Assessment of Genetic Diversity in Walnut (Juglans regia L.) Genotypes from Southern and Southeastern Kazakhstan Using Microsatellite Markers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript Assessment of Genetic Diversity in Walnut (Juglans regia L.)
Genotypes from Southern and Southeastern Kazakhstan Using Microsatellite Markers by Nurzhuma et al. is well written and covers an interesting topic.

This study assessed the genetic diversity of walnut (Juglans regia) in southern Kazakhstan using eight SSR markers. High genetic variation was found, especially in the Turkestan region. Analyses revealed two distinct genetic groups, highlighting the importance of conserving and utilizing local walnut resources for breeding.

Introduction provides important details about the topic of this study. However, remove the paragraphs that are more related to popularization like lines 37-40, 44-47, 58-68, than to the scientific part.

M&M: Table 1 is large and should be moved to supplementary materials.

lines 170-171: indicate the number of trees taken in the study (75?}

Results: Figure 5 - is not clearly visible.

Figure 6 -explain in an additional table what each point in the graph means. leave in the caption what the figure represents only. Further explanations are part of the text.

Discussion: lines 384-390 this paragraph should be moved to the next section, Conclusions.

Author Response

Question 1. Introduction provides important details about the topic of this study. However, remove the paragraphs that are more related to popularization like lines 37-40, 44-47, 58-68, than to the scientific part.

Answer. We partially agree with your comment. We agree that the text in lines 44-47 can be deleted. However, we believe that the information on the current status of walnut cultivation and use in various regions of the world, presented in lines 37-40 and 58-68, is important for understanding the problem. Therefore, we considered it necessary to leave it in the article.

Question 2. M&M: Table 1 is large and should be moved to supplementary materials.

Answer. We agree with your comment. Table 1 is moved to supplementary materials – Supplementary Table 1.

Question 3. lines 170-171: indicate the number of trees taken in the study (75?}

Answer.  In 2024, five young leaves were collected from each of 75 walnut trees during the active growth phase. Thus, the total number of trees included in the study is 75.

Question 4. Results: Figure 5 - is not clearly visible.

Answer . We agree with your comment. To improve clarity, we have improved Figure 5.

Question 5. Figure 6 -explain in an additional table what each point in the graph means. leave in the caption what the figure represents only. Further explanations are part of the text.

Answer.  We did not create an additional table explaining what each point on the graph means. The additional table Table S1 provides information on the studied genotypes. It lists Collection sites: key geographic information for each sampling location in the southern and southeastern regions of Kazakhstan. This table S1 also explains what the abbreviations mean: Turkestan region (Sairam-Ugam, S-U; Mashat, MA; Sarkyrama, SA); Almaty region (Almaty, AL; Baidibek-bi, BA and Esik, ES). Each point in the graph of Figure 6 represents a walnut sample collected in a different geographic region and location: Turkestan region (Sairam-Ugam, S-U; Mashat, MA; Sarkyrama, SA); Almaty region (Almaty, AL; Baidibek-bi, BA and Esik, ES). For example, AL-55 is sample number 55 collected in Almaty city, Almaty region; S-U-1 is sample number 1 collected in Sairam-Ugamб Turkestan region.

Question 6. Discussion: lines 384-390 this paragraph should be moved to the next section, Conclusions.

Answer. We agree with your comment. In the section Discussion: we removed lines 384-390 from the text. However, we did not remove this text to the next section, Conclusions, since these thoughts are already present in the Conclusions section.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The research of this manuscript is very interesting and the theme is also in line with the journal. But some adjustments still need to be made.

Abstract

It is suggested to supplement the explanation on the statistical significance of the data mentioned in the abstract, i.e., "the average expected heterozygosity (He) was 0.704, which was significantly higher than the observed heterozygosity (Ho) of 0.547", such as whether the difference was verified by t-test.

It is suggested that the specific application direction mentioned in the conclusion as "providing a foundation for breeding" be further clarified, such as "it is recommended to prioritize the protection of high-diversity populations in the Turkestan region for the cultivation of stress-resistant varieties".

Keywords

The key words for "walnut" are recommended to be in English instead of the Latin scientific name.

Introduction

It is recommended to use the English term "walnut" throughout the manuscript instead of Latin nomenclature, which can facilitate the search for your research.

It is recommended to replace the data on global walnut production cited from literature published between 2010 and 2019 in the Introduction with the latest production data for the past three years.

It is suggested that the existing statement "genetic resources have not been fully studied" is too vague and should be supplemented with specific details, such as "Kazakhstan has not yet established a national walnut breeding program, and systematic research on polymorphism analysis of SSR markers in local populations is still lacking".

The introduction has too many paragraphs and the content is a bit scattered. It is suggested to condense and summarize it into four to five paragraphs, and at the same time pay attention to the logical connection before and after writing.

Materials and Methods

Please confirm whether the amount and unit of the 192 lines of primers are accurate. If it is impossible to provide an accurate description, it is recommended to delete the unit.

The basis for SSR marker screening needs to be supplemented as follows: "Based on the uniform distribution of the walnut genome and the criterion of polymorphism information content (PIC) >0.5 in previous studies."

Results

The mean genetic distances of the two clusters (0.486 and 0.747) suggest supplementary statistical tests (such as ANOVA) to support the significance of differences between groups.

Almost all the characters in the figure are disproportionate, which affects the expression of the result. It is suggested to make adjustments.

"pop5 has the lowest genetic diversity" can be combined with geographical factors to explain: "The Baidibek Bi population may have allele loss due to isolation of small populations", enhancing the correlation of the results.

Discussion

The analysis of the differences from Turkish walnut (Ne=6.43) can supplement the geographical isolation mechanism: "The Kazakh population is blocked by mountains (such as the Tianshan Mountains), and the gene flow is lower than that in the plain areas of Turkey."

The absence of heterozygosity is attributed to "inbreeding or selection pressure", and it is suggested to further discuss the influence of environmental adaptive evolution in combination with "continental climate screening".

When citing Shamlu et al. (2018), it is necessary to clarify that the research object is "Iranian walnut" to avoid direct analogy with the Kazakh population. It can be supplemented that "the genetic background differences of walnuts in Central Asia may stem from the history of introduction along the Silk Road".

Conclusions

The description of the conclusions is overly macroscopic, lacking the support of key data, and fails to highlight the research outcomes and their necessity.

References

The references are overly outdated, with a little few dating from the past five years.

While there are numerous references to related studies, they are not adequately engaged or sufficiently incorporated into the research.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Abstract

Question 1. It is suggested to supplement the explanation on the statistical significance of the data mentioned in the abstract, i.e., "the average expected heterozygosity (He) was 0.704, which was significantly higher than the observed heterozygosity (Ho) of 0.547", such as whether the difference was verified by t-test.

Answer. We agree with your comment. The average expected heterozygosity (He) was 0.704, which is significantly higher than the observed heterozygosity (Ho) of 0.547. The difference was confirmed by the t-test. Thus, the average expected heterozygosity (He = 0.704) significantly exceeds the observed heterozygosity (Ho = 0.547), which is confirmed by the paired t-test (t = -6.426, p < 0.05). Thus, the difference between He and Ho is statistically significant.

Question 2. It is suggested that the specific application direction mentioned in the conclusion as "providing a foundation for breeding" be further clarified, such as "it is recommended to prioritize the protection of high-diversity populations in the Turkestan region for the cultivation of stress-resistant varieties".

Answer. We agree with your comment. Future research should focus on conserving the highly diverse populations in the Turkestan region to facilitate the development of stress-tolerant varieties. These findings highlight the importance of conserving and harnessing the genetic resources of Kazakhstan’s J. regia populations for future breeding efforts.

Keywords

Question 3. The key words for "walnut" are recommended to be in English instead of the Latin scientific name.

Answer. We agree with your comment. We have replaced the keywords for the word "walnut" in English, not the Latin scientific name.

Introduction

Question 4. It is recommended to use the English term "walnut" throughout the manuscript instead of Latin nomenclature, which can facilitate the search for your research.

Answer. We agree with your comment. We have replaced for the word "walnut" in English, not the Latin scientific name in some places the manuscript.

Question 4. It is recommended to replace the data on global walnut production cited from literature published between 2010 and 2019 in the Introduction with the latest production data for the past three years.

Answer. We agree with your comment. We have provided data for the last 3 years, to the extent that FAO statistics were available. According to the latest data from FAOSTAT, global walnut production has steadily increased over the past three years, reaching approximately 3.1 million tonnes in 2021, 3.2 million tonnes in 2022, and an estimated 3.3 million tonnes in 2023. This growth reflects the rising demand for walnuts worldwide and highlights the importance of sustainable cultivation practices to meet future market needs

 

Question 5. It is suggested that the existing statement "genetic resources have not been fully studied" is too vague and should be supplemented with specific details, such as "Kazakhstan has not yet established a national walnut breeding program, and systematic research on polymorphism analysis of SSR markers in local populations is still lacking".

Answer. We agree with your comment. Kazakhstan has not yet established a national walnut breeding program, and systematic research on polymorphism analysis of SSR markers in local populations is still lacking

Question 6. The introduction has too many paragraphs and the content is a bit scattered. It is suggested to condense and summarize it into four to five paragraphs, and at the same time pay attention to the logical connection before and after writing.

Answer. We agree with your comment. We have condensed and summarized it into five paragraphs.

Materials and Methods

Question 7. Please confirm whether the amount and unit of the 192 lines of primers are accurate. If it is impossible to provide an accurate description, it is recommended to delete the unit.

Answer. We agree with your comment. Made amendments to the table.

Question 8. The basis for SSR marker screening needs to be supplemented as follows: "Based on the uniform distribution of the walnut genome and the criterion of polymorphism information content (PIC) >0.5 in previous studies."

Answer. We agree with your comment. The basis for SSR marker screening needs to be supplemented as follows: "Based on the uniform distribution of the walnut genome and the criterion of polymorphism information content (PIC) >0.5 in previous studies."

Results

Question 9. The mean genetic distances of the two clusters (0.486 and 0.747) suggest supplementary statistical tests (such as ANOVA) to support the significance of differences between groups.

Answer. Thank you for your important comment. As per your request, we performed statistical analysis to assess the significance of differences between the average genetic distances of the two clusters (0.486 and 0.747). ANOVA confirmed statistically significant differences between the groups (F = 2.872, p = 0.00018).

Question 10. Almost all the characters in the figure are disproportionate, which affects the expression of the result. It is suggested to make adjustments.

Answer. We agree with your feedback and have made adjustments.

Question 11. "pop5 has the lowest genetic diversity" can be combined with geographical factors to explain: "The Baidibek Bi population may have allele loss due to isolation of small populations", enhancing the correlation of the results.

Answer. We agree with your comment. The Baidibek Bi population may have allele loss due to isolation of small populations", enhancing the correlation of the results.

Discussion

Question 12. The analysis of the differences from Turkish walnut (Ne=6.43) can supplement the geographical isolation mechanism: "The Kazakh population is blocked by mountains (such as the Tianshan Mountains), and the gene flow is lower than that in the plain areas of Turkey."

Answer. We agree with your comment. The Kazakh population is blocked by mountains (such as the Tianshan Mountains), and the gene flow is lower than that in the plain areas of Turkey.

Question 13. The absence of heterozygosity is attributed to "inbreeding or selection pressure", and it is suggested to further discuss the influence of environmental adaptive evolution in combination with "continental climate screening".

Answer. We agree with your comment. "The observed heterozygosity deficit in Kazakhstani walnut (Juglans regia) populations may be explained not only by inbreeding and selection pressure, but also by adaptation to the region's specific continental climate conditions, characterized by extreme seasonal temperature fluctuations and pronounced aridity, which generate intense selective pressures."

Question 14. When citing Shamlu et al. (2018), it is necessary to clarify that the research object is "Iranian walnut" to avoid direct analogy with the Kazakh population. It can be supplemented that "the genetic background differences of walnuts in Central Asia may stem from the history of introduction along the Silk Road".

Answer. We agree with your comment. The genetic background differences of walnuts in Central Asia may stem from the history of introduction along the Silk Road.

 

Conclusions

Question 15. The description of the conclusions is overly macroscopic, lacking the support of key data, and fails to highlight the research outcomes and their necessity.

Answer. We agree with your comment. We sincerely appreciate the reviewer's valuable feedback regarding our conclusions. Мade a change in the conclusions.

References

Question 17. The references are overly outdated, with a little few dating from the past five years. While there are numerous references to related studies, they are not adequately engaged or sufficiently incorporated into the research.

Answer. We agree with your comment. We are grateful to the reviewers for their constructive feedback, which has enabled us to enhance our manuscript. In the revised version, we have: Expanded the literature review to include recent publications (within the past 5 years) that demonstrate contemporary developments in Juglans regia population genetics.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript 'Assessment of Genetic Diversity in Walnut (Juglans regia L.) Genotypes from Southern and Southeastern Kazakhstan Using Microsatellite Markers' by Nurzhuma et al. has been revised. I noted that the authors made changes that improved the ms. Unfortunately, these were not sufficient.

The introduction includes paragraphs that are not exactly appropriate in a scientific journal. If the authors feel they should maintain these paragraphs, as mentioned, they should at least be minimized.

Results: Figures 5 and 6 are not very clear. Moreover, there is no corresponding legend.

Author Response

Question 1. The introduction includes paragraphs that are not exactly appropriate in a scientific journal. If the authors feel they should maintain these paragraphs, as mentioned, they should at least be minimized.

Answer. We agree with your comment. The original sentences have been revised in lines 37–40 and 58–68 for improved clarity and conciseness.

Question 2. Results: Figures 5 and 6 are not very clear. Moreover, there is no corresponding legend.

Answer. Thank you for your comment. We have improved the brightness of Figures 5 and 6. The corresponding legends are provided below each figure as part of the main text.

Author Response File: Author Response.pdf