Mapping QTL and Identifying Candidate Genes for Resistance to Brown Stripe in Highly Allo-Autopolyploid Modern Sugarcane

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I realize that the parents of the cross are not 'inbred lines' but the F1 versus F2 question needs to be confirmed/  If the plants used were all from different seeds from the cross, F1 is likely OK, just unusual.  In other cases these would be considered F2s.  

Do you have any idea whey there was considerable differences in markers etc. for the 2 parents?   When you say 'plant canes' I think it would be less confusing if you said something like 'plants grown from seeds' to differentiate them from ratoon or soma-clonal plants.   

Comments for author File: Comments.pdf

Comments on the Quality of English Language

There are many corrections for English that should be made.  I have marked some of them on the returned document.

Author Response

Response to Reviewer 1 Comments

 

Dear reviewer,

We are glad to receive your valuable comments and suggestions to our manuscript. Thank you for your kind consideration on this manuscript "Mapping QTL and Identifying Candidate Genes for Resistance to Brown Stripe in Highly Allo-autopolyploid Modern Sugarcane". Without your professional reviews, this manuscript would not be as smooth and more persuasive as what it is now. Thank you very much!

We have amended the manuscript according to all the opinions, suggestions and comments of the reviewers and all the changes have been marked-up in the text by the red fond. The responses to all the comments and suggestions are itemized as follows:

Comment 1: I realize that the parents of the cross are not 'inbred lines' but the F₁ versus F₂ question needs to be confirmed/If the plants used were all from different seeds from the cross, F₁ is likely OK, just unusual. In other cases these would be considered F₂s.

Response 1: Thanks for your professional comment. In terms of the mapping populations used for QTL analysis, sugarcane has no double haploid (DH) and recombinant inbred lines (RIL), but can use F₁ segregating population from crossing or self-crossing because of its characteristics of asexual propagation after the F₁ seedlings. In sugarcane breeding, F₁ generation is often used for heterosis utilization, and diverse progeny are obtained through different parental combinations. For example, according to the target traits of the study, two extreme phenotypes were selected as cross parent materials. The experimental materials used in this study were all from different seeds of the cross parents, so they were regarded as F₁ populations.

Comment 2: Do you have any idea why there was considerable differences in markers etc. for the 2 parents?

Response 2: Thanks for your professional comment. We need to select the appropriate parents for cross to construct the segregating population, such as F₁ population, when screening the associated markers of SBS resistance. Using high-throughput chips to detect and analyze the purity of parents and F₁, after detecting the purity of parents and F₁, we need to use polymorphic markers between parents to detect the genotype of F₁ generation. On the markers with differences between the two parents, F₁ was heterozygous genotype. Individuals in these populations show varying degrees of differences in target traits, and there are also abundant genetic variations in the genome. The mapping population was genotyped based on the high-throughput sugarcane Axiom Sugarcane100K SNP chip. Throughcc² detection, single-dose SNP markers with a separation ratio of 1 : 1 were filtered out ( P < 0.01 ). After screening, a total of 2,743 single-dose SNP markers were obtained for the construction of the genetic map of the two parents, of which the female parent YT93-159 contained 1,814, and the male parent ROC22 contained 929.

Comment 3: When you say 'plant canes' I think it would be less confusing if you said something like 'plants grown from seeds' to differentiate them from ratoon or soma-clonal plants.

Response 3: Thanks for your professional comment. In fact, according to some reported previous studies, plant cane in sugarcane means 'plants grown from seeds ' ; ratoon in sugarcane means 'plants grown from ratoon '. The following lists the DOI address of the reference : ① 10.3389/fpls.2021.817875; ② 10.3390/ijms24032793; ③ 10.1016/j.cj.2021.11.009

Comment 4: There are many corrections for English that should be made.  I have marked some of them on the returned document.

Response 4: Thank you very much for your patient correction. It has been corrected in the uploaded manuscripts and checked in full.

Take this opportunity, again we do want to express our high and great appreciation for your kind words and professional suggestions, which should no doubt help for our future research. Thanks again.

Any questions, we will be more than happy to answer. Looking forward to hearing from you soon.

Regards and Best wishes!

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents an extensive genetic analysis of sugarcane resistance to Sugarcane Brown Stripe (SBS) disease, combining high-density SNP genotyping, linkage map construction, QTL mapping, and candidate gene identification. The study is well-conceived and addresses a critical issue in sugarcane breeding, the molecular basis of disease resistance in a highly polyploid, clonally propagated crop. The results are potentially valuable for marker-assisted selection (MAS) and genomic selection strategies in breeding programs.

The study claims to be the first QTL mapping for SBS resistance in sugarcane. Given the disease’s significance and the complexity of the sugarcane genome, this is a valuable contribution. However, the effect sizes of most QTLs are modest, and the practical applicability of these markers remains uncertain. I recommend the authors contextualize their findings in terms of breeding utility, for example, are the detected QTLs robust enough to be incorporated into MAS pipelines?

Do the authors have plans to functionally validate the role of PR3, DND2, and EDR2 in SBS resistance via overexpression, knockout, or VIGS assays? Have these genes or their homologs been previously implicated in resistance to foliar fungal diseases in sugarcane or related crops?

The six-year phenotypic data are a strength of the manuscript, providing robust evidence that SBS resistance is quantitative. However, the heritability of 0.52 is relatively low, raising concerns about environmental noise. Were genotype-by-environment (G×E) interactions modeled? This would strengthen the claim of stable QTL detection. 

The reported broad-sense heritability is moderate (H² = 0.52). Was this estimate based on a mixed linear model accounting for spatial effects or field design?

Comments on the Quality of English Language

 The text is at times verbose and repetitive. A professional language edit is advised to improve clarity and flow.

Author Response

Response to Reviewer 2 Comments

Dear reviewer,

We are glad to receive your valuable comments and suggestions to our manuscript. Thank you for your kind consideration on this manuscript "Mapping QTL and Identifying Candidate Genes for Resistance to Brown Stripe in Highly Allo-autopolyploid Modern Sugarcane". Without your professional reviews, this manuscript would not be as smooth and more persuasive as what it is now. Thank you very much!

We have amended the manuscript according to all the opinions, suggestions and comments of the reviewers and all the changes have been marked-up in the text by the red fond. The responses to all the comments and suggestions are itemized as follows:

Comment 1: This manuscript presents an extensive genetic analysis of sugarcane resistance to Sugarcane Brown Stripe (SBS) disease, combining high-density SNP genotyping, linkage map construction, QTL mapping, and candidate gene identification. The study is well-conceived and addresses a critical issue in sugarcane breeding, the molecular basis of disease resistance in a highly polyploid, clonally propagated crop. The results are potentially valuable for marker-assisted selection (MAS) and genomic selection strategies in breeding programs.

Response 1: Thank you very much for approving the work of this study. The findings from this study are expected to provide some potential SNP markers for genomic selection and genes for improvement of SBS resistance in sugarcane breeding programs, thus accelerating the release of resistant varieties in future.

Comment 2: The study claims to be the first QTL mapping for SBS resistance in sugarcane. Given the disease’s significance and the complexity of the sugarcane genome, this is a valuable contribution. However, the effect sizes of most QTLs are modest, and the practical applicability of these markers remains uncertain. I recommend the authors contextualize their findings in terms of breeding utility, for example, are the detected QTLs robust enough to be incorporated into MAS pipelines?

Response 2: Thanks for your professional suggestion. The quantitative traits of allo-autopolyploid sugarcane are basically controlled by multiple minor genes or QTLs, and the phenotypic contribution rate of each locus is different. Mapping QTLs associated with important traits on sugarcane and developing linkage markers associated with traits are the key steps in the application of molecular marker-assisted selection (MAS) to sugarcane genetic improvement and variety breeding. Although domestic and foreign researchers have obtained a series of related markers for QTL mapping of sugarcane target traits, only two markers R12H16 and 9020-F4, which are closely linked to the main gene Bru1 of brown rust resistance, have been applied in sugarcane production. In this study, 32 QTLs associated with sugarcane brown stripe (SBS) resistance were detected in 6 different habitats, which could explain 186.53% of the phenotypic variation explained (PVE) in F₁ segregation population. Among them, two major QTLs (QBS-Y38 and qSBS-R46), which could explain 11.47% and 11.64% of the PVE, respectively. In addition, QTL qSBS-Y38-2 was detected in two different habitats (2016 and 2018), QTL qSBS-Y38-1 was detected in two different habitats (2018 and 2020), QTL qSBS-R8 was detected in two different habitats (2016 and 2018), and QTL qSBS-R46 was detected in three different habitats (2015,2016 and 2020). It shows that the QTLs associated with these target traits can be considered as stable QTLs. Therefore, the author 's team plans to verify the detected major QTLs and stable QTLs and their markers in the mapping population and other exogenous populations. If these QTLs are found to be effective in different genetic backgrounds and environments, they can be included in the MAS pipeline.

Comment 3: Do the authors have plans to functionally validate the role of PR3, DND2, and EDR2 in SBS resistance via overexpression, knockout, or VIGS assays? Have these genes or their homologs been previously implicated in resistance to foliar fungal diseases in sugarcane or related crops?

Response 3: Thanks for your professional suggestion. The author plans to verify the gene function of PR3 in SBS resistance through overexpression and VIGS experiments. Fortunately, the author's team has previously reported that the overexpression of ScPR1/PR10 in sugarcane can significantly improve the resistance of sugarcane to Pseudomonas rubrilineans and Nicotiana benthamiana to Ralstonia solanacearum, respectively. These results provide an important theoretical reference basis for the functional identification of PR2 gene. In addition, according to previous studies, DND2 and EDR2 are related to fungal diseases in crops such as grapes (Vitis amurensis), rapeseed (Brassica napus), upland cotton (Gossypium hirsutum), and Arabidopsis. Among them, EDR2 is a negative gene. In the future, the isolation and identification of negative genes in sugarcane may be the main breakthrough in the study of the mechanism of brown streak disease, and also provide a new idea for the cultivation of resistant varieties in sugarcane. In summary, these resistance-related genes or their homologous genes are related to the resistance of sugarcane or other crops to leaf fungal diseases.

Comment 4: The six-year phenotypic data are a strength of the manuscript, providing robust evidence that SBS resistance is quantitative. However, the heritability of 0.52 is relatively low, raising concerns about environmental noise. Were genotype-by-environment (G×E) interactions modeled? This would strengthen the claim of stable QTL detection.

Response 4: Thanks for your professional suggestion. As far as we know, GWAS analysis based on natural populations needs to face the problem of false positive results due to population structure, while QTL mapping of two parents does not need to consider population structure, and the noise of genetic background is relatively small. The six-year phenotypic data indeed revealed that SBS resistance was a quantitative trait. Based on the field phenotypic data, the variance analysis of the fluctuation of SBS resistance traits in different variation sources was carried out. The results showed that the variation sources of genotype and environment had a significant effect on the resistance to SBS disease (P < 0.01), and the interaction between genotype and environment (G × E) also had a significant effect on these traits (P < 0.01) (this part of the results will be added to the Supplementary Materials in the final version.). These results indicate that both the independent effect and the interaction effect can affect the genetic variation of the target trait, and the interaction effect reduces the heritability level of the target trait.

Comment 5: The reported broad-sense heritability is moderate (H² = 0.52). Was this estimate based on a mixed linear model accounting for spatial effects or field design?

Response 5: Thanks for your professional suggestion. Mixed linear model is one of the methods for QTL mapping. In this study, the progeny and bi-parents of the F₁ segregating population were planted in the most suitable areas for the prevalence of SBS disease (Longchuan, Yunnan, China; 24°25′ N, 97°88′ E). This is based on field experimental design. The interaction between genotype and environment (G × E) reduced the heritability level of the target trait. Thus, the heritability of SBS resistance trait was low, indicating that the genetic factors accounted for a small proportion in the decision process of this trait, and the environmental factors accounted for a large proportion.

Comment 6: The text is at times verbose and repetitive. A professional language edit is advised to improve clarity and flow.

Response 6: Thanks for your professional suggestion. All the authors have checked and revised the full text many times. and all the changes have been marked-up in the text by the red fond.

Take this opportunity, again we do want to express our high and great appreciation for your kind words and professional suggestions, which should no doubt help for our future research. Thanks again.

Any questions, we will be more than happy to answer. Looking forward to hearing from you soon.

Regards and Best wishes!

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors promptly responded to all of my inquiries.