Identification and Characterization of Salt-Responsive MicroRNAs in Vicia faba by High-Throughput Sequencing
Round 1
Reviewer 1 Report
Although the topic itself is interesting, the method cannot be said to be described clearly. For example, for DES identification, authors denoted their methods in detail at "2.8. Screening the Differentially Expressed sRNAs (DESs)". On the other hand, there is a description "In this study, we employed the DESeq2 and ExpDiff methods for screening the DESs." at L241. Which is correct?
Also for target prediction "In this study, numerous software were used to identify the possible targets" (L136) "numerous software" cannot help the readers to reproduce the results. Please describe all methods detailed enough to be able to be reproduced, although I am not wiling to list all of them.
Author Response
Reviewers-1 comments | |
Although the topic itself is interesting, the method cannot be said to be described clearly. For example, for DES identification, authors denoted their methods in detail at "2.8. Screening the Differentially Expressed sRNAs (DESs)". On the other hand, there is a description "In this study, we employed the DESeq2 and ExpDiff methods for screening the DESs." at L241. Which is correct? | We corrected this. DESeq2 deleted from the result section |
Also for target prediction "In this study, numerous software were used to identify the possible targets" (L136) "numerous software" cannot help the readers to reproduce the results. Please describe all methods detailed enough to be able to be reproduced, although I am not wiling to list all of them. | We corrected this paragraph and deleted “numerous software were used to identify the possible targets" (L136)” |
Reviewer 2 Report
The authors performed a genome-wide survey of miRNAs possibly involved in the salt response of Vicia faba by RNA-seq. Although the topic itself is of significance, the manuscript is not very informative. The experimental design and data presentation need to be substantially improved.
My major concerns include:
The current manuscript lacks experimental validation of the differentially expressed miRNA and its putative targets. At least, the most important salt-responsive miRNA families and their corresponding targets identified in this study should be validated by qRT-PCR.
Functional analysis (GO and KEGG) of miRNA putative targets is too general, and didn't provide details regarding which physiological/cellular/metabolic processes are, in particular, responsive to the salt treatment.
The authors could further improve the manuscript by comparing the salt-responsive miRNA profiles between the salt-sensitive and salt-tolerant genotypes. This will help to identify the particular miRNA families that contribute to the different salt responses of the two genotypes.
Here are some detailed comments:
Introduction:
Literature is not up-to-date. Recent research progresses in the miRNA-regulated salt responses should be included.
Materials and methods:
How frequent was the salt solution applied to the plants?
Does the salt treatment of 35 days have literature support? I will assume this treatment is too long that the secondary responses/global responses were already induced, resulting in some of the miRNA detected in the study was not directly involved in salt stress, but other secondary responses.
Were all the existing leaves on the plants collected for RNA extraction, or instead, particular leaves were collected?
Result:
Change the sample names to something more indicative like"salt-sensitive-control, salt-sensitive-salt, salt-tolerant-control, salt-tolerant-salt" will make it easier to understand.
Why the percentages of mapped tags in IC-1 and IS-4 samples (~50%) are much less than those of HC-4 and HSA (~70%)? is this due to the genotype differences or sRNA library construction issue?
Discussion:
How was the salt stress-related miRNAs identified in this study compared to those in other plant species, particularly in legume plants? This will provide more insights into the conservations of salt stress-related miRNAs in the phylogenetic context.
Author Response
Reviewers 2 comments | Rebuttal |
The current manuscript lacks experimental validation of the differentially expressed miRNA and its putative targets. At least, the most important salt-responsive miRNA families and their corresponding targets identified in this study should be validated by qRT-PCR.
| As this is a first report of miRNA in faba bean, so we are reporting only initial findings of this study. We will also do the validation of important salt responsive miRNA families and their corresponding targets in details by qRT-PCR and would like to report these in separate article. |
Functional analysis (GO and KEGG) of miRNA putative targets is too general, and didn't provide details regarding which physiological/cellular/metabolic processes are, in particular, responsive to the salt treatment | Details have been given in Supplementary Table S6. |
The authors could further improve the manuscript by comparing the salt-responsive miRNA profiles between the salt-sensitive and salt-tolerant genotypes. | The numbers of differentially expressed miRNA families have been described in Table 4 by comparing the salt-responsive miRNA profiles between the salt-sensitive and salt-tolerant genotypes. |
Literature is not up-to-date. Recent research progresses in the miRNA-regulated salt responses should be included | It was updated. Latest references were added |
How frequent was the salt solution applied to the plants? Does the salt treatment of 35 days have literature support? I will assume this treatment is too long that the secondary responses/global responses were already induced, resulting in some of the miRNA detected in the study was not directly involved in salt stress, but other secondary responses. |
Samples were taken after two weeks of salt treatments. It was incorporated. |
Were all the existing leaves on the plants collected for RNA extraction, or instead, particular leaves were collected? | All leaves were extracted and grounded for RNA isolation. We 100 mg powder for RNA extraction |
Change the sample names to something more indicative like"salt-sensitive-control, salt-sensitive-salt, salt-tolerant-control, salt-tolerant-salt" will make it easier to understand. | Generally the given names were used. |
Why the percentages of mapped tags in IC-1 and IS-4 samples (~50%) are much less than those of HC-4 and HSA (~70%)? is this due to the genotype differences or sRNA library construction issue? | It might be due to genotype differences. |
How was the salt stress-related miRNAs identified in this study compared to those in other plant species, particularly in legume plants? This will provide more insights into the conservations of salt stress-related miRNAs in the phylogenetic context | One comparative paragraph was added |
Round 2
Reviewer 1 Report
The authors addressed all the concerns.
Reviewer 2 Report
Thanks to the efforts the authors have made to improve the manuscript. Most of my concerns were addressed in this version. Although the authors didn't supply experimental validation of the differentially expressed miRNA and its putative targets, it is still acceptable considering the adequate information this study provided. I think the current version is ready for publication on Genes.