DNA Methylation and mRNA Exon Sequence Variations in the Salt Stress Adaptation of Paspalum vaginatum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript evaluates the changes in the DNA methylation pattern and the transcriptome under salt stress in Paspalum vaginatum. I have several concerns, in the title the authors mention that they evaluate RNA mutation and in the text talk about RNA variability when the study is a transcriptomic analysis to search differentially expressed genes in response to salt stress. I feel there is confusion about the terms, which is important to clarify to edit properly in the manuscript.

Moreover, deletions and insertions are mentioned to be detected in transcriptome but I think that these refer to exon variants, its better clarify in the text because just as it is explained seems that RNA mutates

Abstract

line 13, RNA but is mRNA, rRNA or modification of RNA?

line 16, add what was the tissue studied

line 21, "New RNA variants", do you refer to differentially expressed genes, RNA modifications, splicing isoforms or what?

INTRODUCTION

In general, the introduction lacks description of relevant information about the topic, why is the importance of this plant model to understand salt stress epigenetics, and a better description of antecedent related to the topic such as DNA methylation and transcriptome in plant salt stress is poorly described, without mentioning the potential applications of understanding epigenetic mechanisms to response of abiotic stress in management; include relevant transcriptomic analysis on salt stress to link epigenetic changes with transcriptome modulation, so I recommend to include studies in the field; parameters of filtering is lacking;

MATERIAL AND METHODS

Section 2.2 there are lacking several information regarding purification RNA method, details about library to get the reads for transcriptomic analysis

Section 2.3, details about DNA extraction methods, libraries and others are lacking.

RESULTS

lines 220-226, It is not clear for me how the gene quantification was done, I expect that down- and up-regulated genes were identified by comparison of time 1-day or 3-day with day 0; but in day 0 there are differences mentioning that 7266 genes were not expressed but 21446 does. Please clarify because the results are confusing

lines 237-239, delete

lines 251-255, there is no comparison between 3-day and CK transcriptomes, which could be give some interesting results, maybe a Venn diagram can be added for an easy visualization of down and up-regulated genes

section 3.2. 

lines 260, what refers with RNA variants? splicing isoforms or RNA edition? the deletions, insertions and SNPs can be product of splicing or why occurs?

figure 4B, gray color line indicating TE density is poorly seen

There are two figures labeled as 4, but in text is named as 5, please renumbered the figures and correct

The results are focused in showing where methylation was higher in the genome, but what happened in the regions where methylation is reduced? for example, in lines 309-310 is mentioned that elevated methylation is observed in salt tolerance genes such as fatty acids metabolism, but if there is a higher methylation this implicates that this genes are silenced and how contribute with salt resistance? this is contradictory with the logical

In table 2, RNA mutations affect the functional role of protein?

 

Minors

Capital letter where is not required, edit

line 358 variation

361, beened

Table 2 is labeled as 1, adjust the column width

364 thest

Italics in scientific names

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is interesting but has a series of problems that must be revised by authors. In the title and indeed in other parts of the text where used in reference to RNA; the term "mutation" should be replaced by "variants" (as in other sections) or "variation". Mutation is most properly applied to RNA when this nucleic acid is the genetic material of a biological entity, such as a virus.

In Introduction, the construction "capacity to influencing" must be revised by an English corrector with expertise in molecular biology, and the same is valid for the full manuscript (for instance, the concept of "plethora", "gene body", "conjunctiono", ".Based on speculation", "To rapidly respond to salt stress while DNA is less prone to mutation in order to maintain genetic stability, we hypothesized that the superiority of RNA in terms of the number of variations...", "vairations", among others).

Lane 64, Arabidopsis is not a crop but a model plant species. Lane 89, the term "correlation" is mentioned, buy when presenting the methodology in next section, a co-occurrence analysis, no a correlation analysis, was indeed done.

In material and Methods, the experimental design is not clear. There appears in a first assessment that CK, 1d, and 3d are experimental conditions and each of them has their respective controls, but then, in Results, CK appears to be the control and just 1d and 3d the treatments. If so, how were the different transcripts that could be expressed in these treatments according to the time from CK distinguished from those caused by salt? And independently of this fact, which was the number of stolons from which RNA was extracted? Is "SeaIsle200" a uniform germplasm or it has at least an extent of genetic variability? It is an important fact to be clarified because in section 2.4, "variant at the locus", "minimum allele frequency", "population filtering", and "variants on exons of all genes" are mentioned, and these concept are relevant only when genetic variability is present. In section 2.6, the correlation analysis must be revised, as preciously expressed.

In Results, Lane 220 to 225 must be revised according to the correct experimental design that was applied. For instance, the phrase "Quantitative analysis of gene expression levels revealed that, at the initial stage of salt stress (day 0), 7,266 genes were not detected as expressed, whereas 21,446 genes exhibited expression activity." in respect to what experimental controls, provided that, as previously observed, day o (or CK) was the experimental control? In lanes 237-239, the strange phrase "This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn." appears. Please, verify its validity. Figures must be improved, and in Figure 1 B, the percentage of explained variation by each principal component must be indicated, as in Figure 1 A. Also, which was the quantitative variable used for the PCA analysis shown by Figure 1 B must be explicit. In section 3.2, it is explicit that CK was the experimental control; hence a revision of previous information must be done. The same is necessary for section 3.3, according to the correlation or co-occurrence analysis.

Them Discussion must be adjusted to modification in previous sections and improved. It is not a comprehensive discussion and the last phrase is more properly a Conclusion (a section that is absent in the present version). 

Comments on the Quality of English Language

In addition to the mentioned problem, English must be broadly improved. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This new version of the manuscript has been substantially improved by considering all suggestions I had pointed out. The manuscript is now acceptable for publication. Nevertheless, a new assessment of English language scientific quality is recommended.