The Chloroplast Genome of Carya illinoinensis: Genome Structure, Adaptive Evolution, and Phylogenetic Analysis

Round 1

Reviewer 1 Report

Line 52-54 "C. cathayensis, a native nut tree in China, is narrowly distributed in eastern China in the hilly areas between Zhejiang and Anhui provinces [7], which shows a less adaptive capacity than C. cathayensis. "- this sentence should be corrected,
Line 149-152, these 2 sentences should be combined to avoid repetition,
Line 168 "reaching 2559 bp (Table 2)" is not available in Table 2,
Line 183-184 since "Studies have indicated that the migration of infA to nuclear genome ..." then certainly the loss of infA occurred through transfer. These 2 sentences should be corrected,
Line 193 "SSRs belongs to short repeats" this sentence should be deleted because this information is described 2 sentences earlier,
Line 211 just designing primers does not give us any information. At least basic analyzes showing their proper operation should be done,
Line 223-225 what is the explanation of this phenomenon?
Table 3 I suggest to put in supplementary materials,
Line 247-252 please explain and discuss why there are more SNPs in exons than in introns? The next sentence "the comparative result demonstrated that the coding sequences were more conserved than the noncoding sequences" is illogical because it is described that there are 60 SNPs in coding sequences vs. 28 SNPs in introns, i.e. more in conservative structures than in non-coding areas (introns),
Line 271 "In general, the coding regions showed less divergence than the coding sequences." I don't understand the difference between coding regions and coding sequences?
Line 275 was not tested experimentally, which is a pity. It would be good to sequence at least a few of the regions described, confirming the assumption that "could be good candidates for Juglandaceae species identification",
Line 288, 290, 291, 292, 293 - species names and genes should be Italic,
Figure 4 - ndhF in C.kweichowensis should be 2232bp not 2231bp, ycf1 should be 5658bp instead of 5657bp, rpl2 in P.strobilacea can not be 434bp (not divided by 3) and in addition, another copy of rpl2 in IRa does not exist in the original sequence (KX 868670). What is rrn1 in SSC?
In References: Species names should be italic, "PloS one" should be corrected to Pols One (e.g. Line 385, 422 and so on).

Author Response

Point 1: Line 52-54 "C. cathayensis, a native nut tree in China, is narrowly distributed in eastern China in the hilly areas between Zhejiang and Anhui provinces [7], which shows a less adaptive capacity than C. cathayensis. "- this sentence should be corrected. 


 

Response 1: Thanks for your comment. We have rewritten it in the revised manuscript.

 

Point 2: Line 149-152, these 2 sentences should be combined to avoid repetition.

 

Response 2: Thanks for your remind. We have revised it in the latest version.

 

Point 3: Line 168 "reaching 2559 bp (Table 2)" is not available in Table 2.

 

Response 3: Thanks for your remind. We regret for this mistake; we have uploaded our chloroplast sequence along with its annotation to NCBI (MN977124). We prefer to provide it in the website instead of the manuscript.

 

Point 4: Line 183-184 since "Studies have indicated that the migration of infA to nuclear genome ..." then certainly the loss of infA occurred through transfer. These 2 sentences should be corrected.

 

Response 4: Thanks for your comment. We have rewritten these sentences in the revised manuscript.

 

 

Point 5: Line 193 "SSRs belongs to short repeats" this sentence should be deleted because this information is described 2 sentences earlier.

 

Response 5: Thanks for your remind. We have deleted it in the revised manuscript.

 

Point 6: Line 211 just designing primers does not give us any information. At least basic analyzes showing their proper operation should be done.

 

Response 6: Thanks for your comment. The provided PCR primers could be used for future population genetic studies, polymorphism investigations, and evolution analysis. We have revised the related sentence in the revised manuscript.

 

Point 7: Line 223-225 what is the explanation of this phenomenon?

 

Response 7: Thanks for your comment. We have added our explanation for that phenomenon in the revised manuscript.

 

Point 8: Table 3 I suggest to put in supplementary materials.

 

Response 8: Thanks for your suggestion. We have provide in the supplementary materials.

 

Point 9: Line 247-252 please explain and discuss why there are more SNPs in exons than in introns? The next sentence "the comparative result demonstrated that the coding sequences were more conserved than the noncoding sequences" is illogical because it is described that there are 60 SNPs in coding sequences vs. 28 SNPs in introns, i.e. more in conservative structures than in non-coding areas (introns).

 

Response 9: Thanks for your comment.  We have added related explanation for that phenomenon.  We also added interpretation for noncoding sequences (including intergenic regions and intron sequences). Therefore, we still think it is logical for the sentences: coding sequences were more conserved than the noncoding sequences.

 

Point 10: Line 271 "In general, the coding regions showed less divergence than the coding sequences." I don't understand the difference between coding regions and coding sequences?

 

Response 10: Thanks for your comment. We sorry for the mistake. We have revised that sentence to ‘In general, the coding regions showed less divergence than the non-coding sequences.’

 

Point 11: Line 275 was not tested experimentally, which is a pity. It would be good to sequence at least a few of the regions described, confirming the assumption that "could be good candidates for Juglandaceae species identification".

 

Response 11: Thanks for your comment. Indeed, it is a pity that the cp-derived markers have not been validated experimentally in this research. It will be done in our subsequent experiment.

 

Point 12: Line 288, 290, 291, 292, 293 - species names and genes should be Italic.

 

Response 12: Thanks for your remind. We have revised this mistakes throughout the manuscript.

 

Point 13: Figure 4 - ndhF in C.kweichowensis should be 2232bp not 2231bp, ycf1 should be 5658bp instead of 5657bp, rpl2 in P.strobilacea can not be 434bp (not divided by 3) and in addition, another copy of rpl2 in IRa does not exist in the original sequence (KX 868670). What is rrn1 in SSC?

 

Response 13: Thanks very much for your remind. These mistakes in Figure 4 have been revised. Please check in revised manuscript.

 

Point 14: In References: Species names should be italic, "PloS one" should be corrected to Pols One (e.g. Line 385, 422 and so on).

 

Response 14: Thanks for your remind. We have revised these mistake throughout the manuscript.

Reviewer 2 Report

Minor suggestions which would improve greatly the present manuscript:

1) please, upload the figure 1 also as supplementary fig at very high resolution. The map is important, but the quality in manuscript is too low to explore in more detail. This would benefit much for scientists who would be greatly interested in.

2) legends in figure 3 and 5 are hardly readable, please improve (bold, font).

3) some tables are uploaded in manuscript through few pages. Maybe better design of tables (smaller font.., paragraph parameters) would improve this and it would be more readable?

 

Author Response

Point 1: please, upload the figure 1 also as supplementary fig at very high resolution. The map is important, but the quality in manuscript is too low to explore in more detail. This would benefit much for scientists who would be greatly interested in.

 

Response 1: Thanks for your comment. We have also provided Figure 1 in the supplementary material.

 

Point 2: legends in figure 3 and 5 are hardly readable, please improve (bold, font).

 

Response 2: Thanks for your suggestion. All the Figures in our manuscript have been redrawn to make it be more aesthetic. Figure 3~5 are vector graphics in the revised manuscript, they may be somewhat hard to read in the word-formatted file, but are quite clear when converted to the PDF file.

 

Point 3: some tables are uploaded in manuscript through few pages. Maybe better design of tables (smaller font.., paragraph parameters) would improve this and it would be more readable?

 

Response 3: Thanks for your comment. We have make the table be more aesthetic as far as possible. Previous Table 3 has been uploaded as supplementary material.

Reviewer 3 Report

Mo et al. have de novo assembled the cp genome of the C. illinoinensis clone 'Pawnee', and present an in depth analysis of their findings. I believe it is a useful additon to our knowledge on the chloroplast genomics of Carya in general.

Just a few points:

The English would need a final spell check by a native speaker; e.g. already in the first word of the introduction it should read "The chloroplast..." not just "Chloroplast...". Or the sentence starting in the end of line 345 should be re-written to make it more understandable. Please give the GenBank accession no. of the reference genome used and credit the authors of that properly. Line 82: It should be "C. illinoinensis".

Author Response

- The English would need a final spell check by a native speaker; e.g. already in the first word of the introduction it should read "The chloroplast..." not just "Chloroplast...". Or the sentence starting in the end of line 345 should be re-written to make it more understandable. Please give the GenBank accession no. of the reference genome used and credit the authors of that properly. Line 82: It should be "C. illinoinensis".

 

Response : Thanks for your suggestion. We have checked our writing throughout the manuscript. The GenBank accession number (MN977124) have been provided in the latest version.

Reviewer 4 Report

Mo et al 2020 The chloroplast genome of Carya (pecan)

 

The paper describes the chloroplast genome for a cultivar of Pecan, reporting on the structure, genic content, polymorphism and phylogenetic position.

The results and methods are sound and the work is reported with clarity and depth. The cp sequence will be of value to breeders and geneticists and others interested in the improvement, evolution or the ecology of this plant group, and the supporting analysis of the genome structure and polymorphism provides confidence in the quality of the work undertaken given it confirmatory nature.

The paper could be accepted for publication following minor revisions for formatting and minor clarity issues.

Recommended corrections/changes to the ms

L13 Change “Comprehensively” to “Comprehensive”

L22 Insert “each” at end of line after “26,003 bp”

36 Insert “The” at start of sentence

39 Delete “are” before typically and change “consisted” to “consist”

42 Probably should have “most” cp are maternally inherited. Conifers are paternally inherited.

“less” recombination or “no” recombination? This may still be controversial. Is there now good evidence for some recombination?

L47 Delete “in the world” as not required

L54 I think this should be C. illinoinensis rather than C. catayensis here?

L55. Suggest change “Primary” to “In the past” or “Historically”

L59 change “a high demand” to “a need”

L67 Change you provide a better rationale for undertaking the phylogenetic analysis than it has not been done before? What practical difference might that make for the breeder? Better prediction of success of interspecific crossing?

L71 Delete “also” before “conducted”

L77 change “detected” to “analysed”

L80 change “applied” to “used”

L82. “C.illinoinensis” needs a space and italics

L152 Delete “generally” and change “have” to “having”

L160 Table 1. “unique gene number” Do you mean single copy genes? If so can you please replace it as unique gene number could have many different meanings

L166 replace “were with” with “have”

L169 replace “rest” with “other”

L178 Delete “happened in the “Pawnee” cultivar” as doesn’t seem necessary

L181 insert “in” after “included”

L183 replace “participated” with “involved”

L184 Italicise “C. illinoinensis” – check all scientific names for italics as there are others

L193 “unit” instead of “unite”

L193 Delete “SSRs belong to short repeats.” As this sentence does seem to be needed at all.

L198 Delete “were” after SSRs

L222 Insert “the” after “while”

L244 replace “was” with “is”

L291 Italicise scientific names here and L292

L337 Change “unique” genes to “single copy” genes?

L344 Reword sentence to “Phylogenetic reconstruction strongly supported C. ill as a member of Carya genus, and as a sister to the sect. Sinocarya.

 

Author Response

Point 1: L13 Change “Comprehensively” to “Comprehensive”

 

Response 1: Thanks very much for your remind. We have revised it in the latest version.

 

Point 2: L22 Insert “each” at end of line after “26,003 bp”

 

Response 2: Thanks for your remind. We have added it

 

Point 3: 36 Insert “The” at start of sentence

 

Response 3: Thanks for your remind. We have inserted it

 

Point 4: 39 Delete “are” before typically and change “consisted” to “consist”

 

Response 4: Thanks for your remind. We have revised them.

 

Point 5: 42 Probably should have “most” cp are maternally inherited. Conifers are paternally inherited. ‘less’ recombination or ‘no’ recombination? This may still be controversial. Is there now good evidence for some recombination?

 

Response 5: Thanks for your comments. Thanks for your comments. We have revised that sentence. However, the words regarding ‘less recombination’ still make no changes, as a previous report have indicated that the recombination of chloroplasts could be documented in the unicellular algae Chlamydomonas, and in the gymnosperms Pinus, and Cycas [Paternal, maternal, and biparental inheritance of the chloroplast genome in Passiflora (Passifloraceae): implications for phylogenetic studies. American Journal of Botany, 2007, 94(1): 42-46]

 

Point 6: L47 Delete “in the world” as not required

 

Response 6: Thanks for your comments. We have deleted it.

 

Point 7: L54 I think this should be C. illinoinensis rather than C. catayensis here?

 

Response 7: Thanks very much for your remind. We regret for the mistake. It have been revised.

 

Point 8: L55. Suggest change “Primary” to “In the past” or “Historically”

 

Response 8: Thanks for your remind. We have revised it.

 

Point 9: L59 change “a high demand” to “a need”

 

Response 9: Thanks for your remind. We have revised it.

 

Point 10: L67 Change you provide a better rationale for undertaking the phylogenetic analysis than it has not been done before? What practical difference might that make for the breeder? Better prediction of success of interspecific crossing?

 

Response 10: Thanks for your comments. We have added related explanation for undertaking chloroplast genome sequencing.  The complete cp sequence can offer a great number of molecular loci, which is of importance for enhancing phylogenetic accuracy.

 

Point 11: L71 Delete “also” before “conducted”

 

Response 11: Thanks for your remind. We have deleted it.

 

Point 12: L77 change “detected” to “analysed”

 

Response 12: Thanks for your remind. We have revised it.

 

Point 13: L80 change “applied” to “used”

 

Response 13: Thanks for your remind. We have changed it.

 

Point 14: L82. “C.illinoinensis” needs a space and italics

 

Response 14: Thanks for your remind. We have revised the same mistakes throughout the manuscript.

 

Point 15: L152 Delete “generally” and change “have” to “having”

 

Response 15: Thanks for your remind. We have revised them.

 

Point 16: L160 Table 1. “unique gene number” Do you mean single copy genes? If so can you please replace it as unique gene number could have many different meanings.

 

Response 16: Thanks for your comments. ‘unique gene number’ means the different gene number. We added explanation for it (L166, with 112 unique/different ones). As a number of researches used the ‘unique gene number’ to indicate the total different gene number, we still make no change for that words in the revised manuscript.

 

Point 17: L166 replace “were with” with “have”

 

Response 17: Thanks for your remind. We have revised it.

 

Point 18: L169 replace “rest” with “other”

 

Response 18: Thanks for your remind. We have revised it.

 

Point 19: L178 Delete “happened in the “Pawnee” cultivar” as doesn’t seem necessary

 

Response 19: Thanks for your remind. We have deleted it.

 

Point 20: L181 insert “in” after “included”

 

Response 20: Thanks for your remind. We have added it.

 

Point 21: L183 replace “participated” with “involved”

 

Response 21: Thanks for your remind. We have revised it.

 

Point 22: L184 Italicise “C. illinoinensis” – check all scientific names for italics as there are others

 

Response 22: Thanks for your remind. We have revised it throughout the manuscript.

 

Point 23: L193 “unit” instead of “unite”

 

Response 23: Thanks for your remind. We have revised it.

 

Point 24: L193 Delete “SSRs belong to short repeats.” As this sentence does seem to be needed at all.

 

Response 24: Thanks for your remind. We have revised reorganized related sentence.

 

Point 25: L198 Delete “were” after SSRs

 

Response 25: Thanks for your remind. We have deleted it.

 

Point 26: L222 Insert “the” after “while”

 

Response 26: Thanks for your remind. We have revised it.

 

Point 27: L244 replace “was” with “is”

 

Response 27: Thanks for your remind. We have replaced it.

 

Point 28: L291 Italicise scientific names here and L292

 

Response 28: Thanks for your remind. We have revised it.

 

Point 29: L337 Change “unique” genes to “single copy” genes?

 

Response 29: Thanks for your comment. The unique gene means the total different gene. We retain this writing, as numerous reports used the same expression.

 

Point 30: L344 Reword sentence to “Phylogenetic reconstruction strongly supported C. ill as a member of Carya genus, and as a sister to the sect. Sinocarya.

 

Response 30: Thanks for your suggestion. We have revised the related sentences.