Broad-Spectrum Resistance to Leaf Rust in the Argentinean Wheat Cultivar “Klein Proteo” Is Controlled by LrKP Located on Chromosome 2BS
, Jinwei Yang 3, Haitao Yu 4, Shisheng Chen 1,3,* and Xiaodong Wang 2,*Round 1
Reviewer 1 Report
In the present manuscript "Broad-spectrum resistance to leaf rust in an Argentinean wheat cultivar "Klein Proteo" is controlled by LrKP located on Chromosome 2BS" the authors have done bulked segregant RNA sequencing (BSR-seq) to identify the SNPs associated with the phenotype and further developed CAPS markers to map the gene LrKP on chromosome 2BS. The chromosomal region which carries the leaf rust resistance gene LrKP also carry the known gene Lr13.
The following are my observations.
1. You should carry out test of allelism studies with Lr13 NIL to complement your findings.
2. A different susceptible line should be used to generate a mapping population to validate the findings instead of using different generations (F2:3 and F3:4) of the same cross.
3. Make following minor modifications in the paper.
1. Line No 35: Change the test to “World-wide, it occurs more frequently than other rust diseases.” Instead of “It occurs………. Diseases.”
2. Line No 37: Include reference for the statement “under favourable conditions………..40%”.
3. Line No 37: Change the text to “With the trends of global warming during the last decade, leaf rust has expanded its epidemic area to almost all the wheat-planting regions in China.
4. Line No 40: Change the test to “efficient way of controlling the disease.”
5. Line No 105: Insert “and” before Lr13.
6. Reference No 35 is missing
1. In the result section in 3.1 instead of writing about few lines (6 lines: KP, susceptible control ZZ5389, Lr3a-NIL, Lr3bg-NIL, Lr3ka-NIL and Lr10-NIL) tested for 20 pathotypes write about of all the 10 lines and then highlight the importance of six lines.
Author Response
In the present manuscript "Broad-spectrum resistance to leaf rust in an Argentinean wheat cultivar "Klein Proteo" is controlled by LrKP located on Chromosome 2BS" the authors have done bulked segregant RNA sequencing (BSR-seq) to identify the SNPs associated with the phenotype and further developed CAPS markers to map the gene LrKP on chromosome 2BS. The chromosomal region which carries the leaf rust resistance gene LrKP also carry the known gene Lr13.
RE: Thank you for your positive comments on our manuscript.
The following are my observations.
- You should carry out test of allelism studies with Lr13 NIL to complement your findings.
RE: Since Lr13 was cloned, we performed PCR amplification using gene-specific primers Lr13F1/R1 and Lr13F5/R6 to test if the resistant parent “Klein Proteo” carries Lr13. Therefore, we didn’t perform an allelism test using F2 plants derived from the cross between the wheat lines “Klein Proteo” and Lr13-NIL. The absence of this information does not compromise the central results of this paper. An allelism test would be necessary if Lr13 was not cloned.
- A different susceptible line should be used to generate a mapping population to validate the findings instead of using different generations (F2:3 and F3:4) of the same cross.
RE: Fully susceptible line “ZhengZhou 5389” without any known Lr genes has been widely used as susceptible parental line in large number of studies (see below some of the references). F2:3 lines were employed for the BSR-seq whereas F3:4 lines were used to validate the designed CAPS markers. We have added corresponding statement in the M&M section to briefly introduce the susceptible line “ZhengZhou 5389”.
Yan X, Li M, Zhang P, et al. High-temperature wheat leaf rust resistance gene Lr13 exhibits pleiotropic effects on hybrid necrosis[J]. Molecular Plant, 2021, 14(7): 1029-1032.
Zhou H, Xia X, He Z, et al. Molecular mapping of leaf rust resistance gene LrNJ97 in Chinese wheat line Neijiang 977671[J]. Theoretical and Applied Genetics, 2013, 126(8): 2141-2147.
Gebrewahid T W, Zhang P, Zhou Y, et al. QTL mapping of adult plant re-sistance to stripe rust and leaf rust in a Fuyu 3/Zhengzhou 5389 wheat population[J]. The Crop Journal, 2020, 8(4): 655-665.
- Make following minor modifications in the paper.
- Line No 35: Change the test to “World-wide, it occurs more frequently than other rust diseases.” Instead of “It occurs………. Diseases.”
RE: Thank you for this suggestion. Revised.
- Line No 37: Include reference for the statement “under favourable conditions………..40%”.
RE: Added.
- Line No 37: Change the text to “With the trends of global warming during the last decade, leaf rust has expanded its epidemic area to almost all the wheat-planting regions in China.
RE: Revised.
- Line No 40: Change the test to “efficient way of controlling the disease.”
RE: Revised.
- Line No 105: Insert “and” before Lr13.
RE: Revised.
- Reference No 35 is missing
RE: Added.
- In the result section in 3.1 instead of writing about few lines (6 lines: KP, susceptible control ZZ5389,Lr3a-NIL, Lr3bg-NIL, Lr3ka-NIL and Lr10-NIL) tested for 20 pathotypes write about of all the 10 lines and then highlight the importance of six lines.
RE: Revised.
Reviewer 2 Report
The authors have done a commendable amount of work and discovered a new gene for leaf rust resistance. This gene would be helpful for the development of new varieties.
The authors have illustrated how it is possible to exclude the possibility of the presence of a known gene in a stock that carries more than one gene.
Additionally, the markers that were developed for LrKp will be beneficial for marker assisted selection in the future.
The gene displays characteristics of a broad range and is effective against all 20 pathotypes investigated in this work. As a result, the gene can be transferred into superior cultivars so that breeding programmes can make better use of this information.
The experimental design is reasonable, and the experimental procedure has been carried out in its entirety. Including the investigation and characterization of potential candidate genes.
However, the experiment could be improved by doing quantitative real-time PCR on the candidate gene that was discovered.
Author Response
The authors have done a commendable amount of work and discovered a new gene for leaf rust resistance. This gene would be helpful for the development of new varieties. The authors have illustrated how it is possible to exclude the possibility of the presence of a known gene in a stock that carries more than one gene. Additionally, the markers that were developed for LrKp will be beneficial for marker assisted selection in the future. The gene displays characteristics of a broad range and is effective against all 20 pathotypes investigated in this work. As a result, the gene can be transferred into superior cultivars so that breeding programmes can make better use of this information. The experimental design is reasonable, and the experimental procedure has been carried out in its entirety. Including the investigation and characterization of potential candidate genes.
RE: Thank you for all your positive comments on our manuscript.
However, the experiment could be improved by doing quantitative real-time PCR on the candidate gene that was discovered.
RE: Thanks for this suggestion. Lr13 was the only differentially expressed NBS-LRR encoding gene in the LrKP interval. To investigate the possible involvement of all the LrKP-interval DEGs in wheat resistance to leaf rust, we have also profiled the expressions of all the 12 LrKP-interval DEGs in the Lr47- and Lr57-mediated resistance (Figure S3). Certain DEGs were also differentially expressed upon leaf rust infection in association with Lr47 or Lr57, except for the cloned Lr13 gene. All these results indicated that Lr13 was specifically induced and might function in KP upon rust infection. Corresponding statements were added in the Results section.
Reviewer 3 Report
The study confirms the presence of Lr3 and Lr10 genes for resistance to Puccinia triticina in the wheat cultivar ¨Klein Proteo¨. Moreover, the authors identified the Lr13 gene and indicated that ¨Klein Proteo¨ shows a more broad-spectrum to all the P. triticina pathotypes than lines carrying Lr13, speculating about possible reasons.
I think information about resistance genes located in 2BS should be provided in the introduction. The objective of the work is not clear from the introduction. At the end of this section, the paragraph saying "In this study, we found KP showed high resistance to most of the epidemic Pt pathotypes 62 collected in China at the seedling stage. The genetic bases of the leaf rust resistance in "Klein Proteo" were initially investigated¨ is confused as it is a result and the objectives were not clear defined
In materials and methods, without more prior information in the introduction, it is unclear why you chose for inoculation lines carrying Lr13, Lr16, and Lr23. Furthermore, when you started the results section (3.1), you only mentioned the results of Lr3 and Lr10 isogenic lines, omitting the others. In addition to the severity scale 0-4, Cobb's scale should have been used to assess the type of reaction to leaf rust.
English grammar should be carefully revised.
Author Response
The study confirms the presence of Lr3 and Lr10 genes for resistance to Puccinia triticina in the wheat cultivar ¨Klein Proteo¨. Moreover, the authors identified the Lr13 gene and indicated that ¨Klein Proteo¨ shows a more broad-spectrum to all the P. triticina pathotypes than lines carrying Lr13, speculating about possible reasons.
RE: Thank you for your positive comments on our manuscript.
I think information about resistance genes located in 2BS should be provided in the introduction.
RE: We have provided information about designated Lr genes on chromosome 2BS in the introduction section as suggested.
The objective of the work is not clear from the introduction. At the end of this section, the paragraph saying "In this study, we found KP showed high resistance to most of the epidemic Pt pathotypes collected in China at the seedling stage. The genetic bases of the leaf rust resistance in "Klein Proteo" were initially investigated¨ is confused as it is a result, and the objectives were not clearly defined.
RE: We have revised the mentioned statement to define the objectives of the current study.
In materials and methods, without more prior information in the introduction, it is unclear why you chose for inoculation lines carrying Lr13, Lr16, and Lr23.
RE: Necessary explanations were added to the M&M section.
Furthermore, when you started the results section (3.1), you only mentioned the results of Lr3 and Lr10 isogenic lines, omitting the others.
RE: Results about other Lr NILs were demonstrated in section 3.4.
In addition to the severity scale 0-4, Cobb's scale should have been used to assess the type of reaction to leaf rust.
RE: Thank you for this suggestion. Currently, we have used Cobb’s scale to assess the type of reaction to leaf rust. We have revised the corresponding statements in the M&M section.
English grammar should be carefully revised.
RE: We have revised the whole manuscript thoroughly.
Round 2
Reviewer 3 Report
The authors has made the suggested corrections and the manuscript has improved
Author Response
Thank you for your positive comments on our revised manuscript.
