Locating Appropriate Reference Genes in Heteroblastic Plant Ottelia cordata for Quantitative Real-Time PCR Normalization
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for your manuscript entitled "Locating Appropriate Reference Genes in Heteroblastic Plant Ottelia cordata for Quantitative Real-Time PCR Normalization". In this manuscript, you identify the reference genes for Ottelia cordata by selecting from seven different gene families. The information provided in this manuscript is certainly original and the data are not yet present in the literature; therefore, I believe that in this sense the manuscript will be of great interest to the scientific community.
The introduction is well written and offers enough background for readers. In general, the entire manuscript is well structured, the methodology is well detailed, as well as the description of the results. However, all figures need to be improved: the numbers and writing are too small, illegible, and grainy.
Discussion and Conclusions
The first part of the discussion, from lines 335 to 352, seems to be a summary of the results obtained and could be eliminated or streamlined a bit. Instead, the remaining part of the discussion is presented very well, the citations and references are congruent with the topic and with the journal format. The conclusions are clear and appropriate.
Recommendations
I compliment the authors on how they dealt with such laborious and detailed procedures. I have no more suggestions to improve it, although I am not a native English speaker, it seems well written in English. My recommendation is to accept the manuscript for publication after including these minor suggestions (See attached manuscript).
Comments for author File: 
Comments.pdf
Author Response
Commit 1：Thank you for your manuscript entitled "Locating Appropriate Reference Genes in Heteroblastic Plant Ottelia cordata for Quantitative Real-Time PCR Normalization". In this manuscript, you identify the reference genes for Ottelia cordata by selecting from seven different gene families. The information provided in this manuscript is certainly original and the data are not yet present in the literature; therefore, I believe that in this sense the manuscript will be of great interest to the scientific community.
The introduction is well written and offers enough background for readers. In general, the entire manuscript is well structured, the methodology is well detailed, as well as the description of the results. However, all figures need to be improved: the numbers and writing are too small, illegible, and grainy.
Discussion and Conclusions
The first part of the discussion, from lines 335 to 352, seems to be a summary of the results obtained and could be eliminated or streamlined a bit. Instead, the remaining part of the discussion is presented very well, the citations and references are congruent with the topic and with the journal format. The conclusions are clear and appropriate.
Recommendations
I compliment the authors on how they dealt with such laborious and detailed procedures. I have no more suggestions to improve it, although I am not a native English speaker, it seems well written in English. My recommendation is to accept the manuscript for publication after including these minor suggestions (See attached manuscript).
Response 1: Thank you for your patient revisions to the manuscript, all details have been modified as your suggestions in the PDF file and highlighted in the revised version.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsI reviewed the manuscript “Locating appropriate reference genes in heteroblastic plant Ottelia cordata for quantitative real-time PCR normalization” (horticulturae-3449848). The author’s idea is interesting, and the results obtained seem to be sustained with experimental evidence. However, some additional information must be included.
Suggestions:
1) GeNorm, NormFinder, Comparative ΔCt, BestKeeper, and comprehensive analysis were used to evaluate reference genes. Why are required all these evaluations? Is there an official protocol to perform this evaluation?
2) Figure 1. The axes labels are not clear. The quality of the Figure must be improved.
3) Page 7. Line 250, and Figure 4. I suggest defining 2/3 (Is V2/V3?) which corresponds to?
4) Section 3.6. The paragraph indicates that it was estimated SD and CV values, however the CV values are not included. It is important to include the sample size (n=?)
5) PP2A and UBQ were found to be useful for the precise qRT-PCR. What about accuracy? Considering that the technique is quantitative, it would be useful to evaluate the accuracy of the results, or the authors should propose a strategy to evaluate if the results obtained were valid.
Author Response
Comments 1: GeNorm, NormFinder, Comparative ΔCt, BestKeeper, and comprehensive analysis were used to evaluate reference genes. Why are required all these evaluations? Is there an official protocol to perform this evaluation?
Response 1: There was no official protocol to perform this evaluation. The conclusions obtained from each analytical method may vary. In many other similar studies, at least three analytical methods are usually chosen to combine their results and obtain more stable reference genes. We adopt more analytical methods, and by analyzing more data, we can reach a more reliable conclusion.
Comments 2: Figure 1. The axes labels are not clear. The quality of the Figure must be improved.
Response 2: Figure 1 is a screenshot taken directly from the software interface. We have re-edited the group axis labels according to the original information and modified the image.
Comments 3: Page 7. Line 250, and Figure 4. I suggest defining 2/3 (Is V2/V3?) which corresponds to?
Response 3: We have changed “2/3” to “V2 / V3” in text and Figure 4.
Comments 4: Section 3.6. The paragraph indicates that it was estimated SD and CV values, however the CV values are not included. It is important to include the sample size (n=?)
Response 4: The trends of CV and SD are identical; therefore, we use only SD to assess the stability of candidate reference genes. Similar strategies have been employed in other studies, as mentioned in the revised manuscript (Line 271 ~ 273). We have added the sample size in text (Line 282 ~ 284).
Comments 5: PP2A and UBQ were found to be useful for the precise qRT-PCR. What about accuracy? Considering that the technique is quantitative, it would be useful to evaluate the accuracy of the results, or the authors should propose a strategy to evaluate if the results obtained were valid.
Response 5: PP2A and UBQ are indeed more accurate reference genes. We have verified their accuracy in the manuscript (Line 327 ~ 337). As shown in Figure 9, when PP2A or UBQ were used as reference gene, the results were consistent with the RNA-Seq data. In our ongoing work on differential expression gene screening, these two reference genes, PP2A and UBQ, have shown higher accuracy, which can better help us determine the levels of gene expression in different materials.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors
The subject of this Ms is of interest. However, the text need some revision and clarifications. I made suggestions and required clarifications throughout the text. Sample preparation is a critical point as well as the calculation of the reactions' efficiencies.
Kind regards
Comments for author File: Comments.pdf
Needs revision
Author Response
Comments 1: The subject of this Ms is of interest. However, the text need some revision and clarifications. I made suggestions and required clarifications throughout the text. Sample preparation is a critical point as well as the calculation of the reactions' efficiencies.
Response 1: Thank you for your patient revisions to the manuscript. We have incorporated all your suggestions, with changes highlighted in the revised version.
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors
I appreciated the improvements made, but there is still a critical point that I highlighted in the text. I hope that you can give evidences supporting your findings.
Kind regards
Comments for author File: Comments.pdf
Author Response
Comments 1: I cheked reference 16 and found: "...PCR efficiency101/slope1, when the logarithm of the initial template concentration (the independent variable) is plotted on the x axis and Cq (the dependent variable) is plotted on the y axis. The theoretical maximum of 1.00 (or 100%) indicates that the amount of product doubles with each cycle". I cannot understand why you use 5 instead of 10 as is common. Can you clarify? I would also like to request the evidencies that your reactions fulfilled the criteria using the correct formula.
Reply: When constructing the standard curve, we used a 5-fold dilution for the template, rather than a 10-fold dilution, which is why the base of our formula is 5. We also attempted a 10-fold dilution, but when the dilution factor is too large, the Ct values exceed 40 and become unstable. According to reference 16 (7.4.1. PCR efficiency.), “Cq values > 40 are suspect because of the implied low efficiency and generally should not be reported; however, the use of such arbitrary Cq cutoffs is not ideal, because they may be either too low (eliminating valid results) or too high (increasing false-positive results)”, this dilution gradient may not be suitable for our template. After consulting other literature and incorporating advice from technical staff at biotechnology companies, we have learned that a 5-fold dilution is optimal when the template is cDNA, while a 10-fold dilution is more suitable when the template is plasmid DNA. In our study, cDNA was used as template. Additional explanation has been provided in the revised manuscript (Line 129 ~ 133).
Other details have been modified as your suggestions in the PDF file and highlighted in the revised version (Line 143~144, Line 198~199).
