New Variants in the Chloroplast Genome Sequence of Two Colombian Individuals of the Cedar Timber Species (Cedrela odorata L.), Using Long-Read Oxford Nanopore Technology

Round 1

Reviewer 1 Report

Genome analysis of the existing inadequate, I suggest that increase the codon analysis, you can refer to https://doi.org/10.1007/s00606-023-01868-w

Please ensure that the full text format is consistent with the publication requirements

Author Response

Comment 1:

Genome analysis of the existing inadequate, I suggest that increase the codon analysis,  you can refer to https://doi.org/10.1007/s00606-023-01868-w

Answer:

Thanks for your suggestion,

We included the codon usage analysis (Figure 2) and a comparison of both individuals to the reference genome using mVista (Figure S3)

Comment 2:

Please ensure that the full text format is consistent with the publication requirements

Answer:

Thanks for your comment, the format of the whole text was changed to the journal requirements.

Reviewer 2 Report

1. What is the main question addressed by the research?

 How is the diversity of chloroplast genomes of Cedrela odorata inferred from long-read data generated by Oxford Nanopore Technologies? 

2. Do you consider the topic original or relevant in the field? Does it

address a specific gap in the field?

The topic is original and provides additional information for further studies on intraspecific diversity of organelle genomes of land plants.

3. What does it add to the subject area compared with other published

material?

This study added novel information about the genetic variants of Cedrela odorata in Colombia.

4. What specific improvements should the authors consider regarding the

methodology? What further controls should be considered?

The authors used appropriate methods for obtaining the results; therefore, no further improvements are needed.

5. Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed?

The results supported the conclusions of this study and all aims were achieved.

6. Are the references appropriate?

The references are suitable to the content of the manuscript. 

7. Please include any additional comments on the tables and figures.

The tables and figures were used correctly and presented the results.

 

Please check my comments below for minor issues.

1/ Please add the usage of S. mahagoni as the outgroup for phylogenetic analysis in the method part.

2/ Please add more details about the genomic comparison of two new Cedrela odorata chloroplast genomes and related taxa in Cedrela genus regarding gene content and order, genome structure, etc...

Author Response

 

Comment 1:

Please add the usage of S. mahagoni as the outgroup for phylogenetic analysis in the method part

 

Answer:

Thanks for your comment. Details of S. mahagoni were added in materials and methods, section 2.5.

Comment 2:

Please add more details about the genomic comparison of two new Cedrela odorata chloroplast genomes and related taxa in Cedrela genus regarding gene content and order, genome structure, etc

Answer

Thanks for your suggestion. Additional information about the C. odorata chloroplast genome and related taxa in Cedrela previously reported by Mader et al., 2018 was included in the introduction section. The genomic insights of the two new cp genomes compared to the Refseq cp genome is presented in the results section 3.3 suggesting highly similar gene content and order, compared to the reference sequence. mVista (Figure S3) was used to compare both individuals to the Refseq MG724915.1 genome. It will be ideal to compared the gene content of the new cp genomes reported in this manuscript with other related taxa. However, the sequences reported by Finch et al., 2019, from around 20 cp genome assemblies of Cedrela species, are highly fragmented. Thus, not suitable for genome annotation and not valid comparison can be performed.

Reviewer 3 Report

Dear colleagues, in this manuscript, you claim in the abstract that two new chloroplast genomes have been assembled, but only one (OGP2096) is shown in Table 1 ' this study ', which puzzles me. The assembly and quality control of the chloroplast genome should not be placed in the results (3.1 and 3.2), but rather in the materials and methods. The annotation of the chloroplast genome should not only include the structural composition, the number of related genomes, and the length should be counted to explain the variability between populations. It is too simple to consider only SNPs, because it is subject to the setting of subjective thresholds and cannot be convincing. The authors should also analyze the composition of the chloroplast genome-variability analysis (including IR region analysis, polymorphism analysis, comparative genomics analysis, etc.) -select SNPs-phylogenetic analysis, and finally construct a phylogenetic tree based on conserved sequences or whole chloroplast genomes and a phylogenetic tree based on re-series SNPs to illustrate the effectiveness and confidence of SNPs. If possible, the best verification method is rt-PCR verification, and the expression level of SNPs between different species is the most convincing evidence.

1. In 2.5, the selection of evolutionary tree species should be based on the same genus and the same family. Similar taxonomic status, not because they grow in similar living areas.

2. Table 1 does not list the information of out group.

3.Analyzing from Table 1, you seem to have assembled only one species, C.odorata ( OGP2096 ) ? But you seem to claim to assemble two in full? Did you miss it or...

4. Placing 3.1 and 3.2 in the material is a better choice.

In Table 4, the total number of SNPs in OGP2096 and OGP2143 is much lower than that of other species, which is an interesting finding, but you don 't seem to be interested.

5. In Table 4, the total number of SNPs in OGP2096 and OGP2143 is much lower than that of other species, which is an interesting finding, but you don 't seem to be interested.

The statistics of these SVs in Table 5 are meaningless. Because species selection for comparison is not scientific.

6. In general, the manuscript is very rough, and even the fonts are not fully unified ( 1, 2.4, and 4 ). Reporting on the new chloroplast genome is useful for taxonomic research, but the analysis and writing of this manuscript is disappointing. I can 't recommend this manuscript well because his analysis is missing. Only genome annotation, SNPs screening and phylogenetic analysis are not enough.

Dear colleagues, in this manuscript, you claim in the abstract that two new chloroplast genomes have been assembled, but only one (OGP2096) is shown in Table 1 ' this study ', which puzzles me. The assembly and quality control of the chloroplast genome should not be placed in the results (3.1 and 3.2), but rather in the materials and methods. The annotation of the chloroplast genome should not only include the structural composition, the number of related genomes, and the length should be counted to explain the variability between populations. It is too simple to consider only SNPs, because it is subject to the setting of subjective thresholds and cannot be convincing. The authors should also analyze the composition of the chloroplast genome-variability analysis (including IR region analysis, polymorphism analysis, comparative genomics analysis, etc.) -select SNPs-phylogenetic analysis, and finally construct a phylogenetic tree based on conserved sequences or whole chloroplast genomes and a phylogenetic tree based on re-series SNPs to illustrate the effectiveness and confidence of SNPs. If possible, the best verification method is rt-PCR verification, and the expression level of SNPs between different species is the most convincing evidence.

1. In 2.5, the selection of evolutionary tree species should be based on the same genus and the same family. Similar taxonomic status, not because they grow in similar living areas.

2. Table 1 does not list the information of out group.

3.Analyzing from Table 1, you seem to have assembled only one species, C.odorata ( OGP2096 ) ? But you seem to claim to assemble two in full? Did you miss it or...

4. Placing 3.1 and 3.2 in the material is a better choice.

In Table 4, the total number of SNPs in OGP2096 and OGP2143 is much lower than that of other species, which is an interesting finding, but you don 't seem to be interested.

5. In Table 4, the total number of SNPs in OGP2096 and OGP2143 is much lower than that of other species, which is an interesting finding, but you don 't seem to be interested.

The statistics of these SVs in Table 5 are meaningless. Because species selection for comparison is not scientific.

6. In general, the manuscript is very rough, and even the fonts are not fully unified ( 1, 2.4, and 4 ). Reporting on the new chloroplast genome is useful for taxonomic research, but the analysis and writing of this manuscript is disappointing. I can 't recommend this manuscript well because his analysis is missing. Only genome annotation, SNPs screening and phylogenetic analysis are not enough.

Author Response

• Only one (OGP2096) is shown in Table 1:

Thanks for noticing, this was fixed

• Assembly and QC of the chloroplast genome should not be placed in the results:

The reason to include this analysis as part of the results was because not many cp genomes based on ONT Minion seq has been reported. Thus, we consider relevant to include this section as part of results.

• The genome annotation should be counted to explain the variability between populations:

That would be ideal. However, there is a lack of annotated Cedrela chloroplast genomes. Finch et al., 2019 reported around 20 cp genome assemblies of Cedrela, highly fragmented. Thus, not suitable for genome annotation and not valid comparison can be performed.

• Chloroplast genome-variability analysis (including IR region analysis, polymorphism analysis, comparative genomics analysis, etc.).

This was described on the SV analysis (section 3.4), and it has been corrected accordingly.

• RT-PCR verification, and the expression level of SNPs between different species: This is really good suggestion. Unfortunately, this could take a couple of months to obtain reliable results.

This is a really good suggestion. Unfortunately, this could take time and a budget to obtain reliable results. Therefore, we are unable to include such information in the present manuscript, but we will consider this suggestion for further research in this species.

Detailed Comments

1. In 2.5: this comment is in line with another suggestion.

We agree with this and the corresponding modification was performed on the text 

      2. Table 1 does not list the information of out group: the information of the outgroup was included in the table.

The information of the out group was included in materials and methods section 2.5, in order to avoid confusion, considering that Table 1 just includes the Cedrela genus genomes.

 

       3. Analyzing from Table :

 Thanks for identify the mistake, the other sequence was added accordingly

       4. Placing 3.1 and 3.2 in the material is a better choice:

The sequencing assembly results represent a substantial part of the manuscript, as this is the first report for chloroplast genome assemblies in C. odorata using long read sequencing technologies.

       5. In Table 4, the total number of SNPs in OGP2096 and OGP2143 is much lower :

Thanks for pointing this out. We added extra information on the corresponding caption clarifying that those SV were identified by compared 14 cp genomes to the C. odorata refseq. The low number of SV with the new cp genomes might be explained by the complete genome assemblies reported in this study.

        6. The statistics of these SVs in Table 5 are meaningless:

Kindly, we disagree with this statement as we consider a valid comparison (intra-species). The candidate sequence variants could be potentially converted into molecular markers to track illegal timber obtained from natural populations. Thus, we believe this observation is a highly important result of this study.

        7. In general, the manuscript is very rough:

We followed all the suggestions made by all of the reviewers, hoping will be suitable for publication

Reviewer 4 Report

This study investigated novel variations in chloroplast genome sequences of individuals of the Colombian cedar genus (Cedrela odorata). Due to continued overexploitation, Cedrela odorata is listed as vulnerable and therefore conservation efforts are needed. Using the long-read Oxford Nanopore technology, two new chloroplast genomes were assembled and annotated. Both new chloroplast genomes were simultaneously tested for SVs and ultimately screened for a limited number of SNPs. 

Overall, the subject matter itself is interesting and important for the conservation of vulnerable species. However, in order to improve the quality of the paper, additional content needs to be added and the chart format needs to be carefully modified.

 

Major：

1. Why is there no corresponding result for the OGP2143 specimen by Canu software？

2. How many genes are included in the chloroplast genomes of for individuals OGP2143 and OGP2096, and what are their main functions involved?

 

 

1. Why are the fonts and sizes of the third paragraph of the Introduction, 2.4 of the Materials and Methods, and the fourth paragraph of the Discussion inconsistent with those of other parts?

2. The sixth line of the second paragraph in result 3.2 cannot be segmented. 

3. The Materials and Methods section mentions that a total of 15 individuals were collected. Why do Table 1 only show 14 individuals?

4. Why is Table 2 laid out like this? It's a bit confusing. Is Table 2 a three-line table format? If so, please use the correct format. Or you can add a legend to explain it appropriately.

5. The first row and third column should be represented in two rows, consistent with the first two columns

6. The unit abbreviations in Table 3 and throughout the paper should be consistent in capitalization. kb or Kb?

7. Please use a unified format for the first row of the three line tables in Tables 4 and 5.

Author Response

Major：

1. Why is there no corresponding result for the OGP2143 specimen by Canu software? 

Ans:/ 

We believe Canu is one of the most used genome assembler software, however, it performs better for large genomes and not very accurate for short and with inverted repeats such as chloroplasts, we observed this when we tested it with the OGP2096 genome and obtained similar results to Wang et al 2018 in eucalyptus species; therefore, we adapted their chloroplast genome assembly pipeline for long reads based on Hinge software, which resulted in a clear chloroplast genome structure and with a similar size to the previous reported cp genomes for C. odorata.

We included more information in the manuscript on the section 3.2, second paragraph, describing why we did not use Canu to assemble OGP2143 cp genome.

 

2. How many genes are included in the chloroplast genomes of for individuals OGP2143 and OGP2096, and what are their main functions involved?

Ans:/ 

We included additional information about the number of genes and their organization in both chloroplast genomes. We also found similar gene content and order to the reference sequence reported by Mader et al., 2018 (Section 3.3 and table 3)

Detailed comments

 

1. Why are the fonts and sizes of the third paragraph of the Introduction, 2.4 of the Materials and Methods, and the fourth paragraph of the Discussion inconsistent with those of other parts?

Answer

The format of the whole document has been revised accordingly

 

2. The sixth line of the second paragraph in result 3.2 cannot be segmented.

Answer

The format of the whole document has been revised accordingly

 

3. The Materials and Methods section mentions that a total of 15 individuals were collected. Why do Table 1 only show 14 individuals?

 

Answer

Details of the missing individual were added to the Table 1.

 

4. Why is Table 2 laid out like this? It's a bit confusing. Is Table 2 a three-line table format? If so, please use the correct format. Or you can add a legend to explain it appropriately.

 

Answer

Table 2 was formatted in the right orientation

 

5. The first row and third column should be represented in two rows, consistent with the first two columns

 

Answer

Table 2 was formatted in the right orientation

 

6. The unit abbreviations in Table 3 and throughout the paper should be consistent in capitalization kb or Kb?

 

Answer

The units were revised and adjusted throughout the document

 

7. Please use a unified format for the first row of the three line tables in Tables 4 and 5.

 

Answer

Tables 4 and 5 were formatted accordingly

Round 2

Reviewer 3 Report

The authors followed my suggestions. The explanations and changes introduced into the manuscript are satisfactory for me. The paper is ready for publication. Wish you all the best in the future work.

The authors followed my suggestions. The explanations and changes introduced into the manuscript are satisfactory for me. The paper is ready for publication. Wish you all the best in the future work.