Genotyping-by-Sequencing Study of the Genetic Diversity and Population Structure of the Endangered Plant Tsoongiodendron odorum Chun in China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I read with interest the manuscript submitted by colleagues. The study needs great improvement not only in referencing, but also methodology. Advice & suggestions are given below. 

1.      Revise sentence with valid citation: "Tsoongiodendron odorum Chun, a large evergreen tree within the Tsoongiodendrom genus of the Magnoliaceae family, was named in honor of the Chinese botanist Mr. Zhong Guanguang.”

 From the Chinese Study “Isolation and characterization of microsatellite loci in Tsoongiodendron odorum (Magnoliaceae)”, that is not stated at all. Also on 2023 An et al (2023) stated in their paper Tsoongiodendron odorum Chun (syn. Magnolia odora (Chun) Figlar & Noot.) is one of the valuable trees of Vietnamese flora [1]. Please adjust all the information to state the reality.

 Please take into account the following information:

 The homotypic synonyms are:

-           Michelia odora (Chun) Noot. & B.L.Chen in Ann. Missouri Bot. Gard. 80: 1086 (1993)

-           Tsoongiodendron odorum Chun in Acta Phytotax. Sin. 8: 283 (1963)

And the heterotypic synonyms are

-           Michelia gravis Dandy ex Gagnep. in P.H.Lecomte, Fl. Indo-Chine, Suppl. 1: 50 (1938), no Latin descr.

 1.      Give a reference to the geographic distribution: "This species was initially discovered in Hainan, China, and is predominantly found in the tropical to subtropical regions of southern China, encompassing southern Jiangxi, Fujian, Guangdong, Hainan, Guangxi, and southeastern Yunnan."

2.      Also correct the text where says that majority of the studies do not include genetic diversity. Jiang et al (2011) study used microsatellites to study the genetic diversity, add this reference and correct the text to include their study.

3.      Correcting study citation: "The study by Wang et al. was incorrectly cited in the text. Jiang et al. (insert year) reported the development of 12 microsatellite loci for T. odorum to assess current population genetic processes and conservation genetics."

4.      Clarification on natural habitat: "For the endangered T. odorum Chun, which lacks a reference genome, our study focused on 17 populations in their natural habitat. Add that the study was done only in China, as the tree is also natural from Vietnam (provide the reference An et al (2023).

5.      Add references for DNA extraction and digestion methods: "DNA extraction was performed using the cetyltrimethylammonium bromide (CTAB)-based method. For genomic DNA digestion, Nla III+Ecorl I endonucleases were utilized, followed by adaptor and barcode addition at both ends using T4 DNA ligase."

6.      Add reference and link for the CASAVA Base: "CASAVA Base is a software platform commonly used for base calling and data analysis in Next Generation Sequencing (NGS) studies.”

7.      Add references for software used in analysis.

8.      Details on methodology: "In the methodology section, heterozygosity, minor allele frequency, number of clusters (K), and the number of Markov Chain Monte Carlo iterations with burn-in should be clearly indicated. The criteria for determining clusters and admixture need also to be specified."

9.      Re-run the analysis: needs an increase of bootstraps by at least 30,000.

10.  Provide a figure for DeltaK: The figure illustrating the best DeltaK value for the 86 individuals must be provided.

 

11.  Discussion on heterozygosity: In the discussion section, discuss the presence of a high number of heterozygous individuals in light of potential influences such as outcrossing, gene flow, and genetic drift, which can contribute to genetic diversity within populations. Is it possible that this high number of heterozygous individuals is due to the outcross against Vietnam germplasm?

Comments on the Quality of English Language

Needs to be improved

Author Response

Response to Reviewer 1 Comments

 

Dear Reviewers,

Thank you very much for useful comments of our manuscript. We have made careful revisions, and the detailed corrections are listed below point by point. All changes are highlighted in the revised manuscript.

If you have any further questions, please feel free to contact me.

Best regards.

Sincerely,

Yongda Zhong

 

Point 1:Revise sentence with valid citation: "Tsoongiodendron odorum Chun, a large evergreen tree within the Tsoongiodendrom genus of the Magnoliaceae family, was named in honor of the Chinese botanist Mr. Zhong Guanguang.”

 From the Chinese Study “Isolation and characterization of microsatellite loci in Tsoongiodendron odorum (Magnoliaceae)”, that is not stated at all. Also on 2023 An et al (2023) stated in their paper Tsoongiodendron odorum Chun (syn. Magnolia odora (Chun) Figlar & Noot.) is one of the valuable trees of Vietnamese flora [1]. Please adjust all the information to state the reality.

 Please take into account the following information:

 The homotypic synonyms are:

-           Michelia odora (Chun) Noot. & B.L.Chen in Ann. Missouri Bot. Gard. 80: 1086 (1993)

-           Tsoongiodendron odorum Chun in Acta Phytotax. Sin. 8: 283 (1963)

And the heterotypic synonyms are

-           Michelia gravis Dandy ex Gagnep. in P.H.Lecomte, Fl. Indo-Chine, Suppl. 1: 50 (1938), no Latin descr.

Response 1: Thanks for your suggestion. We have made modifications to this sentence and provided a reference. Upon further research, we found that the usage of “Tsoongiodendron Odorum Chun” is more commonly accepted despite some naming disputes.

We revised line 31-32 as“Tsoongiodendron odorum Chun (Magnoliaceae), an endangered evergreen tree, is sporadically distributed in southern China and northern Vietnam[1]”.

Reference: [1] Liu, Y. Magnolias of China. Bright Sparks, China/Beijing. 2004.

Point 2:Give a reference to the geographic distribution: "This species was initially discovered in Hainan, China, and is predominantly found in the tropical to subtropical regions of southern China, encompassing southern Jiangxi, Fujian, Guangdong, Hainan, Guangxi, and southeastern Yunnan."

Response 2: Thanks for your suggestion. We have added reference [2] in revised manuscript.

Reference: [2] Xia, N.; Liu, Y.; Nooteboom, H. P. Flora of China. Science Press, China/ Beijing. 2009:80.

Point 3: Also correct the text where says that majority of the studies do not include genetic diversity. Jiang et al (2011) study used microsatellites to study the genetic diversity, add this reference and correct the text to include their study.

Point 4: Correcting study citation: "The study by Wang et al. was incorrectly cited in the text. Jiang et al. (insert year) reported the development of 12 microsatellite loci for T. odorum to assess current population genetic processes and conservation genetics."

Point 5:Clarification on natural habitat: "For the endangered T. odorum Chun, which lacks a reference genome, our study focused on 17 populations in their natural habitat. Add that the study was done only in China, as the tree is also natural from Vietnam (provide the reference An et al (2023).

Response 5: We fully agree with your point of view. We corrected the sentence as “Therefore, this study used only 17 natural populations within China's natural distribution areas as materials for the endangered plant sightseeing tree, which lacks a reference genome. Based on GBS technology, genome-wide SNP molecular markers were developed to reveal the genetic diversity and inheritance structure of sightseeing trees in various regions.”

Point 6: Add references for DNA extraction and digestion methods: "DNA extraction was performed using the cetyltrimethylammonium bromide (CTAB)-based method. For genomic DNA digestion, Nla III+Ecorl I endonucleases were utilized, followed by adaptor and barcode addition at both ends using T4 DNA ligase."

Point 7: Add reference and link for the CASAVA Base: "CASAVA Base is a software platform commonly used for base calling and data analysis in Next Generation Sequencing (NGS) studies.”

Response 7: Thanks for your suggestion. We have added reference [27] and software links (http://support-docs.illumina.com/SW/ClarityLIMS/ClarityINT/Content/SW/ClarityLIMS/Integrations/CASAVA182SampleSheet.htm) in revised manuscript.

[27] Cock PJA, Fields CJ, Goto N, Heuer ML, Rice PM: The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res 2010, 38(6):1767–1771.

Point 8: Add references for software used in analysis.

Response 8: Thanks for your suggestion. We add a reference “[28]Chen, Shifu, Yanqing Zhou, Yaru Chen, and Jia Gu. 2018. “fastp: An Ultra-Fast All-In-One FASTQ Preprocessor.” Bioinformatics 34: i884–90.” for software used in analysis.

Point 9: Re-run the analysis: needs an increase of bootstraps by at least 30,000.

Response 9: Thanks for your suggestion. We constructed the evolutionary tree using parameters commonly adopted in most articles as references.

Wang, Q., Lan, T., Li, H., Sahu, S. K., Shi, M., Zhu, Y., ... & Hua, Y. (2022). Whole-genome resequencing of Chinese pangolins reveals a population structure and provides insights into their conservation. Communications Biology, 5(1), 821.

Stetter, M. G.; Schmid, K. J. Analysis of phylogenetic relationships and genome size evolution of the Amaranthus genus using GBS indicates the ancestors of an ancient crop. Molecular phylogentics and enolution.2017,109: 80-92.

Point 10:Provide a figure for DeltaK: The figure illustrating the best DeltaK value for the 86 individuals must be provided.

Response 10: Thanks for your good suggestion. We have provided the figure for DeltaK as Figure S1 in the attachment, and the optimal value is K=2.

Point 11: Discussion on heterozygosity: In the discussion section, discuss the presence of a high number of heterozygous individuals in light of potential influences such as outcrossing, gene flow, and genetic drift, which can contribute to genetic diversity within populations. Is it possible that this high number of heterozygous individuals is due to the outcross against Vietnam germplasm?

Response 11: We fully agree with your point. Considering the close geography distance, the germplasm in Guangxi may indeed cross-pollinate with the germplasm in Vietnam, which may induce the high heterozygous among individuals. However, this suggestion requires further confirmation in the future research.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript Genotyping-by-Sequencing Study of the Genetic Diversity and Genetic Structure of Endangered Plant Tsoongiodendron odorum Chun in China by Yiping Wang, Huihu Li, Zhijun Yang, Yuanjie Liu, Bingyu Liu, Yongda Zhong examines the molecular basis of the biodiversity of Tsoongiodendron odorum W.Y. Chun depending on the location in the People's Republic of China. This species is of Indian origin and is used for various needs, in particular for traditional medicine.

The manuscript is compiled according to the rules and contains the necessary parts.

Authors should take into account the botanical features of the species, write the full name, consider options for its appearance in the regions, since some populations apparently have introduced roots, this should be carefully discussed. Unfortunately, the authors do not provide morphological or phenotypic differences between the analyzed populations; there are no photographs of plants and their leaves, sizes, analysis of age and reproduction. What is not clear from the perspective of discussing the issue of reproduction of Тsoongiodendron odorum W.Y. Chun as a valuable species.

The presented data from cluster analysis are interesting, but it is not possible to verify them, and the methodology is not written in sufficient detail. For example, L145 indicates the use of a distance matrix, but the dimensions of the distance and the principle of its selection are not discussed. Meanwhile, I would like to note that the Forest journal is interdisciplinary and should be intuitively understandable to specialists in related fields.

Also, the statement that populations differ must be specified in what way.

For example, how will researchers be able to compare their plants in India or other countries with yours with such information presented, this needs to be made clear.

The last paragraph of the introduction of any experimental article should contain the formulated goals, objectives and hypotheses of the study. I don't see it. Instead, "Therefore, for endangered T. odorum Chun, which lacks a reference genome, we focused on 17 populations in their natural habitat. We employed GBS technology to analyze genetic diversity and structure, to evaluate the levels of species and population genetic diversity, and to explore the sources of genetic variation and causes of this species endangerment, providing a theoretical basis for the further development of effective germplasm resource conservation measures and genetic improvement." Please do not use conclusions, recommendations and reasoning where the purpose should be. Formulate it and then you can understand whether it has been achieved or not. Please note that the article does not propose ways to save trees or reproduce them; you can describe this as relevance in the introduction or as perspectives in the discussion, but this is not relevant to this work.

I ask the authors to pay attention to the need to correct the figures. If you have marked something with color, it means that the caption (or the drawing itself) indicates why (why) it was done. What does the color represent or what does it mean? Figures in MDPI journals can be placed separately from articles and they must contain a full amount of information that is clearly understandable (Figure 2,3). The letters and numbers must be clearly visible (Figure 2, 3). Added green - write what it is (Figure 3).

The conclusion is independent of the results and contains discussion and plans. Shorten and move the discussion to the discussion section.

The article is useful and interesting, but it is unclear why there is no comparison with genotypes outside the country. The article requires revision.

I recommend that the authors make a graphical abstract depicting the plant and cluster analysis, which will attract readers to their valuable data.

Author Response

Response to Reviewer 2 Comments

 

Dear Reviewers,

Thank you very much for useful comments of our manuscript. We have made careful revisions, and the detailed corrections are listed below point by point. All changes are highlighted in the revised manuscript.

If you have any further questions, please feel free to contact me.

Best regards.

Sincerely,

Yongda Zhong

Point 1:Authors should take into account the botanical features of the species, write the full name, consider options for its appearance in the regions, since some populations apparently have introduced roots, this should be carefully discussed. Unfortunately, the authors do not provide morphological or phenotypic differences between the analyzed populations; there are no photographs of plants and their leaves, sizes, analysis of age and reproduction. What is not clear from the perspective of discussing the issue of reproduction of Тsoongiodendron odorum W.Y. Chun as a valuable species.

Response 1: We fully agree with your suggestion and understand the importance of morphological or phenotypic differences between the populations, which will help us conduct the population structure of Тsoongiodendron odorum. In this study, we collected samples from adult trees with a diameter at breast height of more than 15 cm and each sample was collected from trees more than 20 m apart avoid individuals from introduced roots. Moreover, we investigated the genetic diversity population structure with SNP molecular data by GBS, which could more useful than phenotypic data.

Point 2:The presented data from cluster analysis are interesting, but it is not possible to verify them, and the methodology is not written in sufficient detail. For example, L145 indicates the use of a distance matrix, but the dimensions of the distance and the principle of its selection are not discussed. Meanwhile, I would like to note that the Forest journal is interdisciplinary and should be intuitively understandable to specialists in related fields.

Response 2: Thanks for you advice.We have supplemented the data processing methods as follows in revised manuscript. “After SNP detection, the individual SNPs obtained can be used to calculate the distance between populations. The p-distance between two individuals i and j is calculated using the following formula: . In the formula, L represents the length of the high-quality SNPs region and the allele at position 1 is A/C. Therefore  ”

Point 3:Also, the statement that populations differ must be specified in what way.

For example, how will researchers be able to compare their plants in India or other countries with yours with such information presented, this needs to be made clear.

Response 3: Thank you very much for your suggestion. We only compared populations diversity based on SNP molecular data by GBS, which will be help us specified the difference among populations even individuals.

Point 4:The last paragraph of the introduction of any experimental article should contain the formulated goals, objectives and hypotheses of the study. I don't see it. Instead, "Therefore, for endangered T. odorum Chun, which lacks a reference genome, we focused on 17 populations in their natural habitat. We employed GBS technology to analyze genetic diversity and structure, to evaluate the levels of species and population genetic diversity, and to explore the sources of genetic variation and causes of this species endangerment, providing a theoretical basis for the further development of effective germplasm resource conservation measures and genetic improvement." Please do not use conclusions, recommendations and reasoning where the purpose should be. Formulate it and then you can understand whether it has been achieved or not. Please note that the article does not propose ways to save trees or reproduce them; you can describe this as relevance in the introduction or as perspectives in the discussion, but this is not relevant to this work.

Response 4: I completely agree with your suggestion and have made modifications to the results section of the introduction. “Therefore, this study used only 17 natural populations within china's natural distribution areas as materials for the endangered plant sightseeing tree, which  lacks a reference  genome. Based on GBS technology, genome-wide SNP molecular markers  were developed to reveal the genetic diversity and inheritance structure of sightseeing trees in various regions.”

Point 5:I ask the authors to pay attention to the need to correct the figures. If you have marked something with color, it means that the caption (or the drawing itself) indicates why (why) it was done. What does the color represent or what does it mean? Figures in MDPI journals can be placed separately from articles and they must contain a full amount of information that is clearly understandable (Figure 2,3). The letters and numbers must be clearly visible (Figure 2, 3). Added green - write what it is (Figure 3).

Response 5: Thanks for your good suggestion. We have annotated the color numbers in the figure, and the numbers are explained in Table S1 of the attachment.

Figure 2. Neighbor-joining phylogenetic tree of T. odorum Chun based on all SNPs with the evolutionary distances measured by p-distances using TreeBest. The central circle is divided into western China (orange), central China (red), and eastern China (green) based on geographical distribution, whereas the outermost circle is divided into Cluster I (red) and Cluster II (blue).

Figure 3. Population genetic structure of 86 T. odorum Chun trees. Each color represents one cluster. Each individual is represented by a vertical bar and the length of each colored segment in each vertical bar represents the proportion contributed by ancestral populations.

Point 6:The conclusion is independent of the results and contains discussion and plans. Shorten and move the discussion to the discussion section.

Response 6: Thanks for your good suggestion. We have streamlined the discussion section and made modifications to the conclusion section in revised manuscript.

We have deleted sentence“Genetic diversity is closely related to population adaptation [37]. The richer the genetic diversity of a species, the stronger its adaptive ability, which expands the survival environment and enhances the evolutionary potential. Therefore, an in-depth study of biological genetic diversity can assess the survival of the species, reveal their evolutionary history (when, where, and how species originated), and provide an important basis for predicting future development trends [38].”and shorten the discussion“Therefore, the conservation of T. odorum Chun is urgent, and a combination of in situ and ex situ conservation should be adopted in the future to protect the genetic diversity of the existing germplasm resources. At the same time, relevant cultivation and propagation studies should be carried out to explore possible alternative habitats to increase genetic diversity. ”

Point 7: The article is useful and interesting, but it is unclear why there is no comparison with genotypes outside the country. The article requires revision.

Response 7: Thanks for your suggestion. As an ancient relict plant and an endangered species, T. odorum Chun, which is mainly distributed in southern China. Moreover, due to the difficulty in sampling of foreign country, in this study, we only analyzed the population separated by several major mountain ranges in the main distribution areas of China and no comparative analysis was conducted between China and other countries.  

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Article Genotyping-by-Sequencing Study of the Genetic Diversity and Genetic Structure of Endangered Plant Tsoongiodendron odorum Chun in China by Yiping Wang etal. presents data from magnolia leaf analysis to evaluate an effective strategy for using selected trees for reforestation.

The study was well-done and contained a reasonable sample.

Article contains the necessary sections and is well formatted, but the pictures are difficult to read (some letters are not visible).

For this type of research, it is important to clarify the methodology for sampling and storing samples (young leaves, buds...). The preferred method is to collect branches and grow samples in vitro to avoid errors. In this case, it is not clear how the selection was carried out, since it is obvious that this could not happen simultaneously. The rest of the procedure is described in detail, but if the details of the analysis are indicated, the sizes of the fragments should also be given.

In the results section, the caption of Figure 3 should be expanded and it should be explained what K2, K3, K4 mean.

Decipher AMOVA especially in the table; the abbreviations should be clearly understood.

If the authors believe that their research can contribute to conservation as they claim, then work out the application.

If not, remove "Therefore, the conser- 377

vation of T. odorum Chun is urgent, and a combination of in situ and ex situ conservation 378

should be adopted in the future to protect the genetic diversity of the existing germplasm 379

resources. At the same time, relevant cultivation and propagation studies should be car- 380

Ried out to explore possible alternative habitats to increase genetic diversity. The results 381

of this study clarify the current status of genetic diversity and genetic structure of T. 382

odorum Chun, providing a theoretical basis for the development of appropriate conservation- 383

tion measures and genetic improvement strategies. " into the discussion. And remove this section from the abstract, as it is misleading.

I recommend keeping this, but then laying out a clear recommendation and protocol based on your research. Discussion of unfinished work is acceptable in a review but not in a pilot study.

The work is interesting.

Author Response

Response to Reviewer 3 Comments

 

Dear Reviewers,

Thank you very much for useful comments of our manuscript. We have made careful revisions, and the detailed corrections are listed below point by point. All changes are highlighted in the revised manuscript.

If you have any further questions, please feel free to contact me.

Best regards.

Sincerely,

Yongda Zhong

Point 1: Article contains the necessary sections and is well formatted, but the pictures are difficult to read (some letters are not visible).

Response 1: Thanks for your good suggestion. We have made modifications to figure 1 and 2

Figure 2. Neighbor-joining phylogenetic tree of T. odorum Chun based on all SNPs with the evolutionary distances measured by p-distances using TreeBest. The central circle is divided into western China (orange), central China (red), and eastern China (green) based on geographical distribution, whereas the outermost circle is divided into Cluster I (red) and Cluster II (blue).

Figure 3. Population genetic structure of 86 T. odorum Chun trees. Each color represents one cluster. Each individual is represented by a vertical bar and the length of each colored segment in each vertical bar represents the proportion contributed by ancestral populations.

Point 2: For this type of research, it is important to clarify the methodology for sampling and storing samples (young leaves, buds...). The preferred method is to collect branches and grow samples in vitro to avoid errors. In this case, it is not clear how the selection was carried out, since it is obvious that this could not happen simultaneously. The rest of the procedure is described in detail, but if the details of the analysis are indicated, the sizes of the fragments should also be given.

Response 2: Thanks for your good suggestion. We collected samples from different regions in April 2023. Collect 5 healthy tender leaves from each sample, dry them with silica gel on site, store them on dry ice, and bring them back to the laboratory for storage in a -80 degree freezer. After all samples are collected, they will be processed uniformly.

Point 3:  In the results section, the caption of Figure 3 should be expanded and it should be explained what K2, K3, K4 mean.

Response 3: Thanks for your good suggestion. We have made revisions in revised manuscript.

Point 4: Decipher AMOVA especially in the table; the abbreviations should be clearly understood.

Response 4: AMOVA is a molecular analysis of variance.

Point 5: If the authors believe that their research can contribute to conservation as they claim, then work out the application. If not, remove "Therefore, the conservation of T. odorum Chun is urgent, and a combination of in situ and ex situ conservation should be adopted in the future to protect the genetic diversity of the existing germplasm resources. At the same time, relevant cultivation and propagation studies should be carried out to explore possible alternative habitats to increase genetic diversity. The results of this study clarify the current status of genetic diversity and genetic structure of T. odorum Chun, providing a theoretical basis for the development of appropriate conservationtion measures and genetic improvement strategies. " into the discussion. And remove this section from the abstract, as it is misleading. I recommend keeping this, but then laying out a clear recommendation and protocol based on your research. Discussion of unfinished work is acceptable in a review but not in a pilot study.

Response 5: We fully agree with your suggestion and have made modifications to the discussion and conclusion sections of the manuscript accordingly.We corrected the sentence as“Therefore, the conservation of T. odorum Chun is urgent, and a combination of in situ and ex situ conservation should be adopted in the future to protect the genetic diversity of the existing germplasm resources. At the same time, relevant cultivation and propagation studies should be carried out to explore possible alternative habitats to increase genetic diversity. The findings of this study elucidate the present genetic diversity and structure of T. odorum Chun, offering a theoretical foundation for the implementation of suitable conservation measures and genetic enhancement strategies.

5. Conclusions

This study utilized GBS technology to analyze the genetic diversity and structure of 17 natural populations in the primary distribution area of T. odorum Chun for the first time. The phylogenetic tree, population structure, and PCA analysis revealed that the 17 natural populations formed two major clusters: the Fujian population and a mixed cluster of populations from Jiangxi, Guangdong, Hunan, and Guangxi, displaying a clear geographic pattern. The genetic diversity of T. odorum Chun is quite low relative to that of other endangered tree species. AMOVA as well as Fst and Nm values indicated that genetic differentiation among T. odorum Chun populations is at a low level, speculation that a certain degree of gene flow exists among subpopulations. ”（Line 382-398）

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Major:

1) This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style. I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing.

2) Authors should improve figures:

a) Fig. 1 – Use white circle. Also, explain all abbreviations in the figure legend (GXPX, GXNN, etc.).

b) Increase font size in the Fig. 2 – sample names. At the same time, reduce the font size of “Cluster I” and “Cluster II”.

3) Authors should include GeneBank accession numbers of the used reference genome and obtained sequences in the Ms text, in Results or Material and Methods.

Minor:

4) Line 13: Delete “very”.

5) Line 112-113: include reference for used method for DNA analysis.

6) Line 122: include information about used endonucleases – manufacturer.

Comments on the Quality of English Language

This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style. I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing. 

Author Response

Response to Reviewer 1 Comments

 

Dear Reviewers,

Thank you very much for useful comments of our manuscript. We have made careful revisions, and the detailed corrections are listed below point by point. All changes are highlighted in the revised manuscript.

If you have any further questions, please feel free to contact me.

Best regards.

Sincerely,

Yongda Zhong

Point 1:This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style. I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing.

Response 1: This manuscript has been sent to professionals for proofreading.

Point 2:Authors should improve figures:

1. a) Fig. 1 – Use white circle. Also, explain all abbreviations in the figure legend (GXPX, GXNN, etc.).

Response a: Thanks for your good suggestion. We have made modifications to the image, but the white circle is not clear enough, so it was not used.The GXPX, GXNN, etc. are the place names of the sampling sites. Annotations have been made in Figure 1 and Table 1.

1. b) Increase font size in the Fig. 2 – sample names. At the same time, reduce the font size of “Cluster I” and “Cluster II”.

Response b:  Thanks for your good suggestion. We have made modifications to the image.

Figure 2. Neighbor-joining phylogenetic tree of T. odorum Chun based on all SNPs with the evolutionary distances measured by p-distances using TreeBest. The central circle is divided into western China (orange), central China (red), and eastern China (green) based on geographical distribution, whereas the outermost circle is divided into Cluster I (red) and Cluster II (blue).

 

Point 3:Authors should include GeneBank accession numbers of the used reference genome and obtained sequences in the Ms text, in Results or Material and Methods.

Response 3: Due to the lack of whole genome information in Tsoongiodendron Odorum Chun, we based on sample FJYP1 with the highest total number of tags was selected to construct the proposed reference gene using Stacks 1.45 ustacks package (with the parameters of - i 1- m 1- M 6- N 6- p 6) We have made corresponding modifications to lines 134-137 in the article

Point 4: Line 13: Delete “very”.

Response 4:  Thanks for your good suggestion. We have deleted “very”.

Point 5: Line 112-113: include reference for used method for DNA analysis.

Response 5: Thanks for your good suggestion. We have supplemented the references. Our DNA extraction is based on the CTAB extraction method established in this experiment. [25] Gong, X., Yang, A., Wu, Z., Chen, C., Li, H., Liu, Q.; Yu, F.; Zhong, Y. . Employing genome-wide SNP discovery to characterize the genetic diversity in Cinnamomum camphora using genotyping by sequencing. Forests. 2021 12:1511.

Point 6: Line 122: include information about used endonucleases – manufacturer.

Response 6: Thanks for your good suggestion. We have added the manufacturer information about endonuclease in Line 128-134.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Article Genotyping-by-Sequencing Study of the Genetic Diversity and Genetic Structure of Endangered Plant Tsoongiodendron odorum Chun in China by Yiping Wang et al. made the necessary improvements to the images, made corrections and added the necessary clarifications.

 

Author Response

Responses to reviewer comments

Editor's comments:

Thank you very much for useful comments of our manuscript.If you have any further questions, please feel free to contact me.

Best regards.

Sincerely,

Yongda Zhong

Author Response File: Author Response.pdf