Genotyping of Autochthonous Rose Populations in the Netherlands for Effective Ex Situ Gene Conservation Management
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors investigate the genetic diversity of wild rose populations within and among populations in the Netherlands using a set of 10 microsatellite markers to support effective ex situ gene conservation management. The study was well-designed and the article was well-written. It can be considered to accept for publication after addressing the following minor concerns.
1. Tables should be revised according to the requirement of the journal, esp. Tables 1 and 5. Some characters and numbers in several Tables are difficult to recognize. Some abbreviations in the header of Table 1 need to be explained.
2. Some paragraphs in the introduction section contain very few sentences, which can be merged into other relevant paragraphs.
3. There are some sentences in the results section that look like discussions. Please consider if it is possible to transfer these sentences to the discussion section.
Author Response
The authors investigate the genetic diversity of wild rose populations within and among populations in the Netherlands using a set of 10 microsatellite markers to support effective ex situ gene conservation management. The study was well-designed and the article was well-written. It can be considered to accept for publication after addressing the following minor concerns.
Comments 1. Tables should be revised according to the requirement of the journal, esp. Tables 1 and 5. Some characters and numbers in several Tables are difficult to recognize. Some abbreviations in the header of Table 1 need to be explained.
Response 1: A Table note was added to explain the abbreviations in the header of Table 1. The layout of the tables has been revised according to the requirement of the journal
Comments 2. Some paragraphs in the introduction section contain very few sentences, which can be merged into other relevant paragraphs.
Response 2: The following paragraphs have been merged now: the first paragraph on roses in the Netherlands has been merged with the second paragraph on rarity of wild roses in the Netherlands, and the paragraphs on literature on genetic diversity and special features of dogroses have been merged to one paragraph.
Comments 3. There are some sentences in the results section that look like discussions. Please consider if it is possible to transfer these sentences to the discussion section.
Response 3: Although the goals of the studies are different, there is substantial overlap between our study and the study of Reichel, in terms of samples and microsatellite markers (we used 511 samples, of which 409 were included in Reichel et al and we used 10 SSRs, while in their study only 7 SSRs were included), and therefore we considered that it is relevant to start the Results section with referring to their findings, instead of only mentioning their study in the Discussion. We have now transferred the sentence ‘Sharing between species within subsections was explained by misidentification of species, while the sharing of MLGs between species of different subsections may be the result of too low resolution of the marker system’ in Results section 3.1 to the Discussion.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe author has conducted a study on "Genotyping of Autochtonous Rose Populations in the Netherlands for Effective Ex Situ Gene Conservation Management." The paper holds value but also has several issues that need addressing:
1.The abstract lacks the significance of the content.
2. line 38, the Latin name when mentioned the second time should be abbreviated.
3.line 77, plant Latin names need to be italicized, and there are numerous errors throughout the text that need correction.
4. Tables should utilize a three-line format.
5. In MDPI format, results should precede methods.
6. Table 1, all abbreviations should be explained in the table notes. Table 4 lacks annotations, and there are no explanations in the text, making it confusing for the reader.
7.Descriptions like Figure 1.a and Figure 2.b are inappropriate; please format them as a figure panel.
8. The annotations for Table 6 need to be provided.
9. The use of microsatellite markers and PCoA analysis is mentioned but lacks a detailed explanation of the reasons for these choices and how they specifically help achieve the research objectives. Provide more background information for these choices.
10. According to the text, Reichel et al. (2023) have conducted related research. Highlight the innovative aspects and differences between your study and theirs.
11. The discussion is too superficial; MLGs have been applied in many species, and the current discussion is almost limited to roses only, which does not sufficiently highlight the significance of this work.
Author Response
Comments 1. The abstract lacks the significance of the content.
Response 1: We rewrote the abstract and added a concluding remark: ‘The study highlights the value of genetic characterization in guiding sampling strategies for dogroses, revealing clonality and redundancy and optimizing core collections to maximize genetic capture in gene banks’.
Comments 2. line 38, the Latin name when mentioned the second time should be abbreviated.
Response 2: Beyond the first time, all Latin names of Rosa species have been abbreviated to R. now throughout the text.
Comments 3. line 77, plant Latin names need to be italicized, and there are numerous errors throughout the text that need correction.
Response3: The text has been checked for Latin names and all Latin names are now italicized.
Comments 4. Tables should utilize a three-line format.
Response 4: All tables have now been adjusted to the format of the Journal
Comments 5. In MDPI format, results should precede methods.
Response 5: According to the MDPI article template found on the website of Horticulturae M&M precedes Results. For this reason we did not change this.
Comments 6. Table 1, all abbreviations should be explained in the table notes. Table 4 lacks annotations, and there are no explanations in the text, making it confusing for the reader.
Response 6: A Table note was added to explain the abbreviations in the header of Table 1. The header of Table 4 has been adjusted and in the Result section 3.1 the sentences that explain Table 4 for the number of MLGs found in the sample set, have now a reference to Table 4 within brackets.
Comments 7. Descriptions like Figure 1.a and Figure 2.b are inappropriate; please format them as a figure panel.
Response 7: Figure 1 and 2 have now been formatted as a Figure panel.
Comments 8. The annotations for Table 6 need to be provided.
Response 8: The caption of Table 6 has been changed to: Poppr analysis of the different core sets with decreasing sample sizes and the whole sample set (all) for the diversity parameters number of alleles (#alleles) and genetic distance (‘min dist’). The core sets were constructed with Core Hunter using the Average Accession-to-Nearest-Entry distance (A-NE) optimization method.
Comments 9. The use of microsatellite markers and PCoA analysis is mentioned but lacks a detailed explanation of the reasons for these choices and how they specifically help achieve the research objectives. Provide more background information for these choices.
Response 9: As written in the Introduction, microsatellite markers, also known as Simple Sequence Repeat markers, are useful for informing conservation management, and they are particularly effective for identifying clonality. In the introduction we mention Esselink et al. [4] who developed the set of highly polymorphic microsatellite markers in rose. The marker set developed by Esselink et al [4] was chosen for this study as they demonstrated that fingerprinting with a subset of two or three microsatellite loci was already sufficient to distinguish the varieties tested, which make them a useful tool for clone detection and collection management. We added this explanation to the introduction.
The analysis of the PCoA based on the SSR data was added as it illustrates the genetic structure among the rose material studied, e.g. it clearly shows the three distinct clusters separating R. arvensis, R. spinossisima and the species in section Caninae, but also that there is no clear substructering in the subsection Caninae. In that sense, the PCoA analysis was also consistent with the findings of Reichel et al (2023).
Comments 10. According to the text, Reichel et al. (2023) have conducted related research. Highlight the innovative aspects and differences between your study and theirs.
Response 10: The study by Reichel et al. (2023) assessed, amongst others, the degree of clonality within natural populations of dogroses and other native rose species. However, genetic characterization using microsatellites is also a robust tool for the effective management of genebanks, and that is what we focussed on, amongst others with regard to duplicate clones. It has also proven effective for field collections of tree species, such as poplar (Storme et al. 2004), whose populations may partly consist of root suckers (clones) and various fruit trees (Van Treuren et al., 2010; Barreneche et al., 2021), where mistakes in names and name transcriptions occur. We added these two references to the text in the discussion section:
Barreneche T, Cárcamo de la Concepción M, Blouin-Delmas M, Ordidge M, Nybom H, Lacis G, Feldmane D, Sedlak J, Meland M, Kaldmäe H, et al. (2021) SSR-Based Analysis of Genetic Diversity and Structure of Sweet Cherry (Prunus avium L.) from 19 Countries in Europe. Plants 10(10):1983. https://doi.org/10.3390/plants10101983
van Treuren, R., Kemp, H., Ernsting, G. Jongejans, B, Visser, L (2010) Microsatellite genotyping of apple (Malus × domestica Borkh.) genetic resources in the Netherlands: application in collection management and variety identification. Genet Resour Crop Evol 57, 853–865. https://doi.org/10.1007/s10722-009-9525-0
Comments 11. The discussion is too superficial; MLGs have been applied in many species, and the current discussion is almost limited to roses only, which does not sufficiently highlight the significance of this work.
Response 11: The referee is correct in the sense that we did not explicitly indicate that the two-step approach for optimising the genetic composition of an ex situ collection is useful for all species which have sexual and vegetative reproduction in natural populations. We have now added a paragraph to the Conclusion section that explicitly indicates this use. We now also include this in the last sentence of the Abstract.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe topic is interesting and pertinent given the need to maximize the effectiveness of managing the conservation of genetic resources.
The article is well organized, complete and interesting, but there are some items to improve.
In this study the authors used 10 microsatellites loci. The article did not explain (namely in section 2.2) why there was a need to increase from 7 to 10 microsatellite loci.
When discussing the results, the authors also do not comment on whether increasing the number of microsatellites loci improved the quality of the results for Netherlands roses. It is important for the readers to understand whether the effort to include additional microsatellites was relevant, as it could be important in decision-making in other ongoing work.
Lines 77-78; 139; 190, 278, 284, 288: text formatting: the scientific names of the species must be written in italics.
Table 1: The width of the ‘taxa’ column must be greater so that the species name appears in full
In the table footer, I suggest that you include a list of acronyms used in the columns, in order to allow the reader to consult the table without needing to read the text. The size of the information in the table must be larger to make it easier to read. the size must also be increased in tables 2, 3, 4 and 6.
Lin 200: change the sentence to ‘… present in the gene bank.’
Line 209-10: Too low resolution of the marker system could also have happened within subsections?
Line 233: change the sentence to ‘… micrantha and rubiginae-spec)’.
Figure 1.a: suggestion: improve the colours used to identify taxa, as there are several colours that cannot be distinguished from each other (for example, arvensis, canina, Rubiginose-spec.). To improve the reading of the information, we suggest that you write the name of the groups within the image.
Figure 1. b: idem
Figures 2.a and figure 2 b: suggestion: merge the 2 figures into one, the different colours of the MLG are enough to allow interpretation. May also include the location of MLG 72 and MLG 209.
It is also suggested to include the blue of the sea water, in order to facilitate the identification of the islands. Include map scale.
Figure 2 c: Include map scale. Include the identification of the MLG, as they are only 3 different colours, they can appear in a caption within the image.
Line 298: change the sentence to ‘… was 61% and …’.
Line 314: in the methodology section 2.4, the authors refer to an A-NE method. AN method is the same? Please standardize.
Line 325-327: review the text, the sentence seems incomplete. The information is not understood.
Author Response
Comments 1. In this study the authors used 10 microsatellites loci. The article did not explain (namely in section 2.2) why there was a need to increase from 7 to 10 microsatellite loci.
Response 1: As explained in the Introduction, the marker set developed by Esselink et al [4] is a useful set for variety detection. Of the 24 markers two to three SSR loci were already sufficient to distinguish the varieties they tested (Tea hybrids and rootstocks), as these were the result of crosses between diverse parents, while mutant varieties displayed the same MLG (Smulders et al. [5]). The data of our study was created in the frame of the European Generose project, in which 10 SSRs were used, to obtain sufficient discriminative power, also for dogroses with their matroclinal inheritance system (in which sexual offspring is mostly like the mother). The analysis of Reichel et al. [8] combined three data sets (GE, HR and KW) and in order to harmonize these data sets they used only the 7 SSR loci that were common to these three sets, as indicated in M&M.
Comments 2. When discussing the results, the authors also do not comment on whether increasing the number of microsatellites loci improved the quality of the results for Netherlands roses. It is important for the readers to understand whether the effort to include additional microsatellites was relevant, as it could be important in decision-making in other ongoing work.
Response 2: The findings of our study, which utilized the original dataset with 10 polymorphic microsatellites, align with those of Reichel et al., who reduced the number of loci to 7 to harmonize across three datasets. Our overall results indicate that genotyping with either 7 or 10 loci does not result in deviations in the genetic structure or the multi-locus genotypes (MLGs) identified. However, the primary objective of our study was not to compare the impact of the number of loci on genetic diversity results, so we prefer avoiding speculation on the effect of the number of loci on the strength of the conclusions that could be drawn regarding this aspect.
Comments 3. Lines 77-78; 139; 190, 278, 284, 288: text formatting: the scientific names of the species must be written in italics.
Response 3: All scientific names of the species are now in italics.
Comments 4. Table 1: The width of the ‘taxa’ column must be greater so that the species name appears in full
Response 4: The column widths in the tables have been adjusted so that the text appears in full.
Comments 5. In the table footer, I suggest that you include a list of acronyms used in the columns, in order to allow the reader to consult the table without needing to read the text. The size of the information in the table must be larger to make it easier to read. the size must also be increased in tables 2, 3, 4 and 6.
Response 5: As also suggested by other reviewer we added a Table note to explain the abbreviations in the header of table 1 as follow: N: total number of samples per species, MLG: distinct multilocus genotypes, eMLG: expected number of MLG at the lowest common sample size, SE: The standard error for the rarefaction analysis, H: Shannon’s diversity index, G: genotypic richness, E: Evenness, R: clonal richness. We also adjusted the table formatting of table 2, 3, 4 and 6.
Comments 6. Lin 200: change the sentence to ‘… present in the gene bank.’
Response 6: We have changed ‘present the gene bank’ into ‘present in the gene bank’.
Comments 7. Line 209-10: Too low resolution of the marker system could also have happened within subsections?
Response 7: In these lines (now in the Discussion) we cite Reichel et al. [8]. We don’t speculate further as we have not tested using many more markers (or other systems such as an SNP array).
Comments 8. Line 233: change the sentence to ‘… micrantha and rubiginae-spec)’.
Response 8: We have changed the sentence ‘shared between four species (R. gremlii, rubiginosa, micrantha or rubigineae_spec.) into ‘shared between four species (R. gremlii, R. rubiginosa, R. micrantha and rubigineae_spec.)’.
Comments 9. Figure 1.a: suggestion: improve the colours used to identify taxa, as there are several colours that cannot be distinguished from each other (for example, arvensis, canina, Rubiginose-spec.). To improve the reading of the information, we suggest that you write the name of the groups within the image.
Response 9: We have adapted Figure 1a and 1b slightly, and hope that this improved the readability of the Figure. However, as there are many species (17 and 2 unidentified) it remains difficult to assign a distinctive colour to each species.
Comments 10. Figure 1. b: idem
Response 10: See above
Comments 11. Figures 2.a and figure 2 b: suggestion: merge the 2 figures into one, the different colours of the MLG are enough to allow interpretation. May also include the location of MLG 72 and MLG 209. It is also suggested to include the blue of the sea water, in order to facilitate the identification of the islands. Include map scale.
Response 11: We have combined Figure 2a and 2b according to the suggestion of the reviewer, including MLG 72 with MLG209, the blue of the sea, and a map scale, intio one Figure 2a.
Comments 12. Figure 2 c: Include map scale. Include the identification of the MLG, as they are only 3 different colours, they can appear in a caption within the image.
Response 12: We have adapted Figure 2c, now called Figure 2b, and included the identification of the MLGs in the legend and included a map scale.
Comments 13. Line 298: change the sentence to ‘… was 61% and …’.
Response 13: We have changed the sentence ‘was 61% en 28%’ to ‘was 61% and 28%’.
Comments 14. Line 314: in the methodology section 2.4, the authors refer to an A-NE method. AN method is the same? Please standardize.
Response 14: We have standardized the abbreviation A-NE for Average Accession-to-Nearest-Entry distance by replacing AN by A-NE throughout the document.
Comments 15. Line 325-327: review the text, the sentence seems incomplete. The information is not understood.
Response 15: We have changed the sentence ‘In the can group (due to redundancy the whole set only contained 214 genotypes), but reducing from 180 down to 40 had only a slight effect on the genetic distance values’ to ‘In the can group, the whole set only contained 214 genotypes due to redundancy, reducing the sample size from 180 down to 40 had only a slight effect on the genetic distance values.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe submitted manuscript Genotyping of autochthonous rose populations in the Netherlands for effective ex situ gene conservation management deals with the genetic diversity of roses and the selection of new gene sources in the Netherlands.
Genetic diversity has recently become a very important indicator, essentially indispensable for the selection of new genotypes for the gene bank. Due to the large number of varieties, classic phenotypic evaluation is no longer enough, genetic markers are suitable helpers in the selection of new items.
In the presented study, microsatellite markers were used to assess genetic diversity, which are a very useful and still widely used tool for assessing genetic diversity.
I have a few comments about the manuscript:
1) The terms in situ and ex situ should be written in italics in the text.
2) Dividing words at the ends of lines is very distracting when reading the text.
3) All tables lack a sufficient description of the results and especially the parameters, the tables are not uniform, some are not of sufficient quality.
4) In the chapter "Microsatellite Genotyping, it is not clear whether the PCR analysis was performed in a multiplex or individually.
5) I recommend giving group names (spi, arv, rub, can) either in italics or at least in quotation marks. So the names of the groups in the text are very confusing.
6) If possible, I recommend merging figures 2A and 2B, the individual groups are colored differently, so I see no reason to divide the figure.
Author Response
Comments 1) The terms in situ and ex situ should be written in italics in the text.
Response 1: According to the MDPI Style guide (see https://www.mdpi.com/authors/layout) section 3.6 Foreign words do not need to be highlighted or italicized, including Greek/Latin terms, such as i.e., e.g., etc., et al., vs., ca., cf., in vivo, ex vivo, in situ, ex situ, in vitro, in utero, ad hoc, in silico, ab initio, vice versa, and via. We therefore did not put the words ‘in situ’ and ‘ex situ’ in italics.
Comments 2) Dividing words at the ends of lines is very distracting when reading the text.
Response 2: The hyphen to divide words at the end of lines happens automatically in the Word template used. We are not able to change this.
Comments 3) All tables lack a sufficient description of the results and especially the parameters, the tables are not uniform, some are not of sufficient quality.
Response 3: We have adjusted all six tables, including explaining the abbreviations in the header, and made them more uniform according to the template.
Comments 4) In the chapter "Microsatellite Genotyping, it is not clear whether the PCR analysis was performed in a multiplex or individually.
Response 4: We added the word ‘multiplex’ to the sentence: For all samples, we performed multiplex PCRs to amplify allele combinations at 10 microsatellite loci, originally developed by Esselink et al. [4](Table 3).
Comments 5) I recommend giving group names (spi, arv, rub, can) either in italics or at least in quotation marks. So the names of the groups in the text are very confusing.
Response 5: We changed the group names by putting them in quotation marks.
Comments 6) If possible, I recommend merging figures 2A and 2B, the individual groups are colored differently, so I see no reason to divide the figure.
Response 6: As suggested also by other reviewers we have merged Figure 2a and 2b inti one Figure (now called Figure 2a).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have answered my question.
