Stepwise Optimization of the RT-qPCR Protocol and the Evaluation of Housekeeping Genes in Pears (Pyrus bretschneideri) under Various Hormone Treatments and Stresses
Round 1
Reviewer 1 Report
The presented article is devoted to choose the stably expressed reference genes for RT-qPCR of pear plants under various abiotic and biotic treatments. This study is relevant and has some practice novelty. Unfortunately, there are both insignificant and significant remarks, as well as issues that should be taken into account.
1) Add of the author-classifier to the genus and species names plants. In addition, for the first time in the text, the generic name should be presented in full, while further in the text it should be abbreviated.
2) All genes should be written in italics.
3) Be careful when abbreviations are first mentioned, such as ABA, ETH etc. (line 71). For example, the abbreviation first occurs without explanation. On the contrary, the abbreviation with the decoding is found below twice.
4) In addition, the following methodological aspects are absolutely not clear. How many control and treated plants (or leaves?) were used for RNA isolation for each experiment? What order of leaves were used?
5) Line 94: in vitro stem segments?
6) Figure 1. What kind of band (around 500 bp) is detected to the left of track 1?
7) Figs. 3, 5 are of poor quality. Figures are not readable.
8) In the Figures 7C, I recommend specifying scale (bar).
9) Fig. 8. The name of the x-axis is missing.
10) Line 491. B-tubulin → β-tubulin
Best regards.
Author Response
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Reviewer 2 Report
Review of the manuscript “Stepwise optimization of RT-qPCR protocol and evaluation of housekeeping genes in pears (Pyrus bretschneideri) under hormone treatments and various stresses”
The search for reliable reference genes is an urgent problem in molecular biology. The authors tested 8 potential reference genes of pears under various stress conditions and provided recommendations on the use of the most appropriate reference genes for specific experimental treatments.
There some comments to the manuscript:
In Abstract the authors should list all studied reference genes.
In Introduction - lines 70-71 the authors should add full name of hormones ABA, ETH
2. Materials and Methods 2.1. Plant materials and treatments
lines 85-87 – the authors should add full name of hormones MeJA, SA, ABA, ETH
line 100 – the authors should add full name of IBA
In Table S1 - the authors should add the name of the gene product, for example ACT – actin
In Figure 1, Figure 2 and Figure 3 captions – the authors should add the object and conditions (pear leaves / control conditions)
Figure 4, 6, 7, 8 - the authors should add the object
Conclusions – lines 497-498 – abiotic stress was studied not on leaves
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Author Response
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Reviewer 3 Report
This is an interesting paper reporting on the testing of different genes that could be used as expression reference genes in qPCR experiments, especially those looking at stress treatments.
Carefully review results presented in tables with what is written. There are several discrepancies. In Abstract YLS8 is not the most valuable for water deficit, GAPDH is ranked in the bottom 2 for all tested treatments except heat and MeJA. EF1a was not the best for cold treatment and ranked third for some treatments.
Introduction.
First sentence needs a reference.
Spell out the acronyms the first time they are mentioned in the introduction.
Line 69. What genes?
Line 122. not 20 - 23 bp, the primers were 17 - 20 bp in the table.
Thank you for confirming qPCR products with sequencing and PCR efficiency. Excellent work and demonstrates a high standard of experimental design.
Lines 159-169. This section was unclear to me. Can it be reworded for readers that are unfamiliar with the process?
Figure 1 could be cropped and moved to supplementary. If possible to reverse the black/white colouring to make it easier to see.
Table 1 legend. 'List of genes'.....
Figure 2 could also be moved to supplementary.
Lines 225-226. Where do the values come from following the gene?
Table 2 and Table 3.
Suggest moving the RefFinder results to their own table.
Don't number the genes on the left side as the genes are ranked differently for each program and the numbers suggest they might be in the same order.
The values against the genes, is there a way of comparing them, eg Least Significant difference? While there are slight differences in the values maybe for some genes there is no significant difference between the top 3 so any of the genes could be used?
GeNorm results. The top 2 genes have a single number against them. Is there an issue with the styling? If they both have the same number then maybe list it twice.
Figure 4. Put a line across at the 0.15. This will make it easier for the reader to see which values fall above or below. This section needs more explaining. Why are only 7 genes compared? Is this saying that for the mock/controls you only need 2 reference genes, but for SA you should use 3-4 reference genes?
Line 318. Where do the values with the genes come from? Also in section 3.5.2.
Line 347. Put the TUB and WDP together e.g. TUB/WDP so that it is more obvious that they are together for NaCl treatment.
Section. 3.5.3. The values don't match the graph/tables.
Section 3.5.4. WDP was within the top 3 rankings for the abiotic stresses, but not always the most stable, unless a Least Significant Difference shows no difference.
Line 371. Abiotic and Biotic stresses.
Figure 6. Is the vertical axis correct? Put a line across for 0.15.
Section 3.5.5 Check this section that the correct genes are listed for each stress. Genes listed are not always the top and bottom ranked genes, why not? Cold WDP top, but ACT was bottom, SKD1 was ranked 3rd. Heat YLS8 and EF1a are the top/bottom, not WDP and ACT.
Line 398. The Figures are not correctly matched to the labels.
Figure 7. It would be good to include LSD's or trend lines to further highlight differences between stable and unstable reference genes.
Line 450. ACT is ranked at the bottom for cold and water stress.
Line 473. Name the other plant species.
Line 487. While WDP and ACT were suitable for some treatments, alternative reference genes ranked first for other treatments.
Figure S1. Note that for 3 genes only 3 data points were used to calculate the efficiency equations.
Table SB. Include gene names. Is it the same Control as for A?
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear authors,
Thank you for the corrections.
Best regards.
