Intraspecific Genetic Variability of Brassica cretica Lam. (Brassicaceae) Using SSR Markers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper explores genetic variability within Brassica cretica populations using SSR markers, revealing high diversity and significant differentiation. However, comparisons with established baselines are lacking, and more discussion on the practical application of findings for breeding programs is needed.

(1) Could you provide more details on how the SSR markers were selected for this study and how they may be relevant to the broader Brassica genus or other crop species?

(2) The paper mentions a high level of genetic differentiation among populations, but how do these results compare with other Brassica species or crops, especially in terms of evolutionary implications?

(3) Could you elaborate on the potential challenges or limitations of using B. cretica populations for breeding programs, particularly in the context of climate change and environmental adaptation?

 

 

Author Response

(1) Could you provide more details on how the SSR markers were selected for this study and how they may be relevant to the broader Brassica genus or other crop species?

Answer: Thank you for your comment. They were selected according to similar studies (citations; 23, 26-this has been added to Table 2). Moreover, details of selected SSR markers, such as; map position, repeat motif, repeat count, and size range, can be found in the website database.www.brassica.info (we added this part to the materials and methods section-Table 2).

 

 (2) The paper mentions a high level of genetic differentiation among populations, but how do these results compare with other Brassica species or crops, especially in terms of evolutionary implications?

Thank you for this comment. Brassica cretica exhibits unusually high genetic differentiation (Fst = 0.812) compared to other Brassica species, where Fst typically ranges from 0.1 to 0.6. This elevated value, consistent with findings by Edh et al. (2007), likely results from long-term geographic isolation, limited gene flow, and "isolation by distance" in the fragmented Aegean landscape (Allender et al. 2007). Such strong population structure suggests local adaptation and high evolutionary resilience, with each population representing a unique gene pool of significant conservation and breeding value. 

Allender, C. J., Allainguillaume, J., Lynn, J., & King, G. J. (2007). Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n= 9) wild relatives. Theoretical and Applied Genetics, 114, 609-618.

We have already mentioned in the discussion but we have added some things to highlight the evolutionary implications as well as and the aforementioned reference in the discussion (lines 342-350).

 

(3) Could you elaborate on the potential challenges or limitations of using B. cretica populations for breeding programs, particularly in the context of climate change and environmental adaptation?

Thank you for your comment. A paragraph has been added at the end of the discussion section.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors conducted a study on the Intraspecific genetic variability of Brassica cretica Lam. (Brassicaceae) using SSR markers. While the study has a certain significance, several critical issues need revision.

 

The study ultimately employed only 10 SSR markers and a very limited number of intraspecific representative samples. Is this level of data sufficient to support the ambitious goal of “resolving evolution”?

 

Lines 55–62: There is already a substantial body of refs on B. cretica and B. oleracea. The authors should not rely on a single citation to support such a broad claim.

 

The introduction needs to be restructured. A major issue is that the current study only investigates the evolutionary relationship between B. cretica and B. oleracea. It does not include representatives of the A and B genomes, and even within the C genome, only B. oleracea and B. cretica are examined. How can this limited scope justify statements regarding the evolutionary history of “Brassica L. crops” as a whole?

 

Line 64: The abbreviation of Brassica cretica.

 

Line 113: The term “one commercial botanical variety of B. oleracea” is vague. Which specific botanical variety is it?

 

Line 168: Brassica cretica need italicized and abbreviated.

 

Line 177: There is an extraneous comma that should be removed.

 

Line 209: “(Ne) ranged from minimum 2.413 (Na10-F06) to maximum 4,107”, is this value 4107 or 4.107? Please clarify.

 

Line 210: “and the effective number of alleles was 21.9”, did the authors confuse Na with Ne? Please confirm whether this value is accurate.

 

Line 222: Differences in Na and Ne values alone are not sufficient to infer that Population A has higher genetic stability.

 

Lines 233–235: The statement “The genetic differentiation between populations was high (F = 0.812; p > 0.05). AMOVA test proved that differentiation within-population was 80% and among populations was 20% (Figure 2).” presents a contradiction. A high Fst (0.812) typically implies substantial among-population differentiation, yet the AMOVA result suggests the opposite. Have the authors tested the consistency between Fst and AMOVA results? Furthermore, with a p-value > 0.05, even a high Fst cannot be interpreted as statistically significant differentiation among populations.

 

Lines 364–371: The discussion of climate change impacts on Brassica crops appears disconnected from the main theme of the paper. Is this section overly generalized? The authors may consider focusing specifically on how B. cretica, as a wild genetic resource, can be prioritized for conservation and utilization in Brassicaceae.

 

The author should provide pictures of these varieties of Brassica cretica.

 

The manuscript requires careful checking and revision.

 

Author Response

The study ultimately employed only 10 SSR markers and a very limited number of intraspecific representative samples. Is this level of data sufficient to support the ambitious goal of “resolving evolution”?

Answer: Thank you for your comment. Although the number of SSR markers is limited we believe that the level of codominance can provide sufficient evidence for genetic diversity.We initially tested 14 SSR markers taken from several publications (ie Thakur et al. 2018) but we chose the ones (10 in total) that showed the highest level of polymorphism. Generally, in similar studies a similar number of individuals is used and analyzed with a similar number of markers. Edh et. al 2007, accordingly,used fourteen SSR markers, where four of them are chloroplastic SSRs. El-Shaht et al. 2022 used 10 SCOT markers aiming to determine the genetic relationship between the different cultivated and wild genera of the Brassicaceae family. 

The inclusion of additional individuals in the analysis was not possible, as certain natural populations, especially B. cretica subsp. laconica had small population sizes, and the collection of an adequate number of individuals was constrained. Finally,the aims of the study are clearly stated at the end of the Introduction. “Resolving the evolution” of Brassica oleracea crops is certainly not among the stated objectives of this work.

El-Shaht, S., El-Hefnawy, S. F., Hamza, M. K., & Zayed, M. M. (2022). Histological studies of some brassicaceous samples and the genetic variability analysis using SCOT Markers. Journal of Agricultural Chemistry and Biotechnology, 13(1), 9-19.

Thakur, A. K., Singh, K. H., Singh, L., Nanjundan, J., Khan, Y. J., & Singh, D. (2018). SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids. Hereditas, 155, 1-11.

Lines 55–62: There is already a substantial body of refs on B. cretica and B. oleracea. The authors should not rely on a single citation to support such a broad claim.

Answer: Thank you for the comment. We added two more citations listed below to further support our claim and a short paragraph (lines 60-68).

Castillo-Lorenzo, E., Breman, E., Gómez Barreiro, P., & Viruel, J. (2024). Current status of global conservation and characterisation of wild and cultivated Brassicaceae genetic resources. GigaScience, 13, giae050.

Arias, T., & Pires, J. C. (2012). A fully resolved chloroplast phylogeny of the brassica crops and wild relatives (Brassicaceae: Brassiceae): Novel clades and potential taxonomic implications. Taxon, 61(5), 980-988.

The introduction needs to be restructured. A major issue is that the current study only investigates the evolutionary relationship between B. cretica and B. oleracea. It does not include representatives of the A and B genomes, and even within the C genome, only B. oleracea and B. cretica are examined. How can this limited scope justify statements regarding the evolutionary history of “Brassica L. crops” as a whole?

Answer: Thank you for your comment.  To improve clarity and scientific accuracy, we added a paragraph in the introduction that frames the study as a focused contribution to the C genome, specifically exploring the genetic distance and potential evolutionary connection between B. cretica and cultivated cabbage

Reviewer 3 Report

Comments and Suggestions for Authors

Review of manuscript agronomy_3566540

Title: Intraspecific genetic variability of Brassica cretica Lam. (Brassicaceae) using SSR markers

In this study, SSR molecular markers were used to analyze the genetic diversity of five natural populations of Brassica cretica and one B. oleracea botanical variety.

General concept comments
While the use of 2.2% agarose gels for SSR analysis is technically feasible, this approach has inherent limitations in resolution and precision compared to capillary electrophoresis with fluorescently-labeled primers. For enhanced accuracy and reproducibility in allele calling, the study would have been more robust if it had employed capillary electrophoresis (e.g., using an ABI sequencer) with fluorophore-labeled primers.

To support the gel-based SSR genotyping results, raw electrophoresis images should be included as Supplementary Material, showing representative samples with size markers.

Figures and Tables must stand alone, use effective captions: A reader should understand them without reading the main text.

Examples

Table 1. Sampling details of Brassica cretica Lam. populations: collection localities, geographic coordinates (latitude/longitude), and seed collection dates.

Table 2. Characteristics of 10 SSR loci analyzed in Brassica cretica Lam. populations: primer sequences, repeat motifs, expected amplicon size range (bp), and optimized annealing temperatures (Ta).

Specific comments 

Please specify in Table 2 or its caption the source of the primers used to ensure traceability and proper attribution.

Keywords: Keywords should complement, not duplicate, the title by adding new relevant terms to maximize searchability.

In English-language scientific journals, decimal places are always separated by a period (.), not a comma (,). Please conduct a full revision of the manuscript (main text, figures, and tables) ensuring consistent use of periods (.) for decimal places throughout, per English-language scientific conventions.

Lines 39 and 54: In scientific names, the abbreviation of the authority should be in regular font.

Figure 1 caption, the scientific name of the species should be written in italics

Section 2.2, lines 134 to 138:  The sentence has some redundancy. Specifically, the listing of SSR markers could be streamlined, as some markers are repeated in both parts

Line 150 to 160: The description of the PCR amplification conditions is comprehensive but overly long and complex, which may hinder clarity and reproducibility. I strongly recommend breaking the paragraph into two or more shorter, well-structured sentences.

Lines 173 and 174: Please consider revising this sentence for improved flow and precision. A clearer version could be: “Genetic diversity analysis was performed using binary SSR data in GenAlex v. 6.5 [27].”

Line 183: There is a missing space after reference 28.

Line 207: For improved readability, consider replacing “>” with “over”.

Table 4:  Please apply consistent formatting criteria across all tables. the definitions of abbreviations used should be provided in a footnote immediately below the table.

Comments on the Quality of English Language

Overall, the written English is adequate, but certain phrases could be refined to enhance readability and precision. I recommend language editing to optimize expression and ensure unambiguous scientific communication.

Author Response

General concept comments

While the use of 2.2% agarose gels for SSR analysis is technically feasible, this approach has inherent limitations in resolution and precision compared to capillary electrophoresis with fluorescently-labeled primers. For enhanced accuracy and reproducibility in allele calling, the study would have been more robust if it had employed capillary electrophoresis (e.g., using an ABI sequencer) with fluorophore-labeled primers.

To support the gel-based SSR genotyping results, raw electrophoresis images should be included as Supplementary Material, showing representative samples with size markers.

Answer: Thank you for this comment. Indeed, the study would have been even more robust if it had employed capillary electrophoresis with fluorophore-labeled primers. However, this wasn’t applicable due to high cost. 

We made several repetitions of the electrophoresis of each primer pair, and we even used 3% of metaphor agarose for specific SSR primers to double check our results. Some representative images are now included in the Supplementary Material as suggested.

Figures and Tables must stand alone, use effective captions: A reader should understand them without reading the main text.

Answer: Thank you for the comment. We  corrected everything accordingly.

Examples

 

Table 1. Sampling details of Brassica cretica Lam. populations: collection localities, geographic coordinates (latitude/longitude), and seed collection dates.

Answer: Thank you for your consideration. It is corrected according to your suggestions.

Table 2. Characteristics of 10 SSR loci analyzed in Brassica cretica Lam. populations: primer sequences, repeat motifs, expected amplicon size range (bp), and optimized annealing temperatures (Ta).

Answer: Thank you for your consideration. It is corrected according to your suggestions.

Specific comments 

 

Please specify in Table 2 or its caption the source of the primers used to ensure traceability and proper attribution.

Answer: Thank you for your comment. It is corrected.

Keywords: Keywords should complement, not duplicate, the title by adding new relevant terms to maximize searchability.

Answer: Thank you for the comment. They are modified accordingly.

 

In English-language scientific journals, decimal places are always separated by a period (.), not a comma (,). Please conduct a full revision of the manuscript (main text, figures, and tables) ensuring consistent use of periods (.) for decimal places throughout, per English-language scientific conventions.

Answer. Thank you for the comment. It is corrected.

Lines 39 and 54: In scientific names, the abbreviation of the authority should be in regular font.

Answer:Thank you for the comment. It is done.

Figure 1 caption, the scientific name of the species should be written in italics

Answer: Thank you for the comment. It is now in italics.

 

Section 2.2, lines 134 to 138:  The sentence has some redundancy. Specifically, the listing of SSR markers could be streamlined, as some markers are repeated in both parts

Answer: Thank you for the comment. It is done.

 

Line 150 to 160: The description of the PCR amplification conditions is comprehensive but overly long and complex, which may hinder clarity and reproducibility. I strongly recommend breaking the paragraph into two or more shorter, well-structured sentences.

Answer:Thank you for the comment. It is marked with blue letters.

Lines 173 and 174: Please consider revising this sentence for improved flow and precision. A clearer version could be: “Genetic diversity analysis was performed using binary SSR data in GenAlex v. 6.5 [27].”

Answer: Thank you for the comment.Done.

Line 183: There is a missing space after reference 28.

Answer:Thank you for the comment. Done.

Line 207: For improved readability, consider replacing “>” with “over”.

Answer:Thank you for the comment. it is replaced with “more than”.

Table 4:  Please apply consistent formatting criteria across all tables. 

Answer: Thank you for the comment. All tables now have the same format.

the definitions of abbreviations used should be provided in a footnote immediately below the table.

Answer:Thank you for the comment. Done.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been significantly improved. The SSR marker selection and its relevance to Brassica species are well explained. The clarification on genetic differentiation and breeding challenges in the context of climate change enhances the discussion. All reviewer comments have been thoroughly addressed.

Author Response

Reviewer 1: The manuscript has been significantly improved. The SSR marker selection and its relevance to Brassica species are well explained. The clarification on genetic differentiation and breeding challenges in the context of climate change enhances the discussion. All reviewer comments have been thoroughly addressed.

Response to Reviewer 1: We sincerely thank the reviewer for the positive feedback and appreciation of the improvements made. We are glad that the clarifications and revisions addressed the comments effectively.

Reviewer 2 Report

Comments and Suggestions for Authors

Although the authors have addressed my concerns in the revised manuscript, the points should be clearly reflected in a point-by-point manner; otherwise, it can be confusing.

 

A few minor issues: in lines 71, 76, etc., Brassica cretica should be abbreviated after its second mention. In line 270, Brassica cretica should be italicized.

Author Response

Although the authors have addressed my concerns in the revised manuscript, the points should be clearly reflected in a point-by-point manner; otherwise, it can be confusing.

Answer: we provide all the answers to your previous comments which accidentally we did not include in the round one and we apologize for that.

The study ultimately employed only 10 SSR markers and a very limited number of intraspecific representative samples. Is this level of data sufficient to support the ambitious goal of “resolving evolution”?

 

Answer: Thank you for your comment. Although the number of SSR markers is limited we believe that the level of codominance can provide sufficient evidence for genetic diversity.We initially tested 14 SSR markers taken from several publications (ie Thakur et al. 2018) but we chose the ones (10 in total) that showed the highest level of polymorphism. Generally, in similar studies a similar number of individuals is used and analyzed with a similar number of markers. Edh et. al 2007, accordingly,used fourteen SSR markers, where four of them are chloroplastic SSRs. El-Shaht et al. 2022 used 10 SCOT markers aiming to determine the genetic relationship between the different cultivated and wild genera of the Brassicaceae family.

 

The inclusion of additional individuals in the analysis was not possible, as certain natural populations, especially B. cretica subsp. laconica had small population sizes, and the collection of an adequate number of individuals was constrained. Finally,the aims of the study are clearly stated at the end of the Introduction. “Resolving the evolution” of Brassica oleracea crops is certainly not among the stated objectives of this work.

 

El-Shaht, S., El-Hefnawy, S. F., Hamza, M. K., & Zayed, M. M. (2022). Histological studies of some brassicaceous samples and the genetic variability analysis using SCOT Markers. Journal of Agricultural Chemistry and Biotechnology, 13(1), 9-19.

 

Thakur, A. K., Singh, K. H., Singh, L., Nanjundan, J., Khan, Y. J., & Singh, D. (2018). SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids. Hereditas, 155, 1-11.

 

 

 

 

Lines 55–62: There is already a substantial body of refs on B. cretica and B. oleracea. The authors should not rely on a single citation to support such a broad claim.

 

Answer: Thank you for the comment. We added two more citations listed below to further support our claim and a short paragraph (lines 60-68).

 

Castillo-Lorenzo, E., Breman, E., Gómez Barreiro, P., & Viruel, J. (2024). Current status of global conservation and characterisation of wild and cultivated Brassicaceae genetic resources. GigaScience, 13, giae050.

 

Arias, T., & Pires, J. C. (2012). A fully resolved chloroplast phylogeny of the brassica crops and wild relatives (Brassicaceae: Brassiceae): Novel clades and potential taxonomic implications. Taxon, 61(5), 980-988.

 

 

 

 

The introduction needs to be restructured. A major issue is that the current study only investigates the evolutionary relationship between B. cretica and B. oleracea. It does not include representatives of the A and B genomes, and even within the C genome, only B. oleracea and B. cretica are examined. How can this limited scope justify statements regarding the evolutionary history of “Brassica L. crops” as a whole?

 

Answer: Thank you for your comment. To improve clarity and scientific accuracy, we added a paragraph in the introduction that frames the study as a focused contribution to the C genome, specifically exploring the genetic distance and potential evolutionary connection between B. cretica and cultivated cabbage

 

Line 64: The abbreviation of Brassica cretica.

 

 Thank you for the comment it has been changed throughout the manuscript.

 

Line 113: The term “one commercial botanical variety of B. oleracea” is vague. Which specific botanical variety is it?

Thank you for the comment, it has been removed the term botanical variety.

 

 

 

Line 168: Brassica cretica need italicized and abbreviated.

Thank you for the comment, it has been corrected.

 

 
Comment 1 (Line 209):
“(Ne) ranged from minimum 2.413 (Na10-F06) to maximum 4,107”, is this value 4107 or 4.107? Please clarify.

Response: Thank you for your comment. The correct value is 4.107. We will revise the manuscript to reflect the proper format.

Comment 2 (Line 210):
“and the effective number of alleles was 21.9”, did the authors confuse Na with Ne? Please confirm whether this value is accurate.

Response: Thank you for your comment. The value of 21.9 refers to the mean observed number of alleles (Na), not the mean effective number of alleles (Ne), which is 3.049. We will correct the manuscript.

Comment 3 (Line 222):
Differences in Na and Ne values alone are not sufficient to infer that Population A has higher genetic stability.

Response: Thank you for your feedback. We agree that Na and Ne alone do not show genetic stability. We have already clarified this in the discussion.

 

 

Lines 233–235: The statement “The genetic differentiation between populations was high (F = 0.812; p > 0.05). AMOVA test proved that differentiation within-population was 80% and among populations was 20% (Figure 2).” presents a contradiction. A high Fst (0.812) typically implies substantial among-population differentiation, yet the AMOVA result suggests the opposite. Have the authors tested the consistency between Fst and AMOVA results? Furthermore, with a p-value > 0.05, even a high Fst cannot be interpreted as statistically significant differentiation among populations.

Answer: Thank you for the comment, indeed there is a contradiction between the fixation index (Fst) and the AMOVA results. Maybe this is a results due to, low sample sizes, or population imbalance, and we have corrected the part that we interpreted as evidence of meaningful population differentiation.

 

 

Lines 364–371: The discussion of climate change impacts on Brassica crops appears disconnected from the main theme of the paper. Is this section overly generalized? The authors may consider focusing specifically on how B. cretica, as a wild genetic resource, can be prioritized for conservation and utilization in Brassicaceae.

Answer: thank you for the comment, we have changed this part focusing mainly of the breeding potential of B. cretica populations.

 

 

 

The author should provide pictures of these varieties of Brassica cretica.

Thank you for the comment. We are providing some photos as supplementary file2.

 

 

 

The manuscript requires careful checking and revision.

Thank you for the comment. We have carefully checked and revise the whole manuscript.

 

A few minor issues: in lines 71, 76, etc., Brassica cretica should be abbreviated after its second mention. In line 270, Brassica cretica should be italicized.

Answer: We have corrected (abbreviated and/or italicized ) where needed according to your suggestions.