Genetic Diversity of Olive (Olea europaea L.) Cultivars Assessed by Genotyping-by-Sequencing in Southern Peru

1.     The title should be read as one concise sentence with a clear, precise scientific meaning.

Reply:

Thank you for the suggestion. In response, we have revised the title to present a clearer and more concise scientific meaning. The updated title reflects the main objective and methodology of the study while maintaining scientific precision.

2.     As olive varieties are usually propagated by cutting as clones, plants of the same variety have identical genetic material, i.e. genomic DNA. It is referred that the environment of the varieties growing and the depth of sequencing presented the difference to affluence the genotyping results of GS. And another reason, some olive varieties may be genetically more closely related to each other, with less pronounced genetic differentiation and difficult to distinguish at the whole genome level, making their resequencing results somewhat similar, but there may also be subtle differences. It is suggested that these are included in the discussion section.

Reply:

Thank you for this insightful comment. We agree that clonal propagation, environmental conditions, sequencing depth, and close genetic relatedness among certain olive cultivars are critical factors that may influence GBS-based genotyping results. In the revised version of the manuscript, we have expanded the Discussion section to address these aspects in more detail and provide a more nuanced interpretation of the observed patterns of genetic variation. Line 337.

3.     In the results section of the morphological data, it is suggested to include discussions on the possible causes and significance of the morphological variation. It is suggested to analyse how the differences in tree vigour or leaf shape would affect olive growth and yield, or how fruit shape and size could affect olive oil production. And it could present the correlation analysis of the morphological characteristics.

Reply:

We sincerely appreciate this insightful suggestion. In the revised version of the manuscript, we have expanded the discussion of the morphological results by analyzing the potential agronomic implications of key traits such as tree vigor, fruit and endocarp morphology, and leaf shape. Additionally, we recognize the value of performing correlation analyses to identify relationships among phenotypic traits. However, due to limitations in experimental design and the heterogeneous nature of the collected data—primarily qualitative and based on standardized morphological descriptors—we were unable to conduct a statistically robust correlation analysis at this stage. Line 307.

4.     If possible, increase the sample size for genomic analysis to include more olive varieties and individual plants. This would enhance the representativeness of the data and improve the reliability of genetic diversity estimates and population genetic inferences.

Reply:

We appreciate the recommendation to increase the sample size for genomic analysis. We fully agree that including a larger number of varieties and individual plants would enhance the representativeness of the dataset and improve the robustness of genetic diversity estimates and population-level inferences. However, due to logistical constraints during this phase of the study, expanding the sample size was not feasible. Field collection required travel to geographically dispersed cultivation areas, and unforeseen limitations in laboratory access prevented further DNA extraction and sequencing. Additionally, the available time frame to complete the research was limited.

5.     It is suggested to optimize experimental replication, i.e. to increase experimental replication, such as conducting independent DNA extraction and sequencing experiments on different batches of samples, to assess the reproducibility of the results and strengthen their reliability.

Reply:

We appreciate this valuable suggestion. We agree that increasing experimental replication—such as performing independent DNA extractions and sequencing across different batches—would have strengthened the assessment of reproducibility and enhanced the reliability of the findings. However, due to time constraints and limited access to laboratory facilities during the final phase of the study, we were unable to implement this approach.

6.     The results section mentions that Ascolana and Frantoio have high genetic diversity, while Arbequina and Leccino have low diversity. However, the potential applications or implications of these results are not sufficiently emphasised. The value of high-diversity varieties in breeding programmes or the need for conservation strategies for low-diversity varieties would be highlighted. More attention to these key findings would enhance the practical significance of the research results.

Reply:

Thank you for this valuable observation. In the revised manuscript, we have expanded the Discussion section to highlight the practical implications of our findings on genetic diversity. Specifically, we have emphasized the potential use of highly diverse cultivars (Ascolana and Frantoio) in local breeding programs, given their broader allelic base and potential agronomic advantages. Likewise, we have addressed the need to implement conservation strategies for low-diversity cultivars such as Arbequina and Leccino, which, despite their commercial appeal, may be more vulnerable to environmental stress and genetic erosion. Line 360.

7.     The results section describes morphological and genetic data separately, but does not adequately explore the relationships between them. It would be useful to analyse whether certain morphological traits are associated with specific genetic variations, or whether genetic clustering corresponds to morphological similarity. There is room for improvement in the integration of morphological and genetic results, which would provide a more comprehensive understanding of olive cultivar diversity.

Reply:

Thank you for this valuable observation. We fully agree that integrating morphological and genetic data could offer a more comprehensive understanding of olive cultivar diversity. While our study analyzed both data types independently, this approach was primarily due to time and resource constraints. A formal integrative analysis—such as assessing the correspondence between morphological traits and genetic clustering—was beyond the scope of the current work but is recognized as an important direction for future research. We have added a comment in the discussion section to acknowledge this limitation and highlight the potential of combined analyses in subsequent studies. Line 332.

8.     In the discussion section, it is suggested that the findings of this study are compared with those of similar studies to provide a broader research context. This would help readers to better understand the significance and contributions of this research.

Reply:

Thank you for your valuable suggestion. In response, we have incorporated a comparative reference to a similar study in the Discussion section. This addition provides broader research context and helps to emphasize the significance and relevance of our findings in relation to previous work on olive cultivar diversity using genotyping-by-sequencing approaches. Line 382.

9.     Line 33-35, ‘La producción oleícola se concentra principalmente en el sur del país, siendo la egion de Tacna la de mayor crecimiento, especialmente en las zonas de La Yarada, Los Palos, Magollo y Sama[7]’ would be corrected to English writing.

Reply:

Thank you for pointing this out. The sentence has been translated into English to ensure consistency with the rest of the manuscript. Line 34.

10.  Figure 7, the title of X axis and Y axis appeared with non-English language and would be corrected to English writing.

Reply:

Thank you for your comment. The X and Y axis titles in Figure 7 (Now Figure 11) have been corrected and are now presented in English in the revised version of the manuscript.

11.  There are some difference between the abstract of review web and the article manuscript.

Reply:

Thank you for the observation. It is possible that an earlier version of the abstract was retained on the submission platform at the time of upload. We have reviewed and confirmed that the abstract included in the manuscript is the final and updated version. We kindly suggest using this version for evaluation and publication purposes.

1.     Abstract: Please indicate that Ascolana, Frantoio, Arbequina, and Leccino are varieties of olives.

Reply:

Thank you for your suggestion. In response, we have revised the abstract to explicitly indicate that Ascolana, Frantoio, Arbequina, and Leccino are olive cultivars.

2.     Lines 33-35: These sentences are in Spanish.

Reply:

Thank you for pointing this out. The sentence previously written in Spanish has been translated into English in the revised version of the manuscript. Line 34.

3.     Line 84: The morphological description needs to be more explicit. For example, what do terms like "vigor" and "growth habit" mean? How was canopy density determined? Also, regarding leaf traits, please specify whether you used young or old leaves. Since you mention positions in the organs evaluated, consider adding an image that indicates the measurements of these traits.

Reply:

Thank you for this detailed and helpful comment. In response, we have revised the Morphological Characterization section to provide more explicit definitions of key tree traits. Specifically, we clarified that "vigor" refers to the overall abundance of vegetative growth, "growth habit" describes the natural orientation of branches and shoots, and "canopy density" refers to the abundance of canopy vegetation. Line 97.

For canopy density, we included a three-level classification (sparse, intermediate, dense) based on foliage thickness and light penetration. Line 116.

Additionally, we specified that mature leaves, aged between 6 months and 1 year, were used for leaf trait assessment to ensure consistency. Line 134

To further support the evaluation of positional traits and measurement references, we added three labeled figures that illustrate the key morphological characteristics and measurement points for leaves (Figure 1), fruits (Figure 2), and endocarps (Figure 3).

4.     Figure 1: This could include a composite image of the different traits measured and the tissue used for DNA extraction.

Reply:

Thank you for the suggestion. In this study, DNA extraction was performed exclusively from apical shoots, as shown in Figure 4. Therefore, we have not included a composite image of all measured traits and tissues, since morphological characterization and DNA sampling were conducted on different plant organs. To maintain clarity, we retained a specific image illustrating the tissue used for genomic analysis.

5.     Lines 129-131: Please clarify the criteria used for classifying vigor as high, medium, or low.

Reply:

Thank you for your comment. In response, we have added a detailed explanation of the criteria used to classify tree vigor into low, medium, or high categories. The classification was based on tree height and trunk diameter, and applied only to trees aged five years or older to ensure consistent developmental stages. Line 99.

6.     Line 132: A more detailed explanation of how growth habit types are established in the methodology is necessary.

Reply:

Thank you for the comment. We have added a more detailed explanation of how growth habit types were classified in the methodology section. The classification includes “open” and “upright” types, based on branch orientation and apical dominance. Line 109.

7.     Line 137. An image showing the phenotype of each cultivar is required.

Reply:

Thank you for the suggestion. In the revised manuscript, we have added a figure showing the representative phenotype of each olive cultivar included in the study. The image illustrates typical leaf, fruit, and endocarp morphology for all ten varieties, as part of the morphological assessment. Figure 5

8.     Line 141: Is it possible to indicate the standard deviation for the data? Are the endocarp values presented means?

Reply:

Thank you for your comment. We have clarified in the manuscript that the endocarp values presented correspond to the average of six samples per cultivar. Line 122.

9.     Table 2: How was vigor measured? Please include this information in the methodology. I do not fully understand how the categories (small, medium, large) for this trait were established.

Reply:

Thank you for your observation. We have added a detailed explanation in the Materials and Methods section describing how tree vigor was measured and how the categories (low, medium, high) were established. Line 89

10.  Figure 2: This figure is presented without a preceding description in the main text.

Reply:

Thank you for your comment. In the revised manuscript, we have added a description of Figure 2 (Now Figure 6) within the main text. Line 216

11.  Line 145. The calculation method for the Shannon-Weaver diversity index is not described in the methodology.

Reply:

Thank you for your observation. In response, we have added a detailed explanation of how the Shannon-Weaver diversity index was calculated, including the mathematical formula and a reference to the method. Line 136

12.  Figure 4: What does "MQ0" refer to in this image?

Reply:

Thank you for your comment. In the revised version of the manuscript, we have corrected the label in Figure 4 (Now Figure 8) from “MQ0” to “MQ = 0” for clarity. Additionally, we have added an explanation in the main text to clarify the meaning of this term. Line 239

13.  Table 10: This table could be placed in the supplementary files.

Reply:

Thank you for the suggestion. We acknowledge the option to move Table 10 to the supplementary materials; however, we believe this table presents key information that is directly relevant to the main findings and discussion of the manuscript.

14.  Subtitle 3.7: A more thorough description of the results is required. Additionally, there is no description of Figure 8 in the main text.

Thank you for your comment. In the revised manuscript, we have expanded the description under Subtitle 3.7 to provide a more detailed interpretation of the PCA results (Line 270).We have also added a clear reference to Figure 8 (Now Figure 12) in the main text, including an explanation of the grouping patterns and their consistency with the phylogenetic analysis.

1.     Comment 1: Acronyms in parentheses in the title can disrupt the flow and reduce the impact.

Reply:

Thank you for your suggestion. In response, we have revised the title to improve clarity and flow by removing the acronym in parentheses.

2.     Comment 2: The study lacks a clearly stated hypothesis. While the aim to “assess genetic diversity” is valid, articulating a specific hypothesis (e.g., expected clustering based on known origin or phenotypic traits) would strengthen the scientific framework.

Reply:

Thank you for the suggestion. In response, we have added a clearly stated hypothesis at the end of the Introduction section, linking the genetic and morphological components of the study to strengthen the scientific framework. Line 72

3.     Comment 3: Why was Genotyping-by-Sequencing (GBS) selected over other genotyping methods (e.g., SSR, SNP arrays) for this study?.

Reply:

Thank you for your comment. In response, we have added a paragraph at the beginning of the Genotyping-by-Sequencing subsection explaining why GBS was chosen over other genotyping methods such as SSR or SNP arrays. Line 161

4.     Comment 4: How were the 10 olive varieties chosen? Do they represent the full genetic or geographic diversity of olives cultivated in Peru?.

Reply:

Thank you for your comment. We have added a paragraph in the Materials and Methods section explaining the criteria used to select the ten olive cultivars. Line 89

5.     Comment 5: Were technical replicates or validation steps (e.g., duplicate sequencing or PCR confirmation) used to ensure data accuracy?.

Reply:

Thank you for your question. Due to time constraints and limited laboratory resources during the final phase of the study, technical replicates (such as duplicate sequencing or PCR validation) could not be performed.

6.     Comment 6: How do the diversity indices (e.g., observed vs. expected heterozygosity, Fst) compare with those reported in Mediterranean or North African olives?.

Reply:

Thank you for your observation. While we did not calculate specific diversity indices such as observed/expected heterozygosity or F_ST, we have included a comparative discussion referencing a GBS-based study involving Mediterranean cultivars. Our results show consistent patterns of genetic clustering and variability. We have also clarified in the discussion that the inclusion of population-level indices will be addressed in future research to strengthen comparisons with Mediterranean and North African olive germplasm.

7.     Comment 7: How can the results of this study inform breeding, conservation, or expansion of olive cultivation in Peru?.

Reply:

Thank you for this important observation. In response, we have added a paragraph highlighting the practical implications of our findings for breeding, conservation, and the expansion of olive cultivation in Peru. Line 334

8.     Comment 8: Is the raw sequencing data publicly available (e.g., NCBI SRA, Dryad)? If not, do the authors plan to deposit it?

Reply:

Thank you for your question. As indicated in the Data Availability Statement, the raw sequencing data will be made available by the authors upon reasonable request. In addition, we are currently discussing with the research team the possibility of depositing the data in a public repository.

1.     The authors mention shallow sequencing depth, which limits detection of rare variants and may affect genotype calling. Although acknowledged, this limitation should be discussed more explicitly—especially in terms of trade-offs and recommendations for future work. This value looks so strange to me, it is quite low, including for olger NGS technologies.

Reply:

Thank you for your insightful comment. In the revised version of the manuscript, we have expanded the Discussion section to provide a more explicit explanation of the limitations associated with the shallow sequencing depth used in this study. We clarified that the sequencing strategy was chosen based on budgetary constraints and the aim of broad genomic sampling, and we acknowledged its impact on the detection of rare variants and genotype call quality. Line 373

2.     The authors note a high duplication rate in reads but do not elaborate on how this may have affected SNP detection or downstream diversity metrics. A brief discussion of the likely causes (e.g., over-amplification during library prep) and future mitigation strategies (e.g., UMIs) would strengthen the manuscript.

Reply:

Thank you for your comment. We have expanded the Discussion section to include a brief explanation of how high duplication rates may have affected SNP detection and diversity estimates. We also noted that over-amplification during library preparation is a likely cause and suggested future mitigation strategies, including the use of Unique Molecular Identifiers (UMIs) and PCR protocol optimization. Line 348

3.     While both datasets are analyzed, the manuscript would benefit from a combined statistical treatment (e.g., AMOVA, Mantel test, or multivariate ordination) to assess concordance or discordance between morphological traits and genetic clustering.

Reply:

Thank you for your suggestion. We agree that a combined statistical analysis (e.g., AMOVA, Mantel test, or multivariate ordination) could provide additional insights into the relationship between morphological traits and genetic clustering. However, due to time constraints and the scope of the current study, we were unable to perform an integrated analysis..

4.     The study includes three samples per cultivar, but the extent of variation within cultivars is not explored in depth. It would be helpful to clarify whether variation is mostly inter- or intra-cultivar and how that informs the interpretation of clonal stability.

Reply:

Thank you for this observation. The study design included three samples per cultivar to capture representative variability; however, it was not intended to support a robust statistical assessment of intra-cultivar variation. In the revised version of the manuscript, we have clarified that most of the observed genetic variability was distributed between cultivars, with minimal differences detected within each variety. This pattern is consistent with expectations for clonally propagated crops such as olive.

5.     Please consider including a statement indicating where the raw sequencing data and processed variant files will be made available (e.g., NCBI SRA or Zenodo).

Reply:

Thank you for your suggestion. We are currently consulting with the other members of the research team regarding the possibility of depositing the raw sequencing data and processed variant files in a public repository.

6.     Some figures (e.g., Figure 4 and the phylogenetic tree) appear pixelated or have limited resolution. Higher-quality images would improve readability.

Reply:

Thank you for your comment. The resolution of the affected figures, including Figure 4 and the phylogenetic tree, has been improved in the revised version of the manuscript to ensure better readability and presentation quality.

7.     The manuscript is generally well-written, but several long and redundant sentences would benefit from editing for clarity. A light revision by a native English speaker is recommended.

Reply:

Thank you for your observation. The manuscript has been carefully revised to improve clarity and conciseness.