Fine Mapping of BrTCP1 as a Key Regulator of Branching in Flowering Chinese Cabbage (Brassica rapa subsp. chinensis)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The researchers conducted fine mapping of BrTCP1 as a key regulator of branching in flowering Chinese cabbage. They found BraA07g032600.3C (BrTCP1) as the most likely candidate gene for branching regulation. The manuscript requires minor revisions and can be accepted after addressing the comments below.

1. The effect of environmental conditions on BrTCP1 expression and branching phenotypes was not evaluated, though these are known to influence branching. If possible, I suggest authors investigating how environmental factors like light, nutrients, and temperature modulate BrTCP1 expression and branching phenotypes in flowering Chinese cabbage.
2. Authors focused on the BrTCP1 gene as the candidate controlling primary rosette branching. However, branching is a complex trait likely influenced by multiple genes and pathways.
3. In the discussion part, authors should discuss comprehensively on how manipulating BrTCP1 alleles can be utilized in breeding programs for optimal plant architecture under different cultivation systems.

Author Response

Comments 1: The effect of environmental conditions on BrTCP1 expression and branching phenotypes was not evaluated, though these are known to influence branching. If possible, I suggest authors investigating how environmental factors like light, nutrients, and temperature modulate BrTCP1 expression and branching phenotypes in flowering Chinese cabbage.

Response 1:We agree that environmental factors (e.g., light, nutrients, temperature) modulate branching. Although this study primarily focused on the genetic regulation of branching, future research should investigate how environmental stimuli affect BrTCP1 expression and branching phenotypes. Such studies will provide a more comprehensive understanding of how BrTCP1 integrates genetic and environmental signals to regulate shoot architecture in flowering Chinese cabbage under variable cultivation conditions.

Comments 2: Authors focused on the BrTCP1 gene as the candidate controlling primary rosette branching. However, branching is a complex trait likely influenced by multiple genes and pathways.

Response 2: Thank you for your insightful comment. We fully agree that branching is a complex trait regulated by multiple genetic and hormonal pathways. In our study, we focused on BrTCP1 as a major candidate gene based on fine-mapping and functional validation results. While BrTCP1 plays a key role, we acknowledge that other genes may also contribute to the regulation of branching and warrant further investigation in future studies.

Comments 3: In the discussion part, authors should discuss comprehensively on how manipulating BrTCP1 alleles can be utilized in breeding programs for optimal plant architecture under different cultivation systems.

Response 3: We appreciate the reviewer’s insightful suggestion. We have now expanded the discussion to include a detailed perspective on how BrTCP1 alleles may be applied in breeding programs to optimize plant architecture under various cultivation scenarios. This addition aims to strengthen the translational relevance of our findings (see Discussion, final paragraph).

Reviewer 2 Report

Comments and Suggestions for Authors

General Evaluation:

This manuscript presents the identification and fine mapping of BrTCP1, a TCP-domain transcription factor implicated in the regulation of rosette branching in flowering Chinese cabbage. By integrating BSA-seq and fine mapping approaches, the authors delineated a 172 kb genomic region on chromosome A07 harboring BrTCP1. Sequence polymorphisms and gene expression analysis suggest that BrTCP1 functions as a suppressor of branching, a role consistent with orthologous TCP family members in Arabidopsis thaliana and rice.

The research topic is of relevance and aligns well with the scope of journals focused on plant molecular genetics, functional genomics, and crop improvement. Nevertheless, the manuscript requires revision prior to acceptance, as detailed in the following specific comments:

 

1. Abstract

While the abstract concisely summarizes the mapping and candidate gene identification, certain critical aspects require clarification. The aparent inconsistency in BrTCP1 expression—higher in the low-branching line (CX010) but lower in the high-branching mutant (BCT18)—is not addressed. This paradox undermines the clarity of the functional interpretation and should be explicitly explained. Additionally, the claim of functional validation using Arabidopsis is overstated and currently unconvincing, as the presented mutant phenotype is not complemented by overexpression or functional complementation assays.

 

2. Introduction

The Introduction section sufficiently reviews the established roles of TCP transcription factors (TB1, BRC1, MOC1) across plant species and provides a rationale for studying rosette branching. However, there are several notable gaps: 1) the manuscript does not adequately address the hormonal regulatory networks—specifically auxin, strigolactone, and gibberellin pathways—that interact with TCP proteins to control branching. 2) relevant prior studis on branching-related genes in Brassica species (e.g., BnaA09.ELP6, GA2ox1, IAA signaling components) are omitted. 3) the economic significance of rosette branching in flowering Chinese cabbage relative to other Brassica crops is insufficiently justified.

 

3. Materials and Methods

Some methodological details are lacking, such as: 1) biological replication information is incomplete (e.g., number of replicates for qRT-PCR and phenotyping assessments is not provided). 2) The Arabidopsis validation is inadequate: i) functional complementation or overexpression data are absent. ii) the specific phenotype of the AT1G67260 mutant is not fully described; potential functional redundancy of BrTCP1 in Arabidopsis is not addressed. iii) SNP calling parameters (e.g., filtering thresholds, LOD score criteria) are insufficiently described.

 

4. Results

An important contradiction remains unresolved: why is BrTCP1 expression higher in CX010 (low-branching) if the mutation in BCT18 causes an increased branching phenotype? This expression pattern must be reconciled with the proposed functional model. Additionally, no protein functional assay is provided to demonstrate that the mutation affects DNA-binding activity or overall protein function.

 

5. Discussion

The discussion inadequately addresses the expression paradox (BrTCP1 expression higher in CX010 vs. BCT18). This inconsistency must be explicitly discussed and biologically explained.

Missed opportunities include: i) discuscsion of TCP protein interactions with plant hormone networks (auxin, strigolactone, gibberellin). ii) Critical assessment of the limitations of using Arabidopsis as a heterologous validation system. iii) consideration of the translational potential of these findings for marker-assisted selection or genome editing strategies in Brassica breeding programs.

 

Author Response

Comments 1: Abstract. While the abstract concisely summarizes the mapping and candidate gene identification, certain critical aspects require clarification. The aparent inconsistency in BrTCP1 expression—higher in the low-branching line (CX010) but lower in the high-branching mutant (BCT18)—is not addressed. This paradox undermines the clarity of the functional interpretation and should be explicitly explained. Additionally, the claim of functional validation using Arabidopsis is overstated and currently unconvincing, as the presented mutant phenotype is not complemented by overexpression or functional complementation assays.

Response 1: We thank the reviewer for highlighting this important point. We changed the original sentence to: ‘Expression analysis indicated that BrTCP1 was highly expressed in the rosette stems of CX010 compared to BCT18, consistent with its role as a branching suppressor. The heterologous mutants in Arabidopsis confirmed the conserved role of BrTCP1 in branch inhibition’.

Comments 2: The Introduction section sufficiently reviews the established roles of TCP transcription factors (TB1, BRC1, MOC1) across plant species and provides a rationale for studying rosette branching. However, there are several notable gaps: 1) the manuscript does not adequately address the hormonal regulatory networks—specifically auxin, strigolactone, and gibberellin pathways—that interact with TCP proteins to control branching. 2) relevant prior studis on branching-related genes in Brassica species (e.g., BnaA09.ELP6, GA2ox1, IAA signaling components) are omitted. 3) the economic significance of rosette branching in flowering Chinese cabbage relative to other Brassica crops is insufficiently justified.

Response 2: Thank you for this insightful comment. In the revised manuscript, we have expanded the Introduction to include a paragraph discussing the interplay between TCP transcription factors and major phytohormones such as auxin, strigolactones, and gibberellins in the regulation of shoot branching. We have also incorporated a summary of relevant findings on branching-related genes previously identified in Brassica species, including BnaA09.ELP6, GA2ox1, and IAA signaling components. Furthermore, we have emphasized the economic importance of rosette branching in flowering Chinese cabbage, especially in relation to yield formation and cultivation efficiency. These additions help to provide a more comprehensive rationale for our study.

Comments 3: Some methodological details are lacking, such as: 1) biological replication information is incomplete (e.g., number of replicates for qRT-PCR and phenotyping assessments is not provided). 2) The Arabidopsis validation is inadequate: i) functional complementation or overexpression data are absent. ii) the specific phenotype of the AT1G67260 mutant is not fully described; potential functional redundancy of BrTCP1 in Arabidopsis is not addressed. iii) SNP calling parameters (e.g., filtering thresholds, LOD score criteria) are insufficiently described.

Response 3: We appreciate the reviewer’s detailed feedback. We have now clarified that all qRT-PCR and phenotyping experiments were performed with three biological replicates. Regarding Arabidopsis validation, we specified the mutant line (AT1G67260), described its phenotype in more detail. The candidate gene has no highly homologous genes in this species, so there is no problem of functional redundancy. Furthermore, SNP calling parameters, including filtering thresholds and LOD score criteria, have been added to the BSA-seq analysis section (see Materials and Methods, Sections 2.5 and 2.7).

Comments 4: Results. An important contradiction remains unresolved: why is BrTCP1 expression higher in CX010 (low-branching) if the mutation in BCT18 causes an increased branching phenotype? This expression pattern must be reconciled with the proposed functional model. Additionally, no protein functional assay is provided to demonstrate that the mutation affects DNA-binding activity or overall protein function.

Response 4: Thank you for pointing out this contradiction. We now explicitly state that the higher BrTCP1 expression in CX010 suggests its role as a negative regulator of branching. Although our structural analysis indicated mutation-induced changes in protein conformation, we acknowledge that direct protein function assays (e.g., DNA-binding activity) were not performed and represent a valuable direction for future work.

Comments 5: The discussion inadequately addresses the expression paradox (BrTCP1 expression higher in CX010 vs. BCT18). This inconsistency must be explicitly discussed and biologically explained.Missed opportunities include: i) discuscsion of TCP protein interactions with plant hormone networks (auxin, strigolactone, gibberellin). ii) Critical assessment of the limitations of using Arabidopsis as a heterologous validation system. iii) consideration of the translational potential of these findings for marker-assisted selection or genome editing strategies in Brassica breeding programs.

Response 5: BrTCP1 has an inhibitory effect on plant branching, so the expression of BrTCP1 in CX010 is higher than that in BCT18. This was mentioned in the discussion. We acknowledged that while Arabidopsis is a convenient model, functional conservation between species may be incomplete due to evolutionary divergence and species-specific regulatory contexts. Therefore, the branching phenotype observed in the Arabidopsis tcp1 mutant provides supporting, but not definitive, evidence for BrTCP1 function in flowering Chinese cabbage. We have added discussions on the interaction between TCP proteins and plant hormone networks (auxin, estrone, gibberellin), as well as the translation potential for marker assisted selection or genome editing strategies in Brassica breeding programs.

Reviewer 3 Report

Comments and Suggestions for Authors

The MS entitled “Fine Mapping of BrTCP1 As a Key Regulator of Branching in 2 Flowering Chinese Cabbage (Brassica rapa subsp. chinensis)” by Liu et al., describes about identification of a locus for branching in Chinese cabbage using BSA-seq, fine mapping using F2 population, identification of BrTCP1 as a candidate gene for branching and validation. The topic is interesting and the results will pave the way for genetic improvement of Brassica crops with desirable trait. However, there are major concerns about the presentation of their works and the way to draw conclusion (s). Below are some of the points that should be addressed in greater depth for better MS. Also consider lots of minor points that are not described in here.

• LN107: “The two DNA pools, more branching pool (MB) and less branching pool (LB), each consisted of an equal amount of DNA from 50 F2 individuals showing the most extreme branching more and less phenotype, respectively” What are the phenotypes of the selected extreme F2 pools? For example, how many branches in MB? and LB? No. of F2 population mentioned 841(LN99) and 840 (LN186)? Please revise and be consistent.

 

• LN181-188: The authors described “Phenotypic observation and inheritance characteristics of branching in flowering Chinese cabbage” and cited Fig. 1a–d; however, the figures corresponding to “(c) Branching statistics of CX010 and BCT18” and “(d) The primary rosette branching number segregation histogram of the F₂ population” could not be found in the MS. Even Fig. 1a and b are not properly indicated.

 

• The authors claim they have identified a candidate interval for branching on chromosome A07, but I couldn't understand how the authors speculate the indicated interval govern the branching because there is no information about statistical value of delta SNP index that determine the interval is associated with branching. Is Fig. 2a SNP index or delta SNP index? The SNP index of both extreme pools and delta SNP index with statistical significance should be presented together to help the readers understand it easily. what was the window size and sliding window used in SNP index analysis?

 

• In addition, for traditional QTL mapping, the authors said “a significant QTL was defined based on LOD > 3.0 (LN134 -135). However, I couldn’t find the LOD information of the interval in the MS.

 

• Authors genotyped F2 population with Indel markers for fine mapping of the candidate region, but I could not find any data of F2 genotyping with phenotyping data that should be presented.

Author Response

Comments 1: LN107: “The two DNA pools, more branching pool (MB) and less branching pool (LB), each consisted of an equal amount of DNA from 50 F2 individuals showing the most extreme branching more and less phenotype, respectively” What are the phenotypes of the selected extreme F2 pools? For example, how many branches in MB? and LB? No. of F2 population mentioned 841(LN99) and 840 (LN186)? Please revise and be consistent.

Response 1:We thank the reviewer for this constructive suggestion. We have clarified the phenotypes of the extreme F₂ pools by stating that the MB pool consisted of 50 individuals with 10–15 primary rosette branches, while the LB pool had 50 individuals with only 1 branch.  The segregation population size of the F2 generation was unified as 840.

Comments 2: LN181-188: The authors described “Phenotypic observation and inheritance characteristics of branching in flowering Chinese cabbage” and cited Fig. 1a–d; however, the figures corresponding to “(c) Branching statistics of CX010 and BCT18” and “(d) The primary rosette branching number segregation histogram of the F₂ population” could not be found in the MS. Even Fig. 1a and b are not properly indicated.

Response 2:Thank you for pointing this out. We replaced the picture of Fig. 1 to illustrate the problem more clearly.

Comments 3: The authors claim they have identified a candidate interval for branching on chromosome A07, but I couldn't understand how the authors speculate the indicated interval govern the branching because there is no information about statistical value of delta SNP index that determine the interval is associated with branching. Is Fig. 2a SNP index or delta SNP index? The SNP index of both extreme pools and delta SNP index with statistical significance should be presented together to help the readers understand it easily. what was the window size and sliding window used in SNP index analysis?

Response 3: We appreciate the reviewer’s valuable suggestion. Fig. 2a represents the SNP-index, while the Δ(SNP-index). The SNP indices of the extreme pool and the statistically significant delta SNP indices are presented in Figure 2. Additionally, we have specified the sliding window size (1 Mb) and step size (10 kb) used for the analysis in the Methods section.

Comments 4: In addition, for traditional QTL mapping, the authors said “a significant QTL was defined based on LOD > 3.0 (LN134 -135). However, I couldn’t find the LOD information of the interval in the MS.

Response 4:  We added the following content to the original text: ‘The peak LOD score of the QTL between markers 21248 and 21251 was 4.07, exceeding the threshold of 3.0, indicating a statistically significant association with branching traits’.

Comments 5: Authors genotyped F₂ population with Indel markers for fine mapping of the candidate region, but I could not find any data of F₂ genotyping with phenotyping data that should be presented.

Response 5:Thank you for your professional opinion. We have included the corresponding data in Appendix 1.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate the authors for thorough revision of the manuscript. My comments are addressed and incorporated which greatly improved the presentation of their work. The manuscript now looks in better format and clearly presents the identification of BrTCP1 as a key regulator of branching in flowering Chinese Cabbage. Overall, the manuscript will be a valuable input for enhancing molecular breeding in Brassica spp.