Response 3: We appreciate your meticulous review. We have conducted a comprehensive examination of Section 3.3 and the entire manuscript; however,  we could not identify the typographical error “C. ophioCARa”. This error may have appeared in a previous version or been incorrectly referenced. Nonetheless, we have meticulously proofread the entile manuscript to rectify any potential typographical or grammatical errors. We are confident that the revised manuscript now adheres to the required language standards.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a sound genome-announcement manuscript, which in my view would benefit from some additional refinements to the data presentation and discussion in order to place the study in a more appropriate context. Most importantly, I did not find any link to a genebank or other public repository where the authors have deposited their sequences. Without access to these sequences, the study cannot be meaningfully evaluated and make no contribution to the field. In addition, I have several recommendations regarding the presentation of the data that could help the manuscript read as more than a simple genome announcement.

 — lines 72–75. The authors should not jump to conclusions based on the observation that “biosynthesis of these alkaloids involves multiple ATP-consuming enzymatic steps…” by suggesting that Corydalis metabolism and mitochondria must solve unusual energetic tasks. Quantifying the cellular ATP economy is generally challenging; however, it is highly unlikely that any single metabolite, even a massively produced one, accounts for more than a few percent of the total ATP synthesised. It should also be considered that a substantial proportion of ATP is devoted to maintaining ion gradients, vesicular transport, and protein turnover. I therefore recommend that the authors remove this premise from this passage as well as from the abstract and the discussion (lines 430–436).

— lines 168–169. Were there any conflicting long-read scaffolds? If so, how did the authors resolve them?

— Figure 1. The grey-shaded inner part of the circle is not explained. The figure could be improved by adding the contig boundaries; at minimum, the location of ctg3 should be highlighted.

— I suggest the authors provide information on the read depth (for both short reads and long reads) mapped to the final assembly, as well as the average GC content, using a sliding window. This can be done as an additional layer in Figure 1. The average per-contig values given in Table S1 do not convey the full picture.

— To make the codon usage analysis more informative, I suggest comparing codon preference with the availability of tRNAs, given that mitochondria usually do not possess all anticodon variants in the tRNAs. For instance, the authors could highlight codons with perfectly matching tRNA anticodons in Figure 2. In its current form, Figure 2 is not particularly informative and could be removed or moved to the supplementary material.

— Figure 3 is interesting, but I recommend further improvement by presenting a heatmap of the percent identity between plastid and mitochondrial DNAs. Importantly, the authors should overlay genetic features on this figure to allow comparison of which genomic regions are most affected by horizontal transfer of plastid DNA into the mitochondrial genome. The tRNAs and pseudogenised PGCs originating from the plastid genome and located in the mitochondrial DNA should be highlighted, as they are featured in the discussion section of the manuscript.

— Figure 4 (or perhaps Figure 5) could be improved by labelling the mitochondrial genome sizes. Is the sequenced genome the largest within the genus or the family? In addition, the GenBank accession numbers are not legible for all mitochondrial genomes shown in Figure 4.

— I recommend toning down the RNA-editing section (removing it or moving it to the supplement, Figure 6), as the authors did not verify the RNA-editing sites and the entire discussion therefore remains speculative.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The mitochondrial and chloroplast genome records have not yet been made available in GenBank. Specifically, accession PZ380598 has not been released, and accession PZ405204 could not be found in Nucleotide, which returned the message: “The following term was not found in Nucleotide: PZ405204. No items found.” Please ask GenBank staff to check the status of these records and release them if they are ready for public availability.

Author Response

Comment 1: Besides the fact that the raw data Oxford Nanopore long reads and Illumina short reads are not deposited, the assembled mitochondrial genome sequence itself is not deposited, nor are any accession numbers provided, even if the authors stated in the “Data Availability Statement” that all data are within the manuscript and additional files. Therefore, the authors must deposit the complete mitogenome sequence and provide the official accession number so that the community can benefit from this valuable data and verify the results.

Response: We appreciate your critical feedback. We concur that data deposition is crucial for reproducibility and the benefit of the scientific community. Consequently, we have deposited the raw Oxford Nanopore and Illumina short reads in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1473575, with accession numbers to be released upon final processing. The complete mitochondrial and chloroplast genome sequences of C. ophiocarpa have been submitted to GenBank, receiving accession numbers PZ380598 (mitogenome) and PZ405204 (plastome). These entries are currently in "Processing" status and will be publicly accessible on July 15, 2026. We have incorporated the accession numbers and deposition details into the Materials and Methods (Section 2.1, lines 112–116), Results (Section 3.1, line 192), and Data Availability Statement (lines 513-518) of the revised manuscript. Furthermore, we have uploaded the assembled mitochondrial and chloroplast genome FASTA file as Supplementary File S1 to facilitate immediate verification by the reviewers. We believe these revisions comprehensively address the concern. All modifications are highlighted in the revised manuscript using track changes mode.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors reasonably responded to all my comments and questions and improved the manuscript accordingly. I recommend the acceptance. 

Author Response

Comment 1: This is a sound genome-announcement manuscript, which in my view would benefit from some additional refinements to the data presentation and discussion in order to place the study in a more appropriate context. Most importantly, I did not find any link to a genebank or other public repository where the authors have deposited their sequences. Without access to these sequences, the study cannot be meaningfully evaluated and make no contribution to the field.

Response 1: We express our gratitude to the reviewer for these constructive suggestions. In response, we have implemented the following revisions to address the concerns:

(1) Data deposition and accessibility

The raw Oxford Nanopore and Illumina sequencing reads have been deposited in the NCBI SRA under BioProject PRJNA1473575. The complete mitochondrial and chloroplast genome sequences have been deposited in GenBank with accession numbers PZ380598 (mitogenome) and PZ405204 (plastome). These accession numbers have been incorporated to the Materials and Methods (Section 2.1, lines 112–116), Results (Section 3.1, line 192), and Data Availability Statement (lines 513-518). The data will be publicly accessible on July 15, 2026.

(2) Moving beyond a simple genome announcement

In order to more effectively emphasize the conceptual contribution of this study, we have revised the discussion in Sections 4.1 and 4.3. The specific details of these adjustments are presented in lines 325-334 and lines 435-447.

We believe these revisions place the study in a more appropriate context and address the reviewer's concern. We thank the reviewer again for the valuable suggestions.

Comment 2: lines 72– The authors should not jump to conclusions based on the observation that “biosynthesis of these alkaloids involves multiple ATP-consuming enzymatic steps…” by suggesting that Corydalis metabolism and mitochondria must solve unusual energetic tasks. Quantifying the cellular ATP economy is generally challenging; however, it is highly unlikely that any single metabolite, even a massively produced one, accounts for more than a few percent of the total ATP synthesised. It should also be considered that a substantial proportion of ATP is devoted to maintaining ion gradients, vesicular transport, and protein turnover. I therefore recommend that the authors remove this premise from this passage as well as from the abstract and the discussion (lines 430–436)..

Response 2: We express our gratitude to the reviewer for the crucial clarification provided. We have excised the overstated premise from the manuscript as requested. The specific amendments are as follows:

• In the Abstract (line 38), the term “energy‑intensive” has been removed to prevent any misinterpretation suggesting an excessively high ATP consumption for specialized metabolism.
• In the Introduction (lines 73–81), the original assertion that “mt function is almost certainly critical for supporting their production” have been revised to emphasize the role of carbon skeletons produced by the tricarboxylic acid cycle in mitochondria during alkaloid pathways, as supported by previous research.
• In Discussion Section 4.1, the term “energy‑intensive” has been omitted from the description of alkaloid biosynthesis. In Discussion, Section 4.3, the phrase “energetically intensive” and the causal connector “Consequently, it is plausible to hypothesize that” have been deleted.

We believe these revisions adequately address the reviewer’s concern and render the argument more balanced. We extend our thanks once more to the reviewer for the constructive critique.

Comment 3: lines 168– Were there any conflicting long-read scaffolds? If so, how did the authors resolve them?

Response 3: We thank the reviewer for raising this technical question. In our assembly pipeline, we implemented a rigorous validation criterion: a junction was accepted only if it was continuously spanned by a minimum of three independent Nanopore reads. Junctions failing to meet this threshold were excluded. Consequently, no ambiguous or conflicting connections persisted following this filtering step. This information has been incorporated to the Materials and Methods (Section 2.2, lines 128–129) of the revised manuscript. We believe this elucidates our conflict resolution strategy.

Comment 4: Figure 1. The grey-shaded inner part of the circle is not explained. The figure could be improved by adding the contig boundaries; at minimum, the location of ctg3 should be highlighted.

Response 4: We thank the reviewer for the suggestion. We have incorporated the contig boundaries into the revised Figure 1. Consequently, the legend has been updated and can be found on lines 202–209 of the revised manuscript. Additionally, we have prepared a Supplementary Figure S2, which clearly displays the three contigs (ctg1, ctg2, ctg3) and their connections. The position of the 6,383 bp repeat (ctg3) is highlighted with pink annotations and red arrows. We believe these revisions comprehensively address the reviewer’s concerns and enhance the clarity of the genome structure presentation.

Comment 5: I suggest the authors provide information on the read depth (for both short reads and long reads) mapped to the final assembly, as well as the average GC content, using a sliding window. This can be done as an additional layer in Figure 1. The average per-contig values given in Table S1 do not convey the full picture.

Response 5: We thank the reviewer for this valuable suggestion. In response, we have conducted a sliding-window coverage and GC content analysis for both Illumina short reads and Oxford Nanopore long reads, as illustrated in Supplementary Figure S1. Furthermore, we have revised the manuscript to incorporate these analysis. The specific revisions are as follows:

Methods (Lines 169-–179): We have added subsection 2.6, which details the alignment process (BWA for Illumina, minimap2 for ONT) and Qualimap analysis (employing a 1-kb window with 500-bp step).

Results (Lines 195–200): We have included the average coverage depth (693× for Illumina, 237× for ONT) and the average GC content (45.86% and 46.53%). Additionally, a two-fold coverage peak at the 6,383 bp repeat region is now reported.

We are confident that these revisions comprehensively address the reviewer’s concern.

Comment 6: To make the codon usage analysis more informative, I suggest comparing codon preference with the availability of tRNAs, given that mitochondria usually do not possess all anticodon variants in the tRNAs. For instance, the authors could highlight codons with perfectly matching tRNA anticodons in Figure 2. In its current form, Figure 2 is not particularly informative and could be removed or moved to the supplementary material.

Response 6: We thank the reviewer for the valuable suggestion. We concur that an analysis comparing codon usage with tRNA anticodon availability would yield significant insights. Nevertheless, in plant mitochondria, tRNAs undergo extensive RNA editing, potentially altering their anticodon sequences and, consequently, their decoding properties. A roubust comparison necessitates not only genomic tRNA sequences but also experimental validation of RNA editing at the anticodon region, which exceeds the scope of the present study. Consequently, we have relocated Figure 2 to the Supplementary Materials (now Supplementary Figure S4) to maintain the focus of the main text. We trust this modification addresses the reviewer’s concern and appreciate the reviewer's understanding.

Comment 7: Figure 3 is interesting, but I recommend further improvement by presenting a heatmap of the percent identity between plastid and mitochondrial DNAs. Importantly, the authors should overlay genetic features on this figure to allow comparison of which genomic regions are most affected by horizontal transfer of plastid DNA into the mitochondrial genome. The tRNAs and pseudogenised PGCs originating from the plastid genome and located in the mitochondrial DNA should be highlighted, as they are featured in the discussion section of the manuscript.

Response 7: We thank the reviewer for this valuable suggestion. A heatmap, now included as Figure 2B in the  revised manuscript, features annotated genetic elements. Additionally, Supplementary Table S8 encompasses all the requested data for each of the 16 MTPT fragments We are confident that this revised figure, in conjunction with Table S8, supplies the necessary information for assessing the horizontal transfer events.

Comment 8: Figure 4 (or perhaps Figure 5) could be improved by labelling the mitochondrial genome sizes. Is the sequenced genome the largest within the genus or the family? In addition, the GenBank accession numbers are not legible for all mitochondrial genomes shown in Figure 4.

Response 8: We express our gratitude to the reviewer for the constructive suggestions provided. In response, we have revised Figure 3 (previously Figure 4 in the earlier version) as follows: The total genome size is now displayed beneath each species. It is noted that the C. ophiocarpa mitogenome is not the largest within the genus; rather, it is of intermediate size, being larger than C. saxicola at 587,939 bp, but smaller than C. pauciovulata at 675,483 bp. Additionally, the GenBank accession numbers have been enlarged and repositioned to enhance legibility. We are confident that these revisions comprehensively address the reviewer's concerns.

Comment 9: I recommend toning down the RNA-editing section (removing it or moving it to the supplement, Figure 6), as the authors did not verify the RNA-editing sites and the entire discussion therefore remains speculative.

Response 9: We thank the reviewer for the constructive suggestion. The section on RNA editing has undergone substantial revision as follows: Figure 6, illustrating RNA editing distribution, has been moved to the Supplementary Materials (now Supplementary Figure S6). Results section 3.6 has been streamlined to present only the essential factual findings: a total of 775 predicted sites, with 95.4% being non‑synonymous, the highest densities observed in nad4 and nad7, the presence of PTCs in five genes, and a comparison with C. saxicola. Detailed codon‑position data are provided (lines 316-322). Discussion section 4.1 on RNA editing has been removed. A more detailed discusson on RNA editing, particularly the biological function of non-synonymous and synonymous mutations resulted from RNA editing, is included in Discussion section 4.3 (lines 435-445). We believe the revised RNA editing section is appropriately moderated, fact‑based, and devoid of speculation. We trust this revision addresses the reviewer’s concern.

 

Best regards,

Ming Lei and other co-authors

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all of the reviewers’ comments, and I recommend acceptance of the manuscript for publication.