-->PONE-D-26-08710-->-->Uncovering the genetic basis of crown rust resistance in a northern-by-southern oat biparental population-->-->PLOS One

Dear Dr. Klos

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.-->--> -->-->The main issues raised by the reviewers related to sparse crown rust infestation at one of the field sites, the relatively small biparental population size, and the relatively modest number of genetic markers employed in the study. Otherwise, the methodology used to conduct and analyze the study were lauded. Consider these comments carefully as you prepare a revised version for resubmission.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This manuscript provides an analysis on the genetic basis of crown rust resistance in a bi-parental oat population, a useful endeavor to help breeders with the on-going battle against this disease. The manuscript is generally well-written and clear, with the data collection and analysis related to the bi-parental population done well. However, I don’t feel that the “Genotype-phenotype association analysis” part of the manuscript (using the southern and northern panels) makes a compelling case for the markers you identified or is supported by good data. I appreciate the attempt to validate the usefulness of the QTL identified, but I would not put too much trust in marker-phenotype association when disease severity observed in the Winnipeg 2011 nursery was <3%. This is a meaningless level of infection that would be highly subject to human error during rating and chance (in terms of infection establishment across the nursery). Aspects of the manuscript related to this work should be removed.

Other edits and comments are provided below.

Ln 50: suggest “Increased awareness among consumers…”

(I think the awareness is more broadly among consumers, not oat consumers)

Ln 60: if you are going to say that fungicide resistance is likely to develop, I think you need a reference showing this has occurred either in Puccinia coronata, or in other rusts of oat, barley or wheat.

Ln 75: suggest “Under field evaluations at the adult plant stage, …”

Ln 82-83: given that your manuscript relates to APR, I think it is necessary to indicate the location of those APR QTL that have been mapped, such as the APR identified in MN841801.

Ln 85: suggest “… , especially to crown rust.” Also, I don’t think you can start a sentence with an acronym, need to spell out.

Ln 103: I would disagree with the statement that CDC Dancer is susceptible, it is commonly rated Intermediate reaction to crown rust in Canada. Your QTL mapping would also indicate it carries some resistance.

Ln 113: please fix grammar in this sentence, specifically the “as head rows”

Ln 150: was there no criteria related to allele frequency? Would be good to note.

Ln 162-164: Did you attempt to use a premutation test to determine significant LOD score?

Ln 180-182: Were these nurseries naturally infected and were any spreader rows included?

Ln 198: “ There was not much…”

Ln 198-202: you indicate that the population did not show much variation in disease response in BR21 but did in all the 2022 environments. In fact, there appears to be more variation in the BR21 environment than in the other three 2022 environments. Please alter this statement.

Ln 205: appears to be a typo at the end of this line

Ln 207: suggest “…in Figure 1.”

Ln 209: Table S1 is labelled S2 in the supplementary file.

Ln 224: Table S2 is labelled S3 in the supplementary file.

Ln 232: chromosome 1D also had 83 markers, you might want to mention. Also, Table S3 is labelled S4 in the supplementary file.

Ln 229 (Genotyping data and linkage maps Section): I don’t think you need decimal places associated with the cM distances. You don’t have the marker density nor are you narrowing down a region (as per fine mapping) to justify that level of detail.

Ln 252: this sentence should be more specific with respect to which LOD was associated with which trait. You should also mention the PVE for Combined-SEV.

Ln 258: I’m not sure why you consider the 2 QTL on 7D to be different QTL? Their 95% CIs are not mutually exclusive from one another so by your definition should be considered the same QTL. Please correct if you agree or explain why this would not be the case.

Ln 262 (Validation of QTL using genotype-phenotype association Section): some explanation is required regarding why the 4A and 7C QTL were relevant in the Northern Panel when both alleles are derived from the southern parent (LA07065) which would not have been tested in Winnipeg.

Ln 273 (QTL pyramiding effect): for the pyramiding of QTL related to combined severity, when you present the range in combined severity data for the 1 and 2 QTL situations, does this represent each of the 3 single QTL and all combinations of 2 QTL, respectively? Same question for the pyramiding of the IR QTL.

Ln 280: please add units to y-axis.

Ln 302: I disagree with this statement, the 7C QTL was not detected consistently across environments, it was detected in one single environment and the combined environment. The combined environment is not equivalent to every single environment, and detection of the QTL in the combined environment may be largely driven by the BR-22 environment. Please change the wording of this sentence.

Ln 310-111: “Ociepa and Okon [42] reported linkage maps in oat that covered a total genetic distance of

24,209.96 cM…” What does this mean? This number does not make sense with respect to the shortest and longest maps that you then report. If you are reporting the total length of all the maps in the reference you cite, then I don’t see how this is a relevant piece of information. Please remove.

Ln 345-347: Although such genes are Pc48 are technically defeated, it is not possible that they are still influential in providing a small amount of resistance. Given that your QTL explain a limited amount of variation this would be in-line with such small influence of defeated genes. If I compare Canadian varieties (which undoubtedly contain defeated genes like Pc38, Pc68, Pc91 etc) rated as MS, they have a notably better level of resistance compared to many European varieties (a region where crown rust is not activity bred for) which carry no defeated Pc genes and would be rated VS (very susceptible).

Ln 387-392: couple comments here, 1) this is the first time you are mentioning this data so it should be in the results section, 2) you mention rare alleles for both markers but do not indicate if these are the same alleles associated with resistance in the bi-parental mapping population, 3) how rare are they? If below 5% would that not make the results suspect?, 3) I don’t think I’d put too much trust in marker-phenotype association when disease severity was <3%. This is a meaningless level of infection, essentially none, that would be highly subject to human error during rating and chance (in terms of infection establishment across the nursery). No data was presented for the other markers in Table S5, so hard to make a judgement on the reliability of the marker-phenotype association in those cases.

I generally don’t feel that this part of the manuscript (i.e. the “Genotype-phenotype association analysis” using the southern and northern panels) makes a compelling case for the markers you identified or is supported by good data. I suggest removing this portion of the manuscript entirely. However, the information presented between lns 413-422 is useful and should be kept.

Ln 422-426: Again, this is the first time you have mentioned this data so it should be in the results section. Also, is there a statistical analysis to back up your statement regarding the 7D2 QTL marker? If not, please provide one or alter the statement.

Ln 428: this sentence is misleading as you are changing the frame of reference. The preceding studies you quote with respect to the economic impact of crown rust are using disease severity values as the unit of measure, but you report a 26% increase in resistance. Your 26% is based on a relative change in disease severity from 55% to 40%, the actual unit that matters is the 15% change in disease severity. Please alter this sentence to reflect this.

Ln 625 (Fig. 1): suggest that you include the units (i.e. %) on the y-axis label for Severity. I don’t think you need IR after Infection Response on the y-axis of the other boxplot figure. It would be helpful to show where the parental values fall out on each of the boxplots, perhaps with a single letter representing each parent placed on the boxplots?

Ln 649 (Fig. 3): if the length of the bars next to the QTL mean anything then you should mention this in the caption. Also need to insert a space after (RIL) on the second line.

Reviewer #2: It identifies novel QTL and validates them in diverse oat populations.

The statistical methods (ANOVA, heritability, ICIM-ADD mapping) and genotyping approaches are appropriate and well-documented.

It provides actionable information (SNP markers) for oat breeders to improve disease resistance

The authors confirm that all underlying data are available within the manuscript and supporting files

The study employs a solid experimental design, including evaluations across four distinct field environments over two years. This multi-environment approach strengthens the reliability of the identified QTL

A significant strength of this work is the validation of detected QTL using independent oat sets (southern and northern CORE panels). This increases the likelihood that the identified markers will be effective across different germplasm.

The detection of a major, consistent QTL on chromosome 7C (QPca-ars-7C) that explains up to 16.54% of phenotypic variation provides a clear target for marker-assisted selection (MAS) in oat breeding programs.

The research addresses a high-priority issue in oat production—crown rust—and identifies specific markers that can be used to develop cultivars with more durable, non-race-specific resistance.

The manuscript follows standard genotyping and mapping protocols, utilizing the 6K Infinium iSelect SNP array and recognized software like QTL IciMapping and TASSEL.

The RIL population consists of 124 lines. While functional for biparental mapping, this is a relatively small population size by modern standards, which may limit the resolution of the genetic map and the ability to detect minor-effect QTL.

After quality filtering, only 956 polymorphic SNP markers were used for the linkage map. In the current era of high-density genotyping, this density is moderate and may leave some genomic regions underrepresented.

The manuscript notes that disease pressure was relatively low in one environment (BR21). While multi-year data helps mitigate this, low-pressure environments can sometimes skew phenotypic averages.

Although QPca-ars-7C is described as a "major" QTL, it explains 16.54% of the variation. While significant, this suggests that a large portion of the resistance is still controlled by many other unidentified minor loci or environmental factors.

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Reviewer #1: No

Reviewer #2: Yes:   Ziya DUMLUPINAR

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Uncovering the genetic basis of crown rust resistance in a northern-by-southern oat biparental population

PONE-D-26-08710R1

Dear Dr. Klos,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS One

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