Discovery of Four Novel ORFs Responsible for Cytoplasmic Male Sterility (CMS) in Cotton (Gossypium hirsutum L.) through Comparative Analysis of the Mitochondrial Genomes of Four Isoplasmic Lines

Round 1

Reviewer 1 Report

In this manuscript, structures of mitochondrial genomes of three cotton CMS lines and one maintainer line for them are presented. These structures were revealed with the help of next-generation, high-throughput DNA sequencing followed by bioinformatic analyses. The methodology and data presented were interesting to me, and are likely interesting to other readers as well.

However, the manuscript contains minor yet many errors and insufficient descriptions. I am giving them below, but these will not be all. It should be better for the manuscript to be edited by some expert.

1. In Abstract, "CMS 2074A" should be explained.
2. In Line 39, the definition of CMS actually appears to be the definition of male sterility. It should be better to say that mitochondria are involved in CMS.
3. In Line 42, "mt genes" should be "mitochondrial genes".
4. In Line 50, "mt DNA(mtDNA)" should be "mitochondrial DNA (mtDNA)".
5. In Line 58, "Chimeric gene" should be "Chimeric genes". The definition of the chimeric genes described is difficult to understand.
6. In Line 59, "cotranscribe" should be "be cotranscribed".
7. In Line 65, "rape" should not be italicized.
8. In Line 69, "several genes were found to differ at the DNA level or mRNA level" is difficult to understand. (More specific information should be necessary.)
9. In Line 73-92, some descriptions are difficult to read. This is because this part mentions first author's name for each reference. Why does only this part use this citation style?
10. In Line 135, "of clean data was obtained" should be removed.
11. In Line 164, "protein-encoded" should be "protein-coding".
12. In Line 175-176, it is difficult to understand why "Moreover" is used. It is also difficult to understand why the nine nad genes are divided into two groups ((nad3, nad4, nad6 and nad9) and (nad1, nad2, nad4L, nad5 and nad7)).
13. In Line 180, "Repeats are multiple copies of gene sequences" seems awkward.
14. In Line 187, "that existed" should be removed.
15. In Line 191, what is "an unknown sequence"?
16. Line 193 seems to have two sequential spaces (or a tab).
17. In Line 209-212, it is difficult to understand cp1-3. Are they putative plastid-derived sequences found on mt genomes?
18. In Line 218, "27.14 kb-27.40 kb" should be "271.4 kb-274.0 kb" (or this text does not agree with Figure 3).
19. Line 231-233 is difficult to read. Are the 13 ORFs shared between the three CMS lines?
20. In Line 249-257, what is the relevance of categorizing mitochondrial ORFs into the four groups?
21. Line 261-262 is difficult to read. Is the following correct? "orf116b was found to be 193 bp downstream of rpl5, to have a 41 bp overlap with rpl2 and to contain partial repeats"
22. In Line 264, "3" in the beginning of a sentence should be "Three".
23. In Line 275, "SNP variant" should be "single nucleotide polymorphism (SNP)".
24. In Line 295, "these studies" should be "this study" (because only one article is cited here).
25. In Line 304, "typically" should be removed.
26. In Line 321, what does "useless" mean?
27. In Line 330, what does "The results are similar to those of other ORFs" mean?
28. In Line 337-338, "a great relationship with" should be "a role in".
29. In Line 341, "overexpression and knockout" should be "overexpression and knockout of these genes".
30. In Line 346 and elsewhere, "CDS" should be "coding sequence (CDS)".
31. In Line 348, "among different types of plants" can be removed.
32. Line 351 ("and that the genes with non-synonymous mutations deserve special attention") can be removed. The description "deserve special attention" does not seem appropriate.
33. In Line 355, "those in G. hirsutum" should be "those in the reference mt genome of G. hirsutum".
34. In Line 357, "that there were" should be "that were".

Author Response

"Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The MS by Li et al. is on the identification of  CMS-associated genes in the mitochondrial genomes of 4 cotton lines. The authors sequenced, assembled and annotated the mtDNA genomes.

First of all, the text is written in poor English. It would benefit of syntax improvement and revisions by an official translator or a native English-speaker. This compromises easy reading and especially the right interpretation of the main findings. I would suggest the authors to improve the narrative style and text flow.

Even if the question posed by the authors is clear, in my opinion the manuscript includes a great deal of inaccuracies, deficiencies in the description of the methods and errors in the presentation of results.

At some point I had to stop the review because annoyed by the very high number of inaccuracies.

Here a partial  list of suggestions to improve the  overall quality of the manuscript:

The title is unclear in its first part.

Abstract

Line 20-21: Please reword the sentence. Poor English.

The first claim is general. The second sentence (line 21-23) introduces unexpectedly “cotton”. Please, gradually introduce the reader into the issue!

Lines 25-27: Please, use  here a range for mtDNA size. Remember it is an abstract!

Lines 28-30: “Comparative analysis of multiple copies”. What does it mean? It is not clear to me. Please, modify.

Furthermore, which is the association between “Comparative analysis of multiple copies and nonsynonymous mutations” and sterility protein-coding genes?  Not clear.

Indeed authors focused on four ORFs as CMS-associated genes .

Please, rephrase the sentence as it is indecipherable. 

Line 32: what is CMS 2074A? Please, define it. 

 

Introduction

line 42: Please, define mt.  mt genes with several nuclear genes? Please reword the sentence, trying to make the concept understandable to everyone.

line 44: encode for

line 50: mt DNA(mtDNA) . A space is missing.

lines 58-59: Please reword the sentence.Poor English.

 

M&M 

line 95: were their maintainer line ? typo?

line 105: Please specify which sequencing platform has been used.

Line 105-106: The entire mtDNA sequences of the two lines were used for 105 sequence alignment.  Which sequences do the authors refer to?

Line 108: Not clear which is the reference sequence. The citation of the reference should be anticipated in the sentence

Please, provide which settings have been used for SOAP de novo and  SOAP Aligner.

line 111: assembled with the reference genome using BLAST? Never seen anything like this before. Are authors able to assemble mitogenomes with BLAST?

Please, include used BLAST parameters.

line 112: Finally, a mt genome circle map was constructed. How?

line 123: Please replace “were  evaluated” with “were identified”.

line 127: u-fa- C50-bcftoolsview-I-N-b-v-c-g ??? What is that?

line 130: depending on where their location ???? Maybe “depending on their location”?

 

Results

I should avoid the use of “clean data” in a scientific paper. Please, use high quality data throughout the text and table headers.

lines 134-139: Information already present in Table 1. It is a useless repetition. Please be concise. The table is there for supporting your summary.

line 141: Please, remove from the  legend of Table 1 the name of the accessions under study.

lines 144-145: NCBI records, corresponding to the accession numbers the authors provided, are still not available in GenBank. Please, clarify.

Figure 1 is useless as it is unreadable. Please include each mtDNA map in separate figures as Supplementary.

The last row in Table 2 could be deleted. (% of Genome(Intergenic)) this info can be obtained by subtraction from the row Gene length/Genome(%) .

lines 146-152. Again, there is no need to rewrite the content of table 2!

line 173 “had an intron “ should be “had a single intron ”

line 173. Please remove “cotton”.

line 180: “Repeats are multiple copies of gene sequences in the genome. “ Are the authors sure about the definition of repeats they provide?  why do they refer to gene sequences?

lines 180-182: Previous analyses showed that some repeated sequences were involved in rearrangements of the mt genome, which could also results in CMS.  A reference is missing here.

line 182: of the duplicates ????

line 182-186:  Information already present in Table 4. It is a useless repetition. Please be concise. The table is there for supporting your summary.

Figure 2. what is ratio of total length? Ratio is a dictate variable so please avoid continuous line. The name of the line should be  in the title of the graph and not in the Y axis.

I suggest to merge alle barcharts in a single one to facilitate comparison.

line 205: Please, replace “homology” with “similarity”

line 210: Please, replace “were homologous” with “were similar”.

line 211: “no homologous sequences”  should be “no similar sequences”  

and more other minor/major concerns till the end of the text.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Li et al assembled mitochondrial genomes for three cotton CMS and one maintainer lines; and then conducted comparative analysis to study structural differences between these two groups. The authors identified two novel ORFs (orf111f and orf174); and characterized the other two ORFs (orf186a-1, and orf186a-2) that are likely responsible for Cytoplasmic Male Sterility in cotton. I have some minor comments, as below:

1. Line 96: provide affiliation. It the provider is the corresponding author, then I do not think this information is needed in the Methods.
2. Line 102: Delete the word “sample”
3. Line 105: why only two lines, not four? Are they referring to CMS line and maintainer line (considering 2 lines)? Then somewhere in this manuscript, it was three CMS lines and one maintainer line (considering 4 lines). If this is the case, then, the authors should use these consistently.
4. Line 111: correct to “mt reference genome”
5. Regarding assembly of the mt genomes presented in Section 2.2. How many contigs were obtained from SOAPdenovo software for each sample? Or was it only one single contig obtained? If multiple contigs were obtained for each sample, how were the ends of each contig and their directions determined? Were there any overlapped sequences at the ends of two adjacent contigs so that the authors can determine that they can be joined together? I think the section 2.2 should be revised, to clearly present the steps that the authors did to get the mt genome sequences. Currently, it is somewhat not clear whether the authors did de novo assembly, or reference-guided assembly to me.
6. It is not clear which software was used to estimate k-mer content.
7. Line 163: which lines does “both lines” refer to?
8. Line 187, the authors wrote “we found a 9,284 bp repeat sequence (A3R5) that existed only in J4A-3, which was the main cause of the differential sizes among the four lines.”. However, in Table 2, J4A-3 has a smaller mt genome (622,848 bp), compared to J4A-1 and J4A-2 (623,067 and 623,343 bp, respectively). Could the authors elaborate this?
9. Please fix the column alignment in Table 4.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The overall quality and readability of the manuscript have improved considerably.  All my concerns have been addressed.

However, I would like to reiterate that I think authors have not been honest in submitting a manuscript with such a large number of inaccuracies, deficiencies and errors, hoping that the reviewers are lenient and do part of the work instead of authors.

I am very disappointed with this way of doing.