High-Throughput Field Screening of Cassava Brown Streak Disease Resistance for Efficient and Cost-Saving Breeding Selection

Reviewers comments

Author answers

Reviewer 1

Thanks for the general and specific comments of the reviewer. Authors carefully addressed the comments, reviewing the long sentences and accepted the valuable suggestions. We also went through the entire text and reviewed where needed, as reported in these point-by-point answers.

 Frequently using “and” to connect multiple ideas in a single sentence adds complexity and reduces readability.

Sentences were reviewed in general to single sentence to make the paper readable for both scientific and none-scientific communities.

The use of abbreviations does not follow the required format. Always provide the full form followed by the abbreviation in parentheses the first time it is mentioned and use only the abbreviated form in subsequent mentions.

 (Line 26: DRC). Also check others

All the abbreviations were carefully reviewed and spelled out in the first appearance including DRC in line 26.

Line 48: Rewrite the sentence as: In Sub-Saharan Africa, cassava serves as a staple and food security crop [1,2].

The sentence was reviewed.

Can you explain the repetitions in Table 2. Where just 2 samples were considered or from 2 experiments? How many sets of experiment were done to conclude these results?

As indicated in Table 1, 4 sets of experiments were established for this study. However, to cover G x E effect, two leaf samples were collected from each clone. Precision was given in line 180 to clarify this.

Why for some clones were evaluated in leaf and tuber but others only in leaf? Glaziovii20210006, Glaziovii20210006 were only evaluated in leaf.

Manihot Glaziovii is wild species and do not produce root.

Precision was provided in line 256 to clarify.

Why MVZ2017095 was not “selected” though molecular testing shows negative values in both replicates.

The mistake was corrected. MVZ2017095 showed positive response to UCBSV in the leaf and expressed score 3 for leaf severity.

The most important point is about molecular testing. Author mentioned and focused a lot on molecular testing. First of all, clarify either author used RT-PCR or qRT-PCR? In results, only detection was confirmed in the samples. If used qRT-PCR, show it’s data in result about quantification. If RT-PCR was used please correct in the manuscript.

 In our study, we utilized RT-qPCR as the molecular tool for virus detection, a method widely used in several studies due to its sensitivity and reliability, especially when working with resistant or tolerant cassava clones. While RT-qPCR was employed for its high sensitivity in detecting viral presence, it was not used for quantification purposes in this study; instead, the primary objective was to confirm the presence or absence of the target viruses. Additionally, reference genes were used as a secondary measure to ensure RNA quality and validate the reliability of the RT-qPCR results. These details have been clarified in the revised manuscript to ensure accuracy and improve clarity.

Primers information in Table 2 is also vague if no data in result is provided.

All primers were used in the study (please see the comment above)

line 369: please rewrite the sentence. It’s not appropriate to start sentence with citation and make it as subject as “[22] reported similar observations for CMD resistance.” Instead, write “similar observations regarding CMD resistance was reported in yyyy [22]. Same for line 71.

The mistakes related to this comment were corrected in the whole manuscript.

References need to be revised. The abbreviated form of journal names needs to be corrected. Please use same format for all references.

References were reviewed

Figures 3,4,5,6 and Supplementary Figures S1A, S2A are blurred and need to be re-uploaded in good quality.

Original Figures have been added

Supplementary material should be mentioned as “Supplementary Figure S1” and “Supplementary Table S1” instead of (Appendices A1 and A2).

Supplementary materials were reviewed accordingly

Add Supplementary data at the end after References.

Done!

Reviewers comments

Author answers

Reviewer 2

Thanks to the reviewer. Authors carefully addressed the comments, eliminating the use of personalization and accepted the valuable suggestions where needed. We also went through the entire text and reviewed where needed, as reported in these point-by-point answers.

Line 2: Erase inappropriate word High-throughput

High-throughput in this research is not referring to sequencing but used to express the rapid and efficient field screening of large numbers of cassava clones (to scale up) using infection tools and virus indexing for confirmation to identify resistance genotypes.

Line 25: replace preferred by economically valuable

Done!

Line 26: spell out DRC

Reviewed as Democratic Republic of Congo (DRC)

Line 31: reviewer suggested the word RT-qPCR instead of qRT-PCR

Thanks. The suggestion was accepted.

We did the virus indexing test for detection of the CBSVs and not to quantify the load of virus. Therefore, we used qRT-PCR. The same method was previously used by Samar et al. (2022), https://doi.org/10.3390/agronomy12051055   

Line 32: spell out acronym CMD

The first mention of CMD was fully written as cassava mosaic disease.

Line 33: replace Our by the

Done!

Line 35: Replace sensitive by susceptible

Done!

Line 35: We further recorded high heritability

The sentence was reviewed to : High heritability was further recorded

Line 36: values 0.63 to 0.97 is referring to heritability which varied between 0 and 1

Sentence was reviewed for more clarification

Line 37: Insert a between only and few

a was added

Lines 44 and 45: deleted words and suggestion of qRT-PCR

We appreciate the suggestions. However, the words are keys for the study and relevant to the keywords.

Line 48: no verb in the first sentence

Thanks. the verb ‘’serves’’ was added to make the sentence complete.

Lines 56 to 59: Avoid using capital letters for disease names

Sentence was reviewed accordingly

Lines 61 and 62: spelling out the acronyms

These were addressed in the previous responses (Lines 56 to 59).

Line 66: replace sensitivity by susceptible

Done!

Line 67: replace appear healthy by are asymptomatic

Done!

Line 72: deleted cassava disease

Done!

Line 74: Insert M. before Sikirou

Done!

Line 75: Spelling out G X E

Done

Line 77: Replace B. by Bemisia

Done!

Lines 80 and 81: replace have by it was and deleted and seeing

Done and sentence was reviewed

Line 80: the word high-throughput

Authors reported here the previous work as published.

Line 84: Deleted for and review the sentence

Done for more clarity

Line 90: replace a by each

Done!

Lines 92 and 93: eliminate the use of personalization and review lab to laboratory

Sentence was reviewed

Lines 100 to 102: avoid personalizing

It has been reviewed

Line 106: add M. before Sikirou

Done!

Line 111: improve the sentence

Sentence was reviewed for more clarification

Line 118: revise Columbia to Colombia

Done!

Line 132: spell out MAP

Revised to Month after planting (MAP)

Line 134: add author names

Author names were added

Line 135: add M. before Sikirou

Done!

Line 138: delete cut used before into

Deleted!

Line 147: delete sensitive and brackets

Deleted!

Line 147: provide table explaining the scores ‘’ plants with CMD scores greater than 3 and CBSD scores greater than 1 were discarded”

The sentence is referring to the objective of our program to advance the clones to the new breeding stages.

However the details about the cale s were provided in symptom assessment

Line 149: qRT-PCR

Response was provided above

Line 153: use small letter for observing

Done!

Line 157: avoid personalizing

Done!

Line 158: use of the table to explain the scale

Thank you very much. It is true that most of the previous studies were used table to explain the scale. We believe to vary by using our own style to avoid repetition or plagiarism.

Line 160: add ‘’ used’’ between was and to evalate

Done!

Lines 166 to 168: are sure about the description of the percentage?

It was added ‘’multiply by 100 to the sentence’’

Line 171: spell out the acronym U/CBSV

This was reviewed above in the 2.1.2 Field conditions as well as qRT-PCR to RT-qPCR

Line 172: which type of leaf

the sentence was corrected by giving precision on the type of leaves (dried leaf) 

Line 175: quantification of tissue used must be added

The method used is not for quantification but for virus detection. Response provided in above response.

Line 178: replace isolation by extraction

Done!

Line 180: PRX, the name of the company provided the buffer must be added

Done

Lines 187 to 193: qRT-PCR and acronym

Responses were provided above to clarify

Line 218: add a between dried and few

Done!

Line 239: spell out the acronym PYT

PYT was fully written in the first appearance in the text in 2.1.3.

Lines 243 and 244: replace phenotyping and delete recording leaf disease data

Done!

Line 253: replace uninfected by asymptomatic

Done!

Line 257: avoid personalizing

Reviewed! and ‘’it was’’ was added

Lines 277 and 278: Delete the words for and resistance were deleted

Deleted it!

Lines 282 to 287: deleted for and reviewed qRT-PCR

‘’For’’ was deleted from the sentence and response was already provided above regarding qRT-PCR

Line 300: reviewed the words diseases Quality Control assessment to Diseases quality control assessment

Done!

Lines 313-314: improve the sentence

Sentence was reviewed to: no significant correlation was reported

Lines 324 to 326: delete words biology, for infection and by

Dine!

Lines 328 to 336: Delete by and avoid personalizing

Revision was done

Lines 337 to 340: the sentence is not clear

the sentence was rephrased

Lines 342 and 343: how can disease be heritable.

The missing words were added to make i

Line 346: avoid personalizing

Reviewed!

Line 357: avoid personalizing

Reviewed!

Lines 368 and 369: add author names

Author names were added

Line 370: M. glaziovii in italic

Done!

Lines 373 to 382: Move to result

Sentence was moved to 3.1. as last paragraph

Line 383: this following sentence ‘’a consistent correlation between qPCR results’’ was not shown in the result

The result comments were amended and the following sentence ‘’ Therefore indexing assessment showed the consistency between the field assessment based on symptoms and laboratory results based on virus detection with the use of qRT-PCR (Table 3) was added in the section 3.2.

Line 385: the clones that do not show symptoms but are still capable of replicating the virus must be shown as tolerant

Thanks for the advice. we will integrate this while making selection in our breeding program.

Line 388: avoid personalizing

reviewed

Line 398: what is the meaning of ‘’ The results are natural’’

the sentence was reviewed as: The results are from real-world conditions

Lines 399: use the correct name of glaziovii

Sentence was reviewed as follow: The two glaziovii (Glaziovii20210005 and Glaziovii20210006) and DSC

Reviewers comments

Author answers

The manuscript has been revised very well but still I have a few concerns before acceptance for publication. The authors claim the molecular testing of symptomless samples and label them as positive and negative. Only primer data is provided. No actual result has been shown. It's understandable author doesn't show negative results but when you are doing categorization based on molecular testing, results are mandatory to show either positive or negative. So, it is mandatory to show your RT-qPCR results if not in supplementary data at least here in response to the comment.

Thank you for this good comments.

Sure, the authors have provided in addition to the Table 3 of the current manuscript, the details of molecular testing showing either negative or positive results with categorization in the two strains CBSV and UCBSV were shown in Appendix B of the original manuscript. However, other reviewers have recommended to remove which we fine ok because it does not have any negative impact to the quality of the paper as the Table 3 seems enough.

The author claims references have been reviewed and corrected but it's not true. References must be revised according to the Journal's policy:

References reviewed

Journal Articles:

page range.

1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume,

Need to use the same font/style for all references. Hardly any reference is cited correctly.

In the current version Table 2 is not ok. It must be shown in a better way.

The form of Table 2. has been reviewed

Figure 2b is too compressed. Provide it in a way that CBSD symptoms can be visualized easily.

Original photos were added

Line 290: Add space between by and RT-qPCR ... tested negative by RT-qPCR 290 and can be considered virus-free (Table 3).

Done!

Reviewer comment

Author answers

Not all the suggested modification were taken into sufficient consideration and still some

for Authors

personalization was added. Please revise the manuscript again to revised following all the

suggestions enclosed the one for the title that is misleading as it is now.

We really valuable your time to revise this manuscript.

However, we do receive three reviewers’ comments + Associate Editor ones.

The current version is therefore combining of all the 4 comments in addition to the authors expertise input. This brings to make the best choice the submitted version in the round 2 revision. The title has been amended to “High-throughput Field Screening of Cassava Brown Streak Disease Resistance for Efficient and Cost-saving Breeding Selection”, and the references have been adapted to the Agronomy journal style.