Selection of Suitable Reference Genes in Pinus massoniana Lamb. Under Different Abiotic Stresses for qPCR Normalization
Round 1
Reviewer 1 Report
How to validate the reference genes for the expression analysis under abiotic stresses is explained and shown in the article "Selection of suitable reference genes in Pinus massoniana Lamb. under different abiotic stresses for qPCR normalization".
This approach also can be implied in other plant species to find the reference genes. This article is well articulated. However, in the introduction, the last sentence of the second paragraph seems disconnected from the rest of the paragraph. Please rewrite or elaborate it differently.
There are a couple of published articles on the selection of reference genes in different plant species. So, this article has its own merit for the study of Pinus massoniana. However, there are a few points that need to be considered before the publication.
1. On what the basis 12-candidate reference genes were selected? Please elaborate as have done in other published papers: Zhang et al., 2018; Tang et al., 2017, etc.
2. Figures 1 and 3 do not have publication quality, poor resolution, please improve the quality.
3. Tested reference genes are for the 45 old seedlings, can these be used for other parts ofPinus massoniana such as a leaf, shoot, or root or can be used in other plant species for the same abiotic stresses? If not, then reference genes need to be re-evaluated every time depends on the specificity of the experiment, please explain more about this in the discussion section.
4. The author has chosen geNorm, NormFinder, and BestKeeper over the other statistical algorithm such as RefFinder and the Comparative ΔCq method. Is there any specific reason?
Author Response
Please see the attachment.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The manuscript deals with the establishment of reference genes on Pinus massoniana under several abiotic stresses, and after expression genes were determined for the best and worst performing references genes.
The manuscript is very well- written and organised, and technically sound, following statistical applets to address reference stability values.
However, i have concerns, namely:
L93-95- The authors mention that the 12 candidate reference genes were acquired from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), how this was done? Besides, and importantly, why not simply using the primers already developed by reference 22, where they in fact have done a thorough study using 25 reference genes including analysis in abiotic stresses? This fact should be clarified.
How many biological replicates have been done for each condition?
L135- How were this gene of interest selected?please include such information.
L136- have you included the reaction efficiency on the calculation of relative expression by Pffalf method? How was the calculation done with more than one reference gene?
On data analysis sub-section (L116), is lacking the information regarding how was the final ranking of reference genes done as showed in Table 3, after the results obtained from the 3 softwares (GeNorm, NormFinder, BestKeeper).
On Figure 2, it would nice the include a line where the threshold value of 0.15 is represented, for better reading of the figure.
It is not clear the information of the reference genes used to normalize in each condition. For example, L224-227, if we see table 3, genes selected from the ranking of salt tolerance ACT e F-box appear to be the most stable, however, in the abstract is stated actin 2 (ACT2) and cyclophilin (CYP) as the most stable. This issue should be clearly clarified. Have you done the geometric mean of the ranking to achieve the final ranking provided in table 3?
Author Response
Please see the attachment.
Author Response File: Author Response.doc
Round 2
Reviewer 2 Report
The authors have improved significantly the manuscript addressing positively all concerns raised in the first revision.
I would recommend its publication.
