Intraspecific Diversity in the Cold Stress Response of Transposable Elements in the Diatom Leptocylindrus aporus

Round 1

Reviewer 1 Report

I am satisfied that the authors have adequately addressed the (largely minor) concerns raised in my review of the previous submission and thank the authors for their efforts. 

I continue to be a little puzzled by the contents of tables 7 and 8. I now understand that the Total row contains stats on all reported variants (i.e. single nucleotide and indel) but why does the total number of SNPs in transitions/transversions exceed the total number of SNPs in the row that precedes it? 

One of the introduced sections in the Discussion (starting line 578) would benefit from language review. Suggested alternative below:

TE copy number variations (CNV) could also contribute to differences
in expression patterns among strains, an issue that would merit further investigations since CNV, if present, could be related to the strain response to cold. The clusters representation of the data, among
 other things, emphasises the impact of the noise caused by B651. 

Reviewer 2 Report

Dear authors,

Thank you for the new material added to the manuscript and the clarifications brought to the points discussed during the review process. I am now happy to support publication of this manuscript in Genes. 

Kind regards.

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

This manuscript, submitted by Pargana et al., addresses the interesting question of intraspecific diversity in gene and transposable elements transcription in diatoms facing changing environments. This is a timely question, that fits in the broader challenge of assessing how intraspecific genetic and gene expression diversity allows species to adapt to a quickly changing environment, and how this diversity can be harnessed to breed stress resilient species. The authors provide here the transcriptome of three warm water diatom strains subjected to different temperature conditions. Among cold stress-responsive transcripts, the authors identify retrotransposon-related transcripts, that varies considerably between strains and treatments. Further investigation also reveals that these TE-related transcripts disappear after several months of culturing. Based on these observations, the authors argue for high intraspecific diversity in TE-related transcription between diatom strains, and propose this variation might facilitate physiological plasticity of diatoms when facing changing environments.

The authors provide an extensive introduction and set properly their research within the field. The methods are carefully described and allows assessment of the soundness of the data. The data are clearly presented and conclusions are mostly supported by the results. There are nevertheless several points that need to be addressed in order to strengthen the manuscript and reinforce the conclusions before further consideration:

 

Major points:

 

The authors argue that the absence of some TE-related transcripts in 1188A1, 1189A3 and 1189B3, but also increased transcription of some others (TR6356 in 1188A1, 1189A3 by example) supports intraspecific diversity in cold stress response between diatoms. The change of expression could also be the results of copy number variation between strains of the TEs investigated. It would be very interesting to check the copy number of these elements by qPCR to rule out any major burst or purge in those families being the cause for the change in transcription, rather than cold-responsiveness itself. This point would also be worth discussing in Conclusions.

 

Differentially expressed gene analysis reveals that cold stress is the strongest cell response observed in this experiment, and this analysis is the basis for all further analysis in the paper. I would like to have more information about genes from clusters 2, 5, 6, 8, 10, 11 or 13, and having at least some of them validated by qPCR as well. Did you detect any other TE-related genes in these clusters based on repeat annotation? To which cluster (further than cluster 3) belongs the remaining TE-related gene analyzed (L397)?

 

Design of primers specific to TEs is very often challenging due to the repetitive nature of those elements. Some of the qPCR primers used in this study seem to match to 18S rRNA genes and to a rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) gene. While this doesn’t necessary imply low specificity, I recommend to validate the qPCR transcription data with a second, independent set of primer targeting each TE-related gene.

 

The readers would overall benefit from a better description of how significance of the GO terms was assessed (by example L333-335). Especially, the cut-off p-value at 0.1 needs to be supported.

 

The absence of DEG between medium and high temperature treatments is surprising. Could the authors discuss on possible underlying causes?

 

Minor points:

 

L91: I recommend reading and adding as reference Catoni et al., NAR 2019 (https://doi.org/10.1093/nar/gky1196) that provides evidences for a role of TEs in gene shuffling in plants.

 

It appears that variation between acclimation duration is seen between strains. How such variation could influence the observed data? It would be useful for the readers to know how days of acclimation translate in cycles number (Tables 2 and 4).

 

Growth rate seems particularly variable as well (Supplementary Figure 1). Could the author discuss how this could affect their observations?

 

Pairwise distance between TR7186 and TR6586 can’t be obtained based on protein sequences. Did the authors tried using the DNA sequence instead?

 

L535: should read “editing”.

 

There are several issues with references formatting (not exhaustive: 20, 30, 49, 50, 52, 74, 80).

 

Reviewer 2 Report

In the manuscript entitled “Intraspecific diversity in the cold stress response of transposable elements in the diatom Leptocylindrus aporus”, Pargani and colleagues present analysis of the differential expression profiles of multiple strains of the protist, exposed to different temperature stresses. The study is rigorous, and the methods used are appropriate, thorough and carefully interrogate the different components of the observed signal (including assessing the extent to which prolonged culture of a strain may influence results).  The key finding of a major role of transposable elements in the cold temperature response stress response, is discussed in the context of strain-specific differences and is very effectively discussed.

 

English language is generally very good, although I have suggested a  few changes (in the minor edits section).

Specific comments

Data accessibility. Please clarify whether raw data (particularly RNA-seq experiments) will be made available in public databases. Could the custom script noted in line 174 be made available?

 

Line 335: GO ontogeny of differentially expressed transcript: Please briefly mention the two other enriched categories in text (at present they are in Fig 3 only and just DNA integration is discussed in detail).

 

 

Tables 7 and 8: I have not been able to understand how the “total” and “SNP” counts are generated (and consequently not the significance of presenting both).

 

Supplementary Files: Please provide additional descriptions to make the excel file contents more accessible to readers (i.e. expand on meaning of column headers, where appropriate).

 

Minor Edits

Line 47: “such variation”; consider specifying “novel genetic variation”, as I understand this statement to mean.

Line 58: consider replacing “set off” with “respond with”

Line 65: temperatures

Line 110: “In lack of genomic data” reads oddly. Either reword, else simplify statement by deleting clause and starting with “expression differences …”

Line 118: consider rewording slightly “which could help understanding mechanisms´ as it reads a little clunkily.

Formatting of Table 1 is disrupted by the page break (strains #3 and #4 are improperly separated).

Line 161 paired-end

Line 171: specify that CD-HIT threshold is 95% identity.

Line 179: …using the “protists” set of …

Line 323 Suggested edit to “All nine transcripts that were found to be significantly differentially expressed between low  and medium temperature were also contained within the group of significantly differentially expressed genes in the low and high temperature contrast and followed the same direction of expression change as in the less extreme contrast”.

Line 334: is there an alternative to the word “division”?

Line 337: Change “Manually inspected annotation” to either “Manual inspection of annotations”

Line 338: “related to DNA integration, four of which had no further annotation”

Line 403:  “The heterozygous to homozygous ratio,  for both total and SNP 404 specific, was also quite different between 1A1-3A6 and B651

Line 407:  Please reference Table 8 directly in this section.  Also “The intra-strain SNP analysis of B651 showed that there were slightly fewer SNPs in the new transcriptome compared to the original MMETSP transcriptome. Furthermore, the total and SNP specific heterozygous to homozygous ratio has been reduced by half during the three years of culturing.”

Line 429:  “… downregulated at low temperature while in the freshly isolated strain 1189B3 - acclimated 430 for the shortest time - all genes were either expressed at low levels or not detected.”

Line 451: The description of the overlap (or not) of TE homology for DE genes could be clearer.

Line 498: Change “TEs held again” to “TEs again held”

Line 548: please reformat CO2 with subscript