Comparative Transcriptome Analysis of Anthocyanin Biosynthesis in Pansy (Viola × wittrockiana Gams.)

Round 1

Reviewer 1 Report

I don't have more comments. The authors addressed all of my suggestions.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The authors significantly clarified the methodology and overall approach. They addressed most of my concerns, and I feel the manuscript is suitable for consideration. I do think the KEGG pathway analysis is not meaningful and should be removed from the manuscript, however, it is scientifically sound. 

Author Response

Please see the attachment

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In research entitled Comparative transcriptome analysis of anthocyanin biosynthe-2 sis in pansy (Viola×wittrockiana Gams.) authors conducted comparative transcriptome studies for blotched-75 flower (BF) and non-blotched-flower (NBF) of pansy. They compared the expression of genes involved in anthocyanin biosynthesis in pansy and measured the content of anthocyanins and carotenoids.

A major weakness of this article is that the analysis is based only on measurement of anthocyanins and carotenoids and transcriptomic analysis. Certainly, supplementing the study with the measurement of enzymatic activity of selected proteins would be necessary to support the hypotheses. Since gene expression does not give information about the functionality of a particular pathway.

Referring to the methodology and description of results:

1. whether the RNA was digested with DNAase? There is no such information anywhere.
2. How was the expression calculated? What was the calibrator?
3. If qPCR conditions were optimized, what were the reaction efficiencies?
4. L75- it should be: we conducted comparative transcriptome studies...
5. Why was transcriptomics done in only two biological replicates? This is too few, there should be at least three due to the variability of the material.
6. L333-340 - This is obvious. There is no need to write about what should be in the discussion.
7. L341-342  - it's been described in the introduction

 

Reviewer 2 Report

This manuscript performs cytological studies, pigment analyses, and transcriptomic analyses on the petals of two pansy cultivars, blotched flower and non-blotched flower, with a focus on the genes that may be responsible for differences in anthocyanin pigment accumulation. The results are interesting and are for the most part clearly described and presented. However, I have some concerns about how the RNA-sequencing analyses are described and the overall triviality of the gene-expression conclusions, and I think the manuscript would be better served by a more thorough analysis.

 

Major suggestions

Line 72-73 I found it confusing when the different stages of pansy floral development were referred to in this way without more context and explanation. Although this staging was already performed in another paper, it would be helpful to show the same developmental staging with the BF and NBF side by side in Figure 1 of this manuscript, so readers can visualize the differences being studied. In general, I don’t think you have a single image of the NBF in this paper.

Line 73-75 The way you describe anthocyanin accumulation over development makes it seem like stage V might be too late if you’re looking for gene expression differences responsible for different coloration, as the differences appear much earlier than the time profiled in this study. Obviously, that can’t be changed but it would be interesting to use qRT-PCR to check if the MYB identified as differentially expressed is also differentially expressed earlier in the process.

Line 154 To my knowledge, the Trinity toolkit does not have a native way to assess differential expression. How was this differential expression calculated? Also, which transcriptome, BF or NBF, was used as the “reference” “denominator” in the differential expression calculation? It seems that NBF is the reference, but the authors don’t indicate.

This study seems to have been performed with only one replicate library for BF and one replicate library for NBF (unless this is incorrect and the authors failed to indicate replication). This design is really flawed for performing robust differential expression analyses- another replicate would be best for each transcriptome. Some programs have methods for differential expression with only one replicate, but that would have to be clearly described.

Figure 4 This figure is strange. Does it really represent 1 or two genes of each KEGG category (as the legend implies)? If so, this isn’t particularly valuable, and I’m not sure you can claim enrichment from these numbers.

Line 310 Only 754 TFs were annotated, which is likely an underrepresentation. If MYB TFs are of particular interest, a hand-curated BLAST-based approach would be better so the authors can find more MYB TFs in the pansy petal transcriptome. This is also true for annotation of enzymes in the anthocyanin biosynthetic pathway. With the current approach, it is clear the authors may be missing some of the genes of interest.

 

Minor suggestions

Line 26 “late biosynthesis genes” are specifically late anthocyanin biosynthesis genes, this should be clarified

Line 75 What is the difference between BF and NBF? I assume different cultivars but it is never stated.

Line 76 I’m not sure what you mean by “structural genes”

Line 102 It is unclear how many different plants the 3 replicates represent.

Line 156 I believe this is the incorrect citation of the RSEM software used, which should be:

Li, B., Dewey, C.N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011). https://doi.org/10.1186/1471-2105-12-323

Figure 6 The results for this qRT-PCR are presented in the opposite order of the Figure 5 expression analyses. I think the Figure 6 order is more intuitive given the analysis, which is seeking genes upregulated in BF. Either way, they should match. Also, although the transcript IDs are matched to gene names in the text, it would be helpful to see them in the figure too, and to arrange the figure panels by gene family.

Line 347 “previous” not “precious”

Line 400 “is” not “was”