Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLICATION OF HIGH RESOLUTION MELT ANALYSIS (HRM) FOR SCREENING HAPLOTYPE VARIATION IN A NON-MODEL PLANT GENUS: CYCLOPIA (HONEYBUSH)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study presents the development of an approach based upon HRM to screen genetic variation contained in chloroplast DNA (cpDNA) of the non-model plant (Cyclopia subternata), endemic to South Africa (aim 1). Sequence analysis was used to develop a battery of genus-specific primers that target 12 non-coding cpDNA regions. The second aim was to test whether HRM could identify the same haplotypes characterized by Sanger sequencing. To that end, four cpDNA segments were characterized using a battery of eight primer-pair combinations. The authors tested these HRM assays for specificity, sensitivity, and accuracy, and concluded that only segment ATPI-ATPH-IS, targeted with primers MLT S1-S2, displayed 100% accuracy, sensitivity, and specificity when identifying four (A-D) haplotypes. A second segment (NdhAx1-NdhAx2) revealed a fifth haplotype (E), and together, the two HRM assays were used to determine the frequency of the five haplotypes by sampling location (Table 4). However, variation in other cpDNA segments targeted with six HRM assays reveal the presence of seven haplotypes (Table 2 and Fig. 5 inset -incorrectly identified in the text as Fig 4), but those HRM's were not used because of they lacked sufficient specificity, sensitivity or accuracy (although they are included in the discussion). The third aim of this article is to provide a case study to use HRM as a high-throughput alternative to identify cpDNA haplotypes for phylogeographic studies of non-model species, using C. subternata as an example. While for the most part this can be considered a seminal study and a guideline for plants, it is not the first to attempt to advance HRM as an alternative for SNP discovery and genotyping in non-model species of animals (see Smith et al. 2013; Marine Genomics 9:39-49).

In its majority, the article is well-written, although it contains several typos, omissions, inconsistencies, and mislabeled figures that need to be corrected (see attached commented PDF). Among the omissions listed in the commented copy, there are a few references that would be appropriate to include.

In addition to the changes included in the commented copy,  the following changes are strongly recommended to both figures and tables, and corresponding legends.

Fig. 2. Legend. Be consistent throughout in regard to cpDNA segments or loci analyzed. Reference to primer pairs as to denote regions of cpDNA have very little meaning to the reader and cause a constant distraction by having to go back to tables, and even there, then, the usage of acronyms to refer to the segments is not consistent. Throughout the entire manuscript and tables please refer to the loci or segments by their names, followed by primer pair [e.g., atpI-atpH IS (S1-S2)].

Fig. 4 legend. Substitute regions with loci or segments of cpDNA.
Fig. 4. Modify the x-axis to include the names of the cpDNA segments and corresponding primers. Again, the primer names have very little meaning to the reader, except the authors who are familiar with them and their location.

Fig 5. Legend. Mention the inset (SP network). Also, mention that color-coding in the map corresponds to distinct haplotypes in SP network.
Fig. 5. Inset. Two haplotypes in black, are not commented on the text or figure legend. How were these derived? I presume from sequence data. If so, specify in text.
Fig. 5. Locality names of black circles, not given. Suggest changing the color-scheme used as it is difficult to differentiate the tones of blue.

Table 1. Details for primer MLT_S4 not given. Please check names for consistency, throughout. Primers MLT_V1 and V2, appear elsewhere as R1 and R2

Table 2. Similar to comments of Fig. 2 Legend (above), introduce (group?) names of segments/loci by their names (e.g., atpI-atpH IS) to facilitate the placement of the targetted cpDNA loci subject to HRM. Also, sort the order of segment/primers alphabetically. I have a very difficult time understanding the meaning of columns 1-11 (number of times assigned to haplotype cluster). This is not properly explained neither in methods or in the figure legend.
Again, be consistent with names. Primer names should be the same throughout. MLT S1-S4, not S1-4.

Table 4. Data for Tsitzikamma not included.

Table 5. Legend. Correct spelling.
Table 5. Similar to comments above regarding loci names. Please include.
I
Experimental design: This study represents original research in environmental sciences. Largely the experimental design is appropriate and addresses each of the three aims appropriately. The major concern that I have is with the flowchart of work to characterize the five main haplotypes (A-E). Accordingly, they characterize all the samples with two HRM assays, one with primers S1-S2 (atpi-atpH IS) to obtain the identities of haplotypes A-D.  The second HRM using primers U1-U2 (ndh intron) helps identify haplotype E. Since MLT S1-S2 unambiguously determines the identity of haplotypes A-D, and given that the relationship of haplotypes is known through statistical parsimony (SP), then the second HRM should only be applied to individuals belonging haplotype A. Although in this particular example a phylogenetically informative sequential HRM would be of little value as a diagnostic tool (given that Haplotype A dominates most samples), as a generalization for other studies this could be a valuable contribution.

Another area of concern is whether BioRad's High Precision Melt Software normalizes the fluorescent signal, a function of the PCR yield, among samples. Inspection of the left panel of Fig. 2 (HRM) curves suggest that the curves are not normalized, such that some of the differences (and also noise) could result from differential amplification as a function of the quality of DNA of the sample (i.e., sample effect), or as the result that some haplotypes are preferentially amplified over others. Melting curve normalization would remove that noise. prior to calculating the melting curve differences. Further, panel E (also Fig. 2) suggests an incorrect placement in the calculation of the HRM differences, as the amplicon appears to contain two melting domains, with the information of the first compromised by selectin an area still subject to changes in temperature (76-77 degrees), instead of above 84 degrees where all products have been completed melted.
Validity of the findings: Another area of concern regarding the generalization of their approach is the reliance on the automated clustering algorithm of the High Precision Melt Software (BioRad). Not all scientists will utilize the same RT-PCR instrument and software platform and in my experience with four instruments of different brands (HRM-1, Idaho Tech; LC480 & LC96, Roche, and MyGo Mini, IT-IS-LifeSciencies) the reliability to determine similar melting curves vary, and the clustering algorithms can become subjective. Accordingly, the authors should modify lines 437-438  HRM to reflect that the guidelines provided therein to generate markers in non-model species may need to be adjusted according to the clustering algorithm provided by the RT-PCR manufacturer.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLICATION OF HIGH RESOLUTION MELT ANALYSIS (HRM) FOR SCREENING HAPLOTYPE VARIATION IN A NON-MODEL PLANT GENUS: CYCLOPIA (HONEYBUSH)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please check some typos along the text
Lines 42, 90, 136, 153, 172, 189, 190
Experimental design: Overall, the results are interesting and supported by a good experimental design.
Validity of the findings: No comment
Additional comments: My main concern is rather a general comment: it is not clear if the aim of the MS is more methodologic or descriptive. I've found the Material and Methods too long. In Material and Methods, a number of generic information has been described and some methodological results have been discussed. In my opinion, authors should discuss some information provided in M&M such as results and emphasize the primers selection work.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLICATION OF HIGH RESOLUTION MELT ANALYSIS (HRM) FOR SCREENING HAPLOTYPE VARIATION IN A NON-MODEL PLANT GENUS: CYCLOPIA (HONEYBUSH)
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	The article is well written and it contains valuable novel research findings in a field where limited information on Cyclopia species is available. These research findings are contributing meaningful knowledge on population diversity and therefore should be published.
2.	The reference to supplemental Tables, Figures exc. in the article, which is not always available to readers, should rather be included in the article. It will make the article more informative and give a better understanding of the scope of the study.
3.	In line 109 the authors refer to the 14 species used in the study as Table 1, but is actually Table S1. It is recommended that this Table is included as part of the article, (Table 1) since the 14 species are not mentioned anywhere else and it is valuable information to readers and scientists. All other Tables numbering need therefor be changed accordingly.
4.	In line 406 Fig S1 with the population tree is very informative and be included as part of article.
5.	Other reference to supplemental material can be omitted e.g. Table S4 in line 388.
6.	None of the supplemental Tables, Figures etc. had titles to inform reader what it is about.
7.	The supplemental fasta formatted files could not be opened and verified due to not having the necessary software.
8.	The reviewer had not access to verify the permits etc., but they were mentioned in article and it is accepted that they were in order, otherwise collection of wild species would have been impossible.
9.	The following references were not in the reference list – Galuszynski & Potts, 2017; van der Bank et al, 2002 (lines 141 and 142)
10.	References in line 269 (R Core Team 2018) and line 259 (QGIS Development Team 2018) are also not in reference list. In most Journals they are also required to be in reference list with more information on them or articles published on them.
11.	In the abstract under aim - line 6,7 and 8 the word “to” to be added to the aim
a) to develop genus-specific primers for High Resolution Melt analysis (HRM) of members of Cyclopia Vent., b) to test the haplotype discrimination of HRM compared to Sanger sequencing, and c) to provide a case study
12.	Spelling errors –
Please note that Bloukrans is spelled with an “s” and not a “z”. Please change it throughout document
Line 43  - among
Line 172 – primers
Line 314 – Kareedouw and not Kareedow
Line 404 – Langkloof and not Langekloof
13.	Other
For clarity of high ploidy level mentioned in line 420 either say in brackets after ploidy level (hexaploid) or 2n=6x=54
14.	Tables and Figures
•	Please make sure all species names etc. in headings and tables are in Italics.
•	Please remember Tables and Figures headings should be clear and readers should not need to refer back to document to understand the tables and figures
•	Table 1 (should be 2) – reference to Table S2 – is this valid or helpful. TM in Table should be Tm and defined below the Table as melting temperature or heading. Degree missing C at TM in Table.
•	Table 3 heading Tm needs to be defined
•	Table 4 – availability of Table S3?
•	Table 5 – spelling of location
Diepwalle and not Diepwelle
Langkloof and not Langekloof
Kareedouw and not Kareedow
Bloukrans and not Bloukranz
•	Figure 1 – Adding a few main towns (Cape Town, Port Elizabeth, George, Barrydale, Uniondale) into the map will make it easier to identify locations of collections. It would have been nice if the collection sites for the different species could have been indicated as well, but I guess it will make the map too messy.
•	Figure 5  spelling of locations
Diepwalle and not Diepwelle
Langkloof and not Langekloof
Kareedouw and not Kareedow
Bloukrans and not Bloukranz
15.	Results and Discussion – See points at “Validity of the findings”
Authors should also remember ARC only sold seed in the past 7-8 years while cultivation started in 1996. It is still practice of farmers to collect seed in the wild or get from their neighbours and are probably more responsible in spreading plant material to new areas. However, in both cases it should be monitored, especially areas with unique populations.
16.	Acknowledgements
The Agriculture Research Council (ARC) is not acknowledge for making their genebank accessions available to the study. Organisations allowing the authors to collect plant material on their land e.g. SANPARKS, farmers etc. should also be acknowledged. Normally funders are also thanked or acknowledged here unless there is a specific heading for funding.
Experimental design: 1.	The material and methods are clearly described and in length described how primers are developed and chosen.
2.	A wide area was covered and sampled and several individuals in a population were included in the dataset. The collection of plant material alone was a huge and tiresome task. Therefor making the data more reliable and useful.
3.	Again, the availability of supplemental Tables, Figures, files are questionable.
4.	It would have been good to know in which areas the 14 species and their populations were collected, but is not necessarily adding value to this article.
Validity of the findings: 1.	In line 413 the authors made a personal observation stating that the unique population found by Niemandt et al (2018) is C. plicata and not C. subternata. They made no explanation on how they got to that conclusion. Niemandt et al (2018) stated clearly that samples of this population were identified by botanists at SANBI. Furthermore, the leaf shape of these two species are distinct (needle like vs oblong) and the two species cannot be wrongly identified as the other. Locals at Haarlem also identified the population as C. subternata, who are familiar with the species occurring in that area. In Niemandt et al study the SSR markers identified and grouped with these of C. subternata and not with any other species in the study. The chromosome number is also the same as C. subternata, although the chromosome number for C. plicata is still unknown. Both species do occur in the area of collection, but the population collected by the current authors are a few kilometers away from Niemandts sample area. Therefore, authors should omit this statement without any proof. They can rather expand on the phenomenon of unique populations in the Haarlem area found in two studies. It can be speculated that this is perhaps subspeciation and that more studies are required on populations in this area. Definitely something interesting to follow up
2.	Did the authors used the ARC gene bank accessions of C. subternata (breeding population) as a group to compare how they vary from the C. subternata wild populations. This will be a good indication if contamination had took place or may take place. If the breeding population are sharing the same genetic material as the wild populations in an area, the risk for genetic contamination or “pollution” is minimized. This will be a valuable exercise to do and helpful to the honeybush industry.
Additional comments: 1.	The reviewer wants to congratulate the authors with the study and their contribution to science and knowledge on honeybush, and their initiative to use new methodologies to obtain and create new data and understanding of this genus. Honeybush is a valuable niche crop and the more and the better we understand the crop, additional benefit and growth can be added to the industry.
2.	It is with concern that it was noticed the authors uploaded this unreviewed article to an open access website, Researchgate, before the article was accepted. They also didn’t upload the supportive data, Tables etc. that are referred to in the article, thus making the information less usefull e.g. the 14 species used in the study. It is recommended that authors only upload articles to websites after articles are accepted and if it is in accordance to the Journals policies.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLICATION OF HIGH RESOLUTION MELT ANALYSIS (HRM) FOR SCREENING HAPLOTYPE VARIATION IN A NON-MODEL PLANT GENUS: CYCLOPIA (HONEYBUSH)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Line 192. Delete extra period after 'analysis.'
Line 201. Capitalize 't' to upper case in 'table 3'
Line 632. Correct font

Table 3. The right portion of the table does not fit, and thus cannot be read.
Experimental design: This version represents a substantial improvement to all the deficiencies noted in the description of the experimental design and methods.
Validity of the findings: Findings are valid and sound with important ramifications for rapid genotyping and biogeography of non-model plant species.
Additional comments: I am completely satisfied with the manner you addressed that suggestions made in my original review, as well as the way you treated the remarks of the other two reviewers, in particular reviewer number 3.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: APPLICATION OF HIGH RESOLUTION MELT ANALYSIS (HRM) FOR SCREENING HAPLOTYPE VARIATION IN A NON-MODEL PLANT GENUS: CYCLOPIA (HONEYBUSH)
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1.	Still several errors with the names of localities, towns etc. Tsitsikamma is without a z.
        Please make sure all spelling is correct as it appears in roadmaps, google maps etc.
2.	Figure 1 is now much more informative
3.	Please check that all genus and species names are in Italics, especially in Table and
        Figure headings , and spelling of locations
a.	Figure 1 (Cyclopia and C. subternata) Italics
b.	Figure 5 Bergplaas and Tsitsikamma
c.	Table 3 not Landscape
d.	Table 5 Heading –  change “ of each Cyclopia subternata populations” to “are
        given/listed for each Cyclopia subternata population”. It does not read correctly.
e.	In Table 5 – spelling Tsitsikamma
4.	Typing corrections in
a.	Line 185 – delete extra “.”
b.	Line 189 – capital T in table 3
c.	Line 286 – Tsitsikamma with s and not Z
d.	Line 360 – Delete space between SNPS and ,
e.	Line 566 - Delete ?
f.	Line 623-625 – Change Font
Experimental design: No comment
Validity of the findings: 1.	Agree that Haarlem population may need re-evaluation and further study. Not sure if samples were taken in same area as current authors. However, botanist at SANBI is well familiar with Cyclopia species that was part of his PhD studies, using flowering samples for identification and compared it with flowering C. subternata. As mentioned by authors due to environmental changes and stresses species can be misidentified. Schutte 95, 97 mentioned several varieties and includes C. latifolia in her classification of C. subternata. Did Schutte or any botanist verified all populations used in this study?
Paragraph reads better and can be accepted.
2.	It’s a pity the authors lost the ARC genetic material, which could have helped to clarify and support the research findings in this study.
3.	Not familiar with any cultivation activities in the Garcia area.
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FRNAKENSEQ: A FULLY POWERED-BY-CYVERSE CLOUD INTEGRATED RNA-SEQUENCING ANALYSIS TOOL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The author are presenting the workflow fRNAkenseq that runs on the CyVerse platform. This workflow allows to manipulate RNA-Seq sequencing data towards the identification of differentially expressed genes using some standard protocols (but not a RNA-Sequencing platform as expected from the title).

The introduction needs to be improved by a state of the art of the tools that carry on differential expression from RNA-Seq data, and a description of the advantage of Cyverse in a user perspective manner (for instance its facility to upload and store data or to link different tools), and definitely why a workflow for managing those data in CyVerse is appealing.

In a second part of the article, a complete and detailed overview of the pipeline, how it can be used, the (new) functionalities it is offering, its input,  parameters and output, is lacking.

I think that is there is a confusion between enrichment and differential expression analyses. Overall, the article is not properly written, lot of typo are remaining (even the name of the tool is often misspelled). The manuscript needs definitely to be corrected or rewrote correctly. There is too many figures, most of them are screenshots not well described (in the text and in the legend).
Experimental design: The authors focus on the comparison with Galaxy, which should not be the purpose of the article. Indeed, because the fRNAkenseq has been developed in the CyVerse environment, which is probably good because such pipeline was demanding in CyVerse, and also because that it is probably possible to derive fRNAkenseq in Galaxy, as well, by adjusting the tools parameters and adding few other functionalities or wrapped scripts such as the comparison of the results from the various differentially expressed tools.

But again, the purpose of this article is necessary to present more clearly how the pipeline is working, from a user point of view, to propel biologists to use it instead of creating their own pipeline in Galaxy. First of all, I think it is important to show why the use of 3 differential expressions tools would help to define a more specific list of genes in a valuable example. Then give a more detailed the description of why and how the links to CoGE is useful. On the other hand, I would expect that the advantage of such tools in the CyVerse platform should facilitate the analysis of enrichment in Gene Ontology terms. This still could probably be  well illustrated by use of an example.
Validity of the findings: The principal arguments of the authors is that there is no similar pipeline in Galaxy, so far, and at least no one which "synthesize the output of multiple enrichment prediction programs into easy to interpret tab delimited files". But an experienced Galaxy user should probably be able to perform this using the tabular tools. Morevover, the sentence l99-100 "steps of the fRNAkenseq pipeline which cannot be implemented on the Galaxy platform and would otherwise need to be executed manually (for
 example combining the output of multiple tools)." is not exact, because such tool or script could be rapidly developed and wrapped into Galaxy. On the other hand, the sentence l189-l200, "Galaxy commands by default for DESeq2 are likely more stringent for those used by fRNAkenseq" is not demonstrative because the default parameters can be changed in Galaxy, by the administrator, and parameters can be changed by user before running or when creating a workflow.

As a conclusion, comparing fRNAkenseq to Galaxy is not pertinent, because one of the most advantage of Galaxy is that it can be installed and configured in many places, where people can develop their own version of their favourite pipelines and distribute them if they want. By the way, there is also some way to create Cyverse workflows into Galaxy, and making fRNAkenseq working on both platforms should probably be feasible. Nevertheless, the advantage of CyVerse is that they are offering a platform on top of very large resources.

So, I would recommend to move the Galaxy and Cyverse comparison to the discussion, because it is not a central point.
Additional comments: It is important to ease workflows either in Cyverse, SciApps or Galaxy  to be published elsewhere in order to increase their usage and to be cited. This article shows a interesting pipeline that allow users to perform a complete RNA-Seq analysis using a standard mapping strategy and 3 different popular tools for differential expression. The main part of the article consists in comparing fRNAkenseq to an almost similar pipeline generated through Galaxy, while the principal object of this article should be a demonstration of what the pipeline is achieving in a user perspective, enhanced with some technical aspects of the pipeline, and perspectives for a public of computer scientists.

So, in order to increase its readability and probably its impact, I would encourage the authors to reorganize the writing of this manuscript, by simplifying the comparison with Galaxy and moving this part to the discussion, while focusing in the main part to the usage of the pipeline in the CyVerse environment, with a better and clearer illustration, and some technical considerations.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FRNAKENSEQ: A FULLY POWERED-BY-CYVERSE CLOUD INTEGRATED RNA-SEQUENCING ANALYSIS TOOL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presents an overview of a workflow for expression studies, RNA-Seq in particular, that is presented in the context of Cyverse and CoGE.  The workflow seems to be a valuable contribution to the community however the key details are not presented in enough detail for a reader to evaluate this resource.  The authors should provide more literature to describe the best practices in expression analysis for short read data and provide context to the tools selected.  They should also review the limitations of this approach (reference genome only?).  The organization of the paper is problematic in that, key technologies are not introduced early on and important details are scattered through the paper.  The figures are too numerous and not helpful (i.e. screenshots of gene expression outputs). I have provided some feedback on these.  Finally, the comparison with Galaxy is executed with a sample RNA-Seq study however, the results are presented in basic counts of genes and there is no benchmark for this so the emphasis here is not helpful in terms of evaluation.  It would make sense to focus on features, hardware, runtime, flexibility, etc that one would associate with workflow comparisons.
Experimental design: The paper should focus on the details on the workflows (more detail) and less detail on the sample study and sets of gene counts.  The limitations and advantageous of both frameworks should be presented.  Tables comparing features, tools, downstream merging of results, etc would be more helpful to the reader.
Validity of the findings: A restructuring of the background and workflow details will allow a better evaluation of this framework.  I believe it is quite useful but it needs to be explained in more detail and accessible to a broad readership not already familiar with CoGE, Galaxy, or even Cyverse.
Additional comments: Please note the following:

Abstract:
(1) The abstract could be much more informative about the specifics of the problem and the solutions proposed.  Are datasets for RNA-Seq getting larger?  I don’t think that is really the case - the read per library target has been established for some time, and is not particularly high for species with a reference genome.  You are addressing specific challenges but these are not well described here.  I should be very clear why this was developed after reading the abstract.

(2) This sentence needs to be restructured “A strategy to counteract this challenges is developing a modern”

(3) I would avoid citations in the abstract.

(4) The last sentence of the abstract, “Cross-talk with another CyVerse associated tool, CoGe (Lyons et al., 2008), meanwhile,
allows fRNAkenseq users to execute these pipelines on an inventory of 47,000 genomes or upload draft
genomes.” needs to be re-written.

Introduction
(5) The introduction is structured as just one paragraph but should be sub-divided into key sections that would outline the background the reader needs to understand this tool and its benefit.  You go on to compare SciApps, Galaxy etc - these should be at least briefly introduced in the introduction within the scope of cyberinfrastructure.  One potential organization might include:
(a) Cyverse and other cyberinfrastructure
(b) Introduce CoGE
(c) High level overview of challenges with RNA-Seq informatics
(d) Challenges addressed by this application

(6) I would recommend describing the pipeline in detail before moving into the Architecture section.  The applications used in the pipeline would not really fall into the architecture section.

(7) In describing the pipeline applications, I would provide more detail on their selection, role, and possible parameterization.  Literature describing the current best practices for RNA-Seq analysis should be referenced here.

(8) Line 89-90 - “This feature is important, because despite many approaches to differential expression, none is found to be universally superior”.  This is not completely accurate - many of the R packages are recommended in different study designs.  It would be advantageous for the workflow to provide a recommendation for the appropriate applications based upon the replication model.

(9) Figure 1: Data Store is a Cyverse product and should be represented in caps and consistently in Figures, legends, and in text.

(10) Figure 1 should be refactored to clearly indicate the order of operations and where data is initially loaded.  For example, Data Store appears twice.  The starting interface is indicated at the bottom rather than top of the workflow.

(11) Replacement of the Tuxedo package is mentioned briefly in the legend and in the figure but is presented without context.  If important to the reader to know this, it should be described in text and put into context with best practices for RNA-Seq.

(12) Comparison with other tools:  This section seems only to compare with Galaxy, are there other tools that should be included as the section title indicates?

(13) Since Galaxy is not the focus of the paper, figures should not be included depicting their interface.  The snapshot of their workflow GUI is not very helpful in conveying the full picture.

(14) There are packages that exist to connect Galaxy and R now so this statement is not fully accurate.

(15) This section can be much more concise - describe the specific advantages of this system versus Galaxy, including access to genome resources, ability to store data, share data, etc.  You have good points here but they are getting lost in delivery.

(16) Figures 3, 4, and 5 should be combined into 1 - possible with multiple parts (A, B, and C).  They do not fit where currently referenced.  It would make sense to reference these in the first section, after Figure 1.   In addition, what is currently labeled as Figure 5 should be improved visually for clarity.

(17) Figure 6-10 are not appropriate.  They are screenshots of tables of DE values that do not have context with a sample dataset and provide values that the reader cannot interpret.  These would be best shown as a Venn diagram in a single figure (or a venn diagram or simple table of gene counts for both approaches).  The tables of values from each R package should form supplemental tables.

(18) DiffExpress is mentioned for the first time farther down in the paper and in the figure legends.  This should be clearly indicated in Figure 1 since it is important to the workflow described.

(19) An appropriate figure or table might involve a clear comparison of specific features and/or metrics between the two methods.   It should also be made clear that this comparison is executed on the public instance of Galaxy - Galaxy can be installed locally to permit genome upload and abstraction for any command line tool or script.

(20) The way the sample dataset is used in the study does not make a great deal of sense since there is no known truth.  Several tables of listed genes are shown only to say that there is a package that allows you to combine them together and find the intersection.  Neither workflow can evaluate the study design and recommend an appropriate R package (or subset of R packages) - this would be something nice to have and worthy of a comparison.  Otherwise, we are just listing numbers of genes, and more genes is not necessarily a better answer.  I would re-structure this section and think about what you can compare and attempt to quantify this.  I think it would relate more to features than to results from an experiment.

(21) The section labeled Discussion should be denoted as Conclusions


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FRNAKENSEQ: A FULLY POWERED-BY-CYVERSE CLOUD INTEGRATED RNA-SEQUENCING ANALYSIS TOOL
Review round: 2
Reviewer: 1

Output:
Basic reporting: (1) Proper literature references in introduction and for tools mentioned in the paper (HISAT2, Samtools), are not included in the current manuscript
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for providing a revised version of the manuscript is improved however there are organizational details that remain a challenge.  A few notes on the current text:

(1) The Introduction - more citations are needed in the Introduction - external support must be provided for most definitions as well as statements like "Although sequencing costs have decreased substantially for RNA-Seq, the computational footprint of RNA-Seq remains substantial."  Many of these early statements could be quantified and would be more interesting to read.  How many RNA-Seq studies have been completed?  What does the increase look like over the past five years?  These can be compiled from public data.

(2) p.2, line 97: "fRNAkenseq is among the first tool of its kind to employ API-oriented "  Is it the first tool of its kind or?  Define this statement a bit more.

(3) FASTQ should always be capitalized.  Please review that all tools, packages, and file extensions are properly referenced and capitalized.  They are inconsistently referenced through the text.

(4) Order of the Introduction will still cause issue for readers -  make sure to define all terms before referencing your application, Cyverse, CoGE, Agave API, etc must be defined before talking about the tool discussed in this paper.  That portion should come last.

(5) Workflow section: HISAT2 is listed as the mapper - does this take single and paired data - why only default parameter - what about species with very large introns?  No customization possible?

(6) Proper manuscript citations are missing for tools such as HISAT2, Stringtie, Samtools, etc.  Most of these public tools have a paper that goes with them.

(7) StringTie can detect novel isoforms, how is this handled downstream?  It works by default with Ballgown - why is this not used downstream of StringTie?

(8) While I can appreciate that the authors will provide output from multiple R packages, there is literature that provides guidance on when specific packages should be used or should be used differently with different numbers of replicates.  Since the replicate number and study design is known, some guidance should be provided by the application.  This application is geared toward novice users with minimal training in HPC and R, therefore it is important to provide some suggested parameterization or at least suggestion of what subset of packages will be most appropriate given the design.

(9) A little more context is needed in the workflow section describing these tools - why did you choose these tools and parameters - provide some context and supporting literature here.  Later, in the Demonstration, the venn diagram should be referenced to relate back to the importance of including these three tools.  There is some discussion on this further in the paper but the workflow section should contain the general details on the full workflow, from start to finish.  The support is still weak for why these three tools and I do believe it should only run the runs that are

(10) The workflow section does not mention CoGE but I assume this is how one obtains the version of the reference genome - are these pre-indexed for HISAT2?

(11) The Demonstration section starts with describing a chicken RNA-Seq dataset but the text reverts back to general usage (mentioning the ability to select one of 40,000 genomes etc in CoGE).  This description should remain in the general workflow section.  The Demonstration section needs to describe the data, the version of the genome used, the time for execution, etc.  This section should demonstrate to us that this data is telling us something about how this program works and how well it works that could not be conveyed through a general description of the workflow.

(12) The portion on the synteny package for CoGE does not fit in this section.  This could be included in the introduction, where it might be relevant to set the stage for CoGE initially.  Synteny does not relate to RNA-Seq and is not appropriate later in the paper.  The remaining part of this section should also likely go into workflow (where the genome browser visualization comes into view).

(13) The DiffExpress section should also be in the workflow section since this is a general description and not specific to the Demonstration set.  For the Demonstration set, it would be relevant to discuss the study design and how that was passed to the tools (for example).  Essentially, it seems like the Demonstration paragraph is sitting in the middle of the workflow section and breaking up that flow.

(14) The conclusion reads as though it is starting a new comparison with Galaxy.  I would leave this to one to two sentences and instead remind the user of the overall utility of this tool.  Focus in this section on reminding the user of the key features and advantages.  Also, make sure we are aware how to start to using this and where to access it.

(15) The paper currently contains multiple misspellings, punctuation errors, and minor formatting issues.  All co-authors should carefully review the document and correct these.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FULL-LENGTH TRANSCRIPTOME AND TARGETED METABOLOME ANALYSES PROVIDE INSIGHTS INTO DEFENSE MECHANISMS OF MALUS SIEVERSII AGAINST AGRILUS MALI
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, authors reported the identification of genetic and metabolic factors involved on the resistance mechanisms of Malus sieversii (a wild relative of domesticated apple) to the insect Agrilus mali. I think, overall, the article provides some insight into this important issue, although several methodological aspects should be clarified in the manuscript. The structure of the paper is adequate but, in my opinion, the discussion section requires an extensive editing. The metabolomic analysis is not clearly justified and does not seem to provided any new information. Authors declare that they perform a target analysis but it is not clear to me on what type of molecules they are focusing. The figures are very difficult to evaluate due to the poor quality and the lack of figure legends.

Line 412: What is PBD?. In general, acronyms should be introduced the first time used.
Experimental design: I have several questions/comments and concerns about the methodology used.
1.	The experimental design is not clearly explained. As far as I understood authors used two sets of plants (Resistant and Non-resistant) and performed two types of treatments (Infested and Control). Since authors named the non-resistant plant as control (as they did for the control treatments) the results are very confusing, and it is not clear to which comparison authors refer in each case. An example of this possible misinterpretation can be found in the line 402.
2.	It is not clear to me what authors mean by biological replicates. It seems they called biological replicates to different samples collected on the same tree after infestation. If this is the case, these samples cannot be considered biological replicates.
3.	Authors established that a Fold Change ≥ 2 was used as threshold to select DEGs. With this criterion, only up-regulated transcripts would be selected. The limit to detect down-regulated transcripts should be indicated also.
4.	The LC-MS analysis is poorly explained. I cannot understand why, after the extraction, samples were dried and then redissolved in 30% methanol. What is advantage of the reducing the methanol content?. The parameters and equipment used for the mass spectroscopy analysis must be also described.
Validity of the findings: The objective of the paper is clear and well stated and the overall methodology used is adequate. However, how the results improved our knowledge about the resistance mechanism against this pest is barely explained. Most of the information provided in the discussion section must be reallocated to the results section. The discussion section must include only an overall view of the results integrating the results from the different analysis performed and comparing them with those found in the literature. The resistant role of the TIFY genes in apple is not fully discussed. There is not clear conclusion in the manuscript.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FULL-LENGTH TRANSCRIPTOME AND TARGETED METABOLOME ANALYSES PROVIDE INSIGHTS INTO DEFENSE MECHANISMS OF MALUS SIEVERSII AGAINST AGRILUS MALI
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. English writing needs to be much improved, such as wrong grammars, many reduplicate and redundant descriptions.
2. Others comments see blow as general comments for the author.
Experimental design: 1. In experiment design, it seems that the samples of healthy and insect-infection parts were collect from the same plants. As far as I know, many herbivores induced systemic defense response, so I am wondering the gene expression in the “healthy parts” of the infested parts may also be challenged. Therefore the screened genes associated with infestation may be inaccurate.
2. In targeted metabolome analyses, the authors only analyses the metabolites in resistant plants, how about the susceptible plants? Based on our experience, herbivore infestation may also affect the secondary metabolism of susceptible plants. Thus the change of some metabolites may be a stress response of the plants, not the “resistance response” of the resistant plants. I suggest to carry out metabolome analyses of the susceptible plants for fully comparison analyses.
Validity of the findings: 1. The data were poorly analyzed, and more details should be given regarding how to analyze the data. For example, the authors just list the data acquired by omics approaches without further explanation and discussion. In addition, in the materials and methods part, there were no statistical analysis methods regarding the accumulation of secondary metabolites, but the figure 7 showed some statistical significances. Please aid detail methods.
2. The data were not properly explained in discussion. For example, in L420-428, the authors just list some related results, but not deeply discuss the current results.
Additional comments: Specific comments:
1.	L24 There should be a blank in “M.sieversii”, please check through the whole text.
2.	L27 Please give the full terms of “SMRT”.
3.	L179 The cutoff of the significance of GO and KEGG analyses?
4.	L188-189 The results of TIFY family genes should be placed in results part.
5.	L204-205, Table S8? TableS3?
6.	L207 The samples for metabolome analyses are same to transcriptome??
Please give mere details.
7.	L276 The cutoff of Fold Change “≥2 or ≤0.5 and FDR <0.01”?
8.	L365 The unite of the chemicals was “ug/ml”?
9.	L392-394 This information should be placed in results part.
10.	L412 “PBD”?
11.	L434-435 “22 genes in the TIFY domain”? I only found 16 genes in figure 5.
12.	The Figures legends should be clear and concise, please give more details.
1)	Figure 1, I suggest to name the samples with the same treatment with uniform titles like Figure 6A.
2)	Figure 3A, “RvsR”? ““CvsC””?
3)	Figure 4, graph symbol should be “Resistant”?
4)	Figure 7, the statistical methods? Replications? Error bar is SE or SD?
13.	The legend of TableS8 in the main text and the supplemental materials is not consistent.
14.	For reference, there are many inconsistent styles. Please check reference style cite in the main text and reference list carefully. L175, “(Yangyang et al. 2006)”; L217, “Xiangqing et al. 2010”; L587, “Liu X, 2, Mei W, Soltis P, 3, Soltis D, 2,3, and Barbazuk W, 3. 2017.”
15.	…


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FULL-LENGTH TRANSCRIPTOME AND TARGETED METABOLOME ANALYSES PROVIDE INSIGHTS INTO DEFENSE MECHANISMS OF MALUS SIEVERSII AGAINST AGRILUS MALI
Review round: 1
Reviewer: 3

Output:
Basic reporting: Many missing details in the methods section and there was not much substance to the discussion section.  The authors mostly restated their findings and loosely connected their results to what has been observed in previous systems, but their ideas were not well-linked with these previous studies.  I believe this section should be rewritten entirely to improve its impact.

In addition, the quality of the analysis of the differentially expressed genes is extremely poor.  There is no detail about what software was used for the analysis and the authors do not clearly describe how they selected the 266 DEGs for further analysis (they point to a Venn Diagram, but don't explain why this part of the Venn Diagram is relevant to their study).
Experimental design: There are many details of the design that are not clear and I question the validity of the metabolite data since it does not appear that the authors sampled data from infested and uninfested susceptible and resistant trees (only two bars are shown in the graph and it is not clear which treatments are included because the figure legends are missing).  There is no statistical analysis described for the qPCR results and it is also not clear if they used the same samples that were used for the RNA-Seq analysis.
Validity of the findings: Very hard to assess because there are some key details missing in the methods section that are really needed to properly evaluate the design and the results.
Additional comments: The manuscript by Mei et al presents a transcriptome assembly for Malus sieversii and a differential expression analysis of susceptible and resistant plants before and after infestation by Agrilus mali, a devastating pest of apples in China.  Overall, the authors have generated a new genetic resource that can be used to understand responses to biotic and abiotic stresses in apples and that potentially can be used to mine genes associated with insect resistance that can be used to generate more resilient cultivars of apple.

Although the resource will be useful, the experimental methods are lacking numerous details that are needed to evaluate the quality and validity of the analysis.  The figures are confusing and are missing legends and it is unclear how the authors selected differentially expressed genes that are associated with insect-resistance (they point to a Venn Diagram, but fail to show the logic behind why they selected these particular DEGs).  The quality of the results presented are also poor, mostly because it is difficult to say why the authors chose to focus on this particular set of genes, but additionally, the analysis of the TIFYs is unclear (they mention that the TIFYs that are identified show 1:1 orthology with those annotated in the apple genome, but they don’t show any analysis to support that).

Finally, the discussion section 5-6 previous studies and only loosely connects the findings from this study to those from previous studies.  About 75% of the discussion section is repeated from the results section.

Line 20:  What do you mean that when you say this is the original species of domesticated apple?  Is it the wild progenitor for many cultivars of domesticated apple?  Please be more specifc.
Line 21:  However, this valuable species faces a significant threat in the areas north of the Tianshan Mountains in China, by the invasion of Agrilus mali, a destructive pest of apple trees belonging to the family Buprestidae.
Line 23:  has shown that there may be resistance to this insect in M. sieversii plants in the field
Line 24:  change insect-sensitive to insect-susceptible
Line 26 (and throughout):  change insect infection to insect feeding
Line 26:  112,103 non-chimeric full-length reads (FLNC) totaling 10.52 Gb of data were generating with PacBio SMRT sequencing.
Line 28:  the phrase “short-read long sequencing data” doesn’t make sense in this context.  Please rephrase.
Line 29:  Were these alternative splicing events and lcRNAs observed in response to insect feeding or were they just documented in the in the transcriptome assembly?
Line 29:  Functional annotation indicated
Line 31:  and in the synthesis of compounds such as terpenes….
Line 32:  Since TIFY genes are important
Line 33:  in apple species in general or just in this apple species?  Be more specific.
Line 34:  Here and throughout, change Totally to “In total,”
Line 34:  in the apple genome….Also, please specify which apple genome you are referring to.
Line 37:  Through targeted metabolome analysis….showed higher levels of….p-coumaric acid
 Line 47:  Please explain what you mean when you say it shares high “homogeneity” with cultivated apples.
Line 48:  Here and throughout the manuscript, there are numerous places where there are spaces missing between the references and the last word of a sentence.  Please carefully review the manuscript and correct this.
Line 48:  What do you mean when you say it is the most primitive of its kind?  This phrase is ambiguous.
Line 49:  Likewise, this statement also lacks context and is ambiguous.  Its flowers, fruits, nutrients, and growth habits are rich in diversity….please elaborate what you mean here because it is not clear how the the flowers and fruits produced by this tree can be rich in diversity.
Line 49:  These species offers unique genetic resources that can be used in apple breeding programs.
Line 51:  Its larvae, which live underneath the bark of trunks and branches, tunnel through the phloem and xylem in a spiral pattern, resulting in separation of the phloem from the xylem and death of the affected parts of the tree.
Line 57:  Therefore, it will be difficult and/or impossible to recover many unique genes and alleles coded by M. sierversii if it disappeared.
Line 57:  This statement is not entirely true.  Some apples can self-pollinate (ie, not all are self incompatible) and self-incompatibility does not necessarily mean that is the species is doomed if it is threatened by an insect pest.  The species can still persist and reproduce through cross-pollination.  Addition, the inclusion of the term “asexual reproduction” in this statement does not make much sense.  How can this species be self-incompatible and only reproduce by asexual reproduction?  Please check this and reword.  Some apples can produce vegetatively, but this is not the same as saying that they can only produce by asexual reproduction.
Line 58:  When plants are attacked by insects and other herbivores, changes in signaling pathways trigger changes in proteins and metabolites in an attempt to circumvent these stresses.
Line 61:  Morphological, biochemical, and molecular defenses can be triggered through immunological processes.
Line 67:  SA and ethylene can also be induced by some insect species.
Line 67:  I don’t think this is the correct use of the word “attenuate.”  Usually, JA levels initially increase in response to damage by herbivores.  Attenutation is a reduction.  Please revise thiss atement for additional clarity.
Line 69:  Expression levels of several genes associated with JA and ethylene synthesis/response (???) pathways were increased/decreased?….and higher levels of salicylic acid….
This statement is a little confusing.  What are you trying to say about the genes associated with JA and ethylene and how does that relate to what you are saying about the role of salicylic acid in insect defense and response?
Line 71:  Change systemic resistant response to systemic response.  The response does not always lead to resistance
Line 74:  Change “signal” to signaling
Line 82:  In a previous study of the TIFY gene family in apple….30 proteins were identified that contained the TIFY domain that were further classified into the following subfamilies:….
Line 87:  and the accuracy of the gene predictions and annotations
Line 88:  This improved reference genome will enhance our ability to study these gene families in other apple species.
Line 90:  SMRT sequencing,
Line 94:  How do you know that these “resistant” individuals represent different strains?  Could they simply be individuals that are more resistant to the insect than other members of the population?  Please provide some more details here.
Line 96:  Pleas specific pest species here
Line 98:  DEGs were identified between what?  Infested and uninfested trees from the resistant cultivar?  Infested resistant trees and uninfested susceptible trees?  Please be clear what comparisons were made to identify the DEGs.
Line 99:  change “screened out” to “identified.”
Line 109:  Please revise this statement:  We previously identified five individuals that grew at the same elevation (same elevation as what?) that seemed to have resistance against A. mali.
Line 109:  Just because these trees did not appear to have insect damage does not indicate that they have resistance.  Please describe in detail how you determined that they were indeed resistant to this insect.
Line 116:   According to their life cycle, adults lay eggs in the phloem underneath the bark.
Lines 118-124:  This section needs to be rewritten in reorganized for additional clarity.  The larvae begin feeding the follow spring.  The damage (due to what?...larval feeding or adults?) peaks in July.  How can the damage last only until September?  The damage is likely permanent?  Do you mean that the larvae continue to feed until September?  On July 25, 2017, five trees that had shown consistent resistance to A. mali over a 5-year period were infested with larvae as were five control trees (how were the control trees selected?).  Mature larvae (how many) were inserted into each tree through a simulated pore canal (please describe how this canal was created).  After a period of ten days, phloem was sampled from the infested and uninfested parts of the resistant and control trees after the area from the original mechanical damage had healed.  Please describe here what regions were sampled.  A picture as a figure or supplemental figure pointing to the regions that were sampled and diagraming how the trees were infested would be useful.
Line 127:  Please rewrite this section into complete sentences.
Line 135:  by an Agilent Bioanalyzer Nano 6000.
Line 141:  an AMPure
Line 143:  addition of an A-tail
Line 145:  After qualification and quantification of the library,
Line 146: short-read sequencing of each treatment x phenotype combination was performed using the Illumina HiSeq X-ten platform for RNA-Seq analysis.  Please specify what types of reads were obtained (150 x 150 PE reads or other)?  Also, the X-ten platform is not really intended for transcriptome sequencing and was not supported by Illumina when this platformed was launched.  Please explain why this platform was selected.
Line 149:  Do you mean to say that you mixed the RNA from the control/resistant genotypes and the infected and uninfected sites together for long-read sequencing?  Please rewrite these sentences for clarity.
Line 151:  was used to construct full-length cDNA sequences.  Please elaborate on how the BluePippin was used for size selection.  Did you select one size only for the three libraries you sequenced?  Or did you select different sizes for each of the three libraries?  If so, please describe in detail how this was done.
Line 155:  What was this secondary screening for?  Quality check?
Line 156:  What fragment size was selected for sequencing?  Please elaborate by stating in detail what you mean when you say that only samples that complied with a standard were used.
Line 158:  Long-read sequencing was performed using XXXXX.
Line 160:  Please define ROI
Line 160:  ROI sequences were extracted using the following criteria:  >=0 full-pass reading of the polymerase and sequence accuracy >=75.  Readings below 50 bp in length were discarded…
Line 165:  Define the ICE algorithm and explain what parameters were used in the clustering protocol (ie, what values were changed from default parameters)?
Lines 173-179:  Describe the blast searches and HMM searches that were performed to generate these functional annotations.  E-value thresholds need to be provided as well as whether blastp/x was used.  Versions and dates that databases were downloaded also need to be provided.  Please also describe how GO, KOG, eggNOG and KEGG terms were obtained and please specify which transcripts were searched (all transcripts or just the ones that were predicted to code for proteins?).
Line 181:  Why not search your transcriptome for putative TIFY domains using HMMER as opposed to searching known proteins that contain this domain against your transcriptome database?  This may improve your ability to detect divergent TIFY domain proteins that may be dissimilar to those derived from the reference genome.
Line 192:  to validate the results of the differential expression analysis.
Line 193: Please describe in more detail which genes you selected for validation and why.
Line 194:  Total RNA from each sample was
Line 195-196:  The last part of this sentence is not necessary. and we further detected the expression amount by real-time quantitative PCR.
Line 205:  Please describe in detail how the qPCR data were analyzed.  In addition, I don’t see any details in the methods about how the Illumina reads were mapped to the long-read assembly and how differentially expressed genes were identified.  These procedures must be included.
Line 208:  change “at an ultra-low temperature” to “at -80C”
Line 208:  was extracted in 1.0 mL of methanol.
Line 209:  during which the sample was rotated three times to mix the solvent.
Line 212:  then resuspended in a mixture of 30% methanol with vortexing, centrifuged at 12,000 g for 15 minutes, and the supernatant was collected and stored for analysis on an LC/MS.
Line 213-214:  This statement is not necessary. During redissolution, vortex isused to completely dissolve the target substance
Line 221:  The analysis conditions were as follows: (please rewrite all of this into complete sentences).  Please use past tense.  It looks like somebody else wrote this section compared to the rest of the methods section.
Line 234-235:  five resistant and five control trees
Line 239:  and was sequenced on the PacBio RS II platform.
Line 240: and one each for the 2-3K and 3-6K libraries.
Line 244:  Do you mean that of the 64,692 sequences, 47,181 were high-quality full length and the rest were low-quality after correction with the PacBio reads.  These sequences were error corrected with the short-read Illumina data?
Line 254:  What are F01 consensus sequences?  Do you mean FL?
Line 257:  According to your definition in the previous statement, the LQ reads came from the PacBio instrument and not the Illumina instrument.  They were error corrected using the Illumina reads.  Please correct.
Line 261:  Long non-coding RNA (lncRNAs) is defined as RNA >200 nt that does not contain any open reading frames.  To identify putative lcnRNAs, we assessed each transcript for coding potential.
Line 263-264:  Please add details about these analyses to the methods section and clearly define the algorithms that were used to assess coding potential and define any parameters that were used in these programs.

Line 271:  What are p1 and p2?
Line 273-275:  No details regarding the programs that were used for differential expression analysis are supplied in the methods.  Please add these details.
Line 278:  Please explain which parts of the Venn Diagram were considered relevant for insect-resistance and describe your rationale for determining that these were relevant.  For example, genes whose expression levels were upregulated or downregulated in the resistant plant relative to both uninfested resistant and infested and uinfested controls.  Or something to that affect.
Line 286:  How do these ~9500 DEGs differ from the 266-insect related DEGs that were described in the previous section.  Are they just genes that are differentially expressed in all vs all pairwise comparisons?  It would be more beneficial to separate them out by pairwise treatment comparisons because there are important details that are lost by presenting all of the DEGs together.
Line 292:  Were these genes up/down regulated in insect-resistant samples?  More details are needed here..
Line 310:  expression level of a gene annotated as F01.PB7083
Line 318:  change ‘developed’ to ‘were observed’
Line 319:  You can’t be certain that these genes were located on certain chromosomes because there is no reference genome available for the species/cultivars that you analyzed in this study.  It is possible that the gene order may have shuffled or that chromosomal translocations have occurred in this species compared to the reference genome.  Please remove this section from the manuscript.
Line 321:  Are these TIFY genes from your transcriptome assembly?  This was also unclear in the methods section.
Line 323:  These statements are unclear?  What do you mean by TIFY groups?  The subfamilies that were described earlier in the methods?  If so, please be more specific and also provide more details about how the intron/exon structure differed between the different groups/families.
Line 330:  Of course it is not surprising that you were able to find the TIFY domain in all of these proteins because the TIFY domain was used in your searches to identify these genes.
Line 339:  There is not a sufficient level of detail in the methods section to evaluate the quality of this co-expression analysis.   In addition, I do not believe there are a sufficient number of replicates or treatments for any meaningful results to be extracted rom this data.
Line 340:  How were these eight TIFY genes chosen?  Were they the ones that responded to insect feeding?  In the section above, you mention a total of 16 TIFYs.
Line 345:  Please explain how these TIFYs were named in your sample.  Were they annotated based on their orthology with TIFY genes in the apple genome?
Line 346:  Elevated expression levels were also observed in the control samples, although their levels were increased by only 13.9 and 6.9-fold in the infested control plants relative to the uninfested plants.
Line 354:  More details are needed for this analysis.
Line 384:  we performed long-read SMRT sequencing on the PacBio RS II instrument.
Line 389-390:  The lncRNAs were simply annotated and their roles in response to insect infestation were not investigated in this study.  Please remove this statement.
Line 414:  In what plant species and in response to what insect?  More details are needed here.
Line 425:  There are some spaces missing in this sentence.
Line 427:  that are involved in resistance.
Line 432-434:  This statement does not connect well with the rest of the ideas presented in the paragraph.
Figure 4:  Where are the statistical analyses for the qPCR data?  In addition, there is no information about how the data were standardized in the methods section (was ddCT used for DE, how was relative expression level calculated)?  What is normal vs Pet?  Why not just call these infested and uinfested?
Figure legends appear to be missing for all figures and therefore, it is difficult to determine what is being presented in some of these figures.  Sample labeling is also inconsistent across figures.
The raw PacBio and Illumina data need to be deposited into NCBI or some other suitable repository.  The authors should also attempt to make their transcripts available for use


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FULL-LENGTH TRANSCRIPTOME AND TARGETED METABOLOME ANALYSES PROVIDE INSIGHTS INTO DEFENSE MECHANISMS OF MALUS SIEVERSII AGAINST AGRILUS MALI
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although authors have made and effort to improve the manuscript, there are still few things that should be corrected. There are still many grammar mistakes in text. English writing needs to be improved.
Experimental design: The overall methodology used is adequate.
Validity of the findings: I think, overall, the article provides some insight into the plant-insect interaction field.
Additional comments: 1-	In M&M authors state that they used standard solutions to perform absolute quantification of metabolites, however in the figure 4 they reported relative content of phenolic compounds.
2-	Authors should include information in M&M about how they perform the correlation analysis.
3-	I can not see what information is reported in Figure 4D.
4-	Line 276: what authors mean by “In our previous research”, they refer to the Figure 1 of a previous paper?.
5-	Line 246: “It is evident from the Venn graph that 266 DEGs were respond to insect invasion in both resistant and susceptible plants”, according to the Venn diagram this sentence is not correct. The 266 DEGs respond in resistant genotype compared to the susceptible one and the control (non-feeding) samples.
6-	A general discussion about the transcriptomic response must be added.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: FULL-LENGTH TRANSCRIPTOME AND TARGETED METABOLOME ANALYSES PROVIDE INSIGHTS INTO DEFENSE MECHANISMS OF MALUS SIEVERSII AGAINST AGRILUS MALI
Review round: 3
Reviewer: 1

Output:
Basic reporting: In the actual form the paper is suitable for publication
Experimental design: In the actual form the paper is suitable for publication
Validity of the findings: In the actual form the paper is suitable for publication
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Cap making by sponge crab: general and specific comments

Needs some English corrections. FAR TOO LONG! Figure captions need to be shorter.

The actual modeling of the paper is a novel approach and worthy of publication.  But the paper is far too long and contains many references and comparisons that are not pertinent to the study. In the Introduction, I would delete all the references to decoration in families other than the Dromiidae. You could say that the means of decorating in the Majoidea (now split into several families) differs because they attach materials to setae instead of using the hind legs to carry their covering. The reader is lost in all of the references.

Line 71: research spelling; line 72: in field research (research is both singular and plural)
Line 83: we investigated the sponge crab Lauridromia dehaani
The last paragraph on page 2 should be incorporated into the methods. Line 85: We modeled four variates
Line 112: What is melamine and why did you use sponges of this material? ( Or is this the common ingredient of artificial household sponges?)
Line 132 and the rest of the manuscript: the depression in the sponge cap should be called a "cavity", not a "hole". A hole is considered to be deeper than a cavity.
Line 200: a groove
Line 206: I would say "excavating" instead of digging; to make the cavity larger...
Line 210: I think you should omit the line starting at "We will describe" because you say this later.
Line 240: Cap cavity
Line 257: I would confine the discussion to dromiid crabs. Homolids belong to a different family. The sponge crabs that carry shells as mentioned in line 260 do not belong to Lauridomia or related genera.
 Line 261: Bedini et al. (2003) expected (do not repeat the reference)
Line 280: Few marine animals show a camouflaging behavior (decorating is used in reference to the Majoidea, not other crabs). I would delete the lines referring to Oregonia gracilis: there are alternate explanations for why big male crabs don't decorate.
Line 284: crabs were larger
I would delete the first paragraph under "Integrated extended body". Most of this material is not relevant.
Line 309: I would delete the first line. Guinot and Wicksten did not consider camouflaging behavior to be tool use. Indeed, much of the camouflaging behavior of majoid crabs consists of an innate behavioral chain with little evidence of any modification.
The figure captions are FAR TOO LONG! For example: Figure 2: Cap making behavior showing cutting, digging and carrying. A: crab grasps side of sponge, tears pieces to make groove, rotates. B-C: Crab carries artificial sponge cap; B frontal view, C lateral view. (Or something like this).
Line 451 on: be consistent in use of capitals: MUSORSTOM is the abbreviation of an expedition series, but the journal titles should not be in all capitals.
Experimental design: Experimental design is OK as is.
Validity of the findings: Interesting use of a statistical model.
Additional comments: Try to confine your writing directly to your work on sponge crabs and point out the pioneering use of your modeling technique. Your writing should not repeat itself and not contain references that are tangential to your study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See General Comments
Experimental design: No comment
Validity of the findings: No comment
Additional comments: General Comments:
=================
NOTE: My background is in Statistics rather than behavioral studies, and the comments below are limited to my area of experize.

This will be a very useful paper in this particular area because it highlights the need and provides methods to handle grouped data. Moreover it demonstrates the perils of failing to use marginalized likelihood for model comparison. However, I feel that the coverage of this lacks a clear example (see point 3 below), and so will not be meaningful to readers who are not already familiar with the concepts of conditional vs marginal likelihood.

The English grammar is generally good for non-native English speakers, though there were several areas where it caused confusion - these have been listed below.

Specific Comments:
==================
1) There are numerous places where grammar and exposition need improving. A few examples include:
- line 16: From the wording "...including repeated samples, from the same individual crab." I got the initial impression that this study only looked at a single crab. Change to something like "...including repeated samples, from each crab."

- line 19. Change "...with the clustered data." to "...with clustered data."

- line 18. Change "...in statistical modeling. This is because..." to "...in statistical modeling because...".

- line 32. Change "...and studied the individuality in..." to "and studied individuality in..."

- line 65. Add "do", i.e., "...do we need..."

- line 71. Remove the first "the".

- line 84. Replace "one" by "each" or "an".

- line 137. Should "The..." be "We..."?

- lines 150-151. Please reword the sentence beginning "We did in the same way...".

- line 158. The phrase "...and unexpectedly abandoned the choice itself" should be reworded (to say that the crab made no choice).

2) The paragraph spanning lines 50-70 would benefit from a re-write:

- line 52: It would be useful to make it clear that the clustered data is refering to the repeating measurements on an individual.

- lines 61-63. This sentence talks about not being able to appoximate the posterior, and so needing a Bayesian approach. This makes no sense, since if one is attempting to appoximate the posterior then one is already using a Bayesian approach.

3) The second paragraph of the Statistical modeling section does a good job of talking about marginal-level WAIC . However it is not going to be intelligible to many readers without a concrete example. I would suggest that the distinction between conditional and marginal likelihood be made explicit in one of the ensuing models on pages 4 and 5. That is, give the form of (or equation of) the conditional likelihood, and of the marginal likelihood. In fact, the explanation in the above-mentioned paragraph might be better placed by moving it to a position after these explicit examples.

4) There are no citations for the methodology presented on pages 4 and 5, so it is not clear whether these types of model are entirely novel to behavioral studies (which I suspect not, since this is not the focus of the paper), or whether it is the formulation and methods to include repeated measures that is novel.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 2
Reviewer: 1

Output:
Basic reporting: New version is much more concise and to the point. References are good, figures and tables clear.
Experimental design: Reasonable. It would be almost impossible to conduct such a study in the wild.
Validity of the findings: Reasonable. Size-related differences in hiding behavior or camouflage occur in other families of crabs.

I note that the biggest crabs did not carry caps. Did the authors see any morphological change in the gripping dactyls of the pereopods or the chelae that might affect their ability to create a cap? In some  majoid crabs, the largest males lose their hooked setae and also have heavy cheliped with a gape between the fingers, while smaller crabs have the setae and retain forceps-like chelipeds. Do these crabs have a terminal molt? If so, that might explain the lack of a covering--if the crab dies after mating or releasing eggs, there is no advantage to expending energy on camouflage.
Additional comments: Current manuscript is an improvement over first version.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 2
Reviewer: 2

Output:
Basic reporting: See General Comments
Experimental design: See General Comments
Validity of the findings: See General Comments
Additional comments: Review of Revision

Individual behavioral type captured by a Bayesian model comparison in cap making by sponge crab

Comments:
=========
The authors have done a lot of work to meet the concerns of the examiners. The new title is much more appropriate. However some concerns remain for me, and some new concerns have been created. It should be relatively easy to address these concerns.

1) The addition of an explict formulation of conditional vs marginal likelihood is great, but would benefit from being better structured. I found it confusing and doubt that the intended readership would be able to dis-entangle it. For example:
 a) Line 172. There is potential confusion here since the data are said to be iid, yet are then assigned a conditional (upon w) distribution for each individual, and hence can not be iid (at the conditional level).
 b) Lines 172-184. This presentation would be clearer to me if the w latent parameters were given a subscript i. Note that equation (3) only shows a single w value, yet has an index on the x variables. I would recommend using p(x_i|w_i) in line 172.
 c) Note that it is at the marginal level, equation (1), that the data are iid.
 d) Line 177. I think it may be the case that WAIC is estimating the expected log-predictive density, not predictive density.

2) The authors continue to confuse me with their perspectives of Maximum Likelihood and Bayesian approaches, and replied to one of my concerns by writing:
"We are thinking about the maximum likelihood method from a Bayesian point of view, so used the sentence."
Maximum likelihood and Bayesian are two different philosophies! Possibly they view ML as a Bayesian approximation, which seems to be the viewpoint used in Watanabe's book.
However, this failing to distinguish between two fundamental statistical philosophies leads to some very misplaced concepts being presented, for example the justification of using WAIC rather than AIC or Rsq (lines 304-324). At the end of the day, the approach herein is Bayesian and so AIC and Rsq are not relevant since they are Maximum Likelihood measures of model prediction and fit, respectively. I couldn't see any request from the other reviewers for the additional discussion about AIC and Rsq. They can't be used here, so why discuss them? [Aside: Using ML, AIC can be used if it is the marginalized likelihood that is maximized. Rsq is not appropriate for hierarchical models, doubly so if the response is not normal.] See also the mention of Rsq in lines 338-342. Elsewhere?

3) While the formula for conditional likelihood is given, there does not appear to be any presentation or discussion of how the marginal likelihood was calculated. For example, in the Choice of Material Size, how is the marginal likelihood, p_{model}(x|w_0), actually calculated? That is, I'm presuming (?) that the conditional model is specified in Stan, so how was this marginal likelihood calculated. Since use of this marginal likelihood is a focus of this work, it would seem essential that the reader is informed how to evaluate it.

4) The Abstract has doubled in size and would benefit from shortening.

5) Strangely, despite its increased length, the Abstract now fails to mention the need to use marginalized WAIC. This is surprising, since the authors replied to one of my concerns about the orginal manuscript by writing:
"We think the novelty is in the model comparison approach using marginal-WAIC for the repeatedly measured behavioral data."

6) Line 336. Change "However, it is used with a software without the consideration on this point" to something like "However, it is generally more convenient to use conditional-level likelihood within most Bayesian software, with the unfortunate consequence that conditional-level WAIC is often used".


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The wording in the revised manuscript is more concise and accurate than in the first version, and incorporates the suggestions of the editor and other reviewers. For unknown reasons, two references were left off in the text: page 24: Hazlett 1981 (shells used by hermit crabs); Dembowska 1962; page 25.
Experimental design: Very novel experimental design, supplementary videos clear.
Validity of the findings: The authors have incorporated suggestions regarding their statistical analysis as recommended by another reviewer.
Additional comments: A novel approach to crab behavior. Most papers on the subject are strictly observational. The sharp videos are informative and far better than existing film clips that are available on line.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INDIVIDUAL BEHAVIORAL TYPE CAPTURED BY A BAYESIAN MODEL COMPARISON OF CAP MAKING BY SPONGE CRABS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have satisfied my comments. I have no further concerns.
Experimental design: Fine
Validity of the findings: Fine
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF TWO FUNCTIONAL XYLOGLUCAN GALACTOSYLTRANSFERASE HOMOLOGS BRMUR3 AND BOMUR3 IN BRASSICACEOUS VEGETABLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Structure of the manuscript is fine for the scientific content but english and syntax are way below scientific standard and requires improvement
Experimental design: Very good but may be improved by full characterization of the xyloglucan modification in mur3-3 mutant plant overexpressing the Brassica putative galactosyltrasferase.
Validity of the findings: Very good. Data are novels and interesting, worth publications. Discussion is of interest and data supported but impaired by poor syntax. Correction required.
Additional comments: The manuscript by Wang and co-authors described the identification and the functional characterization of two galactosyltransferases from brassicaceous plants acting on xyloglucan (XyG) hemicellulose.
The two genes identified and characterized are BrMUR3 and BoMUR3 from Brassica rapa and Brassica oleracea species, respectively. Galactosyltransferase activity of both genes was demonstrated using a classical functional reverse genetic study where the authors used the mur3-3 mutant from Arabidopsis thaliana which is normally deprived of galactosylated XyG structure. Successfully, using transgenic Arabidopsis thaliana plant over expressing BrMUR3 and BoMUR3, the authors could showed evidence that both genes were able to rescue the mur3-3 mutant back to WT phenotype, and using MALDI mass profiling they showed that XyG profiling was matching the WT.
1.	As displayed, the experimental design is well suited to the study, has been performed rigorously with respect to the protocols employed, which makes the data set presented convincing and meaningful. Although I was satisfied and convinced by the demonstration by the authors of BrMUR3 and BoMUR3 genes encoding XyG galactosyltransferase activities, I was deceived by them not fully characterizing the galactosylated XyG in their transgenic lines. Indeed by combining beta-galactosidase treatment of the XyG from the transgenic lines with linkage analysis they would have clearly demonstrated the type of transfer catalyzed by their putative enzymes. One would also argue that an additional experiment demonstrating in-vitro transfer of UDP-Gal onto AIRs prepared from mur3-3 mutant would have nicely demonstrated the transferase activity of the MUR3 proteins from Brassica species which remains putative without such experiments.
Although the data set is convincing in its actual form, one could point out additional experiments remaining to be done before planning the crystallographic studies of the MUR3 proteins from Brassica.
2.	Another point of pure form but of outmost importance is the quality of the scientific language that is too low for publication in its actual form. Sometimes English mistakes in word spelling or syntax were not too numerous, and one could follow the authors thinking but often writing of poor quality in the manuscript was a limitation for comprehensive understanding of the speculation by the authors.  I will listed hereafter some suggestion of improvement of the introduction section but the discussion would need also to be re-written in its form ( no issue with the data interpretation or speculation that are supported by the data).
a.	For example, it would more clear, line 89 if the authors remind the reader that xxt1 and xxt2 are altered for xylostransferase 1 or 2, as it was clearly said line 85 for xlt2 being altered for a galactosyltransferase.
b.	Line 91 XyG variation does not necessarly lead to “spectacular” variation of plant phenotype would be more accurate.
c.	Line 93 “shorter” instead of short.
d.	Line 94, change “and a little smaller than WT” by “and the double mutant was a little smaller than WT” (more fragile as well in fact).
e.	Line 102 modify ref (L, 1964) put the name of the author for this reference.
f.	Line 114 this implies us that the defiency…check syntax of that sentence.
g.	L117, which “further” KO that AtXXT2…change further or removed it.
h.	L122 That’s means.. no “  ’s  ” needed
i.	L134 eucalyptus instead of eucalypts
j.	L136 were functionally not were functional
k.	L192 change ref M et al 1999 by the name of the author
l.	L194 correct PH5 to  pH5
m.	L267 , why using upper case for GLYCOSYL TRANSFERASE?
n.	L271 check syntax (possibly glycosyl….).
o.	L276 because instead of due to ?
p.	L294 check syntax of that sentence
q.	L303 put double mutant where it is appropriate
r.	L308 syntax to be checked
s.	L310 to 314, I did not get the message clearly for that part
t.	L316_317 are all belong brassicaceous? Check syntax
u.	L323 change complementary for transgenic or other better word
v.	L327 change need to needed
w.	L335 functional to functionaly


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: IDENTIFICATION OF TWO FUNCTIONAL XYLOGLUCAN GALACTOSYLTRANSFERASE HOMOLOGS BRMUR3 AND BOMUR3 IN BRASSICACEOUS VEGETABLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language needs improvement so that an international audience can clearly understand the text. This includes the usage of past and present tense, use of plural or singular, etc. Examples are:
Line 1: Homologs instead of homologies
Line 34: …, which were renamed as BrMUR3….
Line 67: ..wiche are encoded by
Line 68: …are predicted to be….
Line 74….fucosyltransferase that can….
Line 79-82: check the grammar in these sentences
Line 82: …the basic oligosaccharide structure of XyG is (not was) XXXG….
Line 87: use the abbreviation XyG instead of Xyloglucan throughout the whole document
Line 90: reference format not korrekt - [8, 13]
Line 96, 116, 123 and more: cabbage-like or „cabbage-like“ – this should be consitent throughout the text.
Line 100: present tense
Line 101: …exostosins ecoded by…
Line 102, 179, 192: check reference format
Line 102-104: check „specificity“, galactosyl and xylosyl should be followed by residue or moeity
Line 95-126: the whole paragraph needs to be structured more clearly, for example in line 104ff it is stated that mur 3-1 und 3-2 are deficient of XXFG and XLFG, but in line 112 ff  it is stated that they contain F side chains. In line 95 ff it is stated that dysfunction of AtMUR3 results in severe phenotypic changes, but in Line 107 it is stated that mur 3_1 and mur3-2 have similar phenotypes to wild type. Here, a more clear description of the mutants and their phenotypes is necessary.
Line 122: the WT only has 9% of XLXG according to Kong et al. 2015 – I am assuming the authors wanted to refer tot he mur mutants with the 38% proportion? However, here again it is necessary to specify which mutant they are refering to, as the mur3-3 has ~38% XLXG whereas mur3_7 has ~27% XLXG according to Kong et al. 2015.
Line 129: functionally – check if adverb, verb or adjective is correct here.
Line 138: check „…was not in AtMUR3 gene…“
Line 140: rescue, not rescued and report not reported
Line 163: I do not understand the word „acrial“ – maybe „aerial“? also applied to figure 2 legend
Line 164: Check sentence grammar and language
Line 198: …were used….
Line 263: „transfer“ not „transform“
Line 275: here is a cut and hard to follow from the reason of the study to suddenly that some plants are not familiar. This should be improved by stating the phylogenetic relationship first, then giving the reason for the choice of plant species.
Line302: overexpression of OsMUR3 still resulted in cabbage-like phenotype according to liu et al. 2015 – this sentence should be corrected accordingly.
The abstract and the introduction would benefit from a concise description of the reason for the study. This is currently only found in the discussion in Line 269-275, but should be stated earlier already.
Table 1 needs to be improved by 1) units for the cell wall sugars, b) information about statistics that were performed
Figure 4 legend needs to be improved regarding language. Also naming of the transgenic lines is not consitent: BrMUR3-com in the legend versus BrMUR3com in the figure. Also the number of the line is missing. „XyGOs“ should be „XyGs“.
Supplemental Figure 1 and 2: correct for spelling error (e.g. Oryza – Orazy)
Experimental design: The manuscript fits the scope of the journal. Based on already observed functional differences in MUR3 homologs it is of relevance to identify and characterize further MUR3 genes of different species to shed light on the biological variance in xyloglucan biosynthesis.
The methods could benefit of more detail and information for replication with respect to MALDI-TO MS analysis.
I would strongly recommend doing statistical analysis on Hypocotyl length of the MUR3 transgenic lines to see if brMUR3 can fully or only partially complement this phenotype.
In addition the results of the complementation with G.hisutum, N. tabacum and R. sativus should be presented as well even though they did not complement the mur3-3 mutant. However, they are relevant considering the reason of the study (investigating MUR3 homologs regarding specificity of glycosyltransferase activity) and worth showing in supplemental materials.
Validity of the findings: Analyzing the MUR3 clade of GT47 is of high relevance as the different mur3 Mutants in Arabidopsis already show differences in their phenotype and previous research has shown species-specific differences in the ability to complement the AtMUR3 mutant phenotypes. This indicates the biological variance and complex regulation of the xyloglucan biosynthesis pathway in plants. The authors identify 2 genes from Brassica species which can complement the mur3-3 phenotype they describe as cabbage-like.
In this respect the hypocotyl length analysis should be analyzed statistically (or repeated) to shed light if BrMUR3 can fully or only partially complement the short hypocotyl phenotype. From the pictures there seems to be only a partial complementation and this should be clarified and discussed.
The authors also discuss the phenotype complementation with regard to the degree of galactosylation in previous studies (Line 290-309). Therefore, it is important also to determine the proportion of galactosylation for the BrMUR3comp and BoMUR3comp transgenic lines. This is an analysis I would highly recommend in order to verify and justify this part of the discussion.
In line 256 the authors state that XXLG was also lost in mur3-3 mutant which is not in accordance with their figure 4 were the peaks for XXLG and XLXG are still present in mur3-3. This should be corrected.
Additional comments: The manuscript presents interesting new MUR3 genes from two brassica species that are shown to have similar functions as AtMUR3. However, analysis of galactosylation in the transgenic lines is missing which results in this part of the discussion standing alone, unconnected to the findings. I would highly recommend providing this data and include this in the discussion.
I would also recommend to include the negative results for the other three species in supplemental data, as those are still relevant regarding the reason of the study, to shed light on variance of the MUR3-clade of GT47.