Transcriptome Analysis Reveals Putative Genes Involved in the Lipid Metabolism of Chaulmoogra Oil Biosynthesis in Carpotroche brasiliensis (Raddi) A.Gray, a Tropical Tree Species

Round 1

Reviewer 1 Report

Summary

The aim of the  study was to uncover the mechanisms involved in chaulmoogra oil biosynthesis, authors presented a study using a de novo transcriptomic approach to investigate the transcripts involved in the lipid pathways of C. brasiliensis and to identify genes involved in lipid  biosynthesis.  The main contribution and the strength of the paper is the provision of rich genetic information that will be useful to seek insights into the molecular bases of lipid biosynthesis and for understanding the synthesis of chaulmoogra oil.    General concept comments 1) A major weakness of the paper is that the study did not verify the identified genes involved in the  lipid biosynthesis. 2) The overall structure of the paper is not in consistent with that mostly used in the journal, e.g. the results and the discussion are presented in separate sections.   Specific comments 1) Line 2: The title of the paper is rather vague, the name of the species should be  given in the title, rather than 'tropical tree species". 2) Line 207: Results and discussions are better to be presented in separate sections, for easy  understanding by the readers. 3) Line 300: The style of figure 2 in black is not in consistent with other figures which are color figures. 4) Line 410: Figure 4 is a little pressed, at least the width of the figure can be made wider.        

Author Response

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Reviewer 2 Report

Please consider the following comments for revising your manuscript. 

TITLE: 

When I first read the title I assumed that more than one species would be involved in the study, mainly because of the abscence of the artice "a" before "tropical tree species". Therefore, the title should be corrected as simples as this: "Transcriptome analysis reveals putative genes involved in the lipid metabolism of chaulmoogra oil biosynthesis in a tropical tree species". 

INTRODUCTION

- Similarly to the ABSTRACT, please describe the familily to that the species belongs. 

METHODS

Please check the sentence: 

"The samples were col- 88 lected in triplicate to compose the 24 developed libraries, using 12 buds and 12 other plant tissues, namely: 2 leaves, 2 roots, 2 flowers, 3 seeds, pulp and skin of 3 fruits"."

2 + 2 + 2 + 3 + 1 + 3 = 13 samples + 12 buds = 25 

Was there one sample not included? Probably the sentence is misleading my interpretation, so it should be reviewed. How was each library composed? 

Phylogenetic analyses: which criterion was used to define the best substitution model as Tamura-Nei? Did you authors used AIC or BIC methos for accessing that? Moreover, did the authors test their phylogenetic reconstruction through maximum likelihood? It should be better to test a more efficient method, also including the resampling test thereafter (1,000 bootstrap replications). This is because multiple species were included for phylogenetic reconstruction (as in Figure S3), therefore, a distance method such as the neighbor-joining method might not be adequate. 

Figure 1: please stat in the legend that a star is higlighting the column related to lipid metabolism. 

Line 352: "indicated a division of the species". Please rewrite it: indicated a subdivision of all species (...). The way it was written seems to me as information for only one species. 

Line 363: "We we reiterate". We is repeated. 

The phylogeny of figure S3 seems important for the main manuscript. Therefore, why not including it as a main figure? Suggestion. 

Figure 4 is a broad view of the expression patterns. I think that it could be further improved for readability. The clustering patterns are interesting for the type of tissue involved. Further arrows ou subdivisions of lines could improve the impressions the readers have over reading and distinguishing the overall patterns of expressions. It is a suggestion for improvements only. This figure is much to valuable for not working more. 

In the same figure, could the authors provide the right side of the page with derived heatmaps of the main genes that they discussed. I know that they provide further details along the pathway that is presented in figure 5, but it would be interesting and valuable to use the space of figura 4 to highlight better the most important genes. 

CONCLUSION: I think that the authors could give indications that they used the transcriptome to contribute with a model to a pathway that needs completion yet to be achieved. Besides what is already described, it could enhance the importance of such a study. Perhaps, the abstract could also be the place for rehashing the importance of the study for both basic and applied science in the matter ol lipid metabolism. 

GENERAL COMMENT: Please check the manuscript thoroughly for minor English grammar. 

Author Response

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Reviewer 3 Report

The paper by de Vasconcelos and colleagues represents a well-planned and thoroughly conducted attempt at describing the transcriptome of Carpotroche brasiliensis (Raddi) Endl., an important neotropical tree. Even though the work is based on well-established techniques of genomic analysis, I deem it of great interest for the readers of Forests.

I wish I could see answered a small question: the phylogenetic tree of Supplementary Fig. 3 needs to be more defined. In fact, the Authors state that it was built by Neighbour-Joining, but it looks like an UPGMA tree to me. Second, because care was taken to grant some statistical support to the tree (“The consistency of the clustering patterns was assessed by 1,000 bootstrap replications”), the bootstrap values must be added to the Figure, at least for the most important nodes, such as the one leading to Achariaceae. Last, remove one of the two legends provided!

A very minor modification: the reference at line 244 is not properly formatted.

Author Response

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