Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Irradiation-based mutant lines may generate appropriate resistance lines, and some mutations may prevent the fungal infection. However, it must always be ensured with gene network interactions, epigenetic interactions, and epistasis interactions. It's not known whether random mutations through irradiation would also affect the expression of housekeeping genes or other functional genes.

At least you should have tested some house genes, like actin. Please check here further: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472982/

Also, you didn't categorize the alleles based on international allelic nomenclature; please designate the alleles and corresponding SNPs in an appropriate manner. Please check: https://academic.oup.com/pcp/article/57/2/257/2461004

Please represent the SNPs for the given allele locus or loci. For instance, G>C or G>T, etc. Differentiate dominant allele-based SNPs over recessive allele-based SNPs.

Being an autogamous species, wheat is expected to produce progenies with genetic purity unless it is subjected to cross-pollination. Would you expect any segregation pattern of non-mendelain inheritance among mutant progenies?

Your abstract must be very precise; please rewrite it clearly. The abstract should reflect your research content inside the article; there is no need to emphasize much about data points and numerical representation in the abstract unless representing data points is mandatory. This abstract is blended slightly with methodology, results, and discussion. Please thoroughly and precisely rewrite the abstract.

Other comments:

Please change Puccinia triticina, LR, to Puccinia triticina Eriks in the abstract.

When you introduce the abbreviation for the first time, please write the full form of the abbreviation. For instance HSI in abstract. 

It is difficult to have a variety with permanently resistant genes due to co-evolution. Due to co-evolution, the host can't be permanently resistant, and the pathogen can't be either. So, please rewrite.

Please rewrite; one should immediately need to go to the materials section of the abstract to understand that you have used 75 resistant lines. If you write like 75 300... how can the readers understand?

Please correct the inappropriate repetitions. Please write the gene name in italics.

Please rewrite at line 70 as: Generally, SR genes are more effective than APR genes, and the former reportedly lost their activity after several years of utilization.

For Adult Plant Resistance, you have mentioned some time APR (a common term); some time like ARP, please correct it.

At line 75, why are you making so many statements in a huge single line? How can the readers understand? Please split the sentence and write precisely.

You could simply write, "The accurate and right combination with the desired mode of activity would enable durable resistance."

On what basis have you selected the advanced spikes? Have you collected from a single (individual) plant from the original lines or from the mutant lines?

At line 171, There is no connectivity from these sentences of M2 to M3 generations; please write correctly.

Have you developed M3 lines from each dosage of irradiation from the starting resource?

Please clarify how you have tested a large number of Lr genes using mutant lines from virulent fungal isolates. I presume you have derived the results of virulency and avirulency with hyperspectral image analysis, but I didn't see any expression analysis of the given genes. So, it's tough for the researchers to understand how a lump of genes were tested without expression studies. On what basis have you represented those genes in Table 1 in terms of their resistance to fungal isolates?

Which experimental design have you followed, such as CRD or RBD?

You have shown a combined form of irrigation dosage effect. Please show individually for each irradiation corresponding to mutant line, parental line, and time. On what criteria you have normalized the data of both infected and non-infected lines derived data?

 

Why do you show both infected and non-infected data together? Whether the color lines of the peaks emphasize the pooled data of both infected and non-infected. Please redraw the picture and label the X and Y axes. Please differentiate the peaks of infected and non-infected with the visible spectral range in particular.

If you have sequence information for your clones, please verify through the ENSEMBL wheat candidate gene to check the genomic positions for further accuracy of the genes studied and SNP comparisons from database sequences or varieties.

Could you please show the gel pictures of the KASP assay? If you have designated the alleles as "aa" or "bb," they seem all homozygous. Is that so?

There are several typos, and please rewrite the whole manuscript precisely; alter the long sentences to split, shorten them, and be precise. Substantial revision of the manuscript is required.

 

 

Comments on the Quality of English Language

Dear Authors,

The English language can be improved, and the manuscript can be rewritten precisely.

Author Response

Dear Reviewer 1,

 

Thank you very much for providing your suggestion. We have taken into account your valuablecomments and consequently revised the manuscript. Additionally, the manuscript reviewed by the Cambridge Proofreading LLC

 

Comment 1:  Irradiation-based mutant lines may generate appropriate resistance lines, and some mutations may prevent the fungal infection. However, it must always be ensured with gene network interactions, epigenetic interactions, and epistasis interactions. It's not known whether random mutations through irradiation would also affect the expression of housekeeping genes or other functional genes.

At least you should have tested some house genes, like actin. Please check here further: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472982/

 

Response: The aim of our study was to broaden the genetic variation of spring wheat, on the background of rust-resistant variety Kazakhstanskay-19 using induced mutagenesis through 300-, 350-, and 400-Gy doses of gamma irradiation and further to develop new mutant (M3 generation) lines. These lines was used for searching new ones that had the combined the ADP and SR to LR. Additionally, hyper spectral imaging analysis known as high throughput  phenotyping approach was applied the differentiate the different response between non- and infected plants and sustain-able and resistant genotypes.  Your valuable comments as suggested will be the tasks of our future research yet with developed wheat mutant lines.

 

Comment 2: Also, you didn't categorize the alleles based on international allelic nomenclature; please designate the alleles and corresponding SNPs in an appropriate manner. Please check: https://academic.oup.com/pcp/article/57/2/257/2461004

 

Response: As recommended, the appropriate changes have been made.

 

Comment 3: Please represent the SNPs for the given allele locus or loci. For instance, G>C or G>T, etc. Differentiate dominant allele-based SNPs over recessive allele-based SNPs.

 

Response: As recommended, the appropriate changes have been made.

 

 

Comment 4: Being an autogamous species, wheat is expected to produce progenies with genetic purity unless it is subjected to cross-pollination. Would you expect any segregation pattern of non-mendelain inheritance among mutant progenies?

 

Response: Each wheat mutant line was developed from one single spike and is represented genetic purity and therefore the progenies  will be genetic purity. Any segregation pattern cannot be.

Comment 5: Please rewrite at line 70 as: Generally, SR genes are more effective than APR genes, and the former reportedly lost their activity after several years of utilization.

Response: As recommended, we have edited this sentence.

 

Comment 6: Your abstract must be very precise; please rewrite it clearly. The abstract should reflect your research content inside the article; there is no need to emphasize much about data points and numerical representation in the abstract unless representing data points is mandatory. This abstract is blended slightly with methodology, results, and discussion. Please thoroughly and precisely rewrite the abstract.

Response: As recommended, the abstract was thoroughly and precisely rewrite centered on the main results of study.

Other comments:

1. Please change Puccinia triticina, LR, to Puccinia triticina Eriks in the abstract.

Response: as suggested, the appropriate change has been made.

 

2. When you introduce the abbreviation for the first time, please write the full form of the abbreviation. For instance HSI in abstract. 

Response: as suggested, the appropriate changes have been made.

 

3. Please rewrite; one should immediately need to go to the materials section of the abstract to understand that you have used 75 resistant lines. If you write like 75 300... how can the readers understand?

Response: We have rephrased the above sentence as suggested.

 

4. Please correct the inappropriate repetitions. Please write the gene name in italics.

Please rewrite at line 70 as: Generally, SR genes are more effective than APR genes, and the former reportedly lost their activity after several years of utilization.

Response: We have rephrased the above sentence as suggested.

 

5. For Adult Plant Resistance, you have mentioned some time APR (a common term); some time like ARP, please correct it.

Response: following your recommendations, all corrections in terms of using APR have been made.

 

6. At line 75, why are you making so many statements in a huge single line? How can the readers understand? Please split the sentence and write precisely. You could simply write, "The accurate and right combination with the desired mode of activity would enable durable resistance."

 

Response: We have rephrased the above sentence as suggested.

 

7. On what basis have you selected the advanced spikes? Have you collected from a single (individual) plant from the original lines or from the mutant lines?

 

Response: “The single advanced main spike was selected individually from resistant mutant plant based on higher means of grain weight (GWS) and number (GNS) per the main spike compare to that of parent, (WT) cv. Kazakhstanskaya-19. In year of selection, cv. Kazakhstanskaya-19 had  GWS mean of 1.25 ± 0.40 g and a mean GNS of 30.17 ± 4.41. For the M₃ generation, the threshold criteria chosen for selection were GWS > 1.45 g and GNS > 35-38 for the each mutant plant line”. These relevant explanations according to your comments are reflected in the text lines (169-173) and marked in red.

 

8. Have you developed M₃ lines from each dosage of irradiation from the starting resource?

 

Response: All different dosed mutant germplasm was developed from one starting genetic resource, spring wheat cv. Kazakhstanskay-19 using 300, 350 and 400 Gy irradiation doses that was obtained  on analysis of radiosensitivity from that LD50 was 330 Gy for cv. Kazakhstanskay-19.

 

9. Please clarify how you have tested a large number of Lr genes using mutant lines from virulent fungal isolates. I presume you have derived the results of virulency and avirulency with hyperspectral image analysis, but I didn't see any expression analysis of the given genes. So, it's tough for the researchers to understand how a lump of genes were tested without expression studies. On what basis have you represented those genes in Table 1 in terms of their resistance to fungal isolates?

 

Response: The corresponding explanations are given in the lines 141-144 of the manuscript main text.

 

10. Which experimental design have you followed, such as CRD or RBD?

 

Response: In field trials фтв green house experiments the CRD technique was used,

11. You have shown a combined form of irrigation dosage effect. Please show individually for each irradiation corresponding to mutant line, parental line, and time. On what criteria you have normalized the data of both infected and non-infected lines derived data?

 

Response: The corresponding explanations are given in lines 308-315 of the manuscript main text.

 

12. Why do you show both infected and non-infected data together? Whether the color lines of the peaks emphasize the pooled data of both infected and non-infected. Please redraw the picture and label the X and Y axes. Please differentiate the peaks of infected and non-infected with the visible spectral range in particular.

 

Response: In figures 5 and 6 for better comparative clarity, the reflection peaks of non- and infected plants were placed together. As suggested, these figures are redrawed and also labeled by the Y axes.

 

 

13. If you have sequence information for your clones, please verify through the ENSEMBL wheat candidate gene to check the genomic positions for further accuracy of the genes studied and SNP comparisons from database sequences or varieties.

 

Response:

 

14. Could you please show the gel pictures of the KASP assay? If you have designated the alleles as "aa" or "bb," they seem all homozygous. Is that so?

 

Response: As suggested, in Supplement Figure 1. Scatter plot for selected KASP assays showing clustering of genotypes on the Y- and X-axes. Genotypes colored red have a HEX-type allele; genotypes colored blue have a FAM-type allele. Top left and right corners, KASP assays for Lr1–A/G, Lr2 –T/C, respectively. Bottom left and right corner, KASP assays for Lr10 – C/T and Lr14a –C/T, respectively.

 

15. There are several typos, and please rewrite the whole manuscript precisely; alter the long sentences to split, shorten them, and be precise. Substantial revision of the manuscript is required.

 

Response: As suggested, we have thoroughly reviewed the entire manuscript multiple times, making necessary improvements, change the long sentences to split, shorten them, whenever required. Additionally, the manuscript was reviewed by the Cambridge Proofreading LLC

 

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewed manuscript, titled, “Phenotyping and molecular characterization of leaf rust resistance in new spring wheat mutant lines” is a basic molecular characterization of a mutant screen looking at genes that can result in the resistance of the important food crop, wheat, to the fungal pathogen that is commonly referred to as rust.  This work has some strengths, and could contribute relevant information for researchers within this field.  As written, however, this manuscript needs a significant amount of work prior to being suitable for publication.  It is apparent that this was submitted previously – and it seems to reflect that (miss-numbering of sections, copy and pasting that seems to detract from the coherence and the understanding of the work as written).  Here are a list of comments:

·        Line 30: “Results of HSI analysis indicated” – avoid acronyms in the abstract and/or define with the first usage.

·        Line 35: “NDVIs” – same comment as above.

 

·        General comments for the introduction: the structure is very choppy, and the writing is a bit too varied throughout.  There are sections that are incredibly detailed – e.g. lines 89-98 – and other sections that seem out of place – e.g. lines 131-136.  This whole section contains a lot of very necessary and accurate information; however, it should be revised to provide a coherent and sufficient background and introduction to the topic at hand.  What are the big picture and important parts to set the stage for the rest of this work?  Should every single gene be listed with no specification of mechanism (for example, would it be more appropriate to discuss the types of genes that lead to resistance to LR that have been characterized, along the lines of pathway discussion that protects wheat from infection by this fungal pathogen, rather than list a lot of genes with no context?).  Such a re-write would seem reasonable in this reviewers’ opinion and would make the rest of the work accessible to a broader audience.  The specifics are important, just not in the introduction section.  The last paragraph, lines 150-156, are a solid example of clear, focused, and concise writing that is focused and informative.  This style and approach should be the norm in the introduction section – in this manuscript version it is more the exception.

·        Line 190: can the authors elaborate on how the five infection types could/would have been determined or quantified?  This particular reviewer is unfamiliar with this, if it happens to be standard criteria, please provide the citation.

·        Line 286: please provide the details and method for the specific CTAB use DNA extraction.  Provide the details of the KASP master mix used.  The methods should be reproducible, as written they are not.

·        Line 330: reorder the figures in the order that you refer to them in the text.  Figure two is discussed before figure 1.  These should flow in an ascending order based on the rest of the manuscript.

·        Fig 1: set the y-axis to zero.  Even if the SD bars go below 0, it is not possible to have negative HMCs.

·        Fig 2: place the a and b labels in a comparable location (b is offset).  This is mainly for aesthetics, but the figs really should be publication worthy.

·        Line 336: P. triticina should be italicized.  Please review for formatting of Genus and Species names.

·        Line 382-383: “no development of HMCs and uredospore pustules were.” – This is a sentence fragment.

·        Line 377: “with blue balls or dots (indicated by yellow arrows) were observed.”  This figure has no yellow arrows on it.

·        Figure 4: provide images/pictures of the leaf’s for comparison – not just the hyperspectral images.

·        Figure 5, 6, 7: units need to be provided on the graph for both axes. 

·        Tables 2,3: format and simplify for clarity. 

·        Lines 428-452: This section is completely redundant with Table 2.  Table 2 needs to be simplified and formatted for clarity.  As it has the ratios that are used in the analysis, it is not necessary to list every single one of them in the text.  Rather, use the text as an opportunity to highlight the importance components of the results.

·        Discussion: the numbered sections are incorrect (they all start with 3 rather than 4, which is the discussion number).

·        Line 676: “Three resistance alleles have been described at or near the Lr3 locus.” – this is interesting, and consistent with this reviewers work’s.  Clusters of genes are seen in a variety of fungal species and are conserved throughout eukaryotes.  I am providing three representative publications for the authors to consider – however I would like to emphasize that these are not required, and will not impact the recommendations of this reviewer whether the authors choose to include these or other ones that are relevant.  This does seem relevant, hence the suggestions below.

Here are three references: 

1.      Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya; GM Cittadino, J Andrews, H Purewal, P Estanislao Acuña Avila, ... Journal of Fungi 9 (5), 523

2.      Genomic clustering within functionally related gene families in Ascomycota fungi; D Hagee, AA Hardan, J Botero, … Computational and Structural Biotechnology Journal 18, 3267-3277

3.      Functionally related genes cluster into genomic regions that coordinate transcription at a distance in Saccharomyces cerevisiae; A Cera, MK Holganza, AA Hardan, I Gamarra, RS Eldabagh, ... Msphere 4 (2), 10.1128/msphere. 00063-19

 

·        Overall, this work needs a significant rewrite and overhaul.  The overreliance of acronyms, the poor and choppy structure and writing are a bit disjointed.  I would recommend that the authors focus on clarity and being concise.  As written this work is not suitable for publication in this reviewers opinion, however upon major revisions, including those outlined above, I believe that this work can get to that point.

Comments on the Quality of English Language

The phrasing is largely appropriate - the issues are in the structure and style more than grammar, etc

Author Response

Dear Reviewer 2,

 

Response: Thank you for providing your suggestion. We have carefully reviewed the entire manuscript and removed any redundant or colloquial sentences.

 

 

Response: As per your suggestion, we have taken into account the reviewer's comments and consequently revised the manuscript. Additionally, we meticulously reviewed the manuscript multiple times to enhance the language, rectify typos, and correct syntax errors. The manuscript was edited by Cambridge Proofreading LLC.

.

Comment 1: ·Line 30: “Results of HSI analysis indicated” – avoid acronyms in the abstract and/or define with the first usage.

Line 35: “NDVIs” – same comment as above.

 

Response: As recommended, the appropriate changes have been made.

 

Comment 2: Please rewrite at line 70 as: Generally, SR genes are more effective than APR genes, and the former reportedly lost their activity after several years of utilization.

Response: As recommended, we have edited this sentence.

 

Comment 3: For Adult Plant Resistance, you have mentioned some time APR (a common term); some time like ARP, please correct it.

Response: As recommended, we have check the correct using this abbreviation.

Comment 2: At line 75, why are you making so many statements in a huge single line? How can the readers understand? Please split the sentence and write precisely.

You could simply write, "The accurate and right combination with the desired mode of activity would enable durable resistance."

Response: As recommended and following your suggestion, we have substituted this sentence.

 

Comment 3: lines 89-98 and lines 131-136. There are sections that are incredibly detailed – e.g. lines 89-98 – and other sections that seem out of place – e.g. lines 131-136. This whole section contains a lot of very necessary and accurate information; however, it should be revised to provide a coherent and sufficient background and introduction to the topic at hand.  What are the big picture and important parts to set the stage for the rest of this work? 

 

Response: As recommended we had revised these sections.

 

Comment 3:·Line 190: can the authors elaborate on how the five infection types could/would have been determined or quantified? This particular reviewer is unfamiliar with this, if it happens to be standard criteria, please provide the citation.

 

Response: In reference 50 by A.P. Roelfs et al.1992  RUST DISEASES OF WHEAT (p.50).The standard scoring systems for the rust diseases are summarized in Tables 21. Possible rating methods have been added in lines 132 to 138. Further literature regarding the rating systems have been added.

 

Host response (class)	Infection type	Disease symptoms
Immune	o	No uredinia or other macroscopic sign of infection
Very resistant	1	Small uredinia surrounded by necrosis
Moderately resistant	2	Small to medium uredinia often surrounded by chlorosis or necrosis; green island may be surrounded by chlorotic or necrotic border
Moderately susceptible	3	Medium-sized uredinia that may be associated with chlorosis
Susceptible	4	Large uredinia without chlorosis

 

Comment 3:·Line 286: please provide the details and method for the specific CTAB use DNA extraction. Provide the details of the KASP master mix used. The methods should be reproducible, as written they are not.

Response: we added the literature source for the CTAB to the material methods section. You find information at lines 208 to 223. We added furthermore a protocol as supplementary material (now supplementary material 1).

 

Comment 4:·Line 330: reorder the figures in the order that you refer to them in the text. Figure two is discussed before figure 1. These should flow in an ascending order based on the rest of the manuscript.

 

Response: The figures 1 and 2 are reordered as suggested.

 

Comment 5:· Fig 1: set the y-axis to zero. Even if the SD bars go below 0, it is not possible to have negative HMCs.

 

Response: As suggested, in the figure 1 y-axis  is setted to zero.

 

Comment 6:· ·Fig 2: place the a and b labels in a comparable location (b is offset).  This is mainly for aesthetics, but the figs really should be publication worthy.

 

Response: As recommended, in Fig. 2 the labels”a” and “b” are placed in a comparable location.

 

Comment 7: Line 336: P. triticina should be italicized. Please review for formatting of Genus and Species names.

 

Response: As suggested, P. triticina is italicized.

 

Comment 8: ·Line 382-383: “no development of HMCs and uredospore pustules were.” – This is a sentence fragment.

Response: As recommended, the sentence is improved.

 

Comment 9: Line 377: “with blue balls or dots (indicated by yellow arrows) were observed.This figure has no yellow arrows on it.

Response: In the figure 3 the yellow arrows are added.

 

Comment 10: Figure 4: provide images/pictures of the leaf’s for comparison – not just the hyperspectral images.

Response: As suggested, the figure 4 is supplemented by pictures of the leaf’s of non- and infected by LR seedlings.

 

Comment 11:Figure 5, 6, 7: units need to be provided on the graph for both axes. 

Response: As recommended, units are provided on the graph for both axes shown in Figures 5-7. 

 

Comment 12:Tables 2,3: format and simplify for clarity. 

Response: As suggested, the tables 2 and 3 are reformatted.

 

Comment 13: Lines 428-452: This section is completely redundant with Table 2. Table 2 needs to be simplified and formatted for clarity. As it has the ratios that are used in the analysis, it is not necessary to list every single one of them in the text. Rather, use the text as an opportunity to highlight the importance components of the results.

Response: As recommended, this section is changed to highlight the important results.

 

Comment 13: Line 676: “Three resistance alleles have been described at or near the Lr3 locus.” – this is interesting, and consistent with this reviewers work’s. Clusters of genes are seen in a variety of fungal species and are conserved throughout eukaryotes. I am providing three representative publications for the authors to consider – however I would like to emphasize that these are not required, and will not impact the recommendations of this reviewer whether the authors choose to include these or other ones that are relevant.  This does seem relevant, hence the suggestions below.

Here are three references: 

1. Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya; GM Cittadino, J Andrews, H Purewal, P Estanislao Acuña Avila, ... Journal of Fungi 9 (5), 523
2. Genomic clustering within functionally related gene families in Ascomycota fungi; D Hagee, AA Hardan, J Botero, … Computational and Structural Biotechnology Journal 18, 3267-3277
3. Functionally related genes cluster into genomic regions that coordinate transcription at a distance in Saccharomyces cerevisiae; A Cera, MK Holganza, AA Hardan, I Gamarra, RS Eldabagh, ... Msphere 4 (2), 10.1128/msphere. 00063-19

 

Further comments:

Table 1 has now been simplified and further information have been added to the head of table. Resistances, where no differences could be observed have been added to the head of table (lines 145 to lines 149)

 

Figure 4: provide images/pictures of the leaf’s for comparison – not just the hyperspectral images.

We added the RGB pictures which have been taken separately from the same leaf material. Furthermore, we added details to the description (lines 318 to 322).

 

Figure 5, 6, 7: units need to be provided on the graph for both axes. 

All figures have been improved in quality and corrected regarding the numbering (figures 5 and 6). All axes have now units and have been defined by e.g. wavelength to easify comparison between figures.

 

If you have sequence information for your clones, please verify through the ENSEMBL wheat candidate gene to check the genomic positions for further accuracy of the genes studied and SNP comparisons from database sequences or varieties.

Could you please show the gel pictures of the KASP assay? If you have designated the alleles as "aa" or "bb," they seem all homozygous. Is that so?

There are several typos, and please rewrite the whole manuscript precisely; alter the long sentences to split, shorten them, and be precise. Substantial revision of the manuscript is required.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I think they are flaws with testing huge amount of genes according to table 1.

I presume you have just excluded part of genes from table 1, and there is no meaning just excluding genes without appropriate reasons.

Please provide appropriate explanations and evidences for testing huge amount of genes, otherwise with this flaw the manuscript can't be considered. Without expression studies how these much genes can be validated for their action against the fungal pathogen. 

Also on the text, there was no appropriate revision, though you have mentioned: The corresponding explanations are given in the lines 141-144 of the manuscript main text.

Is there any gel picture or KASP marker/SNP ID, please clarify?

 

Please revise thoroughly and present precisely about the results obtained.

 

Author Response

Dear Reviewer 1,

 

Thank you very much for providing your comments.

 

Comment 1:  I think they are flaws with testing huge amount of genes according to table 1. Please provide appropriate explanations and evidences for testing huge amount of genes, otherwise with this flaw the manuscript can't be considered. Without expression studies how these much genes can be validated for their action against the fungal pathogen. 

Response:  Table 1  showed  Virulence/avirulence pattern of leaf rust isolates detected at the seedling stage of near-isogenic wheat lines carrying Lr genes singly. In general, virulence was observed against Lr1, Lr2a, Lr2b, Lr2c, Lr11, Lr12, Lr13, Lr14a, Lr14b, Lr15, Lr16, Lr17, Lr18, Lr20, Lr21, Lr22a, Lr22b, Lr23, Lr30, Lr32, Lr35, Lr36, Lr37, and LrB, and avirulence against Lr9, Lr19, Lr24, Lr25, Lr29, Lr45, Lr51m, Lr53, and LrTm that were studied by other reseaches with relevant references given in the manuscript [1, 53, 54, 55]. In our work  all isolates of leaf rust was used for the SR test  in spring wheat mutant lines and we do not test amount of genes given in Table 1.  

Comment 2:  Also on the text, there was no appropriate revision, though you have mentioned: The corresponding explanations are given in the lines 141-144 of the manuscript main text.

Response: The explanation is given in Response 1.  

Comment 3:  Is there any gel picture or KASP marker/SNP ID, please clarify?

Response: KASP markers are based on the allele-specific competition between two fluorescent labels, FAM and HEX. The detection of the fluorescence for KASP-markers is carried out using qPCR instrument or special detector. However, no gel picture is available because KASP markers do NOT based on the separation of products on the gel. SNP ID is 859166.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did a commendable job revising and addressing the reviewers comments.  I feel that they addressed the overwhelming majority of them.  The only one that was not addressed was:

"Comment 13: Line 676: “Three resistance alleles have been described at or near the Lr3 locus.” – this is interesting, and consistent with this reviewers work’s. Clusters of genes are seen in a variety of fungal species and are conserved throughout eukaryotes. I am providing three representative publications for the authors to consider – however I would like to emphasize that these are not required, and will not impact the recommendations of this reviewer whether the authors choose to include these or other ones that are relevant. This does seem relevant, hence the suggestions below.

 

Here are three references:

 

Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya; GM Cittadino, J Andrews, H Purewal, P Estanislao Acuña Avila, ... Journal of Fungi 9 (5), 523

Genomic clustering within functionally related gene families in Ascomycota fungi; D Hagee, AA Hardan, J Botero, … Computational and Structural Biotechnology Journal 18, 3267-3277

Functionally related genes cluster into genomic regions that coordinate transcription at a distance in Saccharomyces cerevisiae; A Cera, MK Holganza, AA Hardan, I Gamarra, RS Eldabagh, ... Msphere 4 (2), 10.1128/msphere. 00063-19"

As there was no response from the authors to this one, i would like to at least have known that they read and considered this suggestion, as it would be quite beneficial to readers from a broader audience.

Aside from that i feel the authors did an outstanding job and the work has been improved immensely.

 

Author Response

Dear Reviewer 2,

 

Thank you for providing your comment.

 

 

 

Comment 1: Line 676: “Three resistance alleles have been described at or near the Lr3 locus.” – this is interesting, and consistent with this reviewers work’s. Clusters of genes are seen in a variety of fungal species and are conserved throughout eukaryotes. I am providing three representative publications for the authors to consider – however I would like to emphasize that these are not required, and will not impact the recommendations of this reviewer whether the authors choose to include these or other ones that are relevant.  This does seem relevant, hence the suggestions below.

Here are three references: 

1. Functional Clustering of Metabolically Related Genes Is Conserved across Dikarya; GM Cittadino, J Andrews, H Purewal, P Estanislao Acuña Avila, ... Journal of Fungi 9 (5), 523
2. Genomic clustering within functionally related gene families in Ascomycota fungi; D Hagee, AA Hardan, J Botero, … Computational and Structural Biotechnology Journal 18, 3267-3277
3. Functionally related genes cluster into genomic regions that coordinate transcription at a distance in Saccharomyces cerevisiae; A Cera, MK Holganza, AA Hardan, I Gamarra, RS Eldabagh, ... Msphere 4 (2), 10.1128/msphere. 00063-19

Response: Lines 521-523. In our study, three resistance alleles were found near the Lr3 locus. Such clusters of genes are seen in a variety of fungal species, and they are conserved throughout eukaryotes as described in papers published earlier [88, 89.]

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate your response.

So you could have at least verified the few SNPs using PCR-RFLP or ARMS PCR to show the gel picture.

You could write that NILs showed seedling resistance against the virulent fungal strain due to the presence and association of the following genes: Lr9, Lr19, Lr24, Lr25, Lr29, Lr45, Lr51m, Lr53, and LrTm. The other genes Lr1, Lr2a, Lr2b, Lr2c, Lr11, Lr12, Lr13, Lr14a, Lr14b, Lr15, Lr16, Lr17, Lr18, Lr20, Lr21, Lr22a, Lr22b, Lr23, Lr30, Lr32, Lr35, Lr36, Lr37, and LrB were not associated, hence not effective or inactive against the same virulent fungal strain, while comparing race specific manner.

Authors could have also done sequencing of few resistant genes such as Lr2a and Lr3a in mutant lines and original lines to compare the irradiation induced mutations of these genes compare to the wild type of genes.

If you wish, you can still improve or slightly revise the manuscript as suggested unless you are completely satisfied with the content.