PCR-Based InDel Marker Associated with Powdery Mildew-Resistant MR-1
Round 1
Reviewer 1 Report
COMMENTS FOR THE AUTHOR:
General Evaluation
In this study, a PCR based InDel marker that can discriminate MR-1 resistance more efficiently by using information from previously designed BSA12-LI3ECOR1 and BSA12-LI4HIFI CAPS markers, associated with BPm12.1 resistance gene, were developed. It was found that there was an MR-1 specific sequence deletion in the BSA12-L13ECOR1 CAPS marker region, 0.2 cM away from BPm12.1, a major QTL related to PM resistance of MR-1 melon.
The authors used in the study two susceptible and 13 resistant melon lines, and a cross between PM-resistance in MR-1, PM-susceptible Top Mark lines, and F1 progeny.
The study is a contribution to control the powdery mildew through MAS to breed melon cultivars with durable resistance.
The manuscript was well written.
(Note: please see the detail reviewer comments below)
- To date, a total of 12 resistant loci have been reported for P. xanthii race. However, the resistance gene of MR-1 with it’s nonspecific tolerance to powdery mildew has never been previously reported. As authors declare as a result of the study, the resistance provided by the BPm 12.1PM is incompletely dominant. May it mean that the BPm 12.1PM is less valuable for breeding than other reported resistant genes? Are there any other known major dominant resistance genes for melon powdery mildew?
Specific Comments and Suggestions:
- L59 absence of the comma
- L69 wrong font size
- L97 The title 3.1 "Validation of CAPS markers for BSA12-L13ECOR1 and BSA12-L14HINF1" is confusing. At the same time, you mention at L99 “…the CAPS markers BSA12-LI3ECORI and BA12-LI4HINF1…”. It sounds like you design the CAPS markers for the CAPS markers. Please specify when it is the name of the locus or marker.
- L99 misprint of the marker name “BA12-L14HINF1”
- L99 According to Fig.1A. the distance between the BSA12-L14HINF1 and the 1 is 0.28cM, instead of 0.2 cM. It would be easier if you’d use “proximal” and “distal” labelling characterizing markers BSA12-L13ECOR1 and BSA12-L14HINF1.
- L121 Fig.1E and D. Please mark the size of most important bands on the figure.
- L146 Fig.2A. very not easy to read. Please provide better quality picture.
- L168 “the BPm12.1 PM resistance gene” instead of “resistant gene”
- L175 “The degree of infection with Powdery Mildew is indicated as Classes 15” - probably Class 1 – 5?
Author Response
Reviewer 1
(Answer) Thank you for your detailed and kind review. All requested details have been completed as follows. The revised content is marked in red in the revised manuscript.
General Evaluation
In this study, a PCR based InDel marker that can discriminate MR-1 resistance more efficiently by using information from previously designed BSA12-LI3ECOR1 and BSA12-LI4HIFI CAPS markers, associated with BPm12.1 resistance gene, were developed. It was found that there was an MR-1 specific sequence deletion in the BSA12-L13ECOR1 CAPS marker region, 0.2 cM away from BPm12.1, a major QTL related to PM resistance of MR-1 melon.
The authors used in the study two susceptible and 13 resistant melon lines, and a cross between PM-resistance in MR-1, PM-susceptible Top Mark lines, and F1 progeny.
The study is a contribution to control the powdery mildew through MAS to breed melon cultivars with durable resistance.
The manuscript was well written.
(Note: please see the detail reviewer comments below)
To date, a total of 12 resistant loci have been reported for P. xanthii race. However, the resistance gene of MR-1 with it’s nonspecific tolerance to powdery mildew has never been previously reported. As authors declare as a result of the study, the resistance provided by the BPm 12.1PM is incompletely dominant. May it mean that the BPm 12.1PM is less valuable for breeding than other reported resistant genes? Are there any other known major dominant resistance genes for melon powdery mildew?
(Answer) To date, 12 resistance loci have been reported for melon powdery mildew resistance. The exact resistance genes for these loci have not yet been found. Most of them show resistance to PM race-specific. The dominant genes are reported as Edistor47 and MR-1. However, it showed moderate resistance in F1, where we crossed the MR-1 and Top Mark. The cause of this is not clear, but the possibility that the dominance in F1 was weakened due to the simultaneous occurrence of several PM races in spontaneous occurrence cannot be excluded.
Specific Comments and Suggestions:
L59 absence of the comma
(Answer) Comma was added.
L69 wrong font size
(Answer) Font size was fixed.
L97 The title 3.1 "Validation of CAPS markers for BSA12-L13ECOR1 and BSA12-L14HINF1" is confusing. At the same time, you mention at L99 “…the CAPS markers BSA12-LI3ECORI and BA12-LI4HINF1…”. It sounds like you design the CAPS markers for the CAPS markers. Please specify when it is the name of the locus or marker.
(Answer) 3.1 subtitle is changed to “PCR polymorphism analysis of CAPS markers BSA12-L13ECOR1 and BSA12-L14HINF1 in various melon resources”
L99 misprint of the marker name “BA12-L14HINF1”
(Answer) Marker name is corrected to “BAS12-L14HINF1”.
L99 According to Fig.1A. the distance between the BSA12-L14HINF1 and the 1 is 0.28cM, instead of 0.2 cM. It would be easier if you’d use “proximal” an
d “distal” labelling characterizing markers BSA12-L13ECOR1 and BSA12-L14HINF1.
(Answer) 0.2 cM was fixed to “0.28 cM at L99. And “proximal” and “distal” were added to next of each CAPS markets (see Fig.1A).
L121 Fig.1E and D. Please mark the size of most important bands on the figure.
(Answer) The main bands were marked their size at right line in Figure. 1
L146 Fig.2A. very not easy to read. Please provide better quality picture.
(Answer) We replaced new better resolution Figure 2A picture. This new alignment picture has five row and bigger size font than previous one. And added all of each DNA sequence information in Supplementary file.
L168 “the BPm12.1 PM resistance gene” instead of “resistant gene”
(Answer) It is corrected to “the BPm12.1 PM resistance gene”.
L175 “The degree of infection with Powdery Mildew is indicated as Classes 15” - probably Class 1 – 5?
(Answer) It is corrected to “Class 1 – 5”.
Reviewer 2 Report
The manuscript is prepared on an interesting topic, but it is necessary
to add some essential information and improve the quality of some
figures.
Abstract - OK.
Introduction - the highest quality part of the manuscript.
Material and Methods - there is a lack of essential information about
the composition of PCR reactions, the use of restriction enzymes. It is clear from the methodology whether the primers were designed by the authors (no procedure is given) or were taken from the literature (references are missing). This information needs to be supplemented. Is the DNA isolation procedure new or has the procedure already described been used? If so, it is
appropriate to reference. In the case of spontaneous inoculation with
a pathogen, it is appropriate to describe the cultuvation conditions.
It is not clear to what extent the conditions are suitable
for the pathogenic organism.
Results and Discussion - only the first subchapter contains
a discussion. The other two parts do not. These two parts need
to be adjusted. Part of the comments on the facts of Figure 2A cannot be verified due to the very low image quality. Without improving the quality of this image, the text (manuscript content) loses its meaning.
Figures - Figure 1E - samples R4 and R7 - ?*?
Based on the above, I recommend the manuscript for major revision.
Author Response
Reviewer2
(Answer) Thank you for your detailed and kind review. All requested details have been completed as follows. The revised content is marked in red in the revised manuscript.
The manuscript is prepared on an interesting topic, but it is necessary
to add some essential information and improve the quality of some
figures.
Abstract - OK.
Introduction - the highest quality part of the manuscript.
Material and Methods - there is a lack of essential information about
the composition of PCR reactions, the use of restriction enzymes. It is clear from the methodology whether the primers were designed by the authors (no procedure is given) or were taken from the literature (references are missing). This information needs to be supplemented. Is the DNA isolation procedure new or has the procedure already described been used? If so, it is
appropriate to reference.
(Answer) Material and methods were edited more detail. Composition of PCR reaction for genomic DNA was added. The used of restriction enzymes was added in 2.3. section.
Reference for genomic DNA isolation.
“2.4. Design of InDel marker and PCR analysis” section was added to describe more detail in method.
In the case of spontaneous inoculation with
a pathogen, it is appropriate to describe the cultuvation conditions.
It is not clear to what extent the conditions are suitable
for the pathogenic organism.
(Answer) We added growth condition of temperature and RH.
-Results and Discussion - only the first subchapter contains
a discussion. The other two parts do not. These two parts need
to be adjusted.
(Answer) In part “3.2 Nucleotide sequence analysis of PCR product of BSA12-L13ECOR1 CAPS marker”, I added discussion about phylogenetic analysis for DNA sequences among melon resources, like “The phylogenetic tree constructed by the nucleotide sequences of the BSA12_LI3ECORI CAPS marker region divided 15 melon resources into three clades (Figure S1). Clade I included PM-sensitive lines, S1 (IranH) and (S2) Top Mark, and PM-resistant lines, R10 (PMR45_NSL113039), R11 (PMR45_Ames26811), (R13) D-2 Resistant, and (R12) LJ90234. Top Mark, a PM-sensitive line, and PMR45, a resistant line, showed very high genetic similarities. Clade II included the PM-resistant lines, R2 (PMR5), R3 (PMR6), R6 (VIR5682), and R8 (TGR1151). Clade III included R4 (MR-1) and R7 (2563), the parent of MR-1. Phylogenetic analysis suggests that the BPm12.1 locus, the PM-resistant QTL of MR1, diverged from the genes of other PM-resistant melon resources at the beginning of evolution”
In part “3.3 Redesign of BSA12-L13ECOR1-based InDel PCR marker and its validation” , I added discussion about candidate genes of BPm12.1, like " The InDel PCR marker developed in this study is 0.2 cM to the left from the BPm12.1 position, a PM-resistant QTL locus. There are 78 kb between the BSA12-LI3ECOR1 and BSA12-LI4HINFI CAPS marker regions, and 12 genes are present in Cucumis melo L. cv. DHL92, the melon reference genome [29] (Figure S2A). In the future, it remains to find the BPm12.1 locus among 12 candidate genes in the MR-1 melon.”
Part of the comments on the facts of Figure 2A cannot be verified due to the very low image quality. Without improving the quality of this image, the text (manuscript content) loses its meaning.
(Answer) We replaced new better resolution Figure 2A picture. This new alignment picture has five row and bigger size font than previous one. And added all of each DNA sequence information in Supplementary file.
Figures - Figure 1E - samples R4 and R7 - ?*?
(Answer) The meaning of "*" of the band of R4 and R7 is explained in the figure legend.
Based on the above, I recommend the manuscript for major revision
Round 2
Reviewer 2 Report
The authors accepted most of my comments. Nevertheless, I consider it
appropriate to improve the "Results and Discussion" section,
because the discussion is a comparison of achieved results with the results of other authors, which is not 100% fulfilled.
Especially for subchapter 3.2, for other subchapters, the authors
suffice with one citation per subchapter.
Author Response
Thanks again for your review and comments. s. The revised content is marked in red in the 2nd revised manuscript.
The authors accepted most of my comments. Nevertheless, I consider it appropriate to improve the "Results and Discussion" section, because the discussion is a comparison of achieved results with the results of other authors, which is not 100% fulfilled. Especially for subchapter 3.2, for other subchapters, the authors suffice with one citation per subchapter.
(Answer) Three citations were added to Subcapter 3.2, and the relationship between the melon resources and PM race resistance and the relationship between the nucleotide sequence tree and PM race was newly discussed.
In subchapter 3.3, a discussion about the cause of moderate resistance in F1 of MR-1 pointed out by Reviewer 1 has been newly added with citations and Table S1 has been newly added.
