Kompetitive Allele Specific PCR (KASP) Markers for Potato: An Effective Tool for Increased Genetic Gains

Round 1

Reviewer 1 Report

In the study, the authors developed two KASP markers for LB resistance and two for PVY Ryadg from several types of data or markers and through quality control assessment, getting and estimated assay power ranging between 0.65 and 0.88. These data provided a potential use in different breeding population and material. However, following question should be clarified.

1. Comparing with other types of markers, such as SNP, INDEL, SSR and so on, what is the advantages and disadvantages of KASP used as markers? This should be included in the introduction or discussion.
2. One KASP markers for LB resistance, snpST00023 was validated with and estimated assay power of 0.65 and with false positive and negative rates 0.22 and 0.35, the assay power is relatively low and relatively high false positive and negative rates. What are the possible reasons?
3. In LB resistance, the authors used R8 for an example, did you try any other genes for LB resistance? When getting these genes for LB together, what do you think the assay power should be?

Author Response

Dear reviewer,

Thank you very much for reviewing our manuscript.

1- Comparing with other types of markers, such as SNP, INDEL, SSR and so on, what is the advantages and disadvantages of KASP used as markers? This should be included in the introduction or discussion.

Response

KASP markers can be SNP or INDEL. Some benefits of the technology are already described or documented in the introduction but we have now strengthened this by including a sentence to address one more benefit of the KASP assay.

2- One KASP markers for LB resistance, snpST00023 was validated with and estimated assay power of 0.65 and with false positive and negative rates 0.22 and 0.35, the assay power is relatively low and relatively high false positive and negative rates. What are the possible reasons?

Response

This depends on the germplasm used for marker validation. We show later in the paper (Figure 4)  that the marker performs better in the LBHT population than in the LBHTxLTVR population. We have added a more detailed description of the validation plate in materials and methods, and discussed the reason in the results section.

3- In LB resistance, the authors used R8 for an example, did you try any other genes for LB resistance? When getting these genes for LB together, what do you think the assay power should be?

Response

We focused on resistance loci that are present in high frequencies in CIP breeding populations and did not try any other LB resistance genes. More sources of resistance, as explained in the introduction, would probably decrease the odds resistance breakdown.

Reviewer 2 Report

Kante et al - Agronomy-1426657 – Review

As a whole, it is a very sound paper presenting the KASP-marker method to ensure the identity of the clones at key breeding stages, hybrid purity, etc., to alleviate authenticity problems and finally to accelerate the breeding and reduce its costs.

The paper can be considerably supplemented when the authors address the most relevant evidence from a recent publication by Caruana et al. (Genome Enhanced Marker Improvement for Potato Virus Y Disease Resistance in Potato. Agronomy 2021, 11, 832. https://doi.org/10.3390/agronomy11050832). Caruana et al. developed an SNP marker and validated it for a KASP genotyping assay, which was found to perform more accurately than all previously used markers for detecting Ry_adg.

My comments – or let us call them questions - concern the following points of the paper:

• The source of KASP markers and particularly the evidence on their validation by phenotypic resistance. The readers are forwarded to the sources preceding the present paper. I think it is not the most elucidating way to present this crucial information.
• If I am not mistaken, the sequences of both Ry_adg and R8, the focal points of this paper, - or at least the latter - have been established. In this context, one would wonder what kind of wild germplasm was involved in this study. What is the putative position of selected KASP markers towards Ry_adg and R8 gene sequences or their homologs?
• In wild potatoes, the R8 gene is found in the vicinity (cluster?) of several other genes for LB resistance of similar CC-NB-LRR structure. Is the particular QTL of LB resistance employed in this study for marker development specific to R8 – or does it correspond to a large interval covering several genes closely located in this part of chromosome 9? Indeed, the Supplemental Table lists numerous Rpi genes of unknown identity. Which of these Rpi genes correspond to R8 and stand for LB resistance linked to the efficient KASP marker?

Just a trifle: the abbreviation CIP is deciphered only at the second mention. And the MS needs further spellchecking.

Author Response

Dear reviewer,

Thank you very much for reviewing our manuscript and providing very constructive comments.

1- The paper can be considerably supplemented when the authors address the most relevant evidence from a recent publication by Caruana et al. (Genome Enhanced Marker Improvement for Potato Virus Y Disease Resistance in Potato. Agronomy 2021, 11, 832. https://doi.org/10.3390/agronomy11050832). Caruana et al. developed an SNP marker and validated it for a KASP genotyping assay, which was found to perform more accurately than all previously used markers for detecting Ry_adg.

Response

We found the article after submitting our manuscript. A direct comparison is difficult because they did not provide data on the power of the assay. However judging from the supplementary table S2 of Caruana et al paper there are only very few clones with known resistance phenotypes assayed. The marker was developed (just like ours) based on bi-parental crosses and to some extend comparing with known resistant varieties and as such it is expected to work well in the material for what it was developed. As M45 is physically closer to the Ry locus than M6, the chances are that it works better. However, this need to be tested and a routine use of the marker can be envisaged.

2- The source of KASP markers and particularly the evidence on their validation by phenotypic resistance. The readers are forwarded to the sources preceding the present paper. I think it is not the most elucidating way to present this crucial information.

If I am not mistaken, the sequences of both Ry_adg and R8, the focal points of this paper, - or at least the latter - have been established. In this context, one would wonder what kind of wild germplasm was involved in this study. What is the putative position of selected KASP markers towards Ry_adg and R8 gene sequences or their homologs?

Response

Ryadg cloning is on-going work, hence the sequence is not yet available. R8 has been cloned but we haven’t done further sequencing in CIP germplasm to look for better diagnostic markers. R8 and Ryadg originate from S. demissum and S. andigenum, respectively, which are in the pedigrees of CIP germplasm. The M6 marker for PVY locates less than 1cM from the Ryadg gene, while the R8 QTL is a large interval, which has been discussed by Lindqvist-Kreuze et al. 2021. G3, doi.org/10.1093/g3journal/jkab251.

3- In wild potatoes, the R8 gene is found in the vicinity (cluster?) of several other genes for LB resistance of similar CC-NB-LRR structure. Is the particular QTL of LB resistance employed in this study for marker development specific to R8 – or does it correspond to a large interval covering several genes closely located in this part of chromosome 9? Indeed, the Supplemental Table lists numerous Rpi genes of unknown identity. Which of these Rpi genes correspond to R8 and stand for LB resistance linked to the efficient KASP marker?

Response

R8 QTL is a large interval, which has been discussed in the paper [38]. The supplementary Table reference to Rpi gene is a reflection of a phenotypic reaction rather than any specific Rpi gene, as presented in the M&M. We have corrected this in the supplementary file to avoid further misunderstandings (Rpi gene = HR, highly resistant). It is possible there are several R genes in the QTL.

Reviewer 3 Report

The manuscript submitted to Agronomy represents very good research on genomics application in breeding and seed productions conducted by CIP. The paper is well written and clearly presents the case with good graphs and tables. However, there is potential for improvement:

1. The study material (both reference sets and breeding populations) is not well presented in terms of its diversity, parentage and agronomic traits. How unique it is for CIP or how common with the cultivars/breeding material in other parts of the world. The CIP IDs do not possess any information unless they are linked to pedigrees or explained.
2. Supplementary material is very important but lacks accuracy. The paper refers to S1, S2 and so on but these are not identified in the same way in supplement. The sheets lack titles and abbreviations need to be explained where possible.
3. The phenotypic and genomic data for the material is presented in supplement and this is very important. However, it would be beneficial to assemble a table with superior germplasm for both diseases, respective markers and important agronomic traits if available, pedigree/origin explained. This table can be added to the body of the paper or as a supplement.
4. The concluding remarks will benefit from a broader view on the contribution of this study to global efforts in improving resistance. On the other hand, the conclusions also need to be grounded on immediate impact of this study for potato improvement which CIP specific program targets: central America of specific country or other regions or indeed global. In this respect the paper will also benefit from a statement on CIP potato breeding strategy included in introduction.
5. Though scientific style is very professional, additional reading of a paper by an editor would be beneficial to avoid using the same word four times in the same sentence: “Although testing a subset of tested genotypes would prove more efficient than not testing at all, a routine application of QC markers should be defined to test as many genotypes as possible.”

Author Response

Dear reviewer,

Thank you very much for reviewing our manuscript.

The supplemental file had been reviewed. We mistakenly uploaded a working file.

The conclusion was reviewed.