Composition Diversity and Expression Specificity of the TPS Gene Family among 24 Ficus Species

Round 1

Reviewer 1 Report

Terpene synthase (TPS) contributes to the production of specific and diverse terpenoids, which includes the volatile organic compounds attracting pollinators. In this study, the authors identified 248 TPS genes in 24 Ficus species and comprehensively analyzed the sequence characteristics, phylogenic relationships, expression profiles, and selection divergences by RNA-seq. The paper includes many interesting information on molecular diversity among intraspecies. However, it is difficult to discuss about the diversity of VOCs and symbiosis figs and pollinations because the authors did not show any VOC analysis using GC-MS and the behavior of fig pollinators in this paper. The reviewer felt the leap in logic of their conclusion. Please reconsider the last paragraphs of the introduction (p3), discussion (p12), and conclusion (p14). Letters of figure 1 is too small. Please change the layout of panels A-C.

Minor points

1) Keywords. The authors included 'terpene', but they identified the TPS genes, not terpene analysis. Please change to other word. 

2) Keywords. DDXXD domains -> DDXXD motifs

3) Introduction (p3). Selaginella moellendorffii and TPS-a. Why did the authors show in bold?

4) TPS-a encodes sesqiterpenes, and TPS-b and TPS-g encode monoterpenes? The description is very strange because what genes encode are protein, not chemicals.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors assess the diversity of TPS genes across 24 Ficus species because these are involved in the species-specific production of terpenes, which attract pollinators. The study does RNA-seq in replicates, assembles the results, predicts TPS, and compares them bioinformatically. The study is interesting and easy to read.

 

The authors conduct RNA-seq and then assemble the reads into contigs using Trinity, which is not a problem. That said, the result of any assembly that is based only on short reads is that the contig outputs are consensus sequences. This is to say that, if there are two similar alleles in the genome that only differ by a small percent of bases (perhaps the result of a recent duplication event) and both are expressed, the reads will all be pulled to that contig, aligned, and collapsed into a consensus sequence. The genomic differences from the less highly expressed and thus minor population of RNAs will be lost in the consensus, which is now the contig produced as the assembly output (because that is how assemblies work).

 

This work discusses some of the differences in sequences that would indicate homologs to various terpene synthases where the sequences differences indicate the putative products produced. If the authors wanted to actually demonstrate that either their predictions are correct and/or uncover additional allelic/genetic diversity, they could do a SNP analysis on each of their TPS genes. This would show the reader that they have not lost diversity in the assembly (which is a very real possibility in this kind of analysis based on a diploid-polyploid genome with allelic variation) and that they are thoughtful in accounting for the real diversity in genes within their population. While not a requirement for acceptance, anyone who knows something about assemblies and gene prediction from polyploid organisms will know that this analysis is incomplete without such an assessment.

 

The authors mention things in the supplemental material (Fig. S1, Table S1) but there is no supplemental material attached. This makes it impossible to assess these parts of the manuscript.

 

Line 79 – there are terpenes with more than 30 carbons. Add “including” to the sentence for accuracy.

 

Line 80 – what is special about the aromas?

 

Line 131 – what did you resequence? Sequencing?

Author Response

Please see the attachment.

Author Response File: Author Response.docx