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Congratulations, this MS is accepted now.
The author accepted and remodeled all major aspects, except for the we/I aspect. So I do accept changes made by the author.
This MS is a highly detailed SNP analysis which provides important inside in the evolution of Verticillium dahliae. Please address the comments of the reviewer #1, before we formally accept your MS.
This article adheres to PeerJ standards.
It is written in clear English.
The title sounds a little overstated since the paper describes only ONE pathogen while the title may suggest a broader conclusion. I would strongly advise a more precise title.
The article should for sure include some kind of a legend/explanation since the author uses so many definitions: forms, groups, reference strain/assembly etc.. Sometimes it is hard to get through; what is what. :)
The structure is OK.
In my opinion a better figure should present LSs, forms etc., putting it in a broader context. For example, what are the specific combinations of forms in each strain? How do they look? Are they similar? Does the author find repetitive elements and are those transposons (which is not stated in the paper)? I suggest some kind of a map showing similarities, differences and transfers of fragments.
This work clearly defines the problem.
The method is a very clever idea.
The work seems to be conducted rigorously and nicely, but the method and definitions are described partly in Methods section and partly at the beginning of the Results section. Maybe some basic facts may be moved or at least repeated into Methods?
The data is sound and seems to be controlled throughout the process.
As for the Discussion section, I have a few questions/comments that may help the author to improve the article:
- The author states in lines 475-6: "to become adaptable to hundreds of plant species." However he does not explain why so. Why asexual reproduction favours such a broad pathogenecity?
- lines 485-7: The author suggests an influence of transposons, but there are no data that shows the presence of transposons in LS regions;
- at the end of Discussion section the author says that nonhomologous recombination is responsible for LS exchange. On the other hand his discoveries show that these regions are homologous. So why isn't it a homologous recombination while we suspect the involvement of HGT...?
- I see no broader view on the subject. Why the author, with the new tools he built, did not try his method on other pathogens to have a broader perspective? This would strongly enhance his findings.
I read your article with interest and I find it to touch an important point. This type of work needs a broader analysis now to see ahether it is specific or general for eukaryotic pathogens. Congratulations.
I would however suggest a few minor changes that I did not include in the previous sections of the review since there was no space for it:
- Introduction, line 40: you suggest HGT as the only source of variation. Why? Single mutations may also play a role;
- line 97: were mapped not was mapped;
- line 108: "sufficient number" - what do you mean?
- line 151: "were generated": is this an appropriate term in here? Later you suggest an evolutionary term. ;)
- line 159: you find that 30% of SNPs are outside LS regions. I think this finding may lead to a very interesting analysis: either these 30% are dispersed or concentrated in specific areas of the genome. If the latter is true, this may be a crucial finding of novel LS regions or any other specific regions;
- lines 187-8: "Taken together, we conclude that most type 2 SNPs in LS regions are a result of highly similar sequences in LS regions, not a result of sequencing errors in reads". How SNPs may be a result of highly similar sequences? This sentence should be more precise;
- lines 357-8: "These observations show that the variation among strains in sequence type composition contributes to genetic variation in the strains". It sounds like a tautology...
- lines 383-4: a hypothetical protein is suggested but no name/id is given;
- lines 385-90: It would be interesting to learn whether SNPs are more likely to locate in coding or non-coding regions. Is such a c/nc rate important?
- lines 434-437: "For example, the MAT1-2 locus is at positions 348 to 358 kb of supercontig 1.4 of V. dahliae strain VdLs17; the MAT1-1 locus is at positions 1737 to 1747 kb of supercontig1.2 of V. albo-atrum strain VaMs102". Is this info useful in here?
- line 527: "supports" what?
- throughout the article you use the form "we" while there is only one author. I would suggest changing it into "I";
- More generally, I find the notion of reference strain important, however a constant reference to it may dim a bit a broader view on the subject where all strains in fact are equal.
The manuscript is written in clear and good English language. There is sufficient background and introduction, to understand the basics of the manuscript. There is only one figure in the manuscript. All other data are related to the analysis of SNPs.
The manuscript is solely based on SNP analysis. However, as far as I do understand the method it seems to be sound.
I do not understand the SNP methods well enough to be fully sure whether all data are valid. However, the author addresses related problems in the manuscript and it appears that these issues are addressed.
This is a highly detailed SNP analysis which provides important inside in the evolution of Verticillium dahliae. The data indicate that genetic variation in
LS regions appears to be generated by horizontal transfer between strains, and also via chromosomal reshuffling. I particularly like the discussion.
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