Molecular Markers for Identifying Resistance Genes in Brassica napus
, Yueqi Zhang 2, Nur Shuhadah Mohd Saad 2, Hua Yang 2, Elizabeth Sheedy 3, Candace E. Elliott 1 and Jacqueline Batley 2Round 1
Reviewer 1 Report
This manuscript is generally well written in useful to breeders and scientists working in the field. There was no access to Supplementary Figure 2 and beyond for the reviewer and these could therefore not be assessed. However, access to Supplementary Figure 1 and the tables were given.
Regarding Supplementary Table 2 information about the nucleotide positions in the coding sequences could be given to make it easier for the reader to figure out what portions of the genes or transcripts were amplified.
Were the sequences submitted to GenBank? If so, accession numbers should be given to make the information more accessible.
More specific comments follow:
Page 1, line 37: Do the author refer here to qualitative resistance? Qualitative resistance is known to be isolate-specific, quantitative resistance is not.
Page 3, line 99: MiSeq needs to be explained in detail when it is first mentioned. This is a procedure that may not be known to all readers.
Page 4, line 155: “… epistatic effect of AvrLm7”, not “or”.
Page 8, Figure 3: Only Figure 3c shows the expected spread of markers. Fluorescent dyes are not mentioned on page 4, lines 166 – 184. Expected results are: NTC, no florescence; allele 1 (r9/r9), e.g. FAM fluorescence; allele 2 (R9/R9), e.g. HEX fluorescence; heterozygote (R9/r9), both FAM and HEX. Why is it that in Fig. 3a and b the dominant R alleles behave similarly to the heterozygotes?
Page 8, line 253: “DNA samples …”, not DAN samples.
Page 9, lines 286 – 295: The marker used for detection of Rlm4 or Rlm7 acts like a dominant marker in contrast to the other markers described here, which are co-dominant. Would it be worth mentioning this?
Page 9, line 296: “… KASP marker”, not markers.
Page 9, line 317: Italicise “R” as reference is made to genes.
Page 9, lines 316 - 320: These sentences are difficult to understand. Consider revising.
Page 10, lines 334 – 336: What is the evidence? Can the authors refer to any Supplementary material the reviewer does not have access to?
Page 10, line 347: Is reference being made to a “false-positive phenotypic result” and if so, what does this mean – Rlm9 resistance?
Page 10, line 347 – 349: It would be straightforward to test this. Why has this not been done?
Author Response
Dear Editor
Please find below the responses to the reviewers’ comments on the manuscript. We thank both the reviewers for their input on the manuscript.
Thanks
Angela
Reviewer 1 comments:
Reviewer 1: This manuscript is generally well written in useful to breeders and scientists working in the field. There was no access to Supplementary Figure 2 and beyond for the reviewer and these could therefore not be assessed. However, access to Supplementary Figure 1 and the tables were given.
Author response: We are unsure why all the supplementary files were not shared with the reviewers as they were all uploaded with the original submission. We apologise for this error.
Reviewer 1: Regarding Supplementary Table 2 information about the nucleotide positions in the coding sequences could be given to make it easier for the reader to figure out what portions of the genes or transcripts were amplified.
Author response: The primers listed in Supplementary Table 2 were used to amplify the entire open ready frame of the genes, and therefore the positions of some of the primers were actually outside the coding regions therefore we cannot provide the information suggested by the reviewer. However, in Supplementary figure S2 and S3 we have provided the entire coding region.
Reviewer 1: Were the sequences submitted to GenBank? If so, accession numbers should be given to make the information more accessible.
Author response: GenBank accession numbers have now been included in Supplementary Table S1 for those lines that have references. All other sequences were provided in the sequence alignments in the supplementary figures.
Reviewer 1: Page 1, line 37: Do the author refer here to qualitative resistance? Qualitative resistance is known to be isolate-specific, quantitative resistance is not.
Author response: No, we are referring to quantitative resistance as suggested in the text. Recent studies, which are cited, show that for the L. maculans-B. napus interaction, there are isolate-specific reactions associated with quantitative resistance.
Reviewer 1: Page 3, line 99: MiSeq needs to be explained in detail when it is first mentioned. This is a procedure that may not be known to all readers.
Author response: A definition regarding MiSeq has been included in the materials and methods as well as a reference for further reading.
Reviewer 1: Page 4, line 155: “… epistatic effect of AvrLm7”, not “or”.
Author response: This has been amended.
Reviewer 1: Page 8, Figure 3: Only Figure 3c shows the expected spread of markers. Fluorescent dyes are not mentioned on page 4, lines 166 – 184. Expected results are: NTC, no florescence; allele 1 (r9/r9), e.g. FAM fluorescence; allele 2 (R9/R9), e.g. HEX fluorescence; heterozygote (R9/r9), both FAM and HEX. Why is it that in Fig. 3a and b the dominant R alleles behave similarly to the heterozygotes?
Author response: We have included information on the fluorescent dyes for each marker in Table 2. We have also update Figure 3 to specify each of the dyes for each gene.
Reviewer 1: Page 8, line 253: “DNA samples …”, not DAN samples.
Author response: This has been amended
Reviewer 1: Page 9, lines 286 – 295: The marker used for detection of Rlm4 or Rlm7 acts like a dominant marker in contrast to the other markers described here, which are co-dominant. Would it be worth mentioning this?
Author response: The reason they appear dominant is because the marker is positioned within the large insertion that is only present in the Rlm4 and Rlm7 alleles. Therefore, in the susceptible alleles there is no amplification and therefore heterozygotes appear the same as Rlm4 or Rlm7 homozygotes. This has now been mentioned in the text.
Reviewer 1: Page 9, line 296: “… KASP marker”, not markers.
Author response: This has been amended.
Reviewer 1: Page 9, line 317: Italicise “R” as reference is made to genes.
Author response: This has been amended.
Reviewer 1: Page 9, lines 316 - 320: These sentences are difficult to understand. Consider revising.
Author response: These sentences have been changed as follows:
“For the B. napus–L. maculans system, there are a number of examples whereby cultivars were commercially released that were considered to have single novel R genes but upon further characterization were identified as containing up to three resistance genes, one being a novel resistance gene and the others being already deployed resistance genes [21,31,32]. These previously deployed resistance genes were masked due to the presence of the novel sources of resistance; the use of molecular markers would prevent these types of scenarios occurring.”
Reviewer 1: Page 10, lines 334 – 336: What is the evidence? Can the authors refer to any Supplementary material the reviewer does not have access to?
Author response: These alleles were identified and characterised in the recent cloning of Rlm4 and Rlm7. This paper has now been cited here for clarity.
Reviewer 1: Page 10, line 347: Is reference being made to a “false-positive phenotypic result” and if so, what does this mean – Rlm9 resistance?
Author response: Yes, the marker will lead to a false-positive phenotypic result. We have added extra information for clarity.
Reviewer 1: Page 10, line 347 – 349: It would be straightforward to test this. Why has this not been done?
Author response: This has not been done because of MTA regulations associated with commercial lines. We are currently in the process of getting MTA approval from the breeding company to try and establish these experiments.
Reviewer 2 Report
The present study reports the development of reliable markers to discriminate resistant and susceptible alleles to characterize Brassica napus accessions. The work is clear and concise and each section is presented in a quite comprehensible way. In the Introduction section, the main difficulties to infer the resistance genes present in a canola accession when phenotyping with sets of well characterized differential isolates, are shown. The Material and Methods section is justified and well founded. The Results are presented in a clear and understandable way. The developed KASP markers are validated across commercial and advanced breeding lines previously phenotyped. The number of lines in which the markers are validated are high enough as to ensure their validity.
Although the scientific novelty this study is not very high, it stresses the importance of filling the gap between theoretical and applied science. In this sense, the molecular markers developed and validated can aid in the breeding of cultivars by reducing laborious genotyping and assigning unequivocal genotyping to breeding lines and germplasm accessions. Moreover, these molecular markers can help to unmask the real number of resistance alleles present in a cultivar. In the case of the Rlm9 allele in three lines from a single breeding company, some future prospects could be offered in the manuscript regarding this unexpected result. An expression study of this gene could be one proposal. A final overview of the blackleg resistance genes present in the Australian canola cultivars is also of utmost interest.
Author Response
Reviewer 2 comments:
Reviewer 2: The present study reports the development of reliable markers to discriminate resistant and susceptible alleles to characterize Brassica napus accessions. The work is clear and concise and each section is presented in a quite comprehensible way. In the Introduction section, the main difficulties to infer the resistance genes present in a canola accession when phenotyping with sets of well characterized differential isolates, are shown. The Material and Methods section is justified and well founded. The Results are presented in a clear and understandable way. The developed KASP markers are validated across commercial and advanced breeding lines previously phenotyped. The number of lines in which the markers are validated are high enough as to ensure their validity.
Although the scientific novelty this study is not very high, it stresses the importance of filling the gap between theoretical and applied science. In this sense, the molecular markers developed and validated can aid in the breeding of cultivars by reducing laborious genotyping and assigning unequivocal genotyping to breeding lines and germplasm accessions. Moreover, these molecular markers can help to unmask the real number of resistance alleles present in a cultivar. In the case of the Rlm9 allele in three lines from a single breeding company, some future prospects could be offered in the manuscript regarding this unexpected result. An expression study of this gene could be one proposal. A final overview of the blackleg resistance genes present in the Australian canola cultivars is also of utmost interest.
Author response: We have included the following suggested experiments for looking at the Rlm9 discrepancies in further detail:
“Inoculation experiments using avirulent and virulent isolates could be conducted whereby the expression of Rlm9 was determined in these lines compared to lines correctly expressing the Rlm9 and rlm9 phenotypes. Alternatively, mapping populations could be generated between these unusual Rlm9 lines and look at segregation to determine whether a second gene was masking the Rlm9 phenotype.”
