Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

ijms-4569867  comments

 

This study integrates 3D chromatin architecture with comparative genomics across multiple Oryza species. The TCBF-based analysis provides a useful framework for assessing the conservation of topological domain boundaries and their functional features. The associations with RNA metabolism, sequence conservation, and epigenetic signatures are interesting, and the 3D-genomic phylogenetic analysis adds an evolutionary perspective. Overall, the study is well designed and provides useful insights into the evolution of 3D genome organization in plants. Several points should be clarified or corrected before publication. I therefore recommend minor revision.

 

Comments:

1. Lines 24–26: The statement “We created the first phylogenetic reconstruction derived from topological domain data” is too strong. The authors should clarify the scope of this claim, e.g., “to our knowledge, the first in plants” or within the Oryza Also, the NJ tree includes only five taxa and is based on Jaccard distances without bootstrap support. The term “phylogenetic reconstruction” should therefore be used with caution.
2. Lines 64–73: The authors lack sufficient plant-specific background on TAD boundary formation. Unlike mammals, plants lack a CTCF-based boundary system, and the mechanisms of plant TAD organization remain unclear. The authors should briefly discuss this difference and the roles of transcription and epigenetic features in plant TAD boundaries.
3. Lines 133–135: The manuscript provides limited information on why TCBF is needed. The authors should briefly explain its advantages over existing approaches for comparing TAD boundaries across species and clarify what specific limitation of current methods it addresses.
4. Lines 216–218: The mapping of meridionalis boundaries to the Nipponbare genome is unclear. The authors only describe lifting over Azucena, IR64, and O. rufipogon boundaries, although Category 2 also includes O. meridionalis-specific boundaries. Please clarify how these boundaries were mapped for the functional analyses. The 0.95 minimum base overlap should also be justified, particularly for the more divergent O. meridionalis. The numbers of successfully mapped and excluded boundaries should be reported to assess possible mapping bias.
5. Lines 241–243: The reason for not performing bootstrap analysis is not sufficiently justified. Binary presence/absence data can be used for bootstrap analysis, although the small number of taxa and the binarization of conserved boundaries may affect support values. The authors should clarify why bootstrap or another measure of tree stability was not used. Given that the tree includes only five taxa, its stability should be assessed and the 3D tree interpreted with caution.
6. Lines 37–40: This sentence is overly long and difficult to follow, with too many modifying clauses. The authors should simplify and rewrite it for clarity.
7. Lines 48–52: The sentence is overly long, with several examples inserted between the subject and the main verb. The authors should simplify the sentence structure to improve readability.
8. Lines 225–226: “Two-dimensional sequence information” is incorrect. Please change it to “one-dimensional sequence information” to maintain consistency with the rest of the manuscript.

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the article “Evolutionary and Functional Conservation of Topologically Associating Domain Boundaries in Rice Genomes” investigate the extent to which TAD boundaries are evolutionarily conserved across five closely related rice genomes. The central idea is to classify TAD boundaries based on their degree of inter-genomic conservation—ranging from "highly conserved" to "unique"—and to determine whether the level of structural conservation correlates with functional and evolutionary genomic characteristics. The most novel aspect is the attempt to construct an evolutionary tree directly from the presence/absence patterns of conserved TAD boundaries and to compare it with standard whole-genome sequence-based distances.
The evolution of 3D genome architecture in plants has been studied far less extensively than sequence evolution. Furthermore, the very concept of TAD or TAD boundary conservation depends heavily on the computational framework employed. TCBF was specifically designed to identify conserved TAD boundaries across species, utilizing gene synteny and homologous sequences rather than simple coordinate mapping via liftOver; thus, applying this approach to the closely related *Oryza* system is logical.
A previous study by Kurbidaeva et al. (2025) utilized these same five *Oryza* genomes and Micro-C datasets to investigate TAD and TAD boundary evolution. That study demonstrated that global 3D structural divergence correlates with evolutionary distance and that conserved boundaries are characterized by higher gene density, transcriptional activity, and levels of H3K27ac, H3K4me3, FitCons, and PhastCons scores, alongside lower levels of methylation, transposable elements (TEs), and sequence variation. The novelty of the current work needs to be articulated more clearly.
Comments:
1. The distinction between the current work and Kurbidaeva et al. (2025) must be demonstrated much more clearly; currently, this differentiation is insufficiently defined.

2. It is particularly significant that TCBF and the previous liftOver-based analysis yield substantially different results. I would like to see a much more systematic benchmark comparison: TCBF versus the previous liftOver approach.

3. The choice of a 5-kb boundary necessitates a sensitivity analysis. The authors have fixed the boundary length at 5,000 bp. This is understandable, given that the raw Micro-C data were analyzed at that resolution. However, conservation analysis can depend significantly on the size of the boundary interval. It would be advisable to test at least one alternative size/resolution or to provide a clearer justification for the robustness of the specific 5-kb definition.

4. The authors state that TCBF was run three times, with the input files being re-downloaded each time. However, a deterministic computational algorithm should yield the same result given identical data. Re-downloading files does not constitute independent replication, nor does it assess uncertainty. I would either remove this section or replace it with a genuine sensitivity/robustness analysis.

5. The authors tested two background sets but selected the whole Nipponbare genome because it yielded “more biologically informative and reliable results.” One cannot select a background set simply because the results appear more informative. I would recommend presenting both analyses and interpreting them separately, rather than dismissing one as “low-resolution.”

Author Response

Please see the attachment.

Author Response File: Author Response.pdf