Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors show a work to determine the participation of DFR genes in anthocyanin biosynthesis in Allium wallichii, for which they assembled a reference transcriptome and identified the genes coding for dihydrofavonol 4-reductase (DFR). The work is interesting and provides information on the function of these genes in this species. However, several points should be reviewed. I list them from most to least important.

 

The materials and methods section is poor. It must be improved by providing more details of the materials and methods used.

 

Section 2.3: Give more details on the methods and materials used in this section.

Lines 105, 118: Please mention more details about the different stages of flowers collected.

 

Line 119-123: How was the quality and integrity of the RNA checked? How was mRNA purified, identified, and cDNA synthesized? Was double-stranded cDNA synthesis performed? What size fragments were used for sequencing? What type of libraries were constructed for sequencing in PacBio? Was a single library constructed, or were there multiple libraries? The authors must add more details about this section.

 

Section 2.4 It is essential to add the versions and references of the software used.

 

Line 132: What percentage of identity was used to reduce redundancy with CD-HIT?

 

Lines 134-135: How did you define alternative splicing without a gene model available? The blast tool alone is not very sensitive for detecting alternative splicing events. The authors should detail how they identified alternative splicing events using Blast alone. It is suggested to use bioinformatics tools focused on identifying these events without a reference genome, such as DeepASmRNA (https://doi.org/10.1016/j.isci.2022.105345).

 

Lines 144-145: How was the search for DFR genes performed? How many transcripts were found that code for this enzyme?

 

Lines 189-190: What type of promoter was used in the construction?

 

Line 235: What is the n value of the database used in BUSCO analysis, or against which database was the transcriptome compared?

 

Line 238: What about the variant of alternative splicing prediction analysis? How many variants did you find, and what can you say about the theme?

 

Lines 243-246: This section is confusing. If the analysis shows that 85,518 transcripts have CDS, then 21,968 do not have CDS and could probably be non-coding RNAs. Why does your analysis result in 28,224 transcripts that could be non-coding RNAs? What is the explanation for this discrepancy in numbers? What is the range of sizes of these non-coding transcripts? The authors must improve the description of the functional annotation of the transcriptome.

 

Lines 258-259: This section is confusing. Revise wording.

Figure 4B has no relation to this text part.

 

Lines 262: The authors claim that 46,848 transcripts were annotated in the GO database; however, above (lines 255-256), they said that 25,590 transcripts were annotated in this database. The authors should be careful when presenting the results and check for discrepancies. It makes it difficult to understand the meaning of the results.

 

Lines 287-290: The authors claim that based on the transcriptome and metabolome, four probable DFR genes were identified. However, they do not comment on how they based their identification and on why they chose to amplify the DFR1 and DFR2 genes. The authors must improve this information.

 

Figure 8. Information on the species used to construct the DFR genes phylogenetic tree is missing. What functions are associated with the different motifs identified in the analysis? The authors should complete the missing information and comment on the functions of the motifs found in the DFR genes.

 

Line 313: what does preferential expression mean? Please clarify.

 

Lines 315-317: The authors claim that due to the high expression levels of the AwDFR1 gene in petals and their consistency with anthocyanin accumulation, it is very likely that this gene is involved in anthocyanin biosynthesis. However, according to the results shown in Figure 9B, both genes (AwDFR1 and 2) show high expression levels in leaves, scapes, and petals. The DFR2 gene even shows higher expression levels than DFR1. Why could only DFR1 participate in anthocyanin biosynthesis? What other functions could it have in the different tissues? What about the DFR2 gene? The authors must elaborate on the explanation of these results.

 

Figure S2: Authors should explain in panels A and B what lanes 1-4 mean.

 

In general, the authors should improve the quality of the figures. In some of them, it is difficult to distinguish details, or it is complicated to see the names and units of the axes. In addition, they should expand the information in the figure captions to understand better. Also, they add information about the statistical tests used, if applicable.

Figure 1: What units are represented on the X-axis?

Figure 3A: What is the meaning of C and C*?

 

Discussion:

An important contribution of the work is the generation of transcriptomic data of this species. However, the discussion of this section is poor. They comment that they performed an alternative splicing variants analysis, but neither in the results nor in the discussion do they mention it again. The authors should further elaborate on the discussion of these sections and compare them with other species.

 

Lines 414-419: The authors claim that AwDFR 1 and 2 genes have high expression in petals and low expression in roots, suggesting their participation in anthocyanin and proanthocyanidin accumulation. However, as Figure 9B shows, these genes also show similar expression levels in leaves and scapes. How can this be explained? The authors should further discuss this part to expand the knowledge about the function of these genes in other tissues.

 

Lines 442-444: This section is interesting. Before this assertion, the results pointed out that the functions of AwDFR1 and 2 were redundant; however, here, they are talking about independent functions. The authors should deepen this part of the discussion and provide evidence to support this conclusion.

 

In the introduction, the importance of studying metabolism in Allium wallichii is unclear. It is suggested that the authors clarify the importance of studying this species.

 

There are many Error types. Here, list some of them. Please check them.

Line 119: "Total RNA o were extracted..." Delete "o" and change "were" to "was."

Line 170: Scientific name in italics.

Lines 220-222: Revise titles. I guess the first two are wrong.

Lines 255-257: This sentence is repeated: "The data of transcripts annotated with the NR database."  

Caption of figure S2: Correct word "reaction".

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

After reading and analyzing the manuscript, I realized that the authors clearly present a gap in knowledge about the color formation mechanism in A. wallichii, and their objectives revolve around this clarification. The manuscript provides relevant information and contributions to science. However, it should be clear, and the authors should present in the text, how these results can be used in the future and what the contribution to society, the group and researchers is. There is no doubt that the research is robust, the text is clear and well detailed, and the return of this research to the scientific community is relevant. I have other general contributions: the Abstract needs to be reformulated. I would like to see the issues clearly stated: objective, material and methods, and results. The sections are mixed up. What would be "promotion of superior varieties"? This is not addressed throughout the text; In the keywords, replace Allium wallichii and anthocyanins, which are in the title; Introduction and methodology are clear and with robust analyses. Results, lines 220 and 221, check. The images and supplementary material are enlightening.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf