PONE-D-21-06686

High-density DArT-based silicoDArT and SNP markers for genetic diversity and population structure studies in cassava (Manihot esculenta Crantz)

PLOS ONE

Dear Dr. Adu,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: High-density DArT-based silicoDArT and SNP markers for genetic diversity and population structure studies in cassava (Manihot esculenta Crantz)

Header: Take out additional symbols after number superscript

Abstract:

Ln 1: Take out extra spaces throughout the document; just search for space space; Manihot esculenta italix

You need to say somewhere what the germplasm is? Landraces, breeding lines and from where? Before you get into results

Introduction:

Ln 52 due to its ability

Ln 53 Take out “than most staple crops”

Ln 54: contain

Ln56:]

Ln 58: Okogbenin and FrEgene – this work was a long time ago, and much has happened since then. Cassava is no longer an ‘orphan’ crop.

Ln 102: DArTSeq does not involve microarray. It purely involves complexity reduction (restriction digestion), and NGS. The authors need to be aware that before NGS came about, the company used a microarray based genotyping technique called DArT. This is not used much now, if at all. It seems the authors have got confused with the two techniques.

Ln 106: I understand that SilicaDArT are a reflection of epigenetic status. They are epigenetic markers. The difference between DArTseq SNPs and Silico needs to be VERY clearly defined. Microarray is a technology, not a form of marker. It is not used in generating SilicoDArT.

Ln107: Be consistent in how you define DArT-based SNP or DArTseq SNP.

Ln 109/110: With DarT there is no choice in restriction fragments.

Ln 114: Be more specific about the genebank

Materials and methods

Ln120 eighty-seven should be 87

Ln 121: How many were landraces and how many released cultivars?

Ln 124: twenty-five should be 25

Ln 127: This is the fist time you mention morphological. This should be mentioned in introduction.

Table 1: Why choose to report colour of apical leaves? Why dry matter? Also, cassava has storage roots, not tubers.

DArT procedure paragraph: Please be aware that DArT and DArTSeq are different procedures. There is no cloning in DArTSeq. This paragraph needs to be corrected.

Ln 164: You need to be clear that alleles were scored as 1 and 0; provided in two line format; or 0,1 and 2 as single row format with heterozygotes identified. This is critical. Distinguish scoring between Silico DArT and DArtSeq.

Ln 175: Don’t use DArT-based SNPs – these are DArTSeq SNPs

Ln179: I would use the term duplicates rather than redundant.

Ln 178: You need to discuss why you used Gowers

Ln 180: “pvclust” in R (include reference)

Ln 182: SilicoDArT and DArTSeq SNPs.

Ln 198: Why capital letters? I wouldn’t call it a peak; rather a flattening of the curve.

Results:

Ln 207: What do you mean “18 haploid cassava genome”? Explain this. You mean chromosomes?

Fig 1a and b. These figures are not necessary.

Fig. 2 needs to be better quality

Fig 3. Axis labels should not be capitals. Bars must not overlap and needs better quality.

Fig. 4. Cannot read anything from this Figure.

None of the subsequent figures have numbers.

Ln 253 : Use SilicoDArT throughout the document.

Ln 271: As far as I know you are looking for a flattening of the graph where deltaK changes, not a peak.

Ln 275: Please explain the Average Silhouette approach

Ln 276: You need to define which accessions are in which subpop before you start talking about diversity. Individuals are allocated to sub-populations.

Ln 292: Give a reference to average linkage clustering. What is meant by this. Clarfy.

Ln 302: Three rather than 3; and I would call these putative landraces as they may actually be improved lines

Ln 304: all cassava is heterogeneous. This is not a good word. Perhaps use Admixture.

Ln 308: Is there a figure associatd with this?

Ln 316: I wouldn’t indicate left of right as the figure could be switched. Just give colour. Rather say cluster together…

Ln 338: What do you mean 22,516 assigned to specific chromosomes?

Ln 341: I wouldn’t say these markers have to be developed. They are there, they just need to be visualized.

Ln 350: better genome coverage than what??

Ln 353: What about Ferguson et al. (2019) and many studies by Rabbi et al.

Ln 369: This is not two DArT platforms – just two types of data are generated form one platform

Ln 408: two

Ln 411: Rather than heterogeneous, I would say admixture or relatively close relationship.

Ln 416: The authors must understand the basic rules of reporting numbers; 1-10 should be written in full. Anything more than 10 is given in figures. Please correct throughout.

It does not indicate anywhere whether the duplicates were taken out of the dataset before diversity analysis was conducted. This should be done.

Ln 429: You need to find out the background / pedigree of IITA material

Ln 444: You might want to say that DArTSeq SNPs are preferred as they are co-dominant which means heterozygotes can be determined.

Conclusions

Ln 451: It is not an array that is used. Be consistent in naming the technologies throughout the doc.

Ln 459: I don’t think it is defined by geographical differentiation, but more by pedigrees – breeding lines vs landraces.

Reviewer #2: General comments:

The manuscript reports genetic diversity of cassava from Ghana. The study involves more than 80 accessions including improved varieties and landraces that were genotyping using a reduced-representation library sequencing (DArT). The authors presented standard diversity and population genetics analysis results including a report on marker informativeness, pairwise genetic distances among the accessions, hierarchical clustering, PCA and ancestry-based population structure.

Specific comments

1. While the study contributes to the body of knowledge about the diversity of the crop in Ghana, the authors repeatedly put forth statements that are rather outdated when justifying their study in the introduction section of the manuscript. For example, there is repeated mention that cassava is an orphan crop, with no genomic resources available and this is not correct (e.g. lines 28 - 30 and Lines 58 - 61). Indeed, one of the papers used to support this notion is almost two decades old (Okogbenin et al. Theoretical and Applied Genetics. 2003; 107:1452–1462.)

There are many high-quality reference genomes, hundreds of accessions have been whole-genome resequenced to produce hap-map, and several diversity analysis that used genotyping-by-sequencing have been published. Moreover, genomic resources have been used to map QTLs using biparental populations and GWAS. Moreover, genomic selection in cassava is quite advanced and comparable with other major crops. It is therefore not correct for the authors to say that there is very little progress in the crop. A rigorous literature review should address this misperception.

2. The way the manuscript is written is not concise and clear. There are many repetitions and the same thing are being presented multiple times.

Line 42 - 45: seems like a concluding statement that should have been at the end of the abstract.

L58 - 61: fragmented sentence that is not understandable. What is the intended message?

L80 - 81: "... as detailed by some authors" is not necessary. Similar types citation styles are in the manuscript. It should just be "The use of molecular tools in plant genetic analyses and crop improvement cannot be overemphasized [26-29]."

L84 - 92: The authors introduce a description of other types of molecular markers, including anonymous markers such as RFLPs etc. What purpose does this serve? Back when SNPs were new, it was acceptable to describe its advantages compared with the traditional markers but this serves no purpose since SNP have become mainstream.

Instead, the authors should describe DArTseq and compare it with other high-density SNP genotyping methods like GBS which has been extensively used for cassava.

L96-97: Why would one want to use "sequence independent" SNP?

Line 130: Table 1 can go to supplementary file unless readers are interested in individual cassava accessions.

Line 134: "Extraction of DNA and quantification of DNA... " can surely be shortened to "Extraction and quantification of DNA... "

Line 145 - 147: these are unnecessary details - who is interested to know which brand of 96-well PCR plate the samples were packed in?

Line 150 - 151: "As defined by Kilian et al. [38]" is just unnecessary wordiness. Just put the citation at the end of the sentence.

The whole DArT procedure paragraph section is text that needs to be replaced by citing the methods and not repeating them here. For example, the authors say that "genomic representation were generated following the procedure of [38]" and "A detailed procedure is documented by [37]" but still go into details of the laboratory protocols some of which are quite shallow and will not be helpful to the readers. Indeed the objective of the paper is to describe the diversity in the germplasm but not a paper on the genotyping methodology. So the latter should not be the highlight.

L161-162: What value does "Order:162 DCas18-3505 on 01/06/2018" add to the manuscript? How will this information benefit the reader?

L165 - 166: The way SNP calling is mentioned is not clear. "Markers were scored ‘1’ for presence, and ‘0’ for absence and

‘-’ for calls with non-zero count but too low counts to score confidently as “1”." Is this for SNPs or the presence absence DartTag?

L173 - 175: call rate - this level of details is unnecessary. Call rate is % another description of proportion of missing data which is self explanatory.

results:

L207 : which version of the cassava reference genome?

L210: How was reproducibility of the SNPs calculated and based on what?

L228 - 264: This whole section can be shortened by presenting the results graphically e.g. using histogram of pair-wise distance. This can be done for the silicone-dart and the regular SNPs.

L234 - 236: The critical distance threshold to declare whether two genotypes was based on only replicated DNA from 3 samples. In my opinion this is rather small sample size to make an inference about the threshold identifying clones. The authors need to produce a plot of the histogram of pair-wise distance and show if they can pick up the signal of the redundant DNA from these 3 samples.

L244: what is a "mutant" variety ?

The section needs to be made more concise.

L272 - 275: "This means that optimum number of groups that suits the distribution of similar cultivars inside the population was two, indicating that, two different groups (subpopulation1 and subpopulation2) contribute relevant genetic information across the population." should be "The optimum number of subpopulations is two". No need two be repetitive.

L278: How can the expected heterozygosity be close to zero in a non-inbred clonal crop like cassava?

Table 3: what does "inferred clusters" mean? The numbers seem to add up to 1 but is tis the proportion of ancestry to cluster 1 or 2???

L292: Why every time list all accessions in each cluster - or any given result section. This type of information should be in a table or figure for the interested reader to look into but serves no additional value to the manuscript.

Line 313: "The first two axes of the PCoA (Ofigure)..." It seems that the paper was not checked for submission because you cannot have a reference to a figure that is not numbered. The naming of figures/tables in the manuscript does not follow correct convention. Eg. "S3 Table" in Line 231 is actually not a supplementary table; "S1 and S2 Figs" in Line 299.

Discussion: Needs to be made more concise to increase readability.

Figures:

There are too many figures in the manuscript. Some can be joined together.

Figure 1: Represent the distribution of call rate using histograms and not pie-charts.

Figure Dendrograms. These aren't legible and need to be redrawn. There are tons of R-based tutorials on how to generate useful hierarchical clusters that are legible.

Figure 5: What is "average silhouettes" - ? These figures can be sent to supplementary files or be components of the STRUCTURE figure.

The PCA plots have wrong aspect ratio - even though the ranges of PC1 and PC2 are similar.

Figure 8. This plot can be provided as a supplimentary file. The mantel test are already provided in the results and this is redundant.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-06686R1

High-density DArT-based silicoDArT and SNP markers for genetic diversity and population structure studies in cassava (Manihot esculenta Crantz)

PLOS ONE

Dear Dr. Adu,

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.

Please submit your revised manuscript by Aug 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General: The manuscript still requires a major English revision

In. 33. Use expansion of PIC on first use, not on second use.

Ln 55. They are not tuberous roots, they are storage roots. Tubererous roots can sprout from eyes in the roots. Cassava roots do not do this, and are referred to as storage roots.

Ln 56 continue, not continuous

Ln 92. These are SNP markers visualized using DArTSeq technology. At least they should be called DArTSeq SNP markers, not just DArT markers.

Ln 105, what do you mean 1,000 candidate polymorphic clones. Are these distinct cassava clones. If they are, then why are they candidate? All cassava clones are polymorphic, so not necessary to state this.

159, 160: no need to repeat ‘polymorphic information content’ once it has been sued once. Also repeated on line 204.

Ln 292 How did you calculate Net nucleotide distance (add to methodology) and give a reference.

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Reviewer #1: No

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High-density DArT-based silicoDArT and SNP markers for genetic diversity and population structure studies in cassava (Manihot esculenta Crantz)

PONE-D-21-06686R2

Dear Dr. Adu,

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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Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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