Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This submission examines heterosis in triploid hybrids produced by crosses between diploid and tetraploid loquat. Intra and inter-varietal crosses were made. Triploids with different genomic contributions were found to be different from each other for the heterotic response. The experiments appear to be performed carefully.

The authors cast the difference between the two hybrids as a Parent of Origin Effect. The authors attributed this to a dosage-dependent effect, which seems reasonable. In lines 371 to 372, they go further to favor an idea that paternal imprinting is involved. However, outside of the endosperm, there is very little if any evidence across the plant kingdom for an effect of imprinting in the sporophyte. Reciprocal crosses between lines at the diploid level would be different from each other if parental imprinting were significantly involved, but this does not seem to be the case. Rather it seems that genomic dosage involving different doses of allelic variants of dosage sensitive regulatory genes has an impact on the heterotic effect in triploids (Yao et al = reference 16). It is granted that imprinting would fall within this category, but, as noted, there is little evidence for imprinting effects in sporophytes. It would be better to refer to the difference as a genomic dosage effect (GDE) rather than POE.

Author Response

Dear Reviewer,

Thanks for your thorough review and for providing such insightful comments on our manuscript. Your expertise has been invaluable in helping us to refine our arguments and improve the scientific rigor of our work. We agree with your key points and have revised the manuscript accordingly. Below, we address each of your comments in detail.

For clarity and to ensure we address all aspects of your valuable feedback, we have organized our response around what we interpret as three main points within your comment. We will address each in turn.

Comments 1: The reviewer suggests that the observed difference is more accurately described as a "Genomic Dosage Effect (GDE)" rather than a "Parent-of-Origin Effect (POE)," as the latter often implies imprinting, for which there is little evidence in the sporophyte.

Response 1: We agree with your point that the underlying mechanism for the phenomon we observed is indeed a "Genomic Dosage Effect (GDE)," resulting from the imbalanced (1m:2p vs. 2m:1p) genomic contributions from the parents. Our use of the term "Parent-of-Origin Effect (POE)" in the manuscript was based on two main considerations:

(1) Descriptive accuracy: POE is a standard and widely accepted term used to describe the observable phenomenon where the phenotype of an offspring depends on whether an allele or, in this case, a whole genome set, is inherited from the male or female parent. Our reciprocal cross design was specifically set up to detect this effect, making POE a direct and accurate descriptor of our findings.

(2) Field convention: In the study of polyploidy heterosis, POE is commonly used as a standard term to refer to effects arising from imbalanced parental genome contributions in reciprocal interploidy crosses (e.g., as seen in references 11, 12, 17, and 24 cited in our manuscript).

To incorporate your valuable suggestion and clarify the relationship between these terms, we have added a sentence in the Discussion section to state that the POE we report is a specific manifestation of the broader GDE. We believe that retaining POE to describe the observed outcome while acknowledging GDE as the fundamental mechanism is a more scientifically precise approach.

To address this, we have added “It is important to clarify that while we use the term POE to accurately describe the observed phenotypic differences arising from the direction of the cross, this phenomenon is fundamentally a specific manifestation of a broader Genomic Dosage Effect (GDE), driven by the imbalanced contribution of parental genomes." In Discussion section (Lines 366-370). The changes are highlighted in yellow in manuscript.

 

Comments 2: The reviewer points out that there is little evidence for the effect of imprinting in the sporophyte across the plant kingdom and that our suggestion of its involvement (lines 371-372) is not well-supported.

Response 2: We fully accept this criticism. You are correct that evidence for sporophytic imprinting is scarce, and our statement that the mechanism was "likely mediated by paternal imprinting" was an over-speculation not directly supported by our data. Our intention was to propose a possible molecular basis, but the wording was not sufficiently cautious.

Accordingly, we have removed all direct references to "imprinting" from the Discussion and Results sections. We have replaced this speculative language with broader, more defensible terms such as "dosage-dependent gene regulation," "POE-specific gene expression," or "epigenetic modifications linked to parental origin." This revised wording allows us to discuss potential mechanisms without making unsubstantiated claims, thereby improving the scientific integrity of the paper.

To address this, in Result section we have revised “This result indicates that parental genome dosage effects, potentially involving gene expression regulation, epigenetic modification or genomic imprinting, interactively shape triploid heterosis [17,24].” as “This result indicates that parental genome dosage effects, potentially involving dosage-dependent gene regulation and POE-specific epigenetic modifications, interactively shape triploid heterosis [17,24].” (Lines 326-328). In Discussion section we have revised “This phenomenon is best explained by a dosage-dependent mechanism, likely mediated by paternal imprinting that accelerates early growth and cell division, ultimately enhancing overall vigor [34].” (Lines 371-372 in original version) as "This phenomenon points to a dosage-dependent regulatory mechanism where the paternal-excess genome dosage confers a significant growth advantage. This could be mediated by several factors, such as the altered expression of dosage-sensitive genes involved in cell division and growth, or other POE-specific epigenetic modifications, which collectively enhance overall vigor [34]." (Lines 397-402).

 

Comments 3: The reviewer notes that if parental imprinting were significantly involved, reciprocal crosses at the diploid level would also be expected to differ, which was not the case in our study.

Response 3: Thanks for this observation. We believe this point, in fact, strongly supports our central conclusion.

As you noted, our data show no consistent, significant differences between the diploid reciprocal crosses (R2×L2 vs. L2×R2) for the key vigor traits. This is a critical finding because it effectively rules out other potential causes, such as maternal cytoplasmic effects or standard nuclear allelic differences, which would also be present in the diploid crosses.

The fact that the pronounced directional effect only emerges in the interploidy (2x x 4x or 4x x 2x) crosses where the parental genomes are numerically imbalanced strongly implicates the unequal genomic contribution itself as the primary driver. Therefore, the results from the diploid crosses serve as a crucial baseline that isolates and highlights the dosage-dependent nature of the effects observed in our triploids.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract: line 29, “that” should be deleted, or alternatively “…revealed that POE was the main...”

Introduction:

It would be useful to briefly introduce the ploidy level most common in cultivated loquat, add info on diploid loquats, and how many seeds are typically present in fruits. Consider including photographs of the parental plants and cross section of their fruit if available to help readers better visualize the material.

Material and methods:

            2.3. line 119: in which phase of development was the plant when leaves samples were taken for analysis? How many leaves did plant have?

Provide info on protocol for PCR conditions for amplification of InDel markers. Indicate type of gel and concentration used for electrophoresis and differentiation between PCR products of amplified DNA fragments (results that are presented at Figure 2 C and D)

Add information on the distance metric that was applied for cluster analysis.

Please specify which software was used to generate heatmaps and dendrograms.

Results:

Lines 200-202 how many positive results did authors achieve on average with the use of InDel markers in confirmation of true hybrids (how many markers did amplify to confirm hybrids)?

Figure 2 C, please clarify, or recheck on chr6-394 L2 x R2-45, L2 x R2-52, it seems like there is one band only from mother; R2 x L2-6 there are no visible bands. D part of picture chr9-097 R4 x L4-3 resamble selfed R4-1, like in case of chr9-192 where R4 x L4-4 seems like selfed R4-1.

Figure 3A–B: There is inconsistency between the text and the figure. The Results section (3.2) states that L2 and L4 were the closest related parents GDtotal (0.228, panel A) what about 0.22 for L4 and R4? And that L4 and R4 have the smallest GDhomo (0.069, panel B). However, in panel B this lowest value is not circled in the figure, whereas other comparisons are. Figure 3 C and E: Please provide higher resolution heatmaps. Panel E caption, instead of “cluster analysis of F1 hybrid” consider “dendrogram”, or “tree diagram” from cluster analysis.

Lines 265-266. According to figure 4, panel A for leaf area MPH was not positive across all groups, 4x had negative values. In lines 267-268 it is not similar trend, MPH also had negative values for 4x, which is not written in manuscript.

Line 269, “striking” may be overstated for starch heterosis, which is more modest compared to other traits. Consider using more neutral term.

Lines 271-272 rewrite, because it sounds like “except for 3x(p)” relates to BPH, which is not the case.

Discussion:

Line 357, add ploidy level and common name for B. napus.

Appendix A

Figure A1, consider reordering traits in the legend at the bottom right corner. It would be easier to follow if the traits appear in the same order as on heatmap starting from plant height and ending with stomata density.

Table S1 is there available info on chromosome distribution of InDel primers?

Table S4 caption, do you mean data, not date? Insert spaces before brackets in first column with traits, and also after dot in Leaf No. per plant. There is no need for abbreviation of No., however if you decide to keep it, than add meaning of abbreviation in table caption. Ensure consistency in number formatting (without decimals, or with two zeros after decimal point).

Author Response

Dear Reviewer,

We appreciate the time and effort you have dedicated to providing these insightful comments. We have carefully considered each point and have revised the manuscript accordingly. We believe these changes have significantly improved the clarity, rigor, and overall quality of our paper. Below, we address each of your comments point-by-point. All the changes are highlighted in yellow in manuscript.

 

Abstract

Comment 1: line 29, “that” should be deleted, or alternatively “…revealed that POE was the main...”

Response 1: As suggested, we have deleted “that” and revised the sentence as “Quantitative analysis revealed POE as the main positive driver of triploid heterosis...” (Line 29).

 

Introduction

Comment 2: It would be useful to briefly introduce the ploidy level most common in cultivated loquat, add info on diploid loquats, and how many seeds are typically present in fruits. Consider including photographs of the parental plants and cross section of their fruit if available to help readers better visualize the material.

Response 2: Thanks for this excellent suggestion to provide better context for the reader. We have added info on diploid loquats as suggested. " Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a subtropical evergreen fruit crop valued for its fresh fruit and medicinal properties [1]. However, most of its cultivated varieties are diploid (2n=2x=34) producing fruits that typically contain three to five seeds, which significantly hampers its marketability and consumer appeal.” (Lines 39-41).

Furthermore, we agree that photographs would be very helpful. We have added a new supplementary figure (Figure S1) that shows images of the parental plants (L2, R2, L4, R4) and includes cross-sections of their respective fruits to help readers visualize the parental material (Line 41).

Figure S1. Parental plants and their fruit cross-sections. A, Diploid parent L2. B, Tetraploid parent L. C, Diploid parent R2. D, Tetraploid parent R4. Note: The tetraploid parent R4, induced from the white-fleshed cultivar ‘Ruantiaobaisha’, exhibits a yellow-fleshed fruit phenotype. This phenotypic alteration is consistent with somaclonal variation, a phenomenon known to occur during the in vitro polyploidization process. The genetic lineage and ploidy level of all parental materials, including R4, were rigorously confirmed by cytological and genome-wide SNP analyses as described in the main text.

Material and Methods

Comment 3: 2.3 line 119: in which phase of development was the plant when leaves samples were taken for analysis? How many leaves did plant have?

Response 3: Thank you for pointing out this missing information. We have revised the sentence about leaves sample collection to be more specific. “For whole-genome resequencing, a total of 2-3 young, fully expanded leaves were collected from the apical shoots of 6-month-old hybrid progeny and their corresponding parental controls. For each hybrid and parental line, a total of 12 individual plants were selected. These 12 individuals were then randomly assigned to three independent biological replicates, with each replicate consisting of an equal amount of pooled leaf tissue from 4 different plants." (Lines 120-125).

 

Comment 4: Provide info on protocol for PCR conditions for amplification of InDel markers. Indicate type of gel and concentration used for electrophoresis and differentiation between PCR products of amplified DNA fragments (results that are presented at Figure 2 C and D).

Response 4: We apologize for this omission. We have now added the detailed PCR protocol to the Material and Methods section. “PCR amplification for the selected InDel markers was performed in a 20 µL reaction volume containing 0.2 µL of rTaq polymerase (5 U/µL), 2 µL of 10× PCR buffer (containing 15 mM Mg²⁺), 2 µL of dNTP mix (2.5 mM each dNT), 1.6 µL of each forward and reverse primer (10 µM), 2 µL of genomic DNA (approx. 50 ng), and 10.6 µL of nuclease-free water. The PCR cycling conditions were as follows: an initial denaturation at 94°C for 4 min; followed by 33 cycles of denaturation at 94°C for 30 s, annealing at 50-60°C for 50 s, and extension at 72°C for 60 s; and a final extension at 72°C for 10 min. The resulting PCR products were separated and visualized by electrophoresis on a 2.5% high-resolution agarose gel stained with an appropriate DNA dye. A 100 bp DNA ladder was used as a size standard. The DNA fragments were visualized and documented under a gel imaging system.” (Lines 144-154).

 

Comment 5: Add information on the distance metric that was applied for cluster analysis.

Response 5: Thank you for pointing out this missing information. We have added a sentence to describe the clustering method used. "A phylogenetic tree was constructed for cluster analysis based on the total SNP dataset (GD_total) using MEGA X software (version 10.2.6). The tree was inferred using the Neighbor-Joining (NJ) method, with evolutionary distances computed using the p-distance model." (Lines 139-142).

 

Comment 6: Please specify which software was used to generate heatmaps and dendrograms.

Response 6: Thank you for pointing out this missing information. We have added a sentence specifying the software. "The heatmap in Figure A1 was generated using the pheatmap package in R." (Lines 142-143). Regarding the dendrograms, as addressed in our response to Comment 5, the relevant methodological details have been added to the figure legend of Figure 3.

 

Results

Comment 7: Lines 200-202 how many positive results did authors achieve on average with the use of InDel markers in confirmation of true hybrids (how many markers did amplify to confirm hybrids)?

Response 7: To clarify the robustness of our hybrid confirmation, we have added a sentence to the Results section. "Progeny were confirmed as true hybrids only if results from at least two InDel markers consistently showed hybridity patterns. We found that an average of 3 to 4 polymorphic markers were successfully amplified to confirm the hybrid status for each cross combination. Representative examples of these results are shown in Figures 2C and 2D." (Lines 217-221)

 

Comment 8: Figure 2 C, please clarify, or recheck on chr6-394 L2 x R2-45, L2 x R2-52, it seems like there is one band only from mother; R2 x L2-6 there are no visible bands. D part of picture chr9-097 R4 x L4-3 resamble selfed R4-1, like in case of chr9-192 where R4 x L4-4 seems like selfed R4-1.

Response 8: Thanks for your careful examination of the gel images. We have added a sentence to the caption: "In C and D, these markers effectively distinguished true-hybrids from faulse-hybrids based on their banding patterns. True-hybrids were confirmed by the co-inheritance of fragments from both the maternal (F) and paternal (M) parents, resulting in a heterozygous profile (e.g., L2 × R2-46 in C). Conversely, individuals exhibiting only a single parental band were identified as faulse-hybrids arising from self-pollination and were excluded from all subsequent analyses (e.g., L2 × R2-45 in C, which matches the maternal L2 profile)."(Lines 240-245)

 

Comment 9: Figure 3A–B: There is inconsistency between the text and the figure. The Results section (3.2) states that L2 and L4 were the closest related parents GDtotal (0.228, panel A) what about 0.22 for L4 and R4? And that L4 and R4 have the smallest GDhomo (0.069, panel B). However, in panel B this lowest value is not circled in the figure, whereas other comparisons are.

Response 9: We sincerely apologize our oversight and thank the reviewer for your careful attention. We have double-checked our data and made the following corrections:

We have revised that “ ...L4 and R4 were the most closely related parents (GDtotal = 0.220)...” (Lines 251-254). We have added a circle around the the value of 0.069 (for L4 and R4) in Figure 3B.

 

Comment 10: Figure 3 C and E: Please provide higher resolution heatmaps. Panel E caption, instead of “cluster analysis of F1 hybrid” consider “dendrogram”, or “tree diagram” from cluster analysis.

Response 10: We have replaced and checked the heatmaps in Figure 3C and 3E with higher-resolution versions in the revised manuscript. We have also revised the caption for panel E to the more precise term "Dendrogram from cluster analysis of F1 hybrid and parental groups based on GDtotal..." as suggested. (Lines 277-278)

 

Comment 11: Lines 265-266. According to figure 4, panel A for leaf area MPH was not positive across all groups, 4x had negative values. In lines 267-268 it is not similar trend, MPH also had negative values for 4x, which is not written in manuscript.

Response 11: We sincerely apologize our oversight and thank the reviewer for your careful attention. We have revised these sentences to be more precise. “For leaf area, MPH was positive in 2x and 3x groups, peaking at +27.25% in 2x, but was negative for the 4x group. For leaf number per plant, MPH was positive in the 2x and 3x(p) groups, while BPH was positive only in the 2x group.” (Lines 290-293)

 

Comment 12: Line 269, “striking” may be overstated for starch heterosis, which is more modest compared to other traits. Consider using more neutral term.

Response 12: We have replaced the word "striking" with the more neutral term "notable" to describe the heterosis observed for starch content. (Line 293)

 

Comment 13: Lines 271-272 rewrite, because it sounds like “except for 3x(p)” relates to BPH, which is not the case.

Response 13: Thank you for pointing out this ambiguity. We have rewritten the sentence as "In contrast, stomata density presented positive MPH in most groups except for 3x(m), while BPH was negative across all hybrid groups." (Lines 295-296)

 

Discussion

Comment 14: Line 357, add ploidy level and common name for B. napus.

Response 14: We have revised the text as "in allotetraploid canola (Brassica napus) hybrids".(Line 384)

 

Appendix A & Supplementary Tables

Comment 15: Figure A1, consider reordering traits in the legend at the bottom right corner. It would be easier to follow if the traits appear in the same order as on heatmap starting from plant height and ending with stomata density.

Response 15: This is an excellent suggestion for improving the figure's readability. We have reordered the legend in Figure A1 to match the order of the traits presented in the heatmap.

 

Comment 16: Table S1 is there available info on chromosome distribution of InDel primers?

Response 16: Yes, this information is available. We have added a new column to Table S1 that indicates the specific chromosomal location for each InDel primer, as derived from the reference genome. And we have added a sentence to the caption: "Note: The chromosome location of each marker is indicated by the primer name (e.g., 'chr7-404' is located on chromosome 7)."

 

Comment 17: Table S4 caption, do you mean data, not date? Insert spaces before brackets in first column with traits, and also after dot in Leaf No. per plant. There is no need for abbreviation of No., however if you decide to keep it, than add meaning of abbreviation in table caption. Ensure consistency in number formatting (without decimals, or with two zeros after decimal point).

Response 17: We sincerely apologize our oversight and thank the reviewer for your careful attention. We have made all the suggested corrections in Table S4. The typo "date" has been corrected to "data" in the caption. Spaces have been inserted for better formatting (e.g., "Plant height [cm]"). The abbreviation "No." has been changed to "Number" to avoid ambiguity. This change has also been reflected in the figure 4. All numerical values in the table have been reformatted to have a consistent two decimal places.

 

Author Response File: Author Response.pdf