Genetic Diversity of Calligonum L. Species Analyzed Using Newly Developed Genomic Simple Sequence Repeats and Their Application to Conservation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents significant value, with its findings offering potential for an in-depth analysis of genetic diversity within the genus Calligonum, particularly in the context of conservation of the genetic diversity of endemic species and genetic resources of Calligonum species. The article is correct, balanced and formally sound. The work aligns well with the scope of the journal.

In general, I have identified some obstacles to publication and would appreciate it if the Authors made clearer some methodological choices and supported them with additional information.

 

There are certain ambiguities in the criteria used for species selection in the analysis among species of Calligonum genus. While the abstract highlights the importance of the genus' genetic resources in the context of their utility as economically valuable plants, the analysis and discussion extend this significance to the conservation of biodiversity within the natural diversity of endemic Chinese flora. Please ensure a more cohesive connection between the abstract, introduction, and conclusion.

 

Furthermore, in the Introduction section, identifying which of the analyzed species are endemic, which exhibit a broad distribution range, and which have practical applications would enhance the clarity of the paper. These distinctions currently rely on the context.

 

 The article's title suggests that some species were used to analyze genetic diversity of the genus Calligonum. However, the manuscript lacks information on the total number of species within the genus, raising concerns about the representativeness of the findings at the genus level.

 

Essential information on the analyzed population sizes is absent, which is crucial for the methodological development of conservation guidelines.The methodological concerns relate also to the limited sample size used to estimate genetic diversity, especially in cases where species are represented by only four samples.

 

 Given the aforementioned considerations, I suggest choosing one of the options:

 

A.      focusing the analyses on species with a larger sample representation and emphasizing the significance of the findings in the context of conserving the genetic resources of economically and functionally important species within the genus Calligonum. In my opinion, species represented by a small number of individuals (e.g., 4) can serve as valuable examples to demonstrate the potential application of newly developed SSR primers;

 

 or

 

B.      Provide evidence, supported by references and bootstrapping analysis, to demonstrate that a limited number of individuals is sufficient for conducting genetic variation analyses.

 

 

 

The title of the paper is somehow informative, but perhaps not very accurate. I recommend focusing on the primary contribution of the publication, which is the development of SSR markers, eg. „The application of  newly developed genome-SSR markers for genetic diversity analysis and conservation of Calligonum L. species”.

 

Please include the botanical authors' names for the specific taxa mentioned in the 4.1 Experimental Materials section. Some photographs of the plants, fruits, or seeds of the selected species would be beneficial in the presentation of the species of the study.

 

Keywords: do not repeat words that are already in the title.

 

 

Finally, my specific remarks are the following with approximate line number in typeset:

19-20 I think more accurate would be “Their widespread use and breeding efforts highlight the need for conservation of high quality germplasm resources of the genus” instead of “Extensive utilization and associated breeding call for selection and preservation of good germplasm resources of the genus”.

29-30 Please revise the sentence „At the same time, the failure of SSR identification reflects the problematic nature of its present taxonomy” to emphasize that the identified SSR markers lack taxonomic resolving power.

30-32 I would suggest rephrase the sentence „The results of this study can provide reference primers for genus Calligonum, and provide sufficient genetic information for the screening and conservation of the Calligonum germplasm” into „The results of this study provide reference SSR primers for the genus Calligonum, supporting the improvement of its germplasm conservation efforts”.

37-39 Please include information about the number of species or taxa that comprise the genus.

42 Please include references to studies that identify the genus as comprising four sections.

49-58 Please include information about the practical applications or utility of the species analyzed in the study.

60-61 I suggest to rephrase or exclude the sentence “Changes in genetic diversity that occur within species can have important implictions for the subsequent development of populations”.

60-66 Please provide a brief discussion on the use of molecular markers for the conservation of the genus Calligonum, or highlight the scarcity of such studies

67-69 I think that information on the taxonomic discrepancies of the genus should be presented alongside details about the number of species it encompasses. It might be worth considering relocating this sentence to the  section 37-42

69-70 Please, consider moving the sentence “Despite these challenges, Calligonum’s ecological value, especially as a wind and sand-fixing species, and its contribution to desertification control, cannot be denied” to the section between lines 49-58

71-72 Pleaseexplain the “genetic dynamics” or delete the whole sentence.

73-83 Please, rearrange the paragraph, starting with and emphasizing the importance of SSR markers in assessing genetic variability, followed by examples of their application in taxonomic diagnostics.

85-87 sound like results. Please remove it

87-89 I suggest revising the objectives to emphasize: (1) the development of new SSR primers for the genus, (2) their application in genetic diversity analysis of species to support conservation efforts, and (3) their use in taxonomic analysis.

106 please correct the title of Table 2, I suggest “Characteristic of SSR loci of C junceum”

117-118 sounds like introduction, I suggest to remove it.

119-120 Please provide a brief explanation of why PIC was estimated in this study.

128 Please explain what does “saguaro dates” mean

143 Please rephrase the sentence while avoiding the term "gene abundance," as it refers to varying levels of specific genes, which were not analyzed in the scope of this study.

152 Please consider moving it to the relevant discussion section or deleting it if redundant

153-154 sounds like incomplete sentence

179-183 Please revise this part of the results. In my opinion the PCO diagram shows 4–5 groups of samples, with one large group represented by samples from eight species, while a smaller group consists of samples from C. gobicum and C. roborowskii. Additionally, samples from C. pumilum are distinctly separated from the others.

184 The authors reference the genetic diversity results in relation to phenotypic variation; however, no data on phenotypes is provided. I suggest relocating this sentence to the appropriate discussion section.

206 Please consider moving the sentence „indicating that these three species enjoy a common ancestry” into relevant disscussion section.

208-209 Please consider moving into relevant disscussion section.

231 Please provide a reference supporting the use of genomic SSR markers as highly informative and reproducible.

231-232 Please, consider moving the sentence into relevant conclusion section.

244 Consider the another title for paragraph reffereing to the field of failure (eg. Genetic structure and taxonomic identification failure….)

253 Please clarify the meaning of "shako jujube"

265-270 Please, sound like an introduction. Consider moving it into relevant introduction section.

277-283 Explain how lines 277-283 relate to the results or how the findings align with breeding efforts or the ongoing monitoring of populations or remove it.

284 Please replace the “in the in situ and translocation” with “for in situ conservation”

290-291 Please specify the conservation implications of the results, with a particular focus on the conservation of genetic resources of particular analyzed species.

295-300 It sounds like introduction. Please, consider moving to relevant section of introduction or remove it.

307, 311 Please use a capital letter in Table and Fig.

313 Please provide a reference supporting the diploid nature of the analyzed species.

316 Please correct the number of analyzed species (11 or 12?).

320 Please include information on the availability or a reference to the source of the sequenced genome data.

328 Please provide the website address for PCR Primer Statistics along with the date it was accessed.

351 Please clarify: were all DNA samples used for the SSR analysis, or was the entire DNA sample of each individual used for the analysis?

352 Please add the informaction about the concentration of particular PCR components

358 Please add the information about the visulaisation of PCR products (sequencing, capillary electrophoresis or another method?)

371, 381 It seems some spaces in the „STRUCTRUE”

381 The Authors note that the genetic structure of the population was analyzed using the Evanno method through the online STRUCTURE Harvester tool. However, due to the temporary unavailability of this service, it is advisable to consider alternative approaches. These may include performing the analysis using a Python or R script or utilizing an appropriate repository that supports STRUCTURE Harvester functionality. Additionally, it is recommended to update the reference accordingly.

384 please remove „comprehensive”

385 – 390 Please mphasize that the newly developed SSR primers effectively assess the genetic diversity of specific species and hold significant potential for informing detailed conservation strategies for the genus.

393 Please use the italic for „in situ” in manuscript

394 – 396 This content resembles an introduction; please move it to the appropriate section or consider removing it.

 

All the best

A reviewer.

Author Response

Dear Reviewer

 

Thank you for reviewing and commenting, we have revised and responded to the comments below. And marked yellow in the text.

 

Comments and Suggestions for Authors

The manuscript presents significant value, with its findings offering potential for an in-depth analysis of genetic diversity within the genus Calligonum, particularly in the context of conservation of the genetic diversity of endemic species and genetic resources of Calligonum species. The article is correct, balanced and formally sound. The work aligns well with the scope of the journal.

In general, I have identified some obstacles to publication and would appreciate it if the Authors made clearer some methodological choices and supported them with additional information.

 

There are certain ambiguities in the criteria used for species selection in the analysis among species of Calligonum genus. While the abstract highlights the importance of the genus' genetic resources in the context of their utility as economically valuable plants, the analysis and discussion extend this significance to the conservation of biodiversity within the natural diversity of endemic Chinese flora. Please ensure a more cohesive connection between the abstract, introduction, and conclusion.

AN: This is a very good suggestion you have made to improve the quality of the article. We have added content as requested to make it coherent. The additions are the positions marked with yellow.

 

Furthermore, in the Introduction section, identifying which of the analyzed species are endemic, which exhibit a broad distribution range, and which have practical applications would enhance the clarity of the paper. These distinctions currently rely on the context.

AN: We have added this useful information to the text. See it in Line 46-50.

 

The article's title suggests that some species were used to analyze genetic diversity of the genus Calligonum. However, the manuscript lacks information on the total number of species within the genus, raising concerns about the representativeness of the findings at the genus level.

AN: Your comments are very useful. Description has been added. See it in Line 42.

 

Essential information on the analyzed population sizes is absent, which is crucial for the methodological development of conservation guidelines. The methodological concerns relate also to the limited sample size used to estimate genetic diversity, especially in cases where species are represented by only four samples.

Given the aforementioned considerations, I suggest choosing one of the options:

1. focusing the analyses on species with a larger sample representation and emphasizing the significance of the findings in the context of conserving the genetic resources of economically and functionally important species within the genus Calligonum. In my opinion, species represented by a small number of individuals (e.g., 4) can serve as valuable examples to demonstrate the potential application of newly developed SSR primers;

or

1. Provide evidence, supported by references and bootstrapping analysis, to demonstrate that a limited number of individuals is sufficient for conducting genetic variation analyses.

AN: Thank you very much for your valuable input and the way it was resolved. Both of these options are very good angles for asking questions. The option A given by you for us is our original intention for that study. A certain amount of g-SSR from Calligonum junceum was selected to try to see if it was interspecific transferable. The results also indicate the potential applicability of the newly developed SSR primers in this genus. This represents the ability of our selected g-SSR primers to serve as an assessment of the genetic diversity of the 11 plant species selected for the study, which is one of our most important research objectives. In addition to this, another important reason for the low number of samples selected for some species is due to the fact that the identification of plants in the genus is a challenging task. There were a number of species with inherently narrow distributions and low numbers, and we needed to ensure the accuracy of the plants selected, and therefore discarded many samples that were problematic to identify.

 

The title of the paper is somehow informative, but perhaps not very accurate. I recommend focusing on the primary contribution of the publication, which is the development of SSR markers, eg. „The application of newly developed genome-SSR markers for genetic diversity analysis and conservation of Calligonum L. species”.

AN: The title has been revised as requested. See it in Line 2.

 

Please include the botanical authors' names for the specific taxa mentioned in the 4.1 Experimental Materials section. Some photographs of the plants, fruits, or seeds of the selected species would be beneficial in the presentation of the species of the study.

AN: Pictures have been added. See it in Line 102.

 

Keywords: do not repeat words that are already in the title.

AN: Keywords have been modified. See it in Line 33.

 

Finally, my specific remarks are the following with approximate line number in typeset:

19-20 I think more accurate would be “Their widespread use and breeding efforts highlight the need for conservation of high quality germplasm resources of the genus” instead of “Extensive utilization and associated breeding call for selection and preservation of good germplasm resources of the genus”.

AN: Sentences have been revised. See it in Line 19-18.

 

29-30 Please revise the sentence „At the same time, the failure of SSR identification reflects the problematic nature of its present taxonomy” to emphasize that the identified SSR markers lack taxonomic resolving power.

AN: The sentence has been removed.

 

30-32 I would suggest rephrase the sentence „The results of this study can provide reference primers for genus Calligonum, and provide sufficient genetic information for the screening and conservation of the Calligonum germplasm” into „The results of this study provide reference SSR primers for the genus Calligonum, supporting the improvement of its germplasm conservation efforts”.

AN: Sentences have been revised. See it in Line 30-31.

 

37-39 Please include information about the number of species or taxa that comprise the genus.

AN: Description has been added. See it in Line 42.

 

42 Please include references to studies that identify the genus as comprising four sections.

AN: Description has been added. See it in Line 42.

 

49-58 Please include information about the practical applications or utility of the species analyzed in the study.

AN: Description has been added. See it in Line 60-63.

 

60-61 I suggest to rephrase or exclude the sentence “Changes in genetic diversity that occur within species can have important implictions for the subsequent development of populations”.

AN: The sentence has been removed.

 

60-66 Please provide a brief discussion on the use of molecular markers for the conservation of the genus Calligonum, or highlight the scarcity of such studies

AN: Description has been added. See it in Line 82-85.

 

67-69 I think that information on the taxonomic discrepancies of the genus should be presented alongside details about the number of species it encompasses. It might be worth considering relocating this sentence to the  section 37-42

AN: The sentence has been moved. See it in Line 51.

 

69-70 Please, consider moving the sentence “Despite these challenges, Calligonum’s ecological value, especially as a wind and sand-fixing species, and its contribution to desertification control, cannot be denied” to the section between lines 49-58

AN: The sentence has been moved. See it in Line 68-70.

 

71-72 Pleaseexplain the “genetic dynamics” or delete the whole sentence.

AN: The sentence has been removed.

 

73-83 Please, rearrange the paragraph, starting with and emphasizing the importance of SSR markers in assessing genetic variability, followed by examples of their application in taxonomic diagnostics.

AN: The paragraph has been modified. See it in Line 82-85.

 

85-87 sound like results. Please remove it

AN: The sentence has been removed.

 

87-89 I suggest revising the objectives to emphasize: (1) the development of new SSR primers for the genus, (2) their application in genetic diversity analysis of species to support conservation efforts, and (3) their use in taxonomic analysis.

AN: The sentence has been modified. See it in Line 100-102.

 

106 please correct the title of Table 2, I suggest “Characteristic of SSR loci of C junceum”

AN: The sentence has been modified. See it in Line 120.

 

117-118 sounds like introduction, I suggest to remove it.

AN: The sentence has been removed.

 

119-120 Please provide a brief explanation of why PIC was estimated in this study.

AN: Description has been added. See it in Line 133-139.

 

128 Please explain what does “saguaro dates” mean

AN: The word has been modified. See it in Line 147.

 

143 Please rephrase the sentence while avoiding the term "gene abundance," as it refers to varying levels of specific genes, which were not analyzed in the scope of this study.

AN: The sentence has been modified. See it in Line 162.

 

152 Please consider moving it to the relevant discussion section or deleting it if redundant

AN: The sentence has been moved to the discussion section.

 

153-154 sounds like incomplete sentence

AN: The sentence has been modified. See it in Line 172.

 

179-183 Please revise this part of the results. In my opinion the PCO diagram shows 4–5 groups of samples, with one large group represented by samples from eight species, while a smaller group consists of samples from C. gobicum and C. roborowskii. Additionally, samples from C. pumilum are distinctly separated from the others.

AN: The sentence has been modified. See it in Line 201-202.

 

184 The authors reference the genetic diversity results in relation to phenotypic variation; however, no data on phenotypes is provided. I suggest relocating this sentence to the appropriate discussion section.

AN: The sentence has been moved to the discussion section.

 

206 Please consider moving the sentence „indicating that these three species enjoy a common ancestry” into relevant disscussion section.

AN: The sentence has been moved to the discussion section.

 

208-209 Please consider moving into relevant disscussion section.

AN: The sentence has been moved to the discussion section.

 

231 Please provide a reference supporting the use of genomic SSR markers as highly informative and reproducible.

AN: The sentence has been removed.

 

231-232 Please, consider moving the sentence into relevant conclusion section.

AN: The sentence has been moved to the conclusion section.

 

244 Consider the another title for paragraph reffereing to the field of failure (eg. Genetic structure and taxonomic identification failure….)

AN: The sentence has been modified. See it in Line 262.

 

253 Please clarify the meaning of "shako jujube"

AN: The word has been removed.

 

265-270 Please, sound like an introduction. Consider moving it into relevant introduction section.

AN: The sentence has been modified. See it in Line 292.

 

277-283 Explain how lines 277-283 relate to the results or how the findings align with breeding efforts or the ongoing monitoring of populations or remove it.

AN: The sentence has been removed.

 

284 Please replace the “in the in situ and translocation” with “for in situ conservation”

AN: The sentence has been modified. See it in Line 296.

 

290-291 Please specify the conservation implications of the results, with a particular focus on the conservation of genetic resources of particular analyzed species.

AN: The sentence has been modified. See it in Line 298-302.

 

295-300 It sounds like introduction. Please, consider moving to relevant section of introduction or remove it.

AN: The sentence has been removed.

 

307, 311 Please use a capital letter in Table and Fig.

AN: The sentence has been modified. See it in Line 335.

 

313 Please provide a reference supporting the diploid nature of the analyzed species.

AN: References have been added. See it in Line 332.

 

316 Please correct the number of analyzed species (11 or 12?).

AN: The sentence has been modified. See it in Line 136.

 

320 Please include information on the availability or a reference to the source of the sequenced genome data.

AN: The reference genome was assembled by us, the article containing the reference genome is in the process of being submitted, and we will be releasing the resource in the future. If you need this resource now, you can contact us for this data.

 

328 Please provide the website address for PCR Primer Statistics along with the date it was accessed.

AN: The sentence has been modified. See it in Line 348.

 

351 Please clarify: were all DNA samples used for the SSR analysis, or was the entire DNA sample of each individual used for the analysis?

AN: The sentence has been modified. See it in Line 371.

 

352 Please add the informaction about the concentration of particular PCR components

AN: This information has been added. See it in Line 374.

 

358 Please add the information about the visulaisation of PCR products (sequencing, capillary electrophoresis or another method?)

AN: This information has been added. See it in Line 379.

 

371, 381 It seems some spaces in the „STRUCTRUE”

AN: The sentence has been removed.

 

381 The Authors note that the genetic structure of the population was analyzed using the Evanno method through the online STRUCTURE Harvester tool. However, due to the temporary unavailability of this service, it is advisable to consider alternative approaches. These may include performing the analysis using a Python or R script or utilizing an appropriate repository that supports STRUCTURE Harvester functionality. Additionally, it is recommended to update the reference accordingly.

AN: The sentence has been modified. See it in Line 396-400.

 

384 please remove „comprehensive”

AN: The word has been removed.

 

385 – 390 Please mphasize that the newly developed SSR primers effectively assess the genetic diversity of specific species and hold significant potential for informing detailed conservation strategies for the genus.

AN: This information has been added. See it in Line 404-405.

 

393 Please use the italic for „in situ” in manuscript

AN: The word has been modified. See it in Line 411.

 

394 – 396 This content resembles an introduction; please move it to the appropriate section or consider removing it.

AN: The sentence has been removed.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, 17 new molecular markers (SSRs) were developed for the species Calligonum junceum and transferred to 11 other species of the genus. Although with small samples of some species, some genetic analyses are presented, which should be observed with caution. However, in general, the information seems relevant and brings an important molecular tool for the study of species of the genus Calligonum. Nonetheless, the paper needs general improvements in some parts, the mandatory inclusion of GenBank numbers for each SSR marker, and an English review.

Below, I am placing some suggestions or questions at specific points:

Line 20: The term “good” germplasm resources does not seem appropriate (genetic resources of the genus?).

Line 22: 17 microsatellite markers were identified in Calligonum junceum and transferred to 10 other species of the genus...

Line 29-30: I believe this part should not be in the abstract (“At the same time...” ). It is better to use this space to list the species where cross-transferability was effective.

Line 74: Genetic marking technique?

Line 78: Distinguishing species or varieties?

Line 83: Are there SSR markers for Calligonum? Is this the first group? Add a paragraph about some previous molecular studies with species of this genus. (I found this recent one: Genetic diversity of closely related Calligonum species collected from Saudi habitats by analyzing the matK and rpoC1 genes, and SCoT and IRAP markers | Plant Biotechnology Reports

Line 85-87: Seems like results. Remove from this part. Was one of the objectives not to develop primers for the species Calligonum junceum from a reference genome?

Line 95: Reference? From which source was the information taken?

Line 99 and 106: Tables 1 and 2: Numbers sometimes with dots, sometimes with commas.

Line 122-164: Were only 23 primers found, or is this only a part? Explain better here.

Line 124-127: Were 13 or 17 primers used for the final analyses? Clarify.

Line 128: Saguaro dates?

Line 134: Table 3: The complete data with primer descriptions, allele sizes, annealing temperatures, and genetic characterization information should include only those of the species Calligonum junceum since it is the species where the primers were originally developed. It is mandatory to include the deposit number of each sequence in GenBank. Was the same annealing temperature used in the amplification of other species? Since this information is relevant, include this information (annealing temperature) of the other species in the complementary table, if different, or clarify this information in the tables.

Line 142: 2.3-transferability analysis? Start this paragraph explaining that the SSR markers developed for the species Calligonum junceum amplified the DNA of the species xx, xxx, etc.

 

Line 167: In results and discussion, the inclusion of Table S3, which has interesting data, is missing. I suggest bringing this table to the main body of the paper (check the distances of species J (?)).

Lines 213-214: Start the discussion with the success of the development of SSR markers and their transferability to other Calligonum species. Is the frequency and density of SSR in Calligonum similar or different from some species of the genus or related species? (cite 2 or 3 examples).

Line 223: At the beginning of the discussion of the new markers and the genetic analyses of the species, it must be stated that they were the result of the amplification of a few individuals.

Line 228: Better not compare with another type of marker. Better if it is with SSR.

Line 229-230: Are references 29 and 30 of Calligonum sp?

Line 223-235: Review this paragraph. Simplify. Some sentences seem repetitive.

Line 234: Better explain transferability in materials and methods and results.

Line 241-242: Salinity mentioned twice.

Line 243: Are the allele sizes of all these primers similar between species? When alleles are similar in size and number, it is difficult to find interspecific differences since the similarities were exactly found. It would be interesting to analyze if among the SSRs found there are some species-specific alleles that could help interspecific separation.

Line 256: Remove unfortunate. This is a research result; it does not need to be fortunate or unfortunate. It means that the sequences of the analyzed primers are in a genome region with high similarity between the species.

Line 257: Generalizable? (Transferable, Cross-species transferability).

Lines 256-259: Many repetitions.

Line 261-263: Taxonomic data are not necessarily wrong. Sometimes it is necessary to identify species-specific markers.

Line 265-276: Review the English version. There are some redundancies or repetitions.

Line 265: Suggested: “The Calligonum caput-medusae, Calligonum arborescens, Calligonum rubicundum, and Calligonum ebinuricum are used as windbreaks and for sand fixation in the Turpan area of Xinjiang.”.

Line 270-272: Consider that few samples per species were used to make this statement.

Line 281: What is “small species”?

Line 292: Did you carefully analyze these private alleles?

Line 297: Good tree species?

 

 

Line 306: Explain which species was the target for developing the markers and which species were used for cross-transferability. In materials and methods, explain in a short paragraph the transferability of the primers. Were the individuals of the target species characterized first, or were all species analyzed simultaneously?

Line 330: In Figure 5, describe the letters of the locations. Is it possible to include any image of the plant Calligonum junceum (flowers and fruits?) and others related?

Line 368-370: Merge the sentences for this reference.

Line 377-382: Review the verb tense in this part.

Line 378-381: Correct STRUCTURE

Line 383: Here it is necessary to emphasize the importance of the newly developed and transferred primers (Are these the first SSR developed for the Calligonum genus?). Also, reanalyze if the phrase “The results emphasize the limitations of traditional morphological classification in Calligonum, underscoring the need for more integrative approaches” is entirely correct and within the context of the paper.

 

Suggestions for Future Research: ISSR markers are an interesting tool for interspecific separation.

 

Comments on the Quality of English Language

This paper requires an English revision to improve the comprehension of the results.

Author Response

Dear Reviewer

 

Thank you for reviewing and commenting, we have revised and responded to the comments below. And marked blue in the text.

 

In this study, 17 new molecular markers (SSRs) were developed for the species Calligonum junceum and transferred to 11 other species of the genus. Although with small samples of some species, some genetic analyses are presented, which should be observed with caution. However, in general, the information seems relevant and brings an important molecular tool for the study of species of the genus Calligonum. Nonetheless, the paper needs general improvements in some parts, the mandatory inclusion of GenBank numbers for each SSR marker, and an English review.

Below, I am placing some suggestions or questions at specific points:

 

Line 20: The term “good” germplasm resources does not seem appropriate (genetic resources of the genus?).

AN: The word has been modified. See it in Line 20.

 

Line 22: 17 microsatellite markers were identified in Calligonum junceum and transferred to 10 other species of the genus...

AN: The sentence has been modified. See it in Line 22.

 

Line 29-30: I believe this part should not be in the abstract (“At the same time...” ). It is better to use this space to list the species where cross-transferability was effective.

AN: The sentence has been modified. See it in Line 29.

 

Line 74: Genetic marking technique?

AN: The word has been modified. See it in Line 87.

 

Line 78: Distinguishing species or varieties?

AN: The sentence has been modified. See it in Line 92.

 

Line 83: Are there SSR markers for Calligonum? Is this the first group? Add a paragraph about some previous molecular studies with species of this genus. (I found this recent one: Genetic diversity of closely related Calligonum species collected from Saudi habitats by analyzing the matK and rpoC1 genes, and SCoT and IRAP markers | Plant Biotechnology Reports

AN: The sentence has been modified. We have cited that literature. See it in Line 95.

 

Line 85-87: Seems like results. Remove from this part. Was one of the objectives not to develop primers for the species Calligonum junceum from a reference genome?

AN: The sentence has been removed.

 

Line 95: Reference? From which source was the information taken?

AN: We have explained this in the Methods section.

 

Line 99 and 106: Tables 1 and 2: Numbers sometimes with dots, sometimes with commas.

AN: The table1 has been modified.

 

Line 122-164: Were only 23 primers found, or is this only a part? Explain better here.

AN: After considering it irrelevant to the content below, we removed it.

 

Line 124-127: Were 13 or 17 primers used for the final analyses? Clarify.

AN: Writing error, should be 17 pairs. The sentence has been modified. See it in Line 140.

 

Line 128: Saguaro dates?

AN: The sentence has been modified. See it in Line 141.

 

Line 134: Table 3: The complete data with primer descriptions, allele sizes, annealing temperatures, and genetic characterization information should include only those of the species Calligonum junceum since it is the species where the primers were originally developed. It is mandatory to include the deposit number of each sequence in GenBank. Was the same annealing temperature used in the amplification of other species? Since this information is relevant, include this information (annealing temperature) of the other species in the complementary table, if different, or clarify this information in the tables.

AN: The advice you have provided is very useful. As we had previously placed this information in the supplementary table, but after reading your suggestions for its use, we have placed it in the text. See it in table 8.

 

Line 142: 2.3-transferability analysis? Start this paragraph explaining that the SSR markers developed for the species Calligonum junceum amplified the DNA of the species xx, xxx, etc.

AN: The sentence has been modified.

 

Line 167: In results and discussion, the inclusion of Table S3, which has interesting data, is missing. I suggest bringing this table to the main body of the paper (check the distances of species J (?)).

AN: We put the information from the supplemental table in table 6. See it in Line 184.

 

Lines 213-214: Start the discussion with the success of the development of SSR markers and their transferability to other Calligonum species. Is the frequency and density of SSR in Calligonum similar or different from some species of the genus or related species? (cite 2 or 3 examples).

AN: This information has been added. See it in Line 226-230.

 

Line 223: At the beginning of the discussion of the new markers and the genetic analyses of the species, it must be stated that they were the result of the amplification of a few individuals.

AN: This information has been added. See it in Line 241.

 

Line 228: Better not compare with another type of marker. Better if it is with SSR.

AN: This information has been added. See it in Line 243.

 

Line 229-230: Are references 29 and 30 of Calligonum sp?

AN: YES.

 

Line 223-235: Review this paragraph. Simplify. Some sentences seem repetitive.

AN: The sentence has been modified. See it in Line 244.

 

Line 234: Better explain transferability in materials and methods and results.

AN: The sentence has been modified. See it in Line 245.

 

Line 241-242: Salinity mentioned twice.

AN: Extra words have been removed.

 

Line 243: Are the allele sizes of all these primers similar between species? When alleles are similar in size and number, it is difficult to find interspecific differences since the similarities were exactly found. It would be interesting to analyze if among the SSRs found there are some species-specific alleles that could help interspecific separation.

AN: Interesting suggestion you made. We used capillary electrophoresis to detect the size of the allele. The results showed that the size of the alleles was very similar between the species. It is difficult to screen enough specific alleles for interspecific differentiation.

 

Line 256: Remove unfortunate. This is a research result; it does not need to be fortunate or unfortunate. It means that the sequences of the analyzed primers are in a genome region with high similarity between the species.

AN: The sentence has been removed.

 

Line 257: Generalizable? (Transferable, Cross-species transferability).

AN: The sentence has been modified. See it in Line 245.

 

Lines 256-259: Many repetitions.

AN: Duplicates have been removed.

 

Line 261-263: Taxonomic data are not necessarily wrong. Sometimes it is necessary to identify species-specific markers.

AN: The sentence has been modified. See it in Line 267.

 

Line 265-276: Review the English version. There are some redundancies or repetitions.

AN: Duplicates have been removed.

 

Line 265: Suggested: “The Calligonum caput-medusae, Calligonum arborescens, Calligonum rubicundum, and Calligonum ebinuricum are used as windbreaks and for sand fixation in the Turpan area of Xinjiang.”.

AN: The sentence has been modified. See it in Line 60.

 

Line 270-272: Consider that few samples per species were used to make this statement.

AN: This information has been added. See it in Line 291.

 

Line 281: What is “small species”?

AN: Duplicates have been removed. See it in Line 291.

 

Line 292: Did you carefully analyze these private alleles?

AN: We have only counted them and expect to use them in the future when we do a lot of analysis.

 

Line 297: Good tree species?

AN: The sentence has been modified. See it in Line 309.

 

Line 306: Explain which species was the target for developing the markers and which species were used for cross-transferability. In materials and methods, explain in a short paragraph the transferability of the primers. Were the individuals of the target species characterized first, or were all species analyzed simultaneously?

AN: This information has been added. Analyze all species simultaneously. See it in Line 321.

 

Line 330: In Figure 5, describe the letters of the locations. Is it possible to include any image of the plant Calligonum junceum (flowers and fruits?) and others related?

AN: Pictures have been added. See it in Line 101.

 

Line 368-370: Merge the sentences for this reference.

AN: The sentence has been modified. See it in Line 382.

 

Line 377-382: Review the verb tense in this part.

AN: The sentence has been modified. See it in Line 388.

 

Line 378-381: Correct STRUCTURE

AN: The word has been removed.

 

Line 383: Here it is necessary to emphasize the importance of the newly developed and transferred primers (Are these the first SSR developed for the Calligonum genus?). Also, reanalyze if the phrase “The results emphasize the limitations of traditional morphological classification in Calligonum, underscoring the need for more integrative approaches” is entirely correct and within the context of the paper.

AN: The sentence has been modified. See it in Line 398.

 

Suggestions for Future Research: ISSR markers are an interesting tool for interspecific separation.

AN: Thank you for your suggestion, we will try to use this method for interspecific identification in the future.

 

This paper requires an English revision to improve the comprehension of the results.

AN: Thanks to your language suggestion, we have revised the article in English.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors:

Your manuscript presents important data for the genetic studies of the target species and the other 10 species analyzed. However, the current version still has writing issues that could easily be addressed with professional assistance, if possible.

In the first version, I requested the inclusion of the sequences of the 17 SSR primers in GENEBANK (https://www.ncbi.nlm.nih.gov/genbank/). Is there any problem with this inclusion? The importance of making the sequences available is that often the original primers need to be redesigned in other locations where these species are important. Without the sequences, this type of approach would not be possible, and all the effort made by your group would be lost to the scientific community unless there is a reason to keep this information confidential. You can also deposit the sequences in other sources such as TreeBase (http://www.treebase.org), or Dryad (http://datadryad.org/).

Below I am listing some questions and/or suggestions for this version:

• Line 20: Remove “of the genus” from the end of the sentence. 
• Lines 22-23: Remove “In Calligonum junceum.”
• Line 23: After “genus.” add:  “When analyzed together, the 10 species showed significant ….”.
• Line 26: After “at 0.373,” add the diversity data for the target species: “In C. junceum …..”
• Line 28-30: Remove this sentence: "The genomic SSR markers selected….. junceum." . It is repeated from lines 22-23.
• Line 43: are, are.
• Lines 56-60: Merge these sentences; they seem repetitive.
• Line 95: Shakojujube?
• Line 105: This figure is better placed in the materials and methods section.
• Lines 107-114: Round the numbers to two decimal places, both in the text and in Table 1, to avoid confusion.
• Lines 114-119 and 121-130 contain similar information. Delete one of them.
• Line 120: Suggestion: "Characteristic of Simple Sequence Repeats (SSR) of C. junceum."
• Line 121: Suggestion: “The main SSR repeat class types…”
• Lines 138-139: This sentence seems repetitive from the earlier sentences.
• Lines 142-143: Move this paragraph for below paragraphs 144-150 since it analyzes Table 3.
• Line 141-143: Suggestion: "Four of these 17 pairs of SSR primers (Sgz005, Sgz006, Sgz012, Sgz017) were highly polymorphic, and the other 13 pairs were moderately polymorphic. These tools provided a sufficient basis for further studies on the genetic diversity of Calligonum L. germplasm resources. Therefore, we used all 17 pairs of SSR primers for subsequent analysis."

• Lines 156-158: Redundant.
• Line 160: Number of the alleles (Na).
• Lines 171-173: Review this sentence: "C. mongolicum had the largest number of private alleles (35 / 17 loci), C. arborescens (0 / 17 loci), and C. colubrinum (0 / 17 loci) had the least number of private alleles. (?)"
• Lines 183-200: PCA or PCoA. Review and merge sentences from lines 184-188 and 197-200.
• Lines 206-224: Very complex paragraphs. Change “predicted” to “analyzed.”
• Lines 230-242: Delete the first two sentences. Suggestion: “In this study, 380,328 SSR loci with a density of 280.13 SSRs/Mb were obtained from a Calligonum junceum reference genome. The number and density of SSRs identified are higher than that of C. mongolicum cp genome data [30]. It is also higher than the transcriptional data [31] and genomic data [32] of Fagopyrum tibeticum. From the sequences of C. junceum, 17 SSR molecular markers were identified, characterized, and cross-transferred in C. colubrinum, C. leucocladum, C. ebinuricum, C. pumilum, C. trifarium, C. arborescens, C. jeminaicum, C. mongolicum, C. gobicum, and C. roborowskii species. Genetic diversity characterization of this germplasm was achieved with this SSR. Higher genetic diversity shows the evolutionary potential of the species and its ability to adapt to the environment [33]. Genetic diversity is influenced by many factors, including geographic range, seed dispersal, reproductive systems, life history, and evolutionary history of organisms [34, 35], being useful for the future management of germplasm resources and the development of plant breeding measures.”
• Lines 243-250: This part is very confusing and incomplete. The reference to 13 SSRs in line 245?. This paragraph is also placed before the discussion of this work's results. Discuss only with data from other SSRs for related species (if available) and place it after line 258. ISSRs are dominant markers, and it is not advisable to draw analogies with co-dominant markers like those developed in this work. However, you can cited the works carried out with these markers without making comparisons. Also, in line 244, are there 23 or 17 SSRs?
• Line 277: PCA or PCoA?
• Line 306: suitable ... suitable.
• Line 307: Better to use "populations" instead of "plants.".
• Line 308: Better to use "ecological recuperation" instead of "ecological construction.".
• Lines 313-314: "desirable tree species" appears twice.
• Lines 325-327: Suggestion: “We selected Calligonum junceum as the target species for developing the SSR molecular markers and the other 10 species to test the cross-transferability of the identified markers.”
• Line 347: In figure 6, explain the meaning of letters A–J in the description.
• Line 365: Submit the sequences to GENEBANK (https://www.ncbi.nlm.nih.gov/genbank/) and include the number in this table. In GENBANK, you can set a time for the sequences to be reserved until eventual publication. Are there any restrictions on submitting these sequences in China?
• Lines 386-387: "Analysis" appears three times: Suggestion: “Principal coordinate, genetic similarity, and molecular variance (AMOVA) analysis were performed using GenAlEx 6.5 software.”
• Lines 392-394: Confusing.
• Lines 400-402: You did not conduct a study with several markers. It seems this comparison should not be included here.

Comments on the Quality of English Language

The current version still has writing issues that could easily be addressed with professional assistance, if possible.

Author Response

Dear reviewer

 

Thank you very much for your effort in reviewing the manuscript. We have marked them in yellow in the text.

 

In the first version, I requested the inclusion of the sequences of the 17 SSR primers in GENEBANK (https://www.ncbi.nlm.nih.gov/genbank/). Is there any problem with this inclusion? The importance of making the sequences available is that often the original primers need to be redesigned in other locations where these species are important. Without the sequences, this type of approach would not be possible, and all the effort made by your group would be lost to the scientific community unless there is a reason to keep this information confidential. You can also deposit the sequences in other sources such as TreeBase (http://www.treebase.org), or Dryad (http://datadryad.org/).

Below I am listing some questions and/or suggestions for this version:

AN: Thank you for making the important comment to connect that link to the data in order to make sure that other people can use this article of ours smoothly. Spent some time waiting for the data to be reviewed and approved. The query item number is PRJCA036654, and we have consolidated all the primers into one data query number (OMIX009213) in order to quickly obtain the data query number. If you need one data query number per primer information, please contact us and we will re-upload the data. We have also added a note in the Data Availability Statement section. See it in line 412-414.

 

Line 20: Remove “of the genus” from the end of the sentence. 

AN: The word has been removed.

 

Lines 22-23: Remove “In Calligonum junceum.”

AN: The word has been removed.

 

Line 23: After “genus.” add:  “When analyzed together, the 10 species showed significant ….”.

AN: The sentence has been added. See it in line 23.

 

Line 26: After “at 0.373,” add the diversity data for the target species: “In C. junceum …..”

AN: The sentence has been added. See it in line 26.

 

Line 28-30: Remove this sentence: "The genomic SSR markers selected….. junceum." . It is repeated from lines 22-23.

The sentence has been deleted.

 

Line 43: are, are.

AN: Extra words have been removed.

 

Lines 56-60: Merge these sentences; they seem repetitive.

AN: The sentence has been modified. See it in line 55-60.

 

Line 95: Shakojujube?

AN: The redundancy has been removed.

 

Line 105: This figure is better placed in the materials and methods section.

AN: The image has been moved to 4.1. Experimental materials.

 

Lines 107-114: Round the numbers to two decimal places, both in the text and in Table 1, to avoid confusion.

AN: All values are modified to two decimal places. See it in line 99-104.

 

Lines 114-119 and 121-130 contain similar information. Delete one of them.

AN: Duplicates of the paragraph have been deleted. See it in line 105-115.

 

Line 120: Suggestion: "Characteristic of Simple Sequence Repeats (SSR) of C. junceum."

AN: The sentence has been modified. See it in line 109.

 

Line 121: Suggestion: “The main SSR repeat class types…”

AN: The sentence has been modified. See it in line 110.

 

Lines 138-139: This sentence seems repetitive from the earlier sentences.

The sentence has been deleted.

 

Lines 142-143: Move this paragraph for below paragraphs 144-150 since it analyzes Table 3.

AN: The paragraph has been moved. See it in line 136-1342.

 

Line 141-143: Suggestion: "Four of these 17 pairs of SSR primers (Sgz005, Sgz006, Sgz012, Sgz017) were highly polymorphic, and the other 13 pairs were moderately polymorphic. These tools provided a sufficient basis for further studies on the genetic diversity of Calligonum L. germplasm resources. Therefore, we used all 17 pairs of SSR primers for subsequent analysis."

An: The paragraph has been moved. See it in line 128-130.

 

Lines 156-158: Redundant.

An: The sentence has been modified. See it in line 144-145.

 

Line 160: Number of the alleles (Na).

An: The word has been added. See it in line 158.

 

Lines 171-173: Review this sentence: "C. mongolicum had the largest number of private alleles (35 / 17 loci), C. arborescens (0 / 17 loci), and C. colubrinum (0 / 17 loci) had the least number of private alleles. (?)"

AN: The sentence has been modified. See it in line 153-156.

 

Lines 183-200: PCA or PCoA. Review and merge sentences from lines 184-188 and 197-200.

AN: Modify all to PCoA. and consolidate all sentences. See it in line 167-174.

 

Lines 206-224: Very complex paragraphs. Change “predicted” to “analyzed.”

AN: Word has been changed. See it in line 189.

 

Lines 230-242: Delete the first two sentences. Suggestion: “In this study, 380,328 SSR loci with a density of 280.13 SSRs/Mb were obtained from a Calligonum junceum reference genome. The number and density of SSRs identified are higher than that of C. mongolicum cp genome data [30]. It is also higher than the transcriptional data [31] and genomic data [32] of Fagopyrum tibeticum. From the sequences of C. junceum, 17 SSR molecular markers were identified, characterized, and cross-transferred in C. colubrinum, C. leucocladum, C. ebinuricum, C. pumilum, C. trifarium, C. arborescens, C. jeminaicum, C. mongolicum, C. gobicum, and C. roborowskii species. Genetic diversity characterization of this germplasm was achieved with this SSR. Higher genetic diversity shows the evolutionary potential of the species and its ability to adapt to the environment [33]. Genetic diversity is influenced by many factors, including geographic range, seed dispersal, reproductive systems, life history, and evolutionary history of organisms [34, 35], being useful for the future management of germplasm resources and the development of plant breeding measures.”

AN: Thank you for speaking about the paragraph to sort out the logic and embellish the paragraph. We have deleted the first two sentences of this paragraph, as per your comments, and have revised the paragraph as per your suggested changes. This includes merging and adding sentence content. See it in line 215-227.

 

Lines 243-250: This part is very confusing and incomplete. The reference to 13 SSRs in line 245?. This paragraph is also placed before the discussion of this work's results. Discuss only with data from other SSRs for related species (if available) and place it after line 258. ISSRs are dominant markers, and it is not advisable to draw analogies with co-dominant markers like those developed in this work. However, you can cited the works carried out with these markers without making comparisons. Also, in line 244, are there 23 or 17 SSRs?

AN: 1: Thank you for your careful reading of this paragraph. There are 13 primer pairs with PIC values that are greater than or equal to 0.5. In view of the lack of clarity, the table has been deleted. The part about comparing objects. I've tweaked the ISSR case forward, as a quote only. The question about the number of primers was a mistake on our part. Regarding the number of primers, it has been modified to 17. See it in line 230-235.

 

Line 277: PCA or PCoA?

AN: PCoA. See it in line 262.

 

Line 306: suitable ... suitable.

AN: The extra words have been removed. See it in line 291.

 

Line 307: Better to use "populations" instead of "plants.".

AN: The word has been modified. See it in line 292.

 

Line 308: Better to use "ecological recuperation" instead of "ecological construction.".

AN: The word has been modified. See it in line 293.

 

Lines 313-314: "desirable tree species" appears twice.

AN: The extra words have been removed. See it in line 298-300.

 

Lines 325-327: Suggestion: “We selected Calligonum junceum as the target species for developing the SSR molecular markers and the other 10 species to test the cross-transferability of the identified markers.”

AN: The sentence has been modified. See it in line 311-313.

 

Line 347: In figure 6, explain the meaning of letters A–J in the description.

AN: This information has been added. See it in line 335-337.

 

Line 365: Submit the sequences to GENEBANK (https://www.ncbi.nlm.nih.gov/genbank/) and include the number in this table. In GENBANK, you can set a time for the sequences to be reserved until eventual publication. Are there any restrictions on submitting these sequences in China?

AN: We have uploaded the data to NGDC (https://ngdc.cncb.ac.cn/). Spent some time waiting for the data to be reviewed and approved. The project number is PRJCA036654 and the data query number is OMIX009213.

 

Lines 386-387: "Analysis" appears three times: Suggestion: “Principal coordinate, genetic similarity, and molecular variance (AMOVA) analysis were performed using GenAlEx 6.5 software.”

AN: The sentence has been modified. See it in line 376-377.

 

Lines 392-394: Confusing.

AN: The redundant part of the sentence was deleted. See it in line 382-383.

 

Lines 400-402: You did not conduct a study with several markers. It seems this comparison should not be included here.

AN: The sentence has been deleted.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

A few final details.

Abstract line 23: Are there 10 or 11 species analyzed (in my view there are 11 in total).

Line 156-159: private alleles. (Private genotypes?).

Figure 2: Explain the meaning of the letters A-K (refer to Table 7?).

Tables 3 and 4: Consider using only two decimal places in all values.

 

Author Response

Dear reviewer

 

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits. We have marked them in green in the text.

 

Abstract line 23: Are there 10 or 11 species analyzed (in my view there are 11 in total).

AN: SSR loci were screened from one species and tested for transfer effects in 10 other species. The total number of all species was 11. We have revised it to 11 species. See it in line 23.

In order to minimize possible ambiguities in the text, we revised it to 11 species. And check the number of other occurrences of species statistics in the text, all changed to 11.

 

Line 156-159: private alleles. (Private genotypes?).

AN: It should be a private allele. The sentence has been revised. See it in line 155-157.

 

Figure 2: Explain the meaning of the letters A-K (refer to Table 7?).

AN: We have labeled the notes A-K at the bottom of Table 7. See it in line 187-189.

 

Tables 3 and 4: Consider using only two decimal places in all values.

AN: We have left the table of values in all tables to two decimal places. See it in table 5 and table 6. In addition to this we also check the data in the text, whether it is preserved to two decimal places.

Author Response File: Author Response.pdf