Codon Usage Profiling of Chloroplast Genome in Juglandaceae
Keith E. Woeste
Round 1
Reviewer 1 Report
Title should perhaps be “Codon Usage Profiling of Juglandaceae Chloroplast Genomes”
Abstract: Shows signs of cut/paste authorship. Juglandaceae is not a species.
Line 10, what do the authors mean by Woody grains?
Line 15: not clear (short ones? Of what?)
Line 17 “of all” not “all of”
Line 20, 22 and 24 repeat information
Intro;
Line 38, authorities of species names are not italicized
Line 62, I think the authors mean NOT extensively studied
Line 63 capitalize Juglandaceae
Sentence at line 70 is not comprehensible.
Line 75, is this Englehardia?
Table 1, what does dash mean under column “accession number”. Are these distinct genotypes? Where did they come from? What are they? Are they wild? Cultivated?
Line 79 says 49 but Table 1 says 64
Line 107 ‘were to essential’ is incorrect grammar
All tables appear to have formatting issues.
Line 157, do you mean some genotypes were the same? Your text mentions two individuals of the same species, not two species.
Line 171, this sentence is so general that it is meaningless. Section 2.4 appears to be in a smaller font.
Line 260, what does the word ‘it’ refer to?
Figure 1 does not explain what the different colors mean. The figure, and all figures, must stand on their own, explaining every part of every panel. What do the histograms represent?
Line 201, I think the authors mean Juglandaceae
Line 302 what is meant by ‘greening’ effects?
Line 302 – 311 describes the research results. How do these results relate to expected values in other species? What can be learned from these results?
Line 333 needs a citation.
Line 336, we can conclude that selection has affected the genome of Juglandaceae. Are there examples of selection NOT influencing the genome? In other words, is there any way in which this outcome is surprising or unexpected? We need better context, a clearer hypothesis, a better “so what”
Line 356, what does ‘best codons (24 – 30)’ mean?
Line 357, AAU is likely ASN or LYS, what is Lie? What is the biological or practical significance of this finding: we screened for 6 optimal codons shared by the 26 species: GGU 357 (Gly), AAU (Lie), AAU (Asn), CCU (Pro), CGU (Arg), and ACU (Thr).? Since it appears in the summary, I suppose it must have some importance, but the authors need to make clear what that is.
Font change in References
Citation 27 and 25 have spelling problems. Many citations fail to italicize species names and have problems where words were interrupted by line breaks during copy-paste.
When I look at Fig 3, I see that genotypes C and D are different from other J. regia. Do codon usage differences within species matter? If so, to what extent does limited sample size of all species except one affect the outcome of this study?
Sect 3.4 The reader needs more context. Is there a statistical test to indicate whether the observed variation is significantly different from random expectation? What do values from 24 to 61 mean? What is the significance of a point above or below the line in biological terms?
General note about writing and style.
The authors should be encouraged to remove the word ‘Meanwhile’ from this and all future submissions.
I can see how codon usage bias might inform biotechnological applications, but in that case the important data would be species-specific. It appears the only species represented with more than a small number of samples (J. regia) showed diversity in its codon usage bias. In such a case, what do the authors suggest in terms of how others should use their data?
Nuclear and chloroplast genomes are under different mutational and selection constraints. Can the authors say anything about how the observed bias in chloroplast DNA informs or is related to nuclear genome evolution? If selection and evolution shaped bias in the Cp, what selection or evolutionary factors are able to shape codon bias in chloroplasts?
My greatest concern is that the authors have thrown every possible analytical resource at a problem that must be important to someone, but they fail to explain why the problem is important beyond those who are designing vectors to genetically modify Juglans species (a very small group indeed). Do these results permit any conclusions or validate any hypotheses any beyond simply reporting on bias observed in the particular samples they studied? Is it known if the genotypes they evaluated are representative of the species?
What can be learned about evolution or biology by knowing the aggregated codon usage bias of a few individuals of a few species within a genus?
Author Response
Review 1
Abstract: Shows signs of cut/paste authorship. Juglandaceae is not a species.
Response: Dear reviewer,Revised as suggestion.We have changed the description.
Line 10, what do the authors mean by Woody grains?
Response:We have changed the description
Line 15: not clear (short ones? Of what?)
Response:We have checked and changed the description.
Line 17 “of all” not “all of”
Response: Revised as suggestion.
Line 20, 22 and 24 repeat information
Response: We have checked in “line 20, 22 and 24” ,we described the results of different analyses, but we did not find duplicate expressions.
Intro;
Line 38, authorities of species names are not italicized
Response: Revised as suggestion.
Line 62, I think the authors mean NOT extensively studied
Response: Revised as suggestion.
Line 63 capitalize Juglandaceae
Response: Revised as suggestion.
Sentence at line 70 is not comprehensible.
Response:We have changed the description and revised as suggestion.
Line 75, is this Englehardia?
Response: Revised as suggestion.
Table 1, what does dash mean under column “accession number”. Are these distinct genotypes? Where did they come from? What are they? Are they wild? Cultivated?
Response: Assembled by our laboratory (from b to k) ，not uploaded to NCBI . we will update the accession number later.
Line 79 says 49 but Table 1 says 64
Response: The genes in under column “Protein-Coding Genes” are the total number of Protein-coding genes that were not screened, and 49 is the number of gene entries that were eligible for analysis after screening, filtered data has been added to the table1.
Line 107 ‘were to essential’ is incorrect grammar
Response: We checked and revised as suggestion.
All tables appear to have formatting issues.
Response: Revised as suggestion and we have made adjustments to all tables.
Line 157, do you mean some genotypes were the same? Your text mentions two individuals of the same species, not two species.
Response: Revised as suggestion.We have changed the description.
Line 171, this sentence is so general that it is meaningless. Section 2.4 appears to be in a smaller font.
Response: Checked.
Line 260, what does the word ‘it’ refer to?
Response: We have changed the description.
Figure 1 does not explain what the different colors mean. The figure, and all figures, must stand on their own, explaining every part of every panel. What do the histograms represent?
Response: Based on the reviewers' comments, we have annotated the corresponding plates below.
Line 201, I think the authors mean Juglandaceae
Response: We checked and revised as suggestion.
Line 302 what is meant by ‘greening’ effects?
Response:We want to express that "Juglandaceae is a family of plants that are used for food, medicine and greenery. "The sentence had been revised.
Line 302 – 311 describes the research results. How do these results relate to expected values in other species? What can be learned from these results?
Response:We checked and revised as suggestion.
Line 333 needs a citation.
Response:We have checked and cited the literature as suggestion.
References:
XU, C.;CAI, X.;CHEN, Q.;ZHOU, H.;CAI, Y.BEN, A.;Factors Affecting Synonymous Codon Usage Bias in Chloroplast Genome of Oncidium Gower Ramsey. Evolutionary bioinformatics online, 2011,14(7), 271-278. 10.4137/EBO.S8092
LIU, Q.P.XUE, Q.Z.;Comparative Studies on Codon Usage Pattern of Chloroplasts and Their Host Nuclear Genes in Four Plant Species. Journal of genetics, 2005, 84 (1), 55-62. 10.1007/BF02715890
CAMPBELL, W.H.GOWRI, G.;Codon Usage in Higher Plants, Green Algae, and Cyanobacteria. Plant physiology, 1990, 92 (1), 1-11. 10.1104/PP.92.1.1
Line 336, we can conclude that selection has affected the genome of Juglandaceae. Are there examples of selection NOT influencing the genome? In other words, is there any way in which this outcome is surprising or unexpected? We need better context, a clearer hypothesis, a better “so what”
Response:We have checked and cited the literature as suggestion.
References:
SUEOKA, N.;Directional Mutation Pressure and Neutral Molecular Evolution. Proc Natl Acad Sci U S A, 1988, 85 (8), 2653-2657
SHARP, P.M.;ELIZABETH, C.;DESMOND, G.H.;SHIELDS, D.C.;KENNETH, H.W.WRIGHT, F.;Codon Usage Patterns in Escherichia Coli, Bacillus Subtilis,Saccharomyces Cerevisiae, Schizosaccharomyces Pombe, Drosophilamelanogaster and Homo Sapiens; a Review of the Considerable within-Species Diversity. Nucleic Acids Res, 1988, 16 (17), 8207–8011. doi:10.1093/nar/16.17.8207
Line 356, what does ‘best codons (24 – 30)’ mean?
Response: We checked and revised as suggestion.
Line 357, AAU is likely ASN or LYS, what is Lie? What is the biological or practical significance of this finding: we screened for 6 optimal codons shared by the 26 species: GGU 357 (Gly), AAU (Lie), AAU (Asn), CCU (Pro), CGU (Arg), and ACU (Thr).? Since it appears in the summary, I suppose it must have some importance, but the authors need to make clear what that is.
Response: We checked and revised as suggestion.
Font change in References
Citation 27 and 25 have spelling problems. Many citations fail to italicize species names and have problems where words were interrupted by line breaks during copy-paste.
Response: We checked and revised as suggested.
When I look at Fig 3, I see that genotypes C and D are different from other J. regia. Do codon usage differences within species matter? If so, to what extent does limited sample size of all species except one affect the outcome of this study?
Response: We checked and revised as suggested. We have added relative description in 3.3 section.
Sect 3.4 The reader needs more context. Is there a statistical test to indicate whether the observed variation is significantly different from random expectation? What do values from 24 to 61 mean? What is the significance of a point above or below the line in biological terms?
Response: We counted ENC and exp-ENC in Schedule S1. The line was explained in section 2.4.1
General note about writing and style.
The authors should be encouraged to remove the word ‘Meanwhile’ from this and all future submissions.
Response: We accept the reviewers' comments.
I can see how codon usage bias might inform biotechnological applications, but in that case the important data would be species-specific. It appears the only species represented with more than a small number of samples (J. regia) showed diversity in its codon usage bias. In such a case, what do the authors suggest in terms of how others should use their data?
Response: The research data at least proves that there are differences among genera. Due to the limitation of the sample size of the inter-species data, the follow-up research species collected multiple samples of each species of Juglantaceae to prove that its CUB is species-specific.
Nuclear and chloroplast genomes are under different mutational and selection constraints. Can the authors say anything about how the observed bias in chloroplast DNA informs or is related to nuclear genome evolution? If selection and evolution shaped bias in the Cp, what selection or evolutionary factors are able to shape codon bias in chloroplasts?
Response: Revised as suggestion. We have added relative description in Discussion section.
My greatest concern is that the authors have thrown every possible analytical resource at a problem that must be important to someone, but they fail to explain why the problem is important beyond those who are designing vectors to genetically modify Juglans species (a very small group indeed). Do these results permit any conclusions or validate any hypotheses any beyond simply reporting on bias observed in the particular samples they studied? Is it known if the genotypes they evaluated are representative of the species?
Response: Revised as suggestion. We have added relative description in Discussion section.
What can be learned about evolution or biology by knowing the aggregated codon usage bias of a few individuals of a few species within a genus?
Response: Revised as suggestion. We have added relative description in Discussion section.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The manuscript by Zeng et al. describe and analyze codon usage patterns within plastomes of Junglandaceae. Overall, the study is well designed and written.
Minor comments
-
Table 1. All analyzed plastomes of Juglans regia should be submitted to GenBank, even if they are identical.
-
Did authors check annotations of downloaded plastomes in the case when aboriginal records lack particular genes?
-
Figure 2 and 4 captions - Authors should consider adding letters after each sample, it’s hard to read in the present form.
-
I would recommend performing some discrimination analysis like MILC to test inter generic variation of CU pattern.
Author Response
Review 2
Comments and Suggestions for Authors
The manuscript by Zeng et al. describe and analyze codon usage patterns within plastomes of Junglandaceae. Overall, the study is well designed and written.
Minor comments
Table 1. All analyzed plastomes of Juglans regia should be submitted to GenBank, even if they are identical.
Response: We are assembling and annotating the chloroplast genomes of 10 Juglans regia plants sequenced in our laboratory. It has not been uploaded to NCBI yet, we will fill in the Accession No later.
Did authors check annotations of downloaded plastomes in the case when aboriginal records lack particular genes?
Response: We have checked.
Figure 2 and 4 captions - Authors should consider adding letters after each sample, it’s hard to read in the present form.
Response: We accepted the reviewers' suggestions and added the corresponding letter to the name of each species
I would recommend performing some discrimination analysis like MILC to test inter generic variation of CU pattern.
Response: Revised as suggested, We have added MILC analysis in Section 3.7.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Review of Codon usage profiling
Line 32, change ‘has a certain impact on’ to ‘affects’
Line 33, delete word ‘then’
Line 71, change genome to genomes.
Line 107 -108 does not make sense.
Line 110 change calculated to calculating
Line 114 is unclear. What is the outcome that Vice versa refers to? Do you mean values >35 are strong? Are not considered strong?
Line 206, change to “the GC dinucleotide.”
Line 214, fairly weak across what?
Line 241 – 242, delete words “From Fig 3 that” and reference fig 3 at the end of the sentence. Change ‘usages’ to ‘usage’
Line 243 – Differences in optimal codon usage within the same species. That’s interesting and it should be discussed more fully. To me, it means that 1. Results based on a small number of samples in a species can mislead and do not support any general conclusions, and 2. May indicate an association between chloroplast optimality and phytogeography based on where the sample was adapted. The authors would do well to add a sentence or two about this in the discussion.
Line 265, Schedules SI? Do you mean Supplement?
Line 269, change “and other factors were speculated to be” to something more specific. The entire phrase is awkward. I suppose you mean something like “ for this reason we believe factors other than natural selection have driven the complex codon usage preference…”?
Line 284 “disrupted” is not the right word. The frequency was what? Greater than expected? Less?
Line 341, delete word ‘the’
Line 361, change ‘in chloroplast in mitochondria’ to ‘in chloroplast and mitochondria’
Line 379, the phrase ‘a certain amount’ is so vague that it is meaningless. If you don’t know the amount, then say “an unknown amount” , if all you know is that it is more than zero, say “selection has occurred in Juglandaceae chloroplast evolution but we have no data to indicate how much” or something like that.
Line 374, change screen to screened?
Line 375 delete all the commas.
Line 377, “We found have MILC values” does not make sense, the sentence should be written in past tense. Do you mean “ We found that MILC values of our samples indicated high gene expression.”
Line 381 does not say anything – “others have found differences” is so vague that it is meaningless. What differences were found? Line 381 – 382, your results are too limited to suggest global differences between dicots and monocots. Delete this sentence.
Line 389, (Lie) is not an amino acid. Please correct this error.
Line 389, delete word “could” , change ‘provides’ to ‘provide’. This sentence would be much improved if you would say specifically what valuable information your study provides to genetic engineering studies. For example, codon usage and codon optimality data.
There are many genus and species names in the References that are not properly italicized.
Author Response
Line 32, change ‘has a certain impact on’ to ‘affects’
Response: Revised as suggestion.
Line 33, delete word ‘then’、
Response: Revised as suggestion.
Line 71, change genome to genomes.
Response: Revised as suggestion.
Line 107 -108 does not make sense.
Response: We have deleted the sentence “Excel was used to calculate the heat mapping and correlation analyses were performed in R.”
Line 110 change calculated to calculating
Response: Revised as suggestion.
Line 114 is unclear. What is the outcome that Vice versa refers to? Do you mean values >35 are strong? Are not considered strong?
Response: “Vice versa ” means the ENC values > 35, codon bias is weak. We have changed the description in line 116-117.
Line 206, change to “the GC dinucleotide.”
Response: Revised as suggestion.
Line 214, fairly weak across what?
Response: We have described in the text.
Line 241 – 242, delete words “From Fig 3 that” and reference fig 3 at the end of the sentence. Change ‘usages’ to ‘usage’
Response: Revised as suggestion.
Line 243 – Differences in optimal codon usage within the same species. That’s interesting and it should be discussed more fully. To me, it means that 1. Results based on a small number of samples in a species can mislead and do not support any general conclusions, and 2. May indicate an association between chloroplast optimality and phytogeography based on where the sample was adapted. The authors would do well to add a sentence or two about this in the discussion.
Response: We have added the relative description in lines 377-379.
Line 265, Schedules SI? Do you mean Supplement?
Response: Schedules SI are actual ENC values and expENC values.
Yes, we mean supplement.
Line 269, change “and other factors were speculated to be” to something more specific. The entire phrase is awkward. I suppose you mean something like “ for this reason we believe factors other than natural selection have driven the complex codon usage preference…”?
Response: We have changed the description and revised as suggestion in line 272-274.
Line 284 “disrupted” is not the right word. The frequency was what? Greater than expected? Less?
Response: We have changed the description and revised as suggestion in line 291-294.
Line 341, delete word ‘the’
Response: Revised as suggestion.
Line 361, change ‘in chloroplast in mitochondria’ to ‘in chloroplast and mitochondria’
Response: Revised as suggestion.
Line 379, the phrase ‘a certain amount’ is so vague that it is meaningless. If you don’t know the amount, then say “an unknown amount” , if all you know is that it is more than zero, say “selection has occurred in Juglandaceae chloroplast evolution but we have no data to indicate how much” or something like that.
Response: We have changed the description and revised as suggestion in 377-378.
Line 374, change screen to screened?
Response: We have revised as suggestion.
Line 375 delete all the commas.
Response: We have revised as suggestion.
Line 377, “We found have MILC values” does not make sense, the sentence should be written in past tense. Do you mean “ We found that MILC values of our samples indicated high gene expression.”
Response: We have revised as suggestion in line 387-388.
Line 381 does not say anything – “others have found differences” is so vague that it is meaningless. What differences were found? Line 381 – 382, your results are too limited to suggest global differences between dicots and monocots. Delete this sentence.
Response: We have deleted the sentence and revised as suggestion in 391-393.
Line 389, (Lie) is not an amino acid. Please correct this error.
Response: We have revised as suggestion in line 405.
Line 389, delete word “could” , change ‘provides’ to ‘provide’. This sentence would be much improved if you would say specifically what valuable information your study provides to genetic engineering studies. For example, codon usage and codon optimality data.
Response: We have revised as suggestion.
There are many genus and species names in the References that are not properly italicized.
Response: We have checked.
Author Response File: Author Response.docx
