Comparative Analysis of the Complete Chloroplast Genomes of Four Chestnut Species (Castanea)
Daniel Agustinho
Round 1
Reviewer 1 Report
Please check the attachment.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1 Comments
Thank you for your valuable comments. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:
Point 1: Overall
This comparative analysis on plastidal genomes of four Chinese Castanea species was well characterizing the chloroplast genomes. The research methods and the description was adequate to understand the plastidal genomic evolution of Chinese Castanea. However, any research backgrounds why you have studied on it, or any research importance and discussion for forestry is insufficient. You “should” provide the proper forestial research background and importance to publish your manuscript on this journal.
Response: Thank you for your valuable and nice comments. We added the describes as followings: “Chestnut (Castanea Mill.) is a genus of the Fagaceae that includes twelve to seventeen species distributed in deciduous forests in eastern North America, Europe, and Asia [1]. Chestnuts and the other members of the genus are ecologically and economically important nut and wood producing trees [2].” from Line 32-35. We added sentence into INTROCUTION part provide the proper forestial research background and importance as followings: “Our study will provide genetic resources for the resolution of Castanea species classification, forest population genetics, biogeographic analysis, and phylogenetic relationship in Fagaceae.” from Line 80-83 in the revision.
Point 2: Line 35-37: Please add authorities to scientific names when their first mentioning.
Response: Thank you for your valuable comments. We added the scientific names as Castanea Mill. Castanea mollissima Bl., Castanea Seguinii Dode, Castanea Henryi (Skan) Rehd. et Wils., Castanea Sativa Mill., Castanea Crenata Sieb. et Zucc., when their first mentioning in the revision from Line 36-43.
Point 3: Line 44-46: These sentences are uninformative contents. You kindly support these with any other related information, or remove them.
Response: Thank you for your comments. We deleted the sentences “It occupies a unique position in the production of nuts.” in the revision. We changed the sentence “China is the world’s largest producer of edible chestnuts and a major user of the genus Castanea” to “China is the world’s largest producer of edible chestnuts, and the chestnuts were roasted over a hot flame to attraction food for tourists and locals” from Line 46-47 in the revision.
Point 4: Line 47: Please remove improper citation and the information based on it (XX).
Response: Thank you for your comments. We deleted the (XX) in the revision.
Point 5: Line 60-63: There’s no “accepted” classification without any references. Please kindly provide the reference and avoid using the word “accepted”. You may describe like: In the current classification based on (reference flora).
Response: Thank you for your suggestions. We changed the sentence “In the accepted classification of chestnuts,” to “In the current classification of chestnuts based on Flora of China (2000) [10-12, 30]” from Line 62-63 in the revision.
Point 6: Line 62-63: The word for rank “section” was missing on Hypocastanon and Balanocastanon.
Response: Thank you for your valuable comments. We added the word “section” before Hypocastanon and Balanocastanon in the revision.
Point 7: Line 89: Please cite the reference studies on NCBI accessions if you possible.
Response: Thank you for your valuable comments. We added the NCBI accessions into Table 1 as new line in the revision.
Point 8: Line 101-102: Please kindly describe the detailed logic for your manual adjustment on start and end position and splice sites. Unevidenced adjustment on gene prediction may result in a manipulation, not an adjustment.
Response: Thank you for your kindly comments. We revised the sentence as “We adjusted the start and stop codons and boundaries between introns and exons using MAFFT v7.0.0 [39] by comparison with homologous genes from other cp genomes [40]” in the revision.
Point 9: Line 120-122: Please describe the number of repeats of each SSR motif for identifying SSR regions.
Response: Thank you for your valuable suggestion. We added the number of repeats of each SSR motif, and revised the sentence as “We used MISA to detect the microsatellites (SSR) in all 8 chloroplast genomes, including mono-(p1, one motif), di-(p2, two motifs), tri-(p3, three motifs), tetra-(p4, four motifs), penta-(p5, five motifs), hexa-(p6, six motifs) and polynucleotide (complex motifs) repeats [45].” from Line 124-127 in the revision.
Point 10: Line 148-155: Please be sure of GTRGAMMA to GTR+G or others. GTR and gamma value are distinct parameters.
Response: Thank you for your valuable suggestion. We revised “GTRGAMMA” to “GTR GAMMA” for the model phylogenetic tree parameter in Line 156 and 161 in the revision.
Point 10: Line 182: Please kindly make a new line between a figure caption and a main paragraph.
Response: Thank you for your valuable suggestion. We added a new line between a figure caption and a main paragraph in the revision. We also checked other figure and added new line between a figure caption and a main paragraph.
Point 10: Line 225-227: The information related to this sentence is missing in M&Ms. Please kindly provide it with references.
Response: Thank you for your valuable suggestion. We added the describe sentences as “Selective pressure Non-synonymous (KA) and synonymous (KS) substitution rates (KA/KS) was computed with the codeml tool from PAML v4.0 using a YN00 model to test every gene sequence [48].” from Line 414-143. We added the cite as followings: Yang, Z. PAML4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 2007, 24, 1586–1591.
Point 11: Line 238-239: Why the characteristic of more conserved in protein-coding gene “means (i.e.)” less conserved in IR regions? This is not only supported with any evidences but IR regions are usually to be highly conserved than LSC or SSC as they contains rRNAs.
Response: Thank you for your valuable suggestion. Yes, you are right. The protein coding regions (CDS) were more conserved than the intergenic regions (IGS) region. We deleted the sentence “(i.e., LSC and SSC was more conserved than the IR region)” from Line 318 in the revision.
Point 12: Line 263-265: Five independent datasets were phylogenetically analyzed but only the result from the whole genome data was shown. Please kindly provide the rest of result as supplementary data.
Response: Thank you for your valuable suggestion. We added the Supplementary Materials: Figure S1 into the revision as followings: Figure S1. Phylogeny trees of eight Castanea individuals plus four taxa based on (a) protein-coding genes (CDS), (b) inverted repeats (IR) regions, (c) large single-copy (LSC) region, and (d) small single-copy (SSC) region. Bootstrap values (%) are shown above branches. We also cite Figure S1 in the text from Line 278-284.
Point 13: Line 268-269: The word “similar” is not proper to phylogenetic inferences. Please kindly infer them as closer relationships.
Response: Thank you for your valuable suggestion. We changed “greater similarity” to “closer relationships” in the revision.
Point 13: Line 271-272: This sentence can be improved as like: Each family clustered into monophyletic cladesor something.
Response: Thank you for your valuable suggestion. We changed the sentence from Line 287-290 in the revision as followings: “Each family clustered into monophyletic clades. The Fagaceae formed a distinct clade from the Myricaceae, Betulaceae, and Juglandaceae. The Myricaceae and Juglandaceae formed a clade distinct from the Betulaceae.”
Point 14: Line 288-292: The word “pseudogenized” seems to be better than “evolved into a pseudogen”. And no evidence or support was provided on the malfunction of ndhD protein. Please kindly provide them.
Response: Thank you for your valuable suggestion. We changed “evolved into a pseudogen” to “pseudogenized” in the revision. We also added the cite “(Figure S2)” in the revision as followings: Figure S2. The genetic variation of the pseudogene ndhK and ndhD in the eight Castanea chloroplast genomes. (a) The pseudogene ndhK in Castanea henryi 1 chloroplast genome. (b) Alignment of pseudogene ndhD in the eight Castanea chloroplast genomes.
Point 15: Line 308-310. These sentences seem to be uninformative and not supporting any discussion. Please remove them.
Response: Thank you for your valuable suggestion. We removed the sentence and cites in the revision as followings:
It is generally believed that the chloroplast NDH complex is derived from cyanobacteria NDH-1 [60], and the NDH in higher chloroplasts is involved in photosystemâ… [61].
- Munekage, Y.; Hashimoto, M.; Miyake, C.; Tomizawa, K.-I.; Endo, T.; Tasaka, M.; Shikanai, T. Cyclic electron flow around photosystem I is essential for photosynthesis. Nature 2004, 429, 579-582.
- Yang, Y., Tao, Z., D Duan, Yang, J., Feng, L., Zhao, G. Comparative analysis of the complete chloroplast genomes of five quercus species. Frontiers in plant science 2016, 7, 759.
Point 16 Line 313: Typo “analyse”
Response: Thank you for your valuable suggestion. We changed “analyse” to “analysis” in the revision.
Point 17 Line 315 & 319: Same to Line 60-63. Please kindly avoid using the word “accepted”.
Response: Thank you for your valuable suggestion. We changed “accepted” to “current” in the revision.
Point 18 Line 317-318: Same to Line 268-269. Please using “closer relationship” and “early diverged” in phylogenetic inference, not “more similar” and “ancient branch”, respectively.
Response: Thank you for your valuable suggestion. We changed the sentences “Therefore, it can be shown that the C. henryi and C. mollissima are closer in kinship. Although morphologically, C. seguinii and C. mollissima are more similar [6-7], the phylogenetic relationship shows that the C. seguinii cp is an ancient branch in Chinese Castanea species (Figure 6).” from Line 412-415 in the revision as followings: “The strong genomic similarity of C. henryi and C. mollissima was confirmed. Based on morphology, C. seguinii and C. mollissima appear most closely related [6-7], but the genomic evidence is that the C. seguinii cp reflects an early diverged of Chinese Castanea species (Figure 6).”
Point 19 Line 322-333: This part seems to be the result one, not a discussion one. Moreover, DNA barcodes do not mean highly variable sites. DNA barcodes have to contain species-level unique variation information to identify their species. Only just high variation rates cannot be DNA barcodes.
Response: Thank you for your valuable suggestions. We revised the previous discussion part from Line 456-467 in the revision as followings: “DNA barcodes have been widely used in evolutionary and phylogenetical studies for both plants and animals [49]. We compared cp genomes of Castanea using slideAnalyses to calculate genetic distance across a moving 800 bp window [47]. This method identified five regions of the Castanea genome that might be useful for barcoding: trnK-UUU-rps16, psbM-trnD-GUU, rbcL-accD, petA-psbJ, and rpl2 (Figure4, 5). We suggest the sequence of rbcL, matK, petA-psbJ as a barcode. The lengths of the three selected sequences are respectively 1428 bp, 1508 bp, and 1072 bp. There are 4, 14, and 75 mutation sites and 4, 10, and 68 informative sites within these loci. The barcode would be useful for distinguishing American chestnut (C. dentata) and Chinese chestnuts and as such could provide a method to track the sources of hybrid used in breeding. Simple sequence repeats (SSRs) were highly variable among and within Castanea species. As such, these have value for population genetics and other applications requiring high levels of polymorphism.”
Point 20 Figure 1: Only Figure 1A is related to the main contents where it has mentioned. Figure 1B and 1C is not informative and inadequate as Figure 1. You can improve the main contents along the Figure 1A-C, or remove Figure 1B-C.
Response: Thank you for your valuable suggestion. We improved the quality of Figure1, and we removed (b) and (c) as supplementary figures in the revision.
We revised and edited our whole manuscript improved grammatical errors and wrongly spelled words by a professional and native English language writing Dr. Keith Woeste.
Thank you for your consideration our manuscript of “Comparative analysis of the complete chloroplast genomes of four chestnut species (Castanea)" publish on journal of Forests.
Sincerely,
Zhao Peng, Ph.D
Author Response File: 
Author Response.doc
Reviewer 2 Report
The study compares the whole chloroplast genome of four species of the genus Castanea. The work also provides a comparison of genes present in the chloroplast of the different species used. Analyses were performed to reconstruct the phylogenetic relationships using the chloroplast genome, traditionally used for evolutionary purpose. The authors found that the four species of the genus are monophyletic.
The work is notably, and I suggest only minor elements to modify.
Line 36
Replace ; with :
Line 102
Move the citation 39 close to the mention of the program used DOGMA.
Please clarify the abbreviations the first time appear in the text and refer at chloroplast genome (cp genome) and chloroplast genomes (CPgs) always with the same abbreviation (e.g. line 147 Cp, line 190 Cpgs, line 307 and 314 Cpg).
Line 306
Replace cps with chloroplasts.
Figure 1
The figure quality has to be improved, it is not readable.
Figure 2
Modify the font size of the caption to make uniform. Please check the caption, probably a new line is missing to separate caption from text.
Figure 6
Explain each supporting values at the nodes, two in the a and three in b.
References
Check carefully the citations, use the correct abbreviations for all the journal and use the same form for the same journal (e.g. line 403 PloS one and line 419 PLoS ONE). Write correctly the genus and species name in the citations (e.g. line 388 and 467)
Author Response
Response to Reviewer 2 Comments
Thank you for your valuable comments. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:
Comments and Suggestions for Authors
Point 1: The study compares the whole chloroplast genome of four species of the genus Castanea. The work also provides a comparison of genes present in the chloroplast of the different species used. Analyses were performed to reconstruct the phylogenetic relationships using the chloroplast genome, traditionally used for evolutionary purpose. The authors found that the four species of the genus are monophyletic.
The work is notably, and I suggest only minor elements to modify.
Response: Thank you for your nice and valuable comments.
Point 2: Line 36 eplace ; with :
Response: Thank you for your valuable comments. We revised the sentence in the revision as following: “Chinese chestnut (Castanea mollissima Bl.), Seguin chestnut (Castanea Seguinii Dode), and Pearl chestnut or Henry chestnut (Castanea Henryi (Skan) Rehd. et Wils.)” in the revision.
Point 3: Line 102 Move the citation 39 close to the mention of the program used DOGMA.
Response: Thank you for your valuable comments. We moved cite [39] after “program Dual Organellar Genome Annotator (DOGMA)” in the revision.
Point 4: please clarify the abbreviations the first time appear in the text and refer at chloroplast genome (cp genome) and chloroplast genomes (CPgs) always with the same abbreviation (e.g. line 147 Cp, line 190 Cpgs, line 307 and 314 Cpg).
Response: Thank you for your valuable comments. We replaced all word “Cpg” to “cp genome” and word “Cp” to “cp” consistently in the text.
Point 5: Line 306 Replace cps with chloroplasts.
Response: Thank you for your valuable comments. We replaced “cps” to “chloroplasts” in the revision.
Point 6: Figure 1 The figure quality has to be improved, it is not readable.
Response: We improved the quality of Figure1, and removed (b) and (c) as supplementary figures in the revision.
Point 7: Figure 2 Modify the font size of the caption to make uniform. Please check the caption, probably a new line is missing to separate caption from text.
Response: Thank you for your valuable comments. We modified the font size of the caption to make uniform in the revision. We added a new line between a figure caption and a main paragraph in the revision. We also checked other figure and added new line between a figure caption and a main paragraph.
Point 8: Figure 6 Explain each supporting values at the nodes, two in the a and three in b.
Response: Thank you for your valuable comments. Trees were calculated using Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian (BI) methods [50-53]. We revised the figure6 legend as followings: “Phylogenetic trees (Maximum likelihood, Maximum Parsimony) based on whole-plastome sequences. (a) The phylogenetic tree based on 37 individuals using Bayesian (BI) and Maximum Likelihood (ML) methods. (b) Results of phylogenetic analysis of sequence data from the chloroplasts of eight Castanea individuals and four outgroups using Bayesian (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP), methods. Bootstrap values (%) are shown above branches based on two and three methods.”
Point 9: References
Check carefully the citations, use the correct abbreviations for all the journal and use the same form for the same journal (e.g. line 403 PloS one and line 419 PLoS ONE). Write correctly the genus and species name in the citations (e.g. line 388 and 467)
Response: Thank you for your valuable comments. We replaced the journal “PloS one” to “PLoS ONE” in the revision. We checked all the citations for the abbreviations for the journal names, author names, genus and species names, titles, volumes, and pages carefully in the revision.
We revised and edited our whole manuscript improved grammatical errors and wrongly spelled words by a professional and native English language writing Dr. Keith Woeste.
Thank you for your consideration our manuscript of “Comparative analysis of the complete chloroplast genomes of four chestnut species (Castanea)" publish on Forests.
Sincerely,
Zhao Peng, Ph.D
Author Response File: Author Response.doc
Reviewer 3 Report
The authors of "Comparative analysis of the complete chloroplast genomes of four chestnut species (Castanea)" sequenced and compared the chloroplast genomes of eight commercially relevant species of Castanea trees. Their findings might prove useful for that industry, their work is scientifically solid and the methods were appropriate. I recommend acceptance with a few minor suggestions and/or corrections: 1) Figure 3a: In the figure legend, the color for Palindrome is wrong (it shows same color as "Complement"). Change to the same as in the figure (dark green). 2) In the text, figures S1 (line 223) and S2 (line 311) are mentioned, but neither was made available for the reviewers. 3) In line 337, in the Conclusion, the authors say that gene rpl22 "was absented in all analyzed species", but on lines 182 and 310/11 they say that the gene was present as a pseudogene. Please correct. 4) Title of Table S6, the word "individuals" is misspelled.Author Response
Response to Reviewer 3 Comments
Comments and Suggestions for Authors
The authors of "Comparative analysis of the complete chloroplast genomes of four chestnut species (Castanea)" sequenced and compared the chloroplast genomes of eight commercially relevant species of Castanea trees. Their findings might prove useful for that industry, their work is scientifically solid and the methods were appropriate. I recommend acceptance with a few minor suggestions and/or corrections:
Response: Thank you for your valuable and nice positive comments and suggestion. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:
Point 1: 1) Figure 3a: In the figure legend, the color for Palindrome is wrong (it shows same color as "Complement"). Change to the same as in the figure (dark green).
Response: Thank you for your valuable suggestion. We changed to the same color in Figure3 (dark green) in the revision.
Point 2: 2) In the text, figures S1 (line 223) and S2 (line 311) are mentioned, but neither was made available for the reviewers.
Response: Thank you for your valuable comments. We combined Figure S1 into Figure4, and we revised “Figure S1” to “Figure 4a” in Line 301 and Line 302. We revised Figure S2 in the revision as followings: Figure S2. The genetic variation of the pseudogene ndhK and ndhD in the eight Castanea chloroplast genomes. (a) The pseudogene ndhK in Castanea henryi 1 chloroplast genome. (b) Alignment of pseudogene ndhD in the eight Castanea chloroplast genomes.
Point 3: 3) In line 337, in the Conclusion, the authors say that gene rpl22 "was absented in all analyzed species", but on lines 182 and 310/11 they say that the gene was present as a pseudogene. Please correct.
Response: Thank you for your valuable comments. We check our results and conclusion again carefully. The rps22 was absented in “Table 2 Gene contents in eight Castanea individuals’ cp genomes”. We replaced “a pseudogene” to “absented” in Line 236 and Line 454 in the revision. We revised “a pseudogene” to “nonfunction” in Line411 in the revision.
Point 4: 4) Title of Table S6, the word "individuals" is misspelled.
Response: Thank you for your valuable comments. we changed the correct word “individuals” of the title of Table S6 in the revision.
We revised and edited our whole manuscript improved grammatical errors and wrongly spelled words by a professional and native English language writing Dr. Keith Woeste.
Thank you for your consideration our manuscript of “Comparative analysis of the complete chloroplast genomes of four chestnut species (Castanea)" publish on Forests.
Sincerely,
Zhao Peng, Ph.D
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
Your manuscript is now highly improved in contents and English writing compared to the draft, and your responses on my review were acceptable and reasonable. You may check the attachment for several minor options to complete your manuscript.
Comments for author File: Comments.docx
