Peer review of: Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium (2020)


Reviewer #2 (Remarks to the Author): 

I am responding to both my previous comments (Reviewer #2) and those made by Reviewer #1. 
Overall, the authors did an excellent job addressing the majority of my and Reviewer #1 previous 
comments. The manuscript is well written for a broad audience; discussion points are all clearly 
described and outlined. The below point (lines 422-425) and supporting data presented by the authors 
is important -- largely ignored by the polyploid community. I completely agree with the authors. This 
paper will serve as an important reference for the polyploid community, thus I anticipate will be highly 
cited. 

"Thus, simply comparing a polyploid genome to reference genomes from the progenitor species will 
greatly overestimate the changes that occurred after polyploidization and will not provide a full 
understanding of polyploid genome evolution." 

Here are just the last two comments for you to further consider: 

1. "Comparative analysis revealed that the vast majority of B. hybridum whole gene presence/absence 
variation was part of the standing variation in B. distachyon." 

Comment: Same comments as before. If you can't establish orthology, how can you confidently 
conclude that the "vast majority" of PAV in Bh was part of the standing variation in Bd? I don't 
disagree that the data are highly supportive of your model to explain the observed subgenome 
differences in PAV. My recommendation would be to simply replace "revealed" with "suggest" in this 
line and similar statements in the manuscript. 

2. "However, since polyploidization essentially duplicates all the core genes I would expect that core 
genes would be lost at an elevated rate relative to the diploid progenitor. That we do not observe this 
suggests that, in this case, there is selection to maintain intact subgenomes. This is the surprising 
finding." 

Response: Genes are retained in duplicate following polyploidy largely to maintain proper network 
stoichiometry. (See review on the Gene Balance Hypothesis: 
https://www.pnas.org/content/109/37/14746.long). The core genome likely encodes for essential 
functions, thus, this finding is the predicted outcome. 

3. Regarding the additional comments made by Reviewer #1: I completely agree with the authors. I 
am also unaware of similar studies in other polyploids, despite what was suggested by Reviewer #1. 
There are several novel and exciting findings presented by the author in this manuscript (which are 
unique from their previous work). I also agree with the authors response regarding the Tef genome 
paper, comment #2 and comment #3. I don't think it's necessary to cite the Tef genome paper. I will 
leave to the authors and editor to decide. In my opinion, the authors have adequately addressed the 
comments/concerns raised by reviewer #1. 

Reviewer #4 (Remarks to the Author): 

The authors response on most of the points I raised and the adaptations (if any) and updates in the 
ms. are often dissappointing and/or didn't adress the critical points I mentioned. Length and structure 
of the ms. remains an issue and esp. the linking of plastome type/nomenclature and genome-type is 
still somewhat enigmatic and not immedeatedly understandable to the non-expert. 

Reviewer #5: 

[Editor: This reviewer was asked to comment authors responses to previous reviewer #3's 

suggestions. The detailed reviewer's comments were submitted in Remark to Editor section as 
summarized below.] 

The reviewer thinks several of the major concerns remain unaddressed: 

1) The reviewer concerned about the genome assembly approach, together with the concerns about 
the annotation differences among these species raised by other reviewers. The authors only stated 
that high quality genomes are used in this study but didn’t provide any statistics. This certainly affects 
the PAV and the TE/K-mer analyses. 

2) The reviewer also concerned about the number and timings of origin of the diploid progenitors and 
the allotetraploids, which is critical to the study. This question, together with the reviewer #4’s 
comment about the GPS data for the collection of the individual lines, is the key to understand the 
true origination of the genetic diversity (SNVs) in diploid progenitors and polyploids. However, the 
authors didn’t answer/address the concern directly. 

3) Regarding the SNP/SNVs, the author changed the term SNP to SNV, which actually still can’t 
distinguish the variants within the same species and the variants intraspecies. 

In general, the reviewer thinks the authors can improve this manuscript by making much more solid 
evidences. Especially, the authors need to re-think about the divergence timing, the size and 
biogeography of the three populations.