TP-ARMS: A Cost-Effective PCR-Based Genotyping System for Precision Breeding of Small InDels in Crops

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a straightforward but useful method for low-cost genotyping based on a Tri‑Primer Amplification Refractory Mutation System (TP‑ARMS). By targeting small indels that are often difficult to resolve with standard gel-based markers, the approach has clear potential for marker-assisted selection in laboratories that do not have access to sequencing facilities or fluorescence-based platforms such as KASP. The demonstrations in rice and quinoa, especially for gene-based markers in high-value rice genes, make the work relevant to crop improvement and to resource-limited settings.

First, the validation against other methods needs to be more rigorous. At present, the comparisons to sequencing or KASP are not presented as parallel tests on the same samples, which makes it difficult to judge accuracy and robustness. A side‑by‑side evaluation in which the same DNA panel is genotyped by TP‑ARMS, Sanger sequencing (as a gold standard), and, where possible, KASP would allow the authors to report concordance, sensitivity, specificity, and call rates in a way that practitioners can readily interpret.

Second, because the main selling point is cost‑effectiveness in low‑resource laboratories, it would be valuable to test TP‑ARMS using DNA obtained from a simple, low‑cost extraction protocol that has been shown to work reliably for PCR in rice (http://dx.doi.org/10.9787/PBB.2016.4.1.99). Demonstrating that TP‑ARMS still produces clear and consistent genotypes with such DNA would strengthen the argument that it is suitable for laboratories with severe budget constraints.

Finally, the discussion would benefit from a clearer placement of TP‑ARMS within existing marker‑assisted selection workflows. In practice, breeders combine SSRs and gel‑based SNP/indel markers, including previously developed gel‑based functional SNP markers for key rice resistance genes (DOI: 10.1007/s11032-015-0323-4), and adding a brief comparison of how TP‑ARMS complements or improves upon these tools would help readers understand when they should consider adopting this system.

Overall, the technique itself is promising and very likely to be useful in the kinds of laboratories the authors have in mind. With a deeper, more comparative discussion and a more robust validation against existing methods and low-cost DNA extraction protocols, the manuscript would make a stronger contribution.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

Avoid repeating DNA extraction in Materials and methods.

Materials and Methods need additional details, for example, Taq polymerase: high fidelity?

1 mm 1 mm leaves???

 

30 times as 30x

1% (w/v) agarose.  Which buffer?

1 base deletion, should be validated with at least 3 genes (only one is shown, and its reliability is questionable)

Subheadings should be avoided in the Discussion section

Avoid redundant sentences in Introduction and Discussion

Supplement (not Suppliment)

It is cost-effective to validate only the InDel.  Nevertheless, it mandates Sanger sequencing.

What about validation using T7 Endonuclease I, which is widely used in genome editing?

Comments on the Quality of English Language

Needs fine tuning in several places. 

for example, 2.1 To examine the utility of the TRI-ARMS marker we developed, we performed tests.....

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the authors’ detailed responses and the additional work addressing my second and third comments. I am satisfied with those aspects of the revision. Regarding my first comment on validation, the response letter explains that TP‑ARMS genotypes were validated side‑by‑side against Sanger sequencing, but in the current version of the manuscript this comparison is not easy to locate and is only briefly described. It would strengthen the paper if the Sanger validation were presented more explicitly in the main text, with clear indication of the number of loci and samples tested, and with explicit reference to the relevant figure and/or table where these data can be found. Making this clearer will help readers quickly appreciate the robustness of the TP‑ARMS assay.

Author Response

Please see the attachment

Author Response File: Author Response.docx