Phenotypic, Biochemical, and Molecular Diversity Within a Local Emblematic Greek Allium sativum L. Variety

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To the authors

This paper is a detailed evaluation of the genetic and biochemical diversity of garlic in the NV region, and is believed to be very useful in considering the importance of native lineages and their future use as new varieties, given the limited maintenance and breeding of garlic. The future prospects based on the obtained results are considered in great detail, but please provide a more detailed description of the reasons for focusing on this region. Please consider the background of garlic production, propagation history, and growing climate in the NV region and the reason why you decided to carefully select and evaluate these collection areas (10 locations).

In this study, individuals from the 10 regions were not grown in the same environmental condition and their characteristics were evaluated, but individuals grown in each area were collected and used as plant material for the survey. It is thought that the growth characteristics and chemical content characteristics of garlic change depending on the cultivation environment (as described in L65-67). The genetic diversity evaluation obtained this time showed low genetic diversity, but significant differences were confirmed in morphology and chemical component production ability between collection locations. Please consider whether this result was observed in a low genetic diversity (genetic factors) or whether it was due to differences in the environment of each region where the samples were collected (environmental factors). In the case of genetic factors, selection due to human preference or natural selection may be possible. Table S7 shows the heterozygosity (He) of the SSR primers used this time, but what was the actual heterozygosity (Ho) confirmed in each collection region? By evaluating He and Ho in each region, the degree of selection and selection can be estimated. If it is an environmental factor, please describe what factors are thought to affect the productivity of S compounds and phenols.

Also, you have mainly investigated the characteristics of the bulbs, but have you investigated other characteristics? (flower stalk length, flowering pattern, early and late development, etc.). In particular, it has been reported that the flowering pattern and inflorescence development pattern are related to metabolic production ability (S compounds, flavonoids, etc.), so if you have any data on these, please post them.

Please write scientific names in italics on all pages. "Allium sativum", "Solanum lycopersicum", "Triticum turgidum var. durum", etc.

The following are minor revising points.

Introduction

L43: Please write Longicuspis, Subtropical, Ophioscorodon, Mediterranean Sativum in italics. Please follow the references in [5].

L77: Since isozymes and RAPD are methods that have been around for decades, resent years are inappropriate. Please use expressions such as "In the past few decades, surveys have been conducted using...".

L91-94: Please provide more details on why this region is a key region.

Materials and Method

L107-111: It is unclear under what conditions the specimens in each region were cultivated. If you do not know, please state so. Details of each region are listed in Stable, but is there any information on altitude?

L113-133: Abbreviations are listed for each trait. Shouldn't cross sections (SC) be CS? Shouldn't root disc be root basal plate?

Please check other places as well.
L194:μl→μL

L233-236: H' is written in italics

L253: a = 0.05→ p < 0.05

L271ASE; 52% and 42%, respectively→Isn't the total 100%?

L332: p is written in italics

L348: Total phenolic content (TCP) →(TPC)?
L374: p is written in italics

Discussion

L530: H’ italics

L677: SSR91 → AsESSR-91

References
Scientific names are written in italics,

L754: Etoh, T. A. K. E. O. M. I → Etoh, T.

L831: Period
Please correct the capitalization of Reference No. 19, 20, 40, 41, 51, 54, 59, 70, 71, 75, 76, 77, 79.

Figures and Tables

Figure 3
Change the color and shape of the plot to show the collection area information (NV1 to NV10), and evaluate the relationship between each trait and the collection area.

Figure 5, Figure 6
The figures are small overall, so please display them larger.

Supplementary Materials

Supplementary Table S1
Please include altitude information if available. Altitude is related to the plant's phenol production capacity.

Supplementary Figure 2
Total bulb weight BFW→TBW?

Supplementary Table S7
Unbiased expected heterozygosity(He)→(uHe)

Author Response

Comment 1. The future prospects based on the obtained results are considered in great detail, but please provide a more detailed description of the reasons for focusing on this region. Please consider the background of garlic production, propagation history, and growing climate in the NV region and the reason why you decided to carefully select and evaluate these collection areas (10 locations).

Response: We thank the reviewer for the insightful comment. We agree that the background of NV garlic production was not described efficiently. In the revised text, we have included this information (both in introduction and M&M sections) and also added a map with sampling sites as well as altitudinal details. The sampling sites were fields of the members from the Nea Vissa garlic cooperative, and the selection was made by them to span NV region.

Comment 2. In this study, individuals from the 10 regions were not grown in the same environmental condition and their characteristics were evaluated, but individuals grown in each area were collected and used as plant material for the survey. It is thought that the growth characteristics and chemical content characteristics of garlic change depending on the cultivation environment (as described in L65-67).

Response: This is not entirely correct, as the sampling sites were all from the same region, with no particular difference in altitude or climate conditions. Since the sampling sites were fields of the members from the Nea Vissa garlic cooperative, local producers adhere to standardized, certified integrated management systems that govern cultivation practices as well as procedures. The text has been modified to clarify this point.

Comment 3. The genetic diversity evaluation obtained this time showed low genetic diversity, but significant differences were confirmed in morphology and chemical component production ability between collection locations. Please consider whether this result was observed in a low genetic diversity (genetic factors) or whether it was due to differences in the environment of each region where the samples were collected (environmental factors). In the case of genetic factors, selection due to human preference or natural selection may be possible.

Response: We cannot estimate the impact of small microclimatic conditions on morphology and chemical components, but we consider that it won’t be very significant. It could be, indeed as you correctly pointed out, some sort of natural selection. The scope of this study was to 1) to test whether there is some intraspecific diversity related to phenotype, quality and organosulfur compounds within the NV population, and 2) to identify loci able to capture this diversity. In future studies, using more advanced genetic tools, we aim to further explore the potential of improving this popular local variety (uniformity and key bioactive components).

Comment 4. Table S7 shows the heterozygosity (He) of the SSR primers used this time, but what was the actual heterozygosity (Ho) confirmed in each collection region? By evaluating He and Ho in each region, the degree of selection and selection can be estimated. If it is an environmental factor, please describe what factors are thought to affect the productivity of S compounds and phenols.

Response: In our study, we calculated expected heterozygosity (He) rather than observed heterozygosity (Ho) because garlic is predominantly a clonally propagated species with very limited sexual reproduction. In the revised manuscript, we have added the calculations of Ho, as suggested by reviewer, per SSR (Suppl Table S7). Results indicated that “The observed heterozygosity (Ho) for AsESSR-83 was also high but slightly lower than its He value, suggesting the presence of an inbreeding effect. Conversely, for AsESSR-14 and AsESSR-91, He values were lower than Ho values, indicating a possible excess of heterozygotes which may be due to outbreeding. However, their lower He, uHe, and PIC values compared to AsESSR-83, suggested that these markers are less informative for the comprehensive diversity study of the NV population. Loci like AsESSR-78 and AsESSR-107 had very low He and Ho values (close to zero), indicating their limited utility for studying intra-varietal diversity within the NV population.”

Therefore, our results indicated that “AsESSR-83 was the most robust and informative marker for assessing genetic diversity within the NV population, making it a valuable tool for guiding breeding and conservation strategies”.

The text has been amended accordingly, we thank the reviewer for the useful comment. As illustrating in Suppl Figure S1 and also Suppl Table S1, all sampling sites were in the same region, therefore no significant environmental effect was expected. Other traits such as soil properties that could have an impact on garlic quality were not evaluated herein.

Comment 5. Also, you have mainly investigated the characteristics of the bulbs, but have you investigated other characteristics? (flower stalk length, flowering pattern, early and late development, etc.). In particular, it has been reported that the flowering pattern and inflorescence development pattern are related to metabolic production ability (S compounds, flavonoids, etc.), so if you have any data on these, please post them.

Response: Thank you for your valuable feedback regarding other flower-related traits. We completely agree with your observation. However, our current study did not assess these traits, as it was primarily focused on the garlic bulbs, with the goal of enhancing the recognition of NV garlic as a product of superior quality. As a first step, we aimed at exploring whether there is a potential variation within the local variety that could be relevant for future breeding efforts towards nutritional enhancement. Since our results suggest that there is, in upcoming research, we plan to incorporate assessments of floral and vegetative traits, as well as soil data, to more comprehensively understand the sources of the observed variation.

Comment 6. Please write scientific names in italics on all pages. "Allium sativum", "Solanum lycopersicum", "Triticum turgidum var. durum", etc.

Response: The text has been revised thoroughly.

 

The following are minor revising points.

L43: Please write Longicuspis, Subtropical, Ophioscorodon, Mediterranean Sativum in italics. Please follow the references in [5].

Response: The text has been revised accordingly, so does the reference list.

L77: Since isozymes and RAPD are methods that have been around for decades, resent years are inappropriate. Please use expressions such as "In the past few decades, surveys have been conducted using...".

Response: The text has been revised accordingly.

L91-94: Please provide more details on why this region is a key region.

Response: Thank you for pointing this out. The garlic of “Nea Vissa” is probably the most popular garlic in Greece, being cultivated in this region for generations, with traditional farming methods. With more than 30 members, the cooperative is a robust community of garlic farmers. The text has been amended as follows: “Garlic has been cultivated in this region for generations, with traditional farming methods well-suited for the local soil and moderate climate. The local farmers, members of the "Nea Vissa" garlic cooperative, adhere to standardized, certified integrated management systems, ensuring uniformity in the garlic delivered to the market. Besides these protocols, there are still natural variations in morphological and nutritional properties that need to be captured for further improving the local garlic variety”.

Materials and Method

L107-111: It is unclear under what conditions the specimens in each region were cultivated. If you do not know, please state so. Details of each region are listed in Stable, but is there any information on altitude?

Response: In the revised text, we have added a map showing the sampling sites (in the same region). As the reviewer can see in Suppl Figure S1, the fields are very close to each other, so no particular difference in climatic conditions are expected, although the microclimate can indeed have some variations. All of sampling sites were fields cultivated by members of the “Nea Vissa’ cooperative, so the same standardized protocols were employed. Regarding the altitude, as these fields are close to Evros river, the altitude has a range between 27-31m. Unfortunately, we don’t have data on soil properties. All this new information has been added to the revised text.

L113-133: Abbreviations are listed for each trait. Shouldn't cross sections (SC) be CS? Shouldn't root disc be root basal plate?

Response: We have followed CPVO terminology for consistency and standardization in our study. Terms such as "root disc" and "cross sections" are used in accordance with their official guidelines. Additionally, the abbreviation "CS" is specifically used for the color of the scale, based on the CPVO terminology.

Please check other places as well.
L194:μl→μL

Response: Corrected.

L233-236: H' is written in italics

Response: Corrected.

L253: a = 0.05→ p < 0.05

Response: Corrected.

L271ASE; 52% and 42%, respectively→Isn't the total 100%?

Response: That was a typo, thank you for pointing this out.

L332: p is written in italics

Response: Corrected.

L348: Total phenolic content (TCP) →(TPC)?

Response: Corrected.

L374: p is written in italics

Response: Corrected.

Discussion

L530: H’ italics

Response: Corrected.

L677: SSR91 → AsESSR-91

Response: Corrected.

References
Scientific names are written in italics,

Response: The reference list has been revised thoroughly.

L754: Etoh, T. A. K. E. O. M I → Etoh, T.

Response: Removed.

L831: Period

Response: Corrected.

Please correct the capitalization of Reference No. 19, 20, 40, 41, 51, 54, 59, 70, 71, 75, 76, 77, 79.

Response: Corrected.

Figures and Tables

Figure 3
Change the color and shape of the plot to show the collection area information (NV1 to NV10) and evaluate the relationship between each trait and the collection area.

Response: Regarding both qualitative and quantitative traits, we have decided to use the entire population of the ten sampling sites as key representatives of the NV garlic production of the cooperation, and not to split the datasets. We have employed this rule for Figure 2 and 3, as well as Suppl Tables S3 and S4. Our decision was based on the fact that there is a limited morphological variation within the population for the majority of traits, except for clove size and anthocyanin stripes.

Figure 5, Figure 6
The figures are small overall, so please display them larger.

Response: Thank you for your feedback. As per a previous reviewer’s suggestion, we have already resized Manuscript Figures. Additionally, we have split Figure 6 into two parts to enhance clarity and presentation. We hope these changes address your concerns.

Supplementary Materials

Supplementary Table S1
Please include altitude information if available. Altitude is related to the plant's phenol production capacity.

Response: The altitude has been added. However, we believe that the range is too short to justify differences in phenol content.

Supplementary Figure 2
Total bulb weight BFW→TBW?

Response: BFW stands for "bulb fresh weight”, separated from clove fresh weight, which was abbreviated as FW. These terms are consistently used throughout the manuscript to describe the fresh weight of garlic bulbs at harvest.

Supplementary Table S7
Unbiased expected heterozygosity(He)→(uHe)

Response: Corrected.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Article Phenotypic, biochemical and molecular diversity within an emblematic Greek Allium sativum L. local variety by Anastasia Papadopoulou, Anastasia Boutsika, Francesco Reale, Silvia Carlin, Urska Vrhovsek, Eleftheria Deligiannidou, Aliki Xanthopoulou, Eirini Sarrou, Ioannis Ganopoulos, Ifigeneia Mellidou a wide range of methodological applications for the study of local diversity of Allium sativum L. crop.
The manuscript is well structured and contains all the necessary sections and can be accepted for publication with clarification of some points.
Key questions
1. The authors used 10 selections collected in different places of cultivation. However, the principle of sample formation is not explained. Was the origin of the planting material clarified. There is no data on differences in humidity, soil conditions, fertilization, pests, agricultural practices, such as planting dates, watering - all these factors can have such a significant impact on the phenotype that building correlations would be invalid. Clarify details, add a map.
2. The authors describe many phenotypical characteristics, but in the photo they only provide a conventional view from above, with a significant shift, without a bar for identifying the sizes. It is necessary to show the dimensions and add to the article or appendix a photo of all the parameters given in the article. If the photo is of poor quality in the article, provide a diagram and method for taking the parameters (measurement accuracy), in the appendix working materials and information on whether the differences were significant.
3. The structure of the garlic clove is uneven and depends on the size, location (inside, outside), and also on the ratio of the parts. In this regard, the total content will differ significantly even if you compare the internal cloves with the external ones or even an identical genotype with and without watering. Specify the first stages of selection and sample preparation.
4. Genetic analysis of markers was carried out on fresh leaves, but the article does not indicate how and where samples were taken. They were collected in the field, grown from cloves, the age of the plants for this analysis is not indicated.
5. It is quite surprising that the authors mention possible external influences, but do not provide data on the availability of similar products on the market and do not include in the study samples that are potentially probable effectors in the past or at present, why does the experiment ignore your own assumption/preamble?
Minor issues
1. Clarify the reliability ranges for morphological descriptors, in particular in Fig. 2
2. Enlarge or split Figures 5 and 6
3. Reword the last paragraph of the introduction into a problem statement or hypothesis, and move the advertising of the results to the discussion or conclusion
4. Find a result in the manuscript that indicates the application of these results in practical farming, for example, to identify the external influence of genotypes or to forecast in the future...
This work is a high-quality and interesting methodological work, but its conclusions are rather weak for both breeding and monitoring the genetic diversity of the region, as well as for the agronomy of this species in the region. However, careful work with the conclusions and the problem statement from the perspective of practical development can easily make the manuscript useful, so I think it is advisable to review the manuscript after the proposed correction.

Author Response

Comment 1. The authors used 10 selections collected in different places of cultivation. However, the principle of sample formation is not explained. Was the origin of the planting material clarified. There is no data on differences in humidity, soil conditions, fertilization, pests, agricultural practices, such as planting dates, watering - all these factors can have such a significant impact on the phenotype that building correlations would be invalid. Clarify details

Response: We appreciate your insightful feedback. We fully acknowledge the concern that environmental conditions and cultivation practices can have a significant impact on the phenotype of garlic. However, the investigation of these factors was beyond the scope of the present study, which was designed to explore the potential for improving the local variety by leveraging existing genetic diversity. All samples were collected in collaboration with the Vissa Garlic Agricultural Cooperative, exclusively from cooperative producers who adhered to the same agricultural practices. Consequently, the sampling sites were confined to a geographically small and environmentally similar region, thereby minimizing variability due to external conditions.

To further clarify this point, we have incorporated the following revisions into the manuscript:

1. A supplementary map illustrating the sampling locations.
2. A brief discussion on the homogeneity of the sampling sites.

Comment 2. The authors describe many phenotypical characteristics, but in the photo, they only provide a conventional view from above, with a significant shift, without a bar for identifying the sizes. It is necessary to show the dimensions and add to the article or appendix a photo of all the parameters given in the article. If the photo is of poor quality in the article, provide a diagram and method for taking the parameters (measurement accuracy), in the appendix working materials and information on whether the differences were significant.

Response: We appreciate your suggestion concerning the photographic representation of the phenotypic characteristics. To enhance the clarity of the depicted features, we have incorporated a scale bar into the image, which reflects the measurements based on our phenotypic analysis. Unfortunately, we did not obtain additional photographs for the other parameters discussed. Detailed measurements, ranges, and the associated statistical analyses are comprehensively presented in Supplementary Table 3.

Regarding the precision of these measurements, we adhered to the morphological descriptors as established by the Community Plant Variety Office (CPVO). The CPVO provides standardized protocols for the documentation and measurement of plant traits, ensuring consistency and accuracy in our methodology. We trust that these amendments address your concerns.

Comment 3. The structure of the garlic clove is uneven and depends on the size, location (inside, outside), and also on the ratio of the parts. In this regard, the total content will differ significantly even if you compare the internal cloves with the external ones or even an identical genotype with and without watering. Specify the first stages of selection and sample preparation.

Response: We appreciate your attention to the potential variability in clove biochemical composition arising from factors such as clove position (inner vs. outer) or size. To mitigate these concerns and ensure analytical consistency, we adhered to a rigorously standardized sample selection and preparation protocol. For the biochemical analyses, we systematically selected three garlic bulbs from each of the ten sampling sites. These bulbs were chosen based on their visual homogeneity in size, color, and shape, thus serving as representative specimens of each site. From each bulb, we have pooled the majority of the cloves, deliberately excluding those that were outliers in size or exhibited any signs of abnormal growth. This strategy was designed to minimize variability introduced by positional biases among the cloves. To clarify this, we expanded the Methods section to explicitly describe the selection criteria and sample preparation process. Although some degree of variation is inevitable, we are confident that our systematic approach allows for a robust and representative assessment of intra-varietal diversity.

Comment 4. Genetic analysis of markers was carried out on fresh leaves, but the article does not indicate how and where samples were taken. They were collected in the field, grown from cloves, the age of the plants for this analysis is not indicated.

Response: The genetic analysis was conducted on fresh leaf samples collected directly from plants grown in the field at the same locations where the bulbs were harvested. We have revised the Methods section to clearly specify the sampling procedure, plant growth stage, and collection conditions to provide greater transparency on the genetic analysis process.

Comment 5. It is quite surprising that the authors mention possible external influences, but do not provide data on the availability of similar products on the market and do not include in the study samples that are potentially probable effectors in the past or at present, why does the experiment ignore your own assumption/preamble?

Response: Thank you for your thoughtful comment. Within the funded grant we analyzed the biochemical content of additional garlic bulbs from the market (including bulbs originating from the same region). It became evident that it was impractical to draw comparisons between these samples regarding their antioxidant or sulfur content, primarily due to variances in environmental conditions, agricultural practices, and postharvest handling. Consequently, we opted to focus our efforts on assessing the intra-varietal diversity within the Nea Vissa garlic landrace, rather than on the influence of environmental or agronomic factors. While several prior studies have investigated genetic diversity across different garlic landraces and local varieties, including notable research in Greece (Polyzos et al., 2019; Avgeri et al., 2020; Papaioannou et al., 2023), systematic studies on intra-population variation are, to the best of our knowledge, relatively scarce.

Minor issues
Comment 6. Clarify the reliability ranges for morphological descriptors, in particular in Fig. 2

Response: We acknowledge the importance of clarifying the statistical aspects of our diversity analysis. In our study, the Shannon-Weaver Diversity Index (H') was used to quantify phenotypic diversity for both qualitative and quantitative traits. This index measures the richness and evenness of trait distribution, providing a comparative view of variability within the Nea Vissa garlic population. However, as H' is a diversity metric rather than a direct statistical measure of trait variability, it does not have associated confidence intervals or standard deviations. For quantitative traits, we further illustrated variability using violin and box plots, which effectively depict data distribution and spread. While H' does not allow for direct statistical reliability measures, the inclusion of these additional plots provides insight into the dispersion and frequency of values for quantitative traits such as bulb fresh weight (BFW), clove number (CN), fresh weight (FW), dry weight (DW), and total soluble solids (TSS). Further statistical analyses for both qualitative and quantitative traits are provided in the supplementary section.

Comment 7. Enlarge or split Figures 5 and 6

Response: We appreciate your feedback regarding Figures 5 and 6. After careful evaluation, we believe that the current size and layout of Figure 5 effectively present the necessary information without compromising clarity. In the final published version, we are confident that the resolution and readability will be adequate. However, we acknowledge that Figure 6 was overly dense, and to enhance clarity, we have split it into two separate figures and revised the text accordingly.

Comment 8. Reword the last paragraph of the introduction into a problem statement or hypothesis, and move the advertising of the results to the discussion or conclusion

Response: We have revised the last paragraph of the introduction accordingly. Additionally, we have moved the discussion regarding the potential results, such as the certification of NV garlic as a Protected Designation of Origin (PDO) product, to the conclusion section, as recommended.

Comment 9. Find a result in the manuscript that indicates the application of these results in practical farming, for example, to identify the external influence of genotypes or to forecast in the future...

Response: As we don’t have any data (environmental factors or soil properties) that may indicate how different garlic varieties perform under varied conditions, i.e. yield or disease resistance, it is too risky to link specific traits with climate or farming techniques. Nonetheless, as we agree with the reviewer’s suggestion, in the revised version, we have tried to discuss the potential of using our results to guide breeding programs for specific environments and further suggest future research goals based on our findings.

"The detailed characterization of the NV garlic population reveals high variability in morphological traits, such as clove size and anthocyanin stripes, as well as significant biochemical diversity, including total phenolic content (TPC), antioxidant capacity, and various organosulfur compounds (e.g., allyl methyl disulfide, trisulfide, cis-1-propenyl propyl disulfide, cis-1-propenyl methyl disulfide, diallyl sulfide, and trisulfide). This suggests substantial intra-varietal diversity within the population, which can guide selective breeding programs aimed at enhancing specific desirable traits of the local variety or serve as alternative sources of high-value metabolites in novel pharmacological formulations [41]. Future research could build on these findings by explicitly studying how different growing conditions, such as soil type and climate, affect the phenotypic and biochemical profiles of these garlic varieties. Such research could aid in developing robust varieties that thrive under diverse environmental conditions.

On the other hand, the study highlighted low to moderate intra-variety differentiation based on microsatellite markers, indicating potential genetic markers that could be targeted in breeding to enhance traits such as anthocyanins or organosulfur compounds. Although no association study between phenotypic and molecular data was performed, presumably due to the lack of phenotypic characterization of additional genotypes with diverse traits, the results suggest the possibility of selecting garlic clones with high functional value within the population, similar to previous reports [26]. Future studies should aim to identify molecular markers ideal for selecting garlic varieties that exhibit desirable agronomic and nutraceutical traits, improved yield, or disease resistance. By integrating environmental and genetic data into predictive models, it may be possible to forecast yield, quality, and resistance traits, providing invaluable insights for breeding efforts to anticipate the effects of climate change on garlic cultivation."

Comment 10: This work is a high-quality and interesting methodological work, but its conclusions are rather weak for both breeding and monitoring the genetic diversity of the region, as well as for the agronomy of this species in the region. However, careful work with the conclusions and the problem statement from the perspective of practical development can easily make the manuscript useful, so I think it is advisable to review the manuscript after the proposed correction.

Response: Thank you for your fruitful comments. We are confident that our revised manuscript addresses all your concerns regarding clarity and the prospect of practical development.

Reviewer 3 Report

Comments and Suggestions for Authors

Supplementary material: It is recommended that tables that extend over more than one page have the header repeated on all pages.

Figures: It is recommended that the size of the figures be increased to better visualize the results and consequently improve the visualization of the identification of the axes. Many identifications are illegible.

Abstract: It is recommended that the first sentences be summarized. Make the objective of the work clear. Then, the statistical design, the principal results, and the main conclusion of the work were adopted.

Introduction: It is recommended that the research objective be made clear in the last paragraph.

Material and methods: It is recommended that in units of measurement, you use "-¹" instead of "/," for example, "g mL-1" instead of "g/mL." You should add the statistical design, what was considered "treatment," and the number of repetitions.

Results: It is recommended that when presenting a mean, you also show the standard error of the mean.

Discussion: It is recommended to avoid writing sentences that avoid presenting the results described above. It is recommended to focus on quotes that help explain the results obtained.

Conclusion: It is recommended that the first three lines be deleted. Adopt a conclusive tone in the sentences. It is not necessary to describe what was done in the study. This section describes the conclusions obtained from the study.

Author Response

Comment 1.Supplementary material: It is recommended that tables that extend over more than one page have the header repeated on all pages.

Response: Thank you for your suggestion. We have ensured that tables extending over multiple pages now have their headers repeated on each page to improve readability.

Comment 2. Figures: It is recommended that the size of the figures be increased to better visualize the results and consequently improve the visualization of the identification of the axes. Many identifications are illegible.

Response: We have revised the figures and also split some of the figures to improve clarity, as suggested by other reviewers. All the figures have also been uploaded as jpeg of 300dpi to ensure high quality in the final version of our manuscript.

Comment 3. Abstract: It is recommended that the first sentences be summarized. Make the objective of the work clear. Then, the statistical design, the principal results, and the main conclusion of the work were adopted.

Response: We have revised the abstract to better clarify its objective, design, and main conclusions, as suggested.

Comment 4. Introduction: It is recommended that the research objective be made clear in the last paragraph.

Response: We have clarified the research objective in the last paragraph of the Introduction, as recommended.

Comment 5. Material and methods: It is recommended that in units of measurement, you use "-¹" instead of "/," for example, "g mL-1" instead of "g/mL." You should add the statistical design, what was considered "treatment," and the number of repetitions.

Response: We have updated the units of measurement, using ‘-¹’ instead of ‘/’, as recommended. Additionally, since we analyzed different individuals within the Nea Vissa landrace to assess intraspecific variations rather than applying different experimental conditions or treatments, we think it would be more accurate to avoid referring to it as a "treatment. Moreover, we have revised the 21.Plant Material section to clearly state the number of repetitions for each analysis and the method of selection.

Comment 6. Results: It is recommended that when presenting a mean, you also show the standard error of the mean.

Response: We assume that the reviewer refers to the results presented in Figure 2. In our study, the Shannon-Weaver Diversity Index (H') was used to quantify phenotypic diversity for both qualitative and quantitative traits. This index assesses the richness and evenness of trait distribution, offering a comparative view of variability within the Nea Vissa garlic population. However, since H' is a diversity metric rather than a direct statistical measure of trait variability, it does not include associated confidence intervals or standard deviations. All the statics related to qualitative traits are presented in Supplementary Table 3, while for quantitative traits, standard deviations are presented in Supplementary Table 4 and Figure 2D.         

Comment 7. Discussion: It is recommended to avoid writing sentences that avoid presenting the results described above. It is recommended to focus on quotes that help explain the results obtained.

Response: The text has been revised accordingly, especially concerning the environmental effect or soil properties on garlic morphology-quality, as it has not been studied in our study.

Comment 8. Conclusion: It is recommended that the first three lines be deleted. Adopt a conclusive tone in the sentences. It is not necessary to describe what was done in the study. This section describes the conclusions obtained from the study.

Response: Thank you for your valuable feedback. We have revised the Conclusion section as suggested, removing the first three lines and adopting a more conclusive tone.

Reviewer 4 Report

Comments and Suggestions for Authors

Minor comments:

1)Material and Methods- Please, expand the description of polyphenol determination: extraction conditions, solvent, temperature and duration, as these peculiarities greatly affect the value of polyphenol content

2) In Biological systems absolute values of various parameters usually vary greatly. In this respect, ratios of the parameters provide more stable data. Have you measured bulb/clove density (m/V ratio)? What about bulb diameter/height ratio? Or various Sulphur derivative ratio???

3)Figure 2D: add units for the ordinate axis

4) References: use  Italics for Allium sativum (ref 1,2,11,12,13,14,16,17 etc)

- Use bold letters for the number of years

-  number of journals’ volumes should be in Italics

 

Author Response

Comment 1. Material and Methods- Please, expand the description of polyphenol determination: extraction conditions, solvent, temperature and duration, as these peculiarities greatly affect the value of polyphenol content.

Response: The spectrophotometric protocol for total phenol content has been amended as suggested.

Comment 2. In Biological systems absolute values of various parameters usually vary greatly. In this respect, ratios of the parameters provide more stable data. Have you measured bulb/clove density (m/V ratio)? What about bulb diameter/height ratio? Or various Sulphur derivative ratio???

Response: Thank you for the suggestion. Our main goal was to use CPVO descriptors for the morphological description. We agree with your point for additional quantitative traits, but unfortunately, we haven’t measured bulb/clove density or bulb diameter/height ratio.

Comment 3. Figure 2D: add units for the ordinate axis.

Response. This is not possible, as different Units are employed for each quantitative trait. Therefore, the y-axis is numerical, while all the related information is presented in Supplementary Table S4. Nonetheless, to clarify your point, we have included units for each trait in Figure’s legend.

Comment 4. References: use  Italics for Allium sativum (ref 1,2,11,12,13,14,16,17 etc)

- Use bold letters for the number of years

-  number of journals’ volumes should be in Italics 

Response: Reference list has been updated as suggested.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

To the authors

Thank you for the correction.
I believe I have made the corrections to the main text.
As a minor correction, I think it would be better to write the symbols indicating the genetic diversity index (Na, Ho, He, uHe, ΔK, I) in italics. In the main text and S Table S4, 7, etc.

Also, please standardize the numbers after the decimal point in figures and tables throughout the main text to two or three digits.

The following are minor corrections.
L217: μl → μL
L397: P < 0.05 → p< 0.05
L712: AsESSR91 → AsESSR-91

References
No.20: Allium sativum → italics
No.54, 67: Allium → italics

No.35, 42, 43, 46, 47, 48, 49, 52, 53, 56, 62, 68, 70, 78, 79: Allium sativum L., Solanum lycopersicum L., Vigna unguiculata (L.), Allium cepa L., Allium porrum L.

→ Do not use italics for L.

Author Response

We wish to thank the reviewer for the constructive comments that helped us further improve our manuscript.

Comment 1: As a minor correction, I think it would be better to write the symbols indicating the genetic diversity index (Na, Ho, He, uHe, ΔK, I) in italics. In the main text and S Table S4, 7, etc.

Response 1: In the revised text, the symbols related to genetic diversity indices are written in italics.

 

Comment 2: Also, please standardize the numbers after the decimal point in figures and tables throughout the main text to two or three digits.

Response 2: Corrected through the text, figures and tables.

 

Comment 3: The following are minor corrections.
L217: μl → μL
L397: P < 0.05 → p< 0.05
L712: AsESSR91 → AsESSR-91

Response 3: Revised.

 

Comment 4: References
No.20: Allium sativum → italics
No.54, 67: Allium → italics

No.35, 42, 43, 46, 47, 48, 49, 52, 53, 56, 62, 68, 70, 78, 79: Allium sativum L., Solanum lycopersicum L., Vigna unguiculata (L.), Allium cepa L., Allium porrum L.

→ Do not use italics for L.

Response 4: Corrected.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The comments were accepted. Inaccuracies were corrected. The article can be recommended for publication in its present form.

Author Response

Comment: The comments were accepted. Inaccuracies were corrected. The article can be recommended for publication in its present form.

 

Response: Thank you for helping us improve and strengthen our manuscript.