PGENETICS-D-26-00294

A high-throughput sequencing screen to identify apomixis and other modes of reproduction in plants

PLOS Genetics

Dear Dr. Harkess,

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Jae Young Choi

Academic Editor

PLOS Genetics

Quan Wang

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Additional Editor Comments:

The manuscript has been reviewed by four reviewers. The reviewers have found the topic of the study and results interesting, and three of the reviewers were generally positive with the paper. However one reviewer (reviewer 2) does have some concerns that addressing it would greatly improve the current paper. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version that fully addresses each of the points raised by the reviewers.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In the paper “A high-throughput sequencing screen to identify apomixis and other modes of reproduction in plants” by Goeckeritz et al., the authors use low coverage genome sequencing and flow cytometric seed screening (FCSS) from a subset of sequenced Malus (apple) species to identify variations in modes of reproduction/apomixis. Apple is a species where FCSS can be used to identify apomixis in maternal plants. The authors describe that sequencing of the maternal genome at a higher coverage along with low coverage sequencing of offsping, with careful sequencing controls, allows the identification of SNP data which can be parsed into 4 quadrants to give both apomict and additional reproductive phenotyping (ie: outcrossing vs. selfing) of the maternal line. The addition of flow cytometric seed screening can give additional reproductive information within the sequencing quadrants. The generation of numerous SNP genetic and FCSS data shows the plasticity/diversity of reproductive outcomes in Malus. While this paper is informative for apomixis in Malus, it also gives a potential pathway of analysis that could be useful for researchers studying variation for sexual and apomictic reproduction in seedlings. The authors do note that this approach for plants with larger genomes and more repetitive DNA may not continue to be price competitive. Overall, the paper is well organized and written although some places seem overly explained (ie: the legends of Figure 3 and 4) or repetitive between the material and methods section and elsewhere in the paper. In discussion, line 378-79 sounds like an advertisement for a kit. Line 601-608 in materials is an important point and should be moved to the discussion.

Small changes:

Line 64 – need ) after parthenogenesis

Line 79 – need ) after Meiosis

Line 111 – remove ( before [32]

Line 189 – year for Phipps et al.

Fig 2A: move the sexual and Apomict description of the Malus phenotypes next to the name of the species in parentheses. As the figure is – it looks like those words are heads/information for the 2 quadrants on the right.

Line 423-424 – PI should be removed to stay consistent with the rest of the paper.

Line 489 degree symbol should be superscript

Table S3 column Q – polen should be pollen and parthenogenesis – ‘h’ is missing

- Line 244: if there is a question on type then both should be in the same column with an or between

Reviewer #2: This manuscript presents a low-pass whole-genome sequencing framework to identify apomixis and other reproductive modes in Malus by directly comparing maternal and embryo genotypes. The study is potentially valuable for both reproductive biology and breeding applications. However, the novelty of this manuscript is insufficient for publication PLOS Genetics. Several aspects of the analytical framework, interpretation of the quadrants, and the handling of edge cases would benefit from further clarification or strengthening before publication.

1. The title is not sufficiently specific and is somewhat ambiguous.

(1) The use of “high-throughput sequencing” does not reflect the key technical feature of this study, namely low-coverage WGS.

(2) “other modes of reproduction” is too vague and should be described more specifically.

(3) “in plants” is overly broad, as the method was only tested in Malus in this study and was not validated in other species.

2. As a methodological paper, a workflow diagram is suggested to summarize the study as a whole. In contrast, Fig. 3 is somewhat confusing, and its intended message is not very clear. I suggest redrawing it in the style of a graphical summary and placing it at the end of the result section or at the beginning of the discussion section.

3. The criteria for setting the “92.5% heteroallelic and 95% homoallelic” thresholds need further clarification. Since the 95% confidence interval was calculated, why were the boundaries lowered afterwards? These values may themselves be affected by the reference genome and the species being analyzed. Because the boundaries are key parameters for distinguishing different reproductive modes, a more practical and reproducible procedure for defining them should be provided.

4. It is not clear how sample ploidy was determined, whether through WGS data or flow cytometry.

5. Please check Fig. S1, as the legend contains “NA”.

6. Line 312: I could not clearly identify the 21 facultative apomicts or the 9 genotypes complemented with FCSS. Please highlight them more clearly in Fig. 4.

Reviewer #3: The manuscript by Goeckeritz and colleagues presents a strategy to analyze reproduction modes in plants with a particular interest for identifying species and accession that produce apomictic seeds. The strategy relies on low-depth whole-genome sequencing that nevertheless still provides enough informative variant sites to infer reproduction modes after comparing maternal and offspring genotypes. The method appears sound, with carefully controlled thresholds for identifying high confidence SNP sites. In addition to presenting the pipeline to execute this method, the authors used it to screen a large number of Malus genotypes as a proof of concept, and they identified several genotypes showing facultative apomixis. Finally, they compared the results of their screening with a previously established method for inferring reproduction modes (FCSS). Together, the presented strategy appears to be a promising tool that will be useful for the plant sexual reproduction community.

I have only a few questions:

1) The authors show average coverage for each of their samples in Fig 1. What is the average coverage in these samples at informative variant sites? I assume the coverage at such a site has to be higher for it to be confidently called?

2) For accessions with deep whole genome sequencing data available, what is the total number of variant sites compared to the number of variants sites that pass the threshold in low coverage data?

3) For the vast majority of analyzed embryos, thousands of variant sites were available. How robust are the % matching Het/Hom site numbers with regards to the number of informative sites? Would a random selection of 100 sites lead to the same conclusions with regards to the position of the embryo in the quadrants? If not, what is the minimal number of sites needed?

4) What guidelines would the authors offer when adapting this pipeline for other species that potentially have different levels of genetic variation? What is the minimum number of variant sites required (similar to point 3)? Knowing the standing genetic variation, what number of informative sites in low-depth sequencing can be expected if one knows the total number of potential sites (similar to point 2)?

Reviewer #4: Generally the authors developed a very efficient strategy to identify apomixis from embyos. The method is very important to identify typs of reproduction. I would offer serveral suggestions to improve the manuscript.

1. the title need to change. It seems the authors only test this method in apple. So the title should precisely reflect the conclusion in this study.

2. The identifed plants with apomixis need to be confirmed by PCR or allele-specific markers.

3. Abstract Line 40, four of these were detected using internal sequencing controls. It is not clear.

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: None

Reviewer #3: None

Reviewer #4: None

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Dear Dr Harkess,

We are pleased to inform you that your manuscript entitled ": Development of a low-coverage whole genome sequencing screen for apomixis using a diverse set of Malus germplasm" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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PLOS Genetics

Quan Wang

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PLOS Genetics

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Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised manuscript "Development of a low-coverage whole genome sequencing screen for apomixis using

a diverse set of Malus germplasm" uses low coverage sequencing, optimized modeling and flow cytometry of seed to identify Malus accessions which reproduce by apomixis. Their data include previously defined sexual and apomictic accessions as controls along with screening accessions with unknown reproduction or that are potentially classified as apomicts in the literature. As a revised manuscript, the authors took care to reply to the first set of reviewers and have made significant changes to the manuscript based on those suggestions to strengthen the manuscript.

Reviewer #3: In their revised manuscript, Goeckeritz and colleagues improved the clarity of the presentation and the description of the methodology underlying their proposed approach to characterize sexual/apomictic modes of reproduction through a combination of low-coverage whole genome sequencing and flow cytometry. They addressed all my questions in a satisfactory manner. I have no further comments.

Reviewer #4: The authors addressed my concern.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #3: None

Reviewer #4: None

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Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

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