Assembly and Comparative Analyses of the Chloroplast Genomes of the Threatened Plant Rosa anemoniflora

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors I think, the article can be accepted for publication after the following problems are corrected:

1. Line 92. "Alignment to the pseudogenome facilitated genome correction". Please, clarify how you performed the alignment, what you mean by "genome correction" and what tool you used to do the correction.
2. Line 98. "pseudogenome was rearranged to construct a circular chloroplast genome". Please, clarify how you did this rearrangement.
3. Lines 99-100. "For annotation, BLAST [15] was used to compare the CDSs with those of the NCBI chloroplast genome". Please, describe this in more detail. If I understand correctly, you aligned some CDSs from the NCBI database using BLAST to the plastid genome of Rosa anemoniflora. What species did you take the CDSs from?
4. Line 100. "HMMER [16] was used to retrieve rRNA annotations". Please, describe this in more detail. What HMMER profiles did you use to find rRNA-coding genes?
5. Lines 106-107. "The Perl package was used to analyse the length, GC content, normal gene type and quantity". Do you mean the Perl programming language or some specific package? Please, specify.
6. Lines 106-107. "The Perl package was used to analyse the length, GC content, normal gene type and quantity". I don't understand what you mean by "normal gene type". This probably should be rephrased.
7. The instructions for authors of MDPI Forest state "Give the name and version of any software used" (https://www.mdpi.com/journal/forests/instructions). Please, indicate versions of all programs that you used.
8. Lines 193-194. "petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure". I think, there are three problems with this statement: 1) P-value for petD is 0.12. I don't understand why you consider this statistically significant. 2) Since you performed separate tests for selection for each gene, you should also have done a multiple hypothesis testing correction. For example, a Benjamini-Hochberg correction, or a Bonferroni correction. After this correction, the statistical significance will become even lower. 3) The fact that the M0 hypothesis can be rejected, doesn't mean that there is positive selection. The M0 hypothesis is that all branches have the same dN/dS. If the M0 hypothesis is rejected, this means that different branches probably evolve under different selective pressure. But this doesn't mean that selection on some of the branches is positive. To test for presence of positive selection, one should compare M1a and M2a hypotheses. See page 28 in the manual of PAML (http://abacus.gene.ucl.ac.uk/software/pamlDOC.pdf).   To sum up, I think that the statement that petD experienced positive selection is wrong and should be removed from the article, including the abstract.
9. Lines 306-308. "The A–T bond is weaker than the G–C bond, increasing the susceptibility to breakage. Therefore, the likelihood of A–T bonds occurring in the chloroplast genome SSR is greater" Please, add a reference to an article that shows that AT-rich SSRs are more frequent because of weaker bonds. It is not obvious to me that weaker bonds between adenines and thymines lead to increased probability of AT-rich SSRs. I suppose that the higher frequency of AT-rich SSRs in plastid genomes can be entirely explained by the fact that plastid genomes have more adenines and thymines than guanines and cytosines.

 

Author Response

Reviewer 1
Thank you for your nine comments. Our responses are as follows:

• Line 92. "Alignment to the pseudogenome facilitated genome correction". Please, clarify how you performed the alignment, what you mean by "genome correction" and what tool you used to do the correction.

Response: We appreciate the comments. "Alignment to the pseudogenome facilitated genome correction" has been revised to "Bowtie2 was used to align the sequenced sequence to the pseudogenomic sequence, ensuring that the reads coverage of the assembly results was normal."

• Line 98. "pseudogenome was rearranged to construct a circular chloroplast genome". Please, clarify how you did this rearrangement.

Response: We appreciate the comments. "pseudogenome was rearranged to construct a circular chloroplast genome" has been revised to "using the large single-copy region as the starting point and referring to the orientation of the small single-copy region in Rosa lucidissima (MK782979.1.gbk), the pseudogenome was rearranged to construct a circular chloroplast genome sequence."

• Lines 99-100. "For annotation, BLAST [15] was used to compare the CDSs with those of the NCBI chloroplast genome". Please, describe this in more detail. If I understand correctly, you aligned some CDSs from the NCBI database using BLAST to the plastid genome of Rosa anemoniflora. What species did you take the CDSs from?

Response: We appreciate the comments. "For annotation, BLAST [15] was used to compare the CDSs with those of the NCBI chloroplast genome" has been revised to "For annotation, the CDSs were compared with those of the closely related species R. lucidissima on NCBI using BLAST [15]."

• Line 100. "HMMER [16] was used to retrieve rRNA annotations". Please, describe this in more detail. What HMMER profiles did you use to find rRNA-coding genes?

Response: We appreciate the comments. "HMMER [16] was used to retrieve rRNA annotations" has been revised to "The rRNA sequences of the R. lucidissima (MK782979) were extracted and aligned using MAFFT [16] (v, --auto), and then the hidden Markov model was constructed using the hmmbuild tool from the HMMER [17] package. Finally, nhmmer was used to search."

（5）Lines 106-107. "The Perl package was used to analyse the length, GC content, normal gene type and quantity". Do you mean the Perl programming language or some specific package? Please, specify.

Response: During the revision process, we switched to using Geneious software to obtain specific data. The current text is: "The Geneious [18] software was used to calculate the lengths of the four different boundaries (SSC, LSC, and IR regions), the number and types of genes, GC content, and other information of the chloroplast genomes of seven species based on their GenBank annotation files."

• Lines 106-107. "The Perl package was used to analyse the length, GC content, normal gene type and quantity". I don't understand what you mean by "normal gene type". This probably should be rephrased.

Response: . "The Perl package was used to analyze the length, GC content, normal gene type, and quantity" was rewritten as “The Geneious [18] software was used to calculate the lengths of the four different boundaries (SSC, LSC, and IR regions), the number and types of genes, GC content, and other information of the chloroplast genomes of seven species based on their GenBank annotation files.” The phrase "types of genes" refers to coding genes, tRNA genes, rRNA genes, and pseudogenes.

• The instructions for authors of MDPI Forest state "Give the name and version of any software used" (https://www.mdpi.com/journal/forests/instructions). Please, indicate versions of all programs that you used.

Response: We appreciate the comments. The name and version of all software used in the text have been indicated.

（8）Lines 193-194. "petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure". I think, there are three problems with this statement: 1) P-value for petD is 0.12. I don't understand why you consider this statistically significant. 2) Since you performed separate tests for selection for each gene, you should also have done a multiple hypothesis testing correction. For example, a Benjamini-Hochberg correction, or a Bonferroni correction. After this correction, the statistical significance will become even lower. 3) The fact that the M0 hypothesis can be rejected, doesn't mean that there is positive selection. The M0 hypothesis is that all branches have the same dN/dS. If the M0 hypothesis is rejected, this means that different branches probably evolve under different selective pressure. But this doesn't mean that selection on some of the branches is positive. To test for presence of positive selection, one should compare M1a and M2a hypotheses. See page 28 in the manual of PAML (http://abacus.gene.ucl.ac.uk/software/pamlDOC.pdf).   To sum up, I think that the statement that petD experienced positive selection is wrong and should be removed from the article, including the abstract.

Response: I have followed your suggestion and removed the description related to "petD experienced positive selection" from the text. Based on your first two points of feedback, we have revised Table S2, recalculated the p-values, and applied the Benjamini-Hochberg correction. The results indeed show that petD does not exhibit significance. However, in response to the third point, our study mainly focuses on whether there is a significant difference in selection pressure on genes between the foreground branches (endangered plants) and the background branches (non-endangered plants), so we chose the branch model. Our methods also state that, to demonstrate significant selection pressure on a gene in the foreground branches, it is necessary to not only reject the m0 hypothesis but also meet two criteria: the dn/ds value calculated by m0 must be less than 1, and the dn/ds value calculated by m1 must be greater than 1. The M1a and M2a hypotheses you suggested are site models, which are used to identify positively selected sites and do not meet the purpose of this study.

（9）Lines 306-308. "The A–T bond is weaker than the G–C bond, increasing the susceptibility to breakage. Therefore, the likelihood of A–T bonds occurring in the chloroplast genome SSR is greater" Please, add a reference to an article that shows that AT-rich SSRs are more frequent because of weaker bonds. It is not obvious to me that weaker bonds between adenines and thymines lead to increased probability of AT-rich SSRs. I suppose that the higher frequency of AT-rich SSRs in plastid genomes can be entirely explained by the fact that plastid genomes have more adenines and thymines than guanines and cytosines.

Response: We appreciate the comments. It has been modified as suggested.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled: “Assembly and comparative analyses of the chloroplast genomes of the threatened plant Rosa anemoniflora” by Wei Gao and coworkers, submitted to Forests  confirmed that R. anemoniflora belongs to Synstylae section of the Rosa genus, diverged approximately 1.82 million years and similarly to its relatives originated from the Fujian region of China and Taiwan.

I consider the comparative analysis of chloroplast genome structure of Rosa anemoniflora and its relatives as an important subject of investigations from the phylogenetic point of view. It is known that plastid DNAs with their conserved organization are very good markers of phylogenetic background of species and their identification at divers taxonomic levels. Some chloroplast genome sequences are also useful as molecular markers of plant response to stress.  Although the chloroplast genome sequences are commonly used in plant molecular phylogenetics, however establishing the phylogenetic position of certain species is important, especially in the case of  human important species or endangered ones, what is the case of the analyzed species. Therefore, I consider  the undertaken subject of the comparative analysis of chloroplast genomes as a useful tool to study evolutionary relationships in plants and worth to study.

In the present paper, methods for comparative analysis of genome structure between R. anemoniflora and several closely related species were well chosen, performed and clearly described,  including among others, GC content, gene number, selection pressure, and nucleotide diversity. I also highly evaluate the phylogenetic analysis based on the complete chloroplast genome sequences of species within the Rosa genus.

 The authors performed a detailed structural characterization of plastid DNA of selected species, in particular of  LSC, SSC, IR lengths and structures, total number of genes including  tRNA and rRNA genes,  as well as protein-coding genes, and other. In spite of evolutionary conserved genomes of Rosa genus, the comparative analysis revealed several variation hotspots, among them the atpH-atpI region, which was used as a DNA barcode for differentiate R. anemoniflora from its close relatives. Additionally, using the branch model the authors identified only one positively selected in R. anemoniflora chloroplast protein-coding gene  - petD.

In my opinion the following results are the major achievements of this paper:

a) sequencing and assembling of the chloroplast genome of R. anemoniflora and comparative analysis with six other related species in sect. Synstylae and sect. Chinenses;

b) finding specific sequences used as molecular markers to  recognize species identity;

c) performing a comparative analysis of Rosa species cp genome, based on their  interesting regions which allowed to reconstruct the phylogenetic tree;

d) finding, according to the evolutionary tree, that the closest relations of R. anemoniflora was with Rosa taiwanensis and Rosa pricei;

e) due to  the estimation of divergence time of the tested plants that R. anemoniflora and its closest relative, R. taiwanensis, diverged approximately 1.82 million years ago.

I appreciate very much the clarity of presentation;

Concluding:

I highly evaluate the results obtained in the manuscript titled: “Assembly and comparative analyses of the chloroplast genomes of the threatened plant Rosa anemoniflora” by Wei Gao and coworkers.  In the manuscript the taxonomic status of Rosa anemoniflora was clarified together with  evolutionary history of this species.

I recommend the manuscript to be published in Forests.

Author Response

Reviewer 2

Thank you for your time and consideration in reviewing our manuscript. As there were no comments provided, we will proceed accordingly. Please let us know if there are any further steps required.

Reviewer 3 Report

Comments and Suggestions for Authors

There are a few comments about this generally well-written article

Why only 7 from 17 examined Rosa species included in Table1 and Figs. 2, 3, and 12 species for molecular marker discovery?

Table S1, Figs.4, 5, Line 132: Rosa deseglisei belongs to section Caninae

In Figure 2, indicate the sections for each species.

From Table S2 it is not clear that only petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure.

Lanes 291-92: “noncoding sequences appear to experience greater selective pressure than coding regions” – it is a wrong ststement.

I recommend placing Figure S1 in a main text as it contains interesting original information

 

Other notes:

Line 106: “Perl package”- specify URL or origin

Line 119: there are mot 11 but 17 Rosa species in the Table S1.

Line 136: what model was used for phylogenetic tree construction?

Line 141: [23] is a wrong Ref. for PhyloSuite v1.2.1

References for software should be placed immediately after software names but not at the ends of sentences describing details of their use.

Lines 149, 152 add Refs. or URLs for Tracer v1.7 and FigTree

Line 149 delete (Bouckaert et al. 2014).

Line 177: genone > genomes;

Lines 177-78: delete “approximately”

Lines186-87, 239-240: use abbreviation for genus name.

Author Response

Reviewer 3

There are a few comments about this generally well-written article

 

（1）Why only 7 from 17 examined Rosa species included in Table1 and Figs. 2, 3, and 12 species for molecular marker discovery?

Response: In our comparative genomic analysis, our aim was to preliminarily explore whether the chloroplast genomes of R. anemoniflora and its closely related species are conserved, so we selected only seven species for the analysis. When developing molecular markers, the objective was to distinguish R. anemoniflora from other species in the Sect. Synstylae. Therefore, we only chose species of Sect. Synstylae for the analysis.

（2）Table S1, Figs.4, 5, Line 132: Rosa deseglisei belongs to section Caninae

Response: We appreciate the comments. It has been modified as suggested.

（3）In Figure 2, indicate the sections for each species.

Response: We appreciate the comments. It has been modified as suggested.

（4）From Table S2 it is not clear that only petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure.

Response: We have followed your suggestion and removed the description related to "petD experienced positive selection" from the text. We have revised Table S2, recalculated the p-values, and applied the Benjamini-Hochberg correction.

（5）Lanes 291-92: “noncoding sequences appear to experience greater selective pressure than coding regions” – it is a wrong ststement.

Response: We have revised the description to: "These regions are located in noncoding regions, where noncoding sequences appear to experience higher variability than coding regions, providing more critical informative sites."

（6）I recommend placing Figure S1 in a main text as it contains interesting original information

Response: We appreciate the comments. It has been modified as suggested

 

 

Other notes:

 

（7）Line 106: “Perl package”- specify URL or origin

Response: During the revision process, we switched to using Geneious software to obtain specific data. The current text is: "The Geneious [18] software was used to calculate the lengths of the four different boundaries (SSC, LSC, and IR regions), the number and types of genes, GC content, and other information of the chloroplast genomes of seven species based on their GenBank annotation files."

 

• Line 119: there are mot 11 but 17 Rosa species in the Table S1.

Response: When developing molecular markers, the objective was to distinguish R. anemoniflora from other species in the Sect. Synstylae. Therefore, we only chose species of Sect. Synstylae for the analysis. We have revised the description to: “The chloroplast sequences of R. anemoniflora and 10 closely related species be-longing to Rosa sect. Synstylae were selected for analysis.”

 

（9）Line 136: what model was used for phylogenetic tree construction?

Response: We have added the relevant description in Method 2.5: “Determined K3Pu+F+I as the best-fit model using ModelFinder [24].”

（10）Line 141: [23] is a wrong Ref. for PhyloSuite v1.2.1

Response: We appreciate the comments. It has been modified. 

（11）References for software should be placed immediately after software names but not at the ends of sentences describing details of their use.

Response: We appreciate the comments. It has been modified as suggested

（12）Lines 149, 152 add Refs. or URLs for Tracer v1.7 and FigTree

Response: We appreciate the comments. It has been modified as suggested

（13）Line 149 delete (Bouckaert et al. 2014).

Response: We appreciate the comments. It has been modified as suggested

（14）Line 177: genome > genomes;

Response: We appreciate the comments. It has been modified as suggested

（15）Lines 177-78: delete “approximately”

Response: We appreciate the comments. It has been modified as suggested

• Lines186-87, 239-240: use abbreviation for genus name.

Response: We appreciate the comments. It has been modified as suggested

 

Reviewer 4 Report

Comments and Suggestions for Authors

Reviewer’s Comment / Report

The manuscript #forests-2993906 entitled “Assembly and comparative analyses of the chloroplast genomes of the threatened plant Rosa anemoniflora." has been reviewed.

The authors have done significant work on sequencing of the Rosa anemoniflora cp genome and comparative analysis with other Rosa species.

This manuscript reveals that the study assembled and annotated the complete cp genome of Rosa anemoniflora, which is novelty.

The following are some concerns that need to be resolved.

Species names must be corrected italic throughout the manuscript file.

In Introduction, line 31-40, include more information for the species and family.

R. anemoniflora was 38 listed as a near threat (NT) on the IUCN Red List… provide source link or reference

Provide more information for the status and reason for the species under IUCN Red List.

Line 41-49, references information missing for the entire section

In methods,

Line 79-84, provide company information.

Line 106, Perl package was used.. provide package information

Line 112, PAML was used for selection pressure analysis.. provide package information with version used for the analysis.  

In Results

In Line 193, only petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure,… the site of positive selection can be confirmed

In Discussion

Include and discuss more reference related to diversity of rosa species

The author concludes that the cp genome could be used for species identification, taxonomic clarification, and phylogenetic resolution.

Some species-specific markers can be identified and included with this study

 

Author Response

Reviewer 4

 

The following are some concerns that need to be resolved.

 

• Species names must be corrected italic throughout the manuscript file.

Response: We appreciate the comments. All species names have been corrected italic throughout the manuscript file.

• In Introduction, line 31-40, include more information for the species and family.

Response: We appreciate the comments. We have added more information for the species and family in the first paragraph of the introduction.

• anemoniflora was 38 listed as a near threat (NT) on the IUCN Red List… provide source link or reference
• Response: After verification, we have revised the relevant content to: R. anemoniflora was listed as China Biodiversity Red List (https://www.mee.gov.cn/xxgk2018/xxgk/xxgk01/202305/W020230522536560832337.pdf)
• Provide more information for the status and reason for the species under IUCN Red List.

Response: We have supplemented the description of the population distribution characteristics in the first paragraph of the introduction to indicate its endangered status. The exact reasons for its endangerment remain unclear and will be the focus of our next research phase.

• Line 41-49, references information missing for the entire section

Response: We appreciate the comments. Currently, there is a lack of research related to R. anemoniflora, so we did not cite any references when introducing its research status in the second paragraph of the introduction.

 

In methods,

 

Line 79-84, provide company information.

Response: We appreciate the comments. As per convention, we have provided the company's name and location. Please let us know what other information you require.

Line 106, Perl package was used.. provide package information

Response: During the revision process, we switched to using Geneious software to obtain specific data. The current text is: "The Geneious [18] software was used to calculate the lengths of the four different boundaries (SSC, LSC, and IR regions), the number and types of genes, GC content, and other information of the chloroplast genomes of seven species based on their GenBank annotation files."

 

Line 112, PAML was used for selection pressure analysis.. provide package information with version used for the analysis.  

Response: We appreciate the comments. It has been modified as suggested

 

In Results

 

In Line 193, only petD in R. anemoniflora and R. lucidissima experienced significant positive selection pressure,… the site of positive selection can be confirmed

Response: We appreciate the comments. Based on the suggestions of other reviewers and following stricter screening criteria, we found that petD did not experience positive selection pressure. We have accordingly revised the relevant description. Therefore, we did not continue to search for sites under positive selection pressure.

 

In Discussion

 

Include and discuss more reference related to diversity of rosa species

 

The author concludes that the cp genome could be used for species identification, taxonomic clarification, and phylogenetic resolution.

 

Some species-specific markers can be identified and included with this study

Response: We appreciate the comments. We have added a description of the diversity of Rosa species in the third paragraph of the discussion. The content is as follows: “The diversity of Rosa species is vast and remarkable, encompassing approximately 200 species that thrive in various temperate and warm regions of the Northern Hemi-sphere [40]. This diversity is not only reflected in their wide range of morphological traits, such as flower color, size, and fragrance, but also in their complex genetic makeup resulting from frequent hybridizations and polyploidization events [41].” 

When developing molecular markers, the objective was to distinguish R. anemoniflora from other species in the Sect. Synstylae distributed in China. Although only one marker was developed in this study, it is because only 11 roses in Sect. Synstylae distributed in China are currently recorded in the database. This marker is already sufficient to distinguish these plants. If more genomes are published in the future, we will continue to develop new markers based on other highly polymorphic regions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript and, I think, now the manuscript can be accepted for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

The revisions implemented by the author have measurably bolstered the manuscript's clarity, methodological rigor, and overall scholarly caliber. Specifically, the author has meticulously addressed each critique and recommendation raised by the reviewers, resulting in a substantially fortified and comprehensive study.

As a consequence of these enhancements, we deem the manuscript to be of sufficient quality for publication in its present form.