Comprehensive Identification of Reliable Reference Genes for qRT-PCR Normalization of Fusarium oxysporum-Resistant Genes’ Expressions in Lilium sargentiae Wilson
Round 1
Reviewer 1 Report
I have included specific comments in the reviewed PDF document provided. I think the authors need to elaborate more on why this approach should be used instead of RNAseq, since the authors claim that the results obtained by qPCR validation are mostly congruent with the RNAseq results. Also, I have detected several charts that do not correspond with the manuscript's text, which should be revisited and perhaps reinterpreted.
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Author Response
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Reviewer 2 Report
11. How author identified the name of strain isolated from diseased plant?
22. In the line number 89 to 91 author has mentioned filtration of spore suspension form potato sucrose broth. How direct filtration of spore could be achieved and what about the mycelia?
33. The references in the result section are not appropriate and should be removed.
44. In the line number 218 to 220 of result section the interpretation of result is not matched with the Figure 4d.
55. The Ct value above 32 is not considerable. So, better to remove the data of GAPDH gene.
66. In materials and method section, line number 79-80, fungal isolation has been done. Author needs to mention the protocol used for the isolation of fungus and its identification.
77. In material and method section, subheading 2.2 should be replaced by more appropriate one.
88. In line number 88-91, the cultivation of fungus has been directly shown in liquid medium without any prior culture on solid media. Author should explain how spore suspension has been obtained from liquid media containing mycelial growth.
99. In Figure 1, the legend should contain name of all genes alongwith their respective figure number. Also, the graph is not clear. Author should replace the same.
110. In Figure 3b, y-axis is not labelled.
111. In Figure 4, graphs are not clear and should be replaced.
112. The conclusion part should be elaborated and included with some future prospects also.
113. What is the rationale of you research work and how this study fills gap in the existing scenario?
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Response to Reviewer 1 Comments
Point 5: How many plants?
Response: We collected about 20 wild lily plants from the same field. The tissue culture seedlings of wild L. sargentiae with the same genetic background were used in the experiment. The number of tissue culture seedlings was shown in 2.2.
Reply:
This information (20 plants etc.) needs to be in the MS; please write it.
Also in the new text – I assume “molecular fingerprint” means that you have isolated DNA from the fungi, amplified certain genes and sequenced them? If so – this must be written – which genes, according to which protocols. Also, the genes’ sequences must be uploaded to NCBI and referenced in this paper.
Furthermore, can you add a few words about “isolated pathogen was purified”? The protocol for the isolation of F. oxysporum from plant tissue should be desribed here briefly, in a sentence or two.
Point 11: Manufacturer?
Response: Potato sucrose liquid medium was formulated by ourselves according to the references.
Reply:
What reference? Please include it in this sentence:
The F. oxysporum isolates were cultivated in potato sucrose liquid medium on a shaker (100 rpm/min) for 15 days [REFERENCE]
Point 13: I assume you have used cell counting chamber? Which one?
Response: We use the hemacytometer (blood cell counter).
Reply:
There are several types of hemocytometers, like Bürker-Türk, Neubauer, Thoma etc. That was what I was asking.
Point 25: What does miscellaneous in this context mean? "Several" peaks? Or something else?
Response: “miscellaneous” means the non-special peaks outside the main peak in qRT-PCR melting curves.
Reply:
You misunderstood me; “miscellaneous” is not appropriate here. I suggest “secondary peak” or “non-specific peak”.
Point 26: Why do you mean by primer stability?
Response: Because the qRT-PCR melting curves of most candidate reference genes showed a single peak, indicating that the primers are highly specific and stable. However, in some samples, the GAPDH has a miscellaneous peak. So it indicated that the GAPDH primer was not highly stable ( the GAPDH primer cannot exhibit a single peak in all samples.)
Reply:
You mean “primer specificity” and “gene expression stability”. Please change in the text where appropriate.
Point 28: What do green curves represent? I.e. why are all of them red, except for these few at GAPDH which are green?
Response: The green curves represent that the GAPDH has a miscellaneous peak in some samples, indicating that the primer specificity of GAPDH was not very good in some samples. The other 8 genes primers were highly specific with a single peak in all samples, so their melting curves were red.
Reply:
You are never referencing Figure 1 in the text. Please remedy that. I suggest somewhere in 3.1. These green and red coloured curves need to be explained in the Figure 1 caption.
Point 30: Needs much better figure caption. What do boxes represent? And what do whiskers represent? Also y axis is shortened, this needs to be mentioned here. How was this calculated - info needs to be added in M&M
Response: We have annotated Figure 2 in more detail “The box graph represents the 75th to 25th percentiles and the line across the box represents the median. The whiskers on each box represent the minimum and maximum values, circles represent outliers.”. In addition, we have given explanations and references in M&M 2.5 section “The Ct (cycle threshold) values of each gene were computed automatically [21]. ”
Reply:
You have omitted to address the shortened y-axis. Please correct that.
Point 35: Is this necessary? You showed only rankings from other programs and gave the values in table. Perhaps this is enough for GeNorm as well?
Response: It is a very good advice. However, we think that there were some reasons to keep Figure 3(a) : We can get the information “8 of these reference genes had M values lower than 1.0, except for GAPDH.” directly from Figure 3(a). Table 2 provided a more detailed M value and ranking of all reference genes, based on the M value and ranking, we can conclude that “HIS and EF-1α were the two most stable reference genes, with M values of 0.533 and 0.535, respectively”. Certainly, we can also delete Figure 3(a) according to your final suggestion.
Reply:
I understand it is visually impactful. Nevertheless, as I said, the information is given in the table below, so is not necessary here. Please remove Figure 3. a and refer in the text to Table 2.
Point 38: FPKM 6 columns in supplementary? If so, needs to be elaborated. Also, in fig 4 there are only qPCR results, not their comparison with RNA-Seq.
I suggest you add RNA-Seq results in the statistic and compare it and show the results.
Frankly, I do not see that the pattern is the same in RNA-Seq and here. Furthermore, I do not know if I am correctly interpreting FPKM columns from supplementary materials, another issue that needs to be addressed.
Response: Yes, the notes "L_24_T_f and L_24_T_w represent the 24 h samples of L. sargentiae with F. oxysporum and water, respectively; 1, 2 and 3 represent the 3 different replicate samples, respectively" was shown below Table S1.
It is very helpful advice. However, the number of samples for qRT-PCR was different from RNA-Seq. The samples for qRT-PCR were collected 6 different times (0h, 6h, 12h, 24h, 48h, and 72h), while RNA-Seq samples were collected only one time (24 h). So we think that it is not appropriate to add RNA-Seq results and compare with qRT-PCR results. In addition, we have provided more detailed data (FPKM of RNA-Seq) in Table S1, we will have to delete Table s 1, if add RNA-Seq results in the statistic.
We have rewritten this paragraph in the article.
Point 39: I am not seeing it. What do you mean by this?
Response: The meaning of this sentence is that the expression patterns of ftarget genes by qRT-PCR were the same as that of RNA-Seq, on the 24 h time-point. We have rewritten this paragraph in the article.
Reply:
I do not understand, in point 38 you mention “The samples for qRT-PCR were collected 6 different times (0h, 6h, 12h, 24h, 48h, and 72h), while RNA-Seq samples were collected only one time (24 h). So we think that it is not appropriate to add RNA-Seq results and compare with qRT-PCR results.”, but here you state “the expression patterns of ftarget genes by qRT-PCR were the same as that of RNA-Seq, on the 24 h time-point.” It cannot be both ways, either the 24h point is taken into account and compared with qPCR results statistically or not. If not, it should be completely omitted.
Point 40: Statistics? Needs to be added in M&M.
Response: We have provided statistical analysis method and references “Their relative expression levels were computed according to the 2−ΔΔCt method [34]” in 2.5 of M&M section.
Reply:
That is not the explanation of the statistics I have asked for. See my comment below.
Point 42: I do not understand what do asterisks represent? Stat. sign difference between what exactly?
Response: P-values reflect the likelihood of an event occurring. P < 0.05: the probability of occurrence by chance is less than 5%, there are generally statistically different between two groups; P < 0.01: the probability of occurrence by chance is less than 1%, there are statistically significantly different between two groups; P < 0.001 : the probability of occurrence by chance is less than 0.1%, there are statistically extremely significantly different between two groups. In Figure 4, we use *, ** and *** represent p<0. 05, p<0. 01 and p< 0. 001, respectively.
Reply:
You misunderstood me. I am quite well aware of what statistical significance, p-values and asterisks mean generally. I am asking you what they specifically mean here to you here? What did you compare with what to get these statistical differences (p-values)? What statistical test did you use? Yes, I know, you have written “Microsoft Excel 2016 and Data 159 Processing System were used for data analysis and statistics [37,38].”. However, that is not enough. I reiterate – please write specifically which values were compared with which (0 h vs. other time points? W vs. f inoculations? Or something completely different. Ref [37] mentions one-way ANOVA followed by post hoc Tukey HSD. Did you use this here as well? Did you compare geometric means of Ct values at different time points? As you can see, it is completely unclear what did you do here and this must be remedied.
Author Response
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Author Response File: Author Response.docx
Reviewer 2 Report
The authors have provided the rebuttal for the minor comments, however major concerns still remains unaddressed.
Author Response
We have improved the article again according to your previous suggestions.
Round 3
Reviewer 1 Report
Only a few smaller points remain:
Response: Yes, the ribosomal DNA-ITS (GenBank: GU371875) sequence of Fol-2 was isolated and used to identify F. oxysporum strains. We have added this information and a new reference to the Materials and Methods section.
This is the reference gene, not de novo sequenced IST region from your isolated samples. I assume you have isolated several F. oxysporum samples from 20 plants that you have mention and thet you have confirmed what they are by sequenceing? Well, these sequences must be uloaded to NCBI.
Or are these 20 plants sampled in 2009 and the NCBI entry GU371875 is actually the sample from 2009 with which you are working now? Either way, in result you are no longer mention this isolation of the fungal pathogen, the isolates obtained, or any result regarding the section 2.1. from M&M.
Response: We have added the sentence “The pathogen was isolated and purified by conventional tissue separation and agar plate dilution” and a new reference to the Materials and Methods section.
Reference [34] is in Chinese and unavailable as a full text (only the abstract is available). So I am trusting you that in the reference the procedures are described in detail.
Response: We have added the manufacturer information ( Shanghai Qiujing Biochemical Reagent and Instrument Co., Ltd, China) of the hemacytometer in the Materials and Methods.
My original comment: There are several types of hemocytometers, like Bürker-Türk, Neubauer, Thoma etc.
My new comment: Types, not manufacturer, but honestly, whatever.
Response: I'm sorry I still don't fully understand what you mean. I guess you mean the smallest number should be 0 on the Y-axis. In this study, the Ct values of the 9 reference genes varied from 20.21 to 34.82, areas below 20 are blank. As a consequence, we primarily show this local area of 20 to 35 in Figure 2. If we offer all local areas from 0 to 35, the box graph and the cross line will not display clearly in Figure 2. Now, at your suggestion, we have replaced the original Figure 2 with a new one in the text.
Yes, exactly, you cannot and should not shorten y axis for any reason. It is gross misrepresentation of the results and not only should it be avoided, but actually never used. I understand that it does not look spectacular as it would have been with shortened (20-35) y axis, but this is the only correct way to represent these results.
Response: I apologize for misunderstanding you.
In theory, the gene expression level is the lowest in 0 h time-point with sterile water treatment (0 hW), so 0 hW was used as the control in Figure 4,
Not the case for CHI, but OK, 0h is a good point of reference. It is understandable now. I see you have written this in Figure 4 caption, please add this explanation to statistics in M&M section.
other time points(each time period inoculation with F. oxysporum or sterile water) was compared with 0 hW. The p-value was the difference between the sample marked with asterisks and the control (0 hW). We use the same statistical method as Ref [37] which mentions one-way ANOVA followed by post-hoc Tukey HSD.
This as well should be explained in the text, in the M&M section where you explain the statistics, not only in figure caption.
Author Response
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Author Response File: Author Response.docx
Reviewer 2 Report
Accepted for publication.
Author Response
Thank you for your help in revising our article.
