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I believe that this latest version tried to deal with the issues associated with concerns of gene expression measurement. The rebuttal addressed this, the manuscript was updated accordingly, and an updated supplemental file set was supplied. This may provide what was needed to move the publication forward for publication. Once approved, the manuscript may be considered accepted. Thank you for your efforts.
[# PeerJ Staff Note - this decision was reviewed and approved by Julin Maloof, a PeerJ Section Editor covering this Section #]
Thank you for considering the suggested edits to your manuscript. The data provided within the supplemental files helps to address many of the earlier concerns.
Upon further review there was some question about the differential expression analysis requiring some further clarification. The manuscript states that “Gene expression was evaluated using fragments per kilobase of transcript per million mapped reads (FPKM), and the expression abundance of unigenes was obtained (Trapnell et al., 2010)." And yet in the manuscript you used DEseq to analyze the data. DEseq requires raw counts, not FPKM.
The DEseq manual states "The count values must be raw counts of sequencing reads. This is important for DESeq’s statistical model to hold, as only the actual counts allow assessing the measurement precision correctly. Hence, please do do not supply other quantities, such as (rounded) normalized counts, or counts of covered base pairs – this will only lead to nonsensical results."
You may need to clarify these finding a bit further and how they impact your findings I will place this manuscript under the minor revision cntext until this can be resolved.
The manuscript provided good discussion of gene families expressed during different phases of expression using multiple annotation methods, yet the ability for the reader to connect the discussion to actual sequence data, or a repository with the sequence data appears to be missing. It is valuable to know that the measurements were a range of the over 43,000 unigenes; but some of the assembled sequences should at least be presented. It would be of value to have the raw assembled data placed at a repository such as the European Nucleotide Archive (ENA) where assembled annotations can be stored; there may be other similar repositories.
Within the Supplemental data there appears to be some issues. I was not able to completely read the content of Table S1; is it complete? There are references to gene names, but which allele, GI reference, or other sequence reference of the gene is referred to? Especially in the context of the data provided in Supplemental table S9, and additional FASTA file should be made available (or direct links to the data). The reader needs to be able to make such comparisons.
Other comments refer to the basic language of the manuscript. It should be thoroughly reviewed with someone familiar with language context for clarity. There are additional comments in the reviews which should be noted. I will place a “major revision” status at this point; however, the issues should be readily addressed to provide a well-developed manuscript. We look forward to your revisions. I was very impressed with the good usage of metadata annotations.
This paper aims to provide new information on the genetic basis of senescence development for future applications in plant breeding.The authors provide annotation method with transcriptomics tools in order to observe and offer a novel interpretation of the molecular mechanisms of leaf senescence in alfalfa. The bioinformatics pipeline involves several standard transcriptomics tools such as bowtie2, primer primer 5, and DESeq2. The provided annotations deserve attentions in this respect. However, there are several questions that should be answered in order to improve this manuscript, and the authors should revise it with incorporating the answer of these questions:
Extensive editing of English language and style are required. English used in this manuscript was not clear and ambiguous. As for example: Line: 15,16, 28, 71, 80 (maize should not be italic) 81, 100, 101 and many other lines. The author should use professional English Language.
The background was sufficient. But the references in many places were old. Authors should use more related new references.
Besides this fundamental criticism, in the Material and Method section, the authors need to specify in more detail the plant growth conditions and the age of the samples leaves.
In line 159 :Did you use default parameters for Primer Primer 5? Please expose all the parameters that you used!
Figures were relevant and well described to the content of this article. But the resolution should be better. The title of Y-axis of Fig 2A should be in right direction.
1. Is there any biological explanation why those KEGGS have the most significant enrichment?
2. What is the version of your reference genome? For example, human genome has version number as well (eg.hg19).
3. Line 169-171 and Line 175-177 should be moved to materials and method part
4. Please change the title at line: 187
5. Why did you take 12 genes not more?
6. The discussion is weak. The conclusions are vague. Line 377-379: How the novel ribosomal protein genes expressed at high levels in mature leaves and are expressed at lower levels in young and senescent leaves are related to leaf senescence.
7. Extensive editing of English language and style are required for all part of this manuscript with new reference.
In general, the manuscript meets most of the acceptability criteria. It is only suggested to revise the writing of English by a specialized service to improve understanding in some passages, especially in the Discussion section.
It is suggested to omit from the Introduction section the paragraph that explains the methodology and the preliminary results (lines 84 to 95). That information can be considered in the Summary.
It is also suggested to enrich the background with a description of the transcriptomic analyzes in alfalfa related publications, the status of the reference genome and its complexity, among other aspects that account for the state of the RNASeq technique in this plant model.
It is generally recommended to leave the conclusions of each analysis presented in the Results section as part of the discussion.
In the DEG analysis (Fig 2) it is necessary to declare which is the reference condition in each comparison.
It is recommended to incorporate a table that can summarize the abundant functional information coming from the classification of significant GO terms in all the comparisons as part of the main tables of the manuscript. The supplementary figure in tree format by category CC, MF and BP could remain but the size of the letter needs to be increased.
Finally, as a general recommendation, it is known that numbers greater than 10 can be expressed in digits (example: line 424)
The experimental design of the study is appropriate. I only have one comment about the plant material. Only the quantification of chlorophyll is mentioned as an indicator for the stage of development of the three study conditions of alfalfa. Fig. 1 shows the same values of chlorophyll a and b between the Young and Senescent stages. It is suggested to improve the description of the phenological characterization of the three conditions studied, in order to demonstrate a clear physiological and metabolic differentiation between the three selected stages of development. In any case, the low amount of DEG shared among the three conditions (Fig 2B, 60 genes) suggests that the three transcriptomes are sufficiently different.
No comment
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