Morphological and Molecular Insights into Genetic Variability and Heritability in Four Strawberry (Fragaria × ananassa) Cultivars

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The research paper primarily analyzes genetic variability and heritability of four Strawberry (Fragaria × ananassa) Cutivars by assessment morphological and molecular feature. However, The research paper lacks key findings and innovation. There are serious issues with the writing of the article, including incomplete description of results, absence of relevant research methods, and a discussion that merely summarizes the results without exploring important conclusions. Therefore, both the results and writing of this paper require further improvement.

1. The abstract section should focus on the description of results, rather than experimental methods. For example, ‘Phylogenetic analysis was performed using MEGA 11 software, while statistical evaluations included AMOVA (GenAIEx), correlation (OriginPro), and descriptive statistics based on standard agronomic methods. Additionally, the degree of cross-compatibility and pollen viability among the cultivars were studied, and their significance in cultivar hybridization was analyzed.’
2. The Introduction section should be improved. Authors should be written around the main research content of the article. Line 83-89, it seems unrelated to the research content. In this section, authors should introduction the aim and objective of the article.
3. The section 2.2 and 2.3 should be written together. Line 140, the detailed evaluation method needs to be described.
4. There is a lack of experimental Such as, AMOVA Analysis/ Pollen Fertility/ Intercrossing of Strawberry Cultivars. Please provide a detailed introduction
5. The results of Figure2 loss, where is the description?
6. AMOVA Analysis section, It is basically a reference, rather than a result.
7. Line 327-334, where is the support results?
8. Pollen Fertility section, It is basically a reference.
9. The Discussion section should be improved, authors should be discuss innovative aspects, difference and explain the reason.

Author Response

Response to Reviewer 1:

General comment: The research paper primarily analyzes genetic variability and heritability of four Strawberry (Fragaria × ananassa) Cutivars by assessment morphological and molecular feature. However, The research paper lacks key findings and innovation. There are serious issues with the writing of the article, including incomplete description of results, absence of relevant research methods, and a discussion that merely summarizes the results without exploring important conclusions. Therefore, both the results and writing of this paper require further improvement.

General response: Thank you for your constructive feedback. In response, we have substantially revised the manuscript, rewriting approximately 60% of the content to improve clarity, highlight key findings, and enhance the scientific contribution. Major updates were made to the Results, Methods, and Discussion sections to provide deeper analysis, correct methodological gaps, and better articulate the study's novelty and implications.

Comment #1: The abstract section should focus on the description of results, rather than experimental methods. For example, ‘Phylogenetic analysis was performed using MEGA 11 software, while statistical evaluations included AMOVA (GenAIEx), correlation (OriginPro), and descriptive statistics based on standard agronomic methods. Additionally, the degree of cross-compatibility and pollen viability among the cultivars were studied, and their significance in cultivar hybridization was analyzed.’

Response #1: The suggested revision to the abstract was implemented accordingly and can be found on page 1, lines 41-43.

Comment #2: The Introduction section should be improved. Authors should be written around the main research content of the article. Line 83-89, it seems unrelated to the research content. In this section, authors should introduction the aim and objective of the article.

Response #2: Additional information has been added into the “Introduction” to better align it with the main research focus. The revised content reflecting the study’s aim and objectives can be found on page 2, lines 86-93 and 110-116. The unrelated content previously located in lines 83-89 has been removed.

Comment #3: The section 2.2 and 2.3 should be written together. Line 140, the detailed evaluation method needs to be described.

Response #3: Sections 2.2 and 2.3 have been merged into a single section titled “Experimental Design and Morphological Evaluation Method”. Additionally, the detailed evaluation method referenced in line 140 has been fully described in Section 2.2 “Experimental Design and Morphological Evaluation Method ” now found on lines 151-182.

Comment #4: There is a lack of experimental Such as, AMOVA Analysis/ Pollen Fertility/ Intercrossing of Strawberry Cultivars. Please provide a detailed introduction

Response #4: Detailed information regarding the AMOVA analysis has been provided on page 5 in the relevant lines 229-234. The section on Pollen Fertility has been elaborated in lines 235-251 of page 5. Additionally, Explanations related to intercrossing have been incorporated into the revised manuscript in lines 252–262 to address this comment.

Comment #5: The results of Figure2 loss, where is the description?

Response #5: The detailed description of the data presented in Figure 2 has been provided on pages 12, lines 399-403 of the revised manuscript.

Comment #6: AMOVA Analysis section, It is basically a reference, rather than a result.

Response #6: The literature review portion within the AMOVA analysis section has been removed, and the section was revised to focus solely on experimental findings and interpretations.

Comment #7: Line 327-334, where is the support results?

Response #7: Supporting data and relevant evidence have been included in the revised version on page 16, lines 500-512 to substantiate the statements made in lines 327-334.

Comment #8: Pollen Fertility section, It is basically a reference.

Response #8: The reference-based discussion in the Pollen Fertility section has been removed. This part has been revised to present only the results and observations derived from the current study.

Comment #9: The Discussion section should be improved, authors should be discuss innovative aspects, difference and explain the reason.

Response #9: Revisions have been made to the Discussion section on pages 20, lines 630-637 and 654-663, The section was enhanced by elaborating on the innovative findings of the study, interpreting differences between cultivars, and providing explanations based on morphological and genetic data.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

• Line 40 Removed the unnecessary comma after using
• Line 41 polymorphism analysis, which revealed polymorphism
• Line 47 and their significance for cultivar hybridization was analyzed
• Line 58 cultivated worldwide for its attractive aroma
• Line 68 an allo-octoploid species (2n = 8x = 56)
• Line 74 diversity among subgenomes
• Line 86 terpenes, contribute to the unique flavor
• Line 93 remain a major challenge due
• Lines 211–213 The text states that Alba flowers measure only 1.2 × 1.2 cm, whereas in Seolhyang, Cinderella, and Vitaberry the average diameter is 3.5–4.0 cm. This is inconsistent and likely a typographical error (perhaps 3.2 × 3.2 cm or 4.2 × 4.2 cm). Review the measurements of Alba flowers; there is a discrepancy compared with the other cultivars.
• Lines 200–206 and 237–240: The qualitative text describes 12–15 flowers in Cinderella and 15–18 in Vitaberry. However, in Table 2 (line 247 onward), the mean values are 15.2 flowers (Cinderella) and 21.4 flowers (Vitaberry). Clarify which data were used for the discussion: the qualitative description or the statistical analysis?
• Abstract, Lines 49–51 reports a global negative correlation (r = –0.688) but in Results, Lines 275–277 for Alba, a positive correlation appears (r = 0.501). Clarify that the negative correlation is general, but that differences exist by cultivar, highlighting the case of Alba.
• Lines 284–287 - 89 markers tested, 31 polymorphic, 22 monomorphic, and 36 with no amplification. The text does not clarify whether the 36 were discarded due to technical failure (DNA, primers, PCR) or if they are genuinely not applicable. Include a discussion of the causes for the lack of amplification in 36 markers.
• In addition, only 50 alleles were detected (lines 287–288), well below the 7–16 alleles per locus range reported in the literature. Justify why allele diversity was low and how this impacts the robustness of the analysis.
• Lines 312–317 and 326 onward - The text uses the term “populations,” but only four commercial cultivars were evaluated. Replace with “within cultivars” or justify the definition of “population” in the context of this study.
• Lines 353–357 and Table 3 (line 358 onward): Cinderella shows a coefficient of variation (CV) of 12.77%, much higher than the others (3–6%).
• The text does not discuss this heterogeneity. Explain the high variation in pollen fertility in Cinderella and its implications for its recommendation as a parent in crosses.
• The study uses only four cultivars (lines 120–126), three from Korea and one from Italy. Justify how these results can be extrapolated to broader breeding programs for × ananassa.
• I believe the conclusion text is too long.
• He/FST is in italics
• Line 567 - Journal names need to be in italics – check all
• Line 591 - Author names should not appear in bold
• There are journals that are not abbreviated (eg., Line 493, 502, 526, 550, 579)

Author Response

Comment #1: Line 40 Removed the unnecessary comma after using

Response #1: The punctuation was corrected as suggested.

Comment #2: Line 41 polymorphism analysis, which revealed polymorphism

Response #2: The sentence was revised to eliminate redundancy and improve clarity (Lines 41-42).

Comment #3: Line 47 and their significance for cultivar hybridization was analyzed

Response #3: This part of the sentence was edited for clarity and accuracy (Line 50).

Comment #4: Line 58 cultivated worldwide for its attractive aroma

Response #4: The sentence was revised to improve grammatical flow (Page 2, Line 61).

Comment #5: Line 68 an allo-octoploid species (2n = 8x = 56)

Response #5: The ploidy information was confirmed and reformatted for clarity (Page 2, Line 71).

Comment #6: Line 74 diversity among subgenomes

Response #6: The sentence was revised to enhance scientific precision (Page 2, Line 77).

Comment #7: Line 86 terpenes, contribute to the unique flavor

Response #7: This sentence was removed due to its limited relevance to the research focus.

Comment #8: Line 93 remain a major challenge due

Response #8: The sentence was corrected for better clarity and structure (Page 3, Lines 104-105).

Comment #9: Lines 211–213 The text states that Alba flowers measure only 1.2 × 1.2 cm, whereas in Seolhyang, Cinderella, and Vitaberry the average diameter is 3.5–4.0 cm. This is inconsistent and likely a typographical error (perhaps 3.2 × 3.2 cm or 4.2 × 4.2 cm). Review the measurements of Alba flowers; there is a discrepancy compared with the other cultivars.

Response #9: The correct measurement was confirmed to be 3.2 × 4.2 cm. The reviewer’s observations were fully addressed, and all relevant corrections were made in the manuscript to ensure consistency across cultivars and accuracy of data presentation (Page 10, Line 363).

Comment #10: Lines 200-206 and 237-240: The qualitative text describes 12-15 flowers in Cinderella and 15-18 in Vitaberry. However, in Table 2 (line 247 onward), the mean values are 15.2 flowers (Cinderella) and 21.4 flowers (Vitaberry). Clarify which data were used for the discussion: the qualitative description or the statistical analysis?

Response #10: The inconsistency has been resolved. The text was revised to ensure that the reported values reflect the statistical means derived from the data, not general qualitative descriptions. The corrected values for the Cinderella cultivar appear on Page 9, Line 351, and for Vitaberry on Page 10, Line 370.

Comment #11: Abstract, Lines 49-51 reports a global negative correlation (r = -0.688) but in Results, Lines 275-277 for Alba, a positive correlation appears (r = 0.501). Clarify that the negative correlation is general, but that differences exist by cultivar, highlighting the case of Alba.

Response #11: This issue has been clarified in the Results section (Page 14, Lines 452-456). The revised text explains that the negative correlation (r = -0.688) represents a general trend across cultivars, while Alba exhibited a positive correlation, indicating cultivar-specific variation.

Comment #12: Lines 284-287 - 89 markers tested, 31 polymorphic, 22 monomorphic, and 36 with no amplification. The text does not clarify whether the 36 were discarded due to technical failure (DNA, primers, PCR) or if they are genuinely not applicable. Include a discussion of the causes for the lack of amplification in 36 markers.

Response #12: A clarification has been added to address the amplification failure of the 36 markers. As stated in the revised text, the lack of amplification could be attributed to several factors including poor primer binding, suboptimal PCR conditions, or DNA template quality. This discussion is now included in the Molecular Analysis section to better explain the technical basis for excluding these markers from the analysis.

Comment #13: In addition, only 50 alleles were detected (lines 287-288), well below the 7-16 alleles per locus range reported in the literature. Justify why allele diversity was low and how this impacts the robustness of the analysis.

Response #13: Thank you for your constructive comment regarding the low level of allele diversity observed in our study. Indeed, only 50 alleles were detected across the analyzed loci, which is below the 7-16 alleles per locus range commonly reported in the literature. This discrepancy may be attributed to several key factors: 1) our analysis was based on a relatively narrow genetic base, as only four commercial strawberry cultivars were included. These cultivars often share common breeding backgrounds, resulting in limited polymorphism across standard SSR loci. And 2) only 67 SSR markers were employed in the current study (not 89 as initially misstated), and among these, only 31 were found to be polymorphic. A considerable number of markers (36) did not yield amplification, potentially due to mismatches in primer binding sites, poor DNA quality, or suboptimal PCR conditions. These non-amplified loci were excluded from further analysis to ensure the reliability of the results. Additionally, it should be noted that genotyping in this study was conducted using conventional gel electrophoresis techniques. While effective for basic allele scoring, this method has lower resolution compared to capillary electrophoresis, which can detect subtle size variations and often reveals a higher number of alleles per locus. It is likely that the use of capillary electrophoresis would have resulted in a greater number of detected alleles, thereby increasing the overall allelic richness and sensitivity of the genetic analysis. Despite these limitations, the polymorphic SSR markers used in this study were informative enough to distinguish among the cultivars and assess inter-genotypic variation. Future studies will aim to improve resolution by incorporating a larger and more diverse panel of genotypes and employing high-throughput genotyping technologies to capture a more comprehensive picture of allele diversity and genetic structure.

Comment #14: Lines 312-317 and 326 onward - The text uses the term “populations,” but only four commercial cultivars were evaluated. Replace with “within cultivars” or justify the definition of “population” in the context of this study.

Response #14: The term “populations” was indeed used inappropriately given that the study only analyzed four distinct commercial cultivars. All instances of “population” in the manuscript (Page 16, lines 495-496) were replaced with “within cultivars” to accurately reflect the structure of the genetic analysis. The terminology has been revised to avoid misinterpretation.

Comment #15: Lines 353-357 and Table 3 (line 358 onward): Cinderella shows a coefficient of variation (CV) of 12.77%, much higher than the others (3-6%).

Response #15:  The coefficient of variation (CV) in pollen viability for the Cinderella cultivar was indeed higher (12.77%) compared to the other cultivars (3-6%). This elevated variation is now explained in the revised manuscript (page 18, lines 567-574), highlighting that the broad range of observed pollen viability in Cinderella (67.31%-96.91%) likely stems from environmental heterogeneity during flowering and the high number of pollen grains analyzed. This variability, while notable, does not diminish its potential use as a male parent due to the high average viability and successful compatibility observed in hybridizations.

Comment #16: The text does not discuss this heterogeneity. Explain the high variation in pollen fertility in Cinderella and its implications for its recommendation as a parent in crosses.

Response #16: The observed heterogeneity in pollen fertility within the Cinderella cultivar can be attributed to both genetic and environmental influences. Factors such as elevated temperatures during anthesis, fluctuating humidity, or suboptimal agro-technical practices are known to impact cytoplasmic activity in pollen grains, which may result in partial sterility. Despite this variation, the compatibility index between cultivars remained high, indicating that fertilization success was largely unaffected. Thus, Cinderella retains strong potential as a pollen donor for future breeding programs due to its overall reproductive stability and favorable cross-compatibility profile.

Comment #17: The study uses only four cultivars (lines 120-126), three from Korea and one from Italy. Justify how these results can be extrapolated to broader breeding programs for × ananassa.

Response #17: We appreciate the reviewer’s concern regarding the representativeness of the selected cultivars. This study constitutes one of the first molecular investigations of strawberry (Fragaria × ananassa) conducted at our institute, and therefore serves as a foundational step in establishing molecular breeding infrastructure for this crop. Initially, over 30 cultivars and advanced breeding lines were selected for preliminary screening. However, due to inconsistent or suboptimal molecular data obtained from many of the accessions, primarily caused by poor DNA quality, low amplification success, or marker non-responsiveness, we narrowed our focus to the four cultivars (Seolhyang, Cinderella, Vitaberry, and Alba) that consistently yielded high-quality morphological and molecular results. Although limited in number, these cultivars represent distinct geographical origins (three Korean and one Italian), morphological variation, and agronomic traits. As such, the findings offer valuable insights into genotype-specific performance and inter-cultivar genetic variability, which can inform future breeding programs and assist in selecting suitable parental combinations. This work is intended as a baseline for broader studies involving more diverse germplasm collections.

Comment #18: I believe the conclusion text is too long.

Response #18: The Conclusion section was revised.

Comment #19: He/FST is in italics

Response #19: All typographic inconsistencies, including the formatting of He and F_ST, have been corrected.

Comment #20: Line 567 - Journal names need to be in italics - check all

Response #20: Corrected.

Comment #21: Line 591 - Author names should not appear in bold

Response #21: Edited (page 25, line 903).

Comment #22: There are journals that are not abbreviated (eg., Line 493, 502, 526, 550, 579)

Response #22: Revised.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In general, the manuscript is well-written, clear, and readable. The English language is appropriate and understandable. However, there are several shortcomings related to the Materials and Methods and Results sections.

The Materials and Methods section needs to be reorganized and supplemented. All analyzed parameters should be clearly listed, and the methodology used for their assessment should be described in detail.

In the Results section, it is necessary to avoid duplicate presentation of certain data. Additionally, some numerical values presented in the text and figures should be cross-checked for consistency. Certain subheadings appear to be redundant and could be removed to improve the clarity and flow of the manuscript.

Considering the number of parameters studied, the discussion should be expanded accordingly.

All observed issues have been clearly indicated in the PDF version of the manuscript, with detailed suggestions provided for their correction.

The manuscript can be considered for publication only after major revisions have been made to address all the identified shortcomings.

Comments for author File: Comments.pdf

Author Response

General comment: In general, the manuscript is well-written, clear, and readable. The English language is appropriate and understandable. However, there are several shortcomings related to the Materials and Methods and Results sections.

General response: We sincerely thank Reviewer 3 for the positive assessment of our manuscript's clarity and language quality. In response to the valuable suggestions regarding the Materials and Methods and Results sections, we undertook a comprehensive revision of the manuscript. Nearly 60% of the entire text was rewritten to enhance clarity, logical flow, and scientific rigor. The Materials and Methods section was substantially edited to ensure it meets the required academic and methodological standards. Similarly, the Results section was refined to provide a more precise and comprehensive presentation of findings, with improved alignment between text, figures, and tables.

 Comment #1: The Materials and Methods section needs to be reorganized and supplemented. All analyzed parameters should be clearly listed, and the methodology used for their assessment should be described in detail.

 

Response #1: We have thoroughly revised the Materials and Methods section to improve clarity, structure, and completeness. Specifically, all analyzed parameters, including morphological traits (plant height, crown width, flower number, fruit number, fruit weight), pollen viability, inter-cultivar compatibility, and molecular markers, have been explicitly listed and organized into relevant sub-sections. We have also provided detailed descriptions of the methodologies used for their assessment. Morphological evaluations were conducted following standard UPOV guidelines, pollen viability was assessed via acetocarmine staining and microscopy, and cross-compatibility was analyzed based on fruit set rates after controlled pollination. Additionally, SSR marker analysis procedures, including DNA extraction, PCR amplification, electrophoresis conditions, and scoring criteria, have been elaborated. These revisions can be found on Pages 5 to 6, Lines 192-233, ensuring transparency and reproducibility of the experimental design.

 

Comment #2: In the Results section, it is necessary to avoid duplicate presentation of certain data. Additionally, some numerical values presented in the text and figures should be cross-checked for consistency. Certain subheadings appear to be redundant and could be removed to improve the clarity and flow of the manuscript.

Response #2: We have revised the Results section to eliminate duplicated information and ensured consistency between numerical values reported in the text and those shown in the figures and tables. Redundant subheadings have been removed or consolidated to improve the logical flow and readability of the manuscript.

Comment #3: Considering the number of parameters studied, the discussion should be expanded accordingly.

Response #3: The Discussion section has been expanded to address a broader range of evaluated parameters, including morphological, reproductive, and molecular traits. Additional interpretations and literature comparisons were incorporated (pp. 19-22) to strengthen the scientific context and highlight key findings relevant to breeding and cultivar improvement.

Comment #4: All observed issues have been clearly indicated in the PDF version of the manuscript, with detailed suggestions provided for their correction.

Response #4: We have accepted and incorporated most of these valuable suggestions. Some comments, however, require clarification, and our responses to them are provided below.

Comment: This statement is incorrect; according to the data presented in Graph 3, a negative correlation was also observed in the cultivar Alba (r = -0.279).

Response: In the correlation analysis, the cultivar Alba showed a positive correlation between fruit number and fruit weight (r = 0.501). The reviewer's reference to a negative value (r = -0.279) appears to be a misunderstanding. We have re-examined our statistical results and the positive correlation reported in the manuscript is correct. (Page 14, Lines 454-455)

Comment: The authors should clearly specify the method used to assess pollen viability, including the criteria applied to distinguish viable from non-viable pollen grains, as well as the number of pollen grains analyzed.

Response: The white arrows indicate viable dust particles, and the black arrows indicate sterile dust particles. This separation was performed using acetocarmine staining, and this method and the criteria based on it are included in the 'Materials and Methods' section of our manuscript with clarification. The number of dust particles analyzed is also clearly indicated. (Page 18 Lines 559-561).

Comment: ”from a total of 1301 pollen grains counted”. -This information belongs to the Materials and Method.

Response: This information – specifically, the highest pollen viability percentage observed in the 'Cinderella' cultivar and the total pollen count – represents the findings of our analysis. As such, we believe it is appropriately placed within the 'Results' section of the manuscript, as it directly addresses our research objectives.

Upon a careful review of this data, we have also identified and corrected a technical error: the total number of pollen grains counted was 1501, not 1301. This inaccuracy has been rectified in the revised manuscript (Page 17, line 554).

Comment: The fruit setting rate is the only parameter that requires analysis in the Results section, as the remaining parameters were used solely for its calculation. Accordingly, the authors should provide a clear explanation of how the fruit setting rate was calculated, without presenting the intermediate data.

Response: Thank you for your comment. Your comment about the misuse of the term "fruit setting rate" is very relevant. The purpose of our study was, in fact, to determine the "crossing rate", and your attention helped to clarify this ambiguity. We are pleased with your attention.

In accordance with your recommendation, the phrase "fruit setting rate" in the table has been changed to the phrase "crossing rate".

Additionally, a more detailed explanation of how the cross-fertilization efficiency was calculated is provided in the "Results" section of the manuscript. We have highlighted the main indicators without providing intermediate data in this process.

Comment #5: The manuscript can be considered for publication only after major revisions have been made to address all the identified shortcomings.

Response #5: The suggestions and identified shortcomings will undoubtedly help improve the quality of our work. We have addressed all the major revisions you requested. We've carefully considered each comment individually and have worked to resolve them completely and precisely. Thank you once again for your time and consideration.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Your manuscript “Morphological and Molecular Insights into Genetic Variability and Heritability in Four Strawberry (Fragaria × ananassa) Cultivars” presents a valuable integrative study combining morphological, molecular, and reproductive analyses. The work can contribute significantly to strawberry breeding research.  Please consider the following comments.

- Add clarity on replication details for molecular analysis.

- Consider including multivariate analyses (e.g., PCA) to complement correlations.

- Expand discussion on Alba and Vitaberry’s breeding value.

- Minor English editing for fluency and consistency. Thanks

Author Response

Response to Reviewer 4:

General comment: Your manuscript “Morphological and Molecular Insights into Genetic Variability and Heritability in Four Strawberry (Fragaria × ananassa) Cultivars” presents a valuable integrative study combining morphological, molecular, and reproductive analyses. The work can contribute significantly to strawberry breeding research.  Please consider the following comments.

General response: We appreciate your recognition of the integrative nature of our study. All your comments have been carefully addressed, and the corresponding sections of the manuscript have been revised to enhance clarity, scientific rigor, and overall impact.

Comment #1: Add clarity on replication details for molecular analysis.

Response #1: The manuscript has been updated to clarify that molecular analysis was conducted using DNA extracted from three independent biological replicates per cultivar. These replicates were pooled to ensure consistency and minimize sampling error. This clarification is now included in the revised Materials and Methods section (Lines 197-202).

Comment #2: Consider including multivariate analyses (e.g., PCA) to complement correlations.

Response #2: While a PCA analysis would provide additional insight, due to the limited number of cultivars (n = 4), the dataset lacked sufficient resolution for robust multivariate analysis. Nevertheless, we agree with its value and plan to incorporate PCA in future studies with expanded genotype sets.

Comment #3: Expand discussion on Alba and Vitaberry’s breeding value.

Response #3: The Discussion section (Lines 669-674) has been revised to elaborate on the high flower and fruit counts observed in Alba and Vitaberry. These traits underline their potential as promising donor parents for improving fruit set and yield stability in breeding programs.

Comment #4: Minor English editing for fluency and consistency. Thanks

Response #4: The manuscript has undergone comprehensive language editing to improve grammar, fluency, and consistency throughout. This has enhanced overall readability and clarity of presentation.

Author Response File: Author Response.pdf