Cytological and Molecular Characterization for Ploidy Determination in Yams (Dioscorea spp.)
, Denisa Šimoníková 3, Rajneesh Paliwal 1, Asrat Asfaw 1, Michael Abberton 1, Gueye Badara 1, Robert Asiedu 1, Jana Čížková 3, Eva Hřibová 3, Jaroslav Doležel 3, David DeKoeyer 4, Victor Adetimirin 5 and Ranjana Bhattacharjee 1,*Round 1
Reviewer 1 Report
The authors have performed very important research on plant species (Dioscorea spp.) that are of great importance in the practice of tropical and subtropical crop production. Although further research will be needed on the subject, the research work of authors is forward-looking and will support further scientific research on Dioscorea spp. in order to produce this species more efficiently.
Comments for author File: 
Comments.docx
Author Response
Response to Reviewer 1 comments/suggestions
Do the authors know of any artificial or natural interspecific hybrid yams that outperform the growth performance of underground tubers or ariel bulbils of the parent species?
Response: We thank the Reviewer for this valuable observation. Several attempts have been made at the International Institute of Tropical Agriculture to make interspecific crosses, mainly between D. rotundata and D. alata as well as D. rotundata and D. cayenensis, and D. alata and D. bulbifera. However, there has been no success in generating viable progenies from these crosses. Most of the times, there has been embryo abortion and successful crossing could not be established. The embryo rescue technique was also applied but this also didn’t give any success.
General recommendation
For future research, I also suggest to the authors the karyotyping of some intra- and interspecific hybrids, which may also be important in the breeding program of different Dioscorea species.
Response: We appreciate this recommendation and now that we know the ploidy status of the accessions across different species, and we have identified internal standards to routinely check the ploidy status of target accessions/lines, it will be easier to carry out intra- and interspecific crosses and check their ploidy status using flow cytometry and karyotyping.
Review
Line 27: “intra- and inter-specific hybridization” – unnecessary hyphen between inter-specific words - correct: interspecific.
Response: The change has been affected in the revised manuscript.
Line 28: Key words: Dioscorea spp. Is included in the title, so it should no longer be included in the keywords, I would recommend the term “hybridization” instead.
Response: Dioscorea spp. has been removed from the keywords. We did not use hybridization as one of the keywords since it is not a key part of this manuscript.
Line 30: Intruduction: this section should write about the effect of heterosis, with particular reference to the fact that the progeny generation(s) of parents with different levels of ploidy may exceed or underperform performance of their parents. This may even be a consequence of hybrid sterility from different ploidy levels.
Response: We appreciate the reviewer for this suggestion, and we have revised the Introduction to include a section on heterosis related with polyploidy and issues of hybrid sterility in crossing among parents of different ploidy levels.
Line 58: “which result in incompatibility, preventing the successful transfer of genes” –correct: “could preventing the successful transfer of genes”, because the progeny generation may be only partially sterile.
Response: We agree with the reviewer and added to the sentence.
Line 203: Figure 1. - for the figures in Figure (y axis) it should be written “accessions”
Response: We agree and added ‘accessions’ in the y-axis of the figure.
Line 318: Figure 4: Why is the species D. esculenta missing from this figure?
Response: We thank the reviewer for this keen observation, this was an accidental miss from our side. The figure has been corrected and the accessions of D. esculenta has been clearly mentioned in the figure.
Line 367-380: Evaluation of D.esculenta, D. bulbifera, D. dumetorum and D. cayenensis and comparison of data with other literature data, if available, are missing from this section and should be wrote in here.
Response: The authors appreciate this suggestion and has added necessary information in the revised manuscript.
Line 396: “intra- and inter-specific hybridization” – unnecessary hyphen between inter-specific words - correct: interspecific.
Response: Necessary change has been made in the revised manuscript.
Reviewer 2 Report
To present the basis for the determination of the ploidy of yam, authors studied using chromosomal microscopy, FCM, and DArT.
In the introduction, there seems to be insufficient persuasion as to why this study should be conducted.
In Materials and Methods, some of the results mentioned are omitted.
In the result, the content that should be included in the discussion is included.
Overall, I think manuscript needs to be rewritten more logically.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 2 comments/suggestions
Do you mean 40% higher compared to that of cassava?
Response: We mean the production is 40% lower than that of cassava and this has now been added in the revised manuscript.
Please clarify if seed is a proper term. Consider using planting materials or tubers.
Response: We thank the reviewer for this observation, and 'seed' has been changed to 'planting materials'.
What is the rationale for this sentence? Try to cite examples from other crops.
Response: We fully agree with the reviewer, which has also been raised by another reviewer. We have now included a section on relation of polyploidy to heterosis and how tradition yam breeding has been creating only diploids. We also provided example from rose wherein careful selection of parents with similar ploidy level are being done as a breeding strategy.
Authors didn't mention why you should apply molecular character (SNP) to ploidy determination in Introduction. Why is this study needed?
Response: We appreciate the reviewer for this valuable suggestion and a section has now been added explaining why molecular genotyping was carried out.
Please rephrase this ‘in good agreement’ as this is vague and does not elucidate your results. What does good agreement mean? How is this defined?
Response: We have rephrased the sentence and deleted 'in good agreement'.
Figure legends should look better in the upper right corner of your figure. Likewise, please polish the legends as there is a misplaced blue thread.
Response: We appreciate this observation of the reviewer and revised the figure accordingly.
This part (Line 281-287) should described in the discussion.
Response: This paragraph has been shifted to Discussion section in the revised manuscript.
On what basis and by what method did you construct the phylogeny relationship? There isn't mention of this part in Materials and Methods.
Response: We thank the reviewer for this observation. A section has been added in Materials and Methods section to reflect the method used to construct phylogenetic relationships.
This sentence is very general information, but how does your research relate to reproduction? A review of yam breeding and ploidy is absolutely necessary in the introduction.
Response: We fully agree the suggestion of the reviewer and a section on importance of ploidy and yam breeding has been added in Introduction section.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors determined ploidy level of Dioscorea accessions by chromosome counting and flow cytometry, and compared these values with estimates from genotyping-based DArTseq data. In my opinion, the analyzes were correctly performed and the presented metaphases are of good quality, enabling the counting of chromosomes. I recommend some minor changes in the manuscript:
- Lines 73-75: 'The flow cytometry technique has been used for determining the ploidy levels of various plant species, including yams.' - It would be good to add citation here
- Lines 90-94: I would be more precise in describing your objectives:
(i) much data on yam ploidy has already been published. Can you please explain better why more ploidy research is needed for these species? Do you mean the germplasm collections haven't been fully screened for ploidy?
(ii) what comparison or results do you mean? I recommend to rewrite this sentence.
- Lines 102-103: Is there any data on their origin? Do you know their collection sites, especially for the two wild species you studied? Is there an online database? Or maybe you have some internal data on these accessions that you can add to the supplement? I understand that you do not studied accessions in terms of ploidy and geographical distribution but in the introduction and discussion you stated that understanding the ploidy of these species is important for developing strategies for crossbreeding and genetic improvement. I think the origin of these accessions will also be important to breeders.
- All latin names should be in italics.
Author Response
Response to Reviewer 3 comments/suggestions
The authors determined ploidy level of Dioscorea accessions by chromosome counting and flow cytometry, and compared these values with estimates from genotyping-based DArTseq data. In my opinion, the analyzes were correctly performed and the presented metaphases are of good quality, enabling the counting of chromosomes. I recommend some minor changes in the manuscript:
- Lines 73-75: 'The flow cytometry technique has been used for determining the ploidy levels of various plant species, including yams.' - It would be good to add citation here
Response: We appreciate the suggestion of the reviewer and we have added citations in the revised manuscript.
- Lines 90-94: I would be more precise in describing your objectives:
(i) much data on yam ploidy has already been published. Can you please explain better why more ploidy research is needed for these species? Do you mean the germplasm collections haven't been fully screened for ploidy?
Response: We like to thank the reviewer for this observation. More ploidy research is required to screen the germplasm collection of different Dioscorea spp using a known internal standard from within the species. Several studies have been carried out on ploidy analysis, however, the internal standards were not from the same species and this can have an impact on assessment of ploidy status of the target individual. This has been explained in the revised manuscript.
(ii) what comparison or results do you mean? I recommend to rewrite this sentence.
Response: The sentence has been re-written.
- Lines 102-103: Is there any data on their origin? Do you know their collection sites, especially for the two wild species you studied? Is there an online database? Or maybe you have some internal data on these accessions that you can add to the supplement? I understand that you do not studied accessions in terms of ploidy and geographical distribution but in the introduction and discussion you stated that understanding the ploidy of these species is important for developing strategies for crossbreeding and genetic improvement. I think the origin of these accessions will also be important to breeders.
Response: We thank the reviewer for this observation. The geographical origin of the accessions have been included in the Supplementary Table 1. There is an online database (genesys-pgr.org/c/yam) where the information on all accessions is provided and can be made available to anyone who requests for information. This information has been included in the revised manuscript. We also have internal morphological data on these accessions since routine characterization of the accessions are carried out at the Genetic Resources Unit, IITA however we did not include any additional data for this study as it is beyond the scope of the manuscript.
- All latin names should be in italics.
Response: We appreciate this observation of the reviewer, and all latin names have been italicized in the revised manuscript.
