Molecular Identification of Genetic Diversity and Population Structure in Moroccan Male Date Palm (Phoenix dactylifera L.) Using Inter-Simple Sequence Repeat, Direct Amplification of Minisatellite DNA, and Simple Sequence Repeat Markers
, Ilham Amghar 1, Fatima Gaboun 1, Aicha Habach 4, Reda Meziani 5, Jamal Elfadile 5, Rabha Abdelwahd 1 and Ghizlane Diria 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIntroduction
Some results about the patterns of genetic diversity of palm in the world and especially in North Africa (Tunisia and Algeria) must be added.
L 53. Please add reference
Line 62. It should the genetic diversity and differentiation
L 69. The ideal molecular marker must be polymorphic, reproducible, and most importantly co-dominant.
MM
MM were well described
Results
L 238 – 241. This part is already mentioned in MM section. It can be deleted from the results section and please rephrase the rest of the paragraph accordingly.
In tables, I think (,) must changed by (.) to be uniform with the text
In table 9, what are M and F?
Figure 5 can be removed since the results are already available in table 8.
In table 10, the author showed the genetic distance based on the three markers. As both dominant and co-dominant markers were used, the authors must analyze each maker separately.
The same for PCoA, it is better to check the patterns of differentiation based on each marker separately and then make a comparison
Later, a combination between all markers can be used and discussed
In the PCoA an cluster analysis, I see the that the populations are well differentiated so I expect higher value of PhPiT. In the results I see the this value is relatively small (6%)
Discussion
L 371 – L 374. The authors repeated the results. These should not be shown in the discussion section. They should be discussed
L 377. The authors must discuss why markers were good enough
L 404. Please use the past. Please check the whole text
L 409. Tunis should be Tunisia
English should revised in the whole text
Comments on the Quality of English LanguagePoor English. It must be improved
Author Response
Dear Reviewer 1,
We are grateful to you, for your careful review and the valuable feedback provided. Your insightful comments have been considered to improve current version. Please find below our responses to your comments:
Comment 1: Some results about the patterns of genetic diversity of palm in the world and especially in North Africa (Tunisia and Algeria) must be added.
Response: We appreciate the suggestion to enhance the introduction by including specific results related to the genetic diversity of palms in the world and especially in North Africa. We have made the change throughout our article.
Research on the genetic diversity of the date palm (Phoenix dactylifera L.) has been conducted in various regions. Some of this research has investigated genetic diversity among date palm pollinizers in southern Tunisia, revealing high genetic diversity, and accessions did not group according to their geographical origin (El Kadri et al., 2019).In Iran, a moderate level of genetic diversity has been observed among the studied cultivars (Dehghanian and Sheidai, 2022). In Ethiopia, genetic variations of date palms have been identified as crucial for crop improvement and conservation programs (Ahmed et al., 2021). A study conducted on 102 Algerian date palm samples from the oases of the Biskra region found a high degree of genetic diversity (Abdelkrim et al., 2023).Another study assessed the genetic diversity among thirty-nine date palm cultivars from Tunisia and India using six AFLP (Amplified Fragment Length Polymorphism) markers. The findings indicated a clear separation between Tunisian and Indian cultivars, with accessions grouping according to their geographical origin (Karim et al., 2023).
In Tunisia, a genetic analysis of wild African date palms revealed strong genetic differentiation between Western and Eastern date palms, demonstrating high diversity among North African accessions(Saffar et al., 2022). Indeed, the assessment of genetic diversity provides valuable insights crucial for breeding programs across different regions worldwide.
Comment 2: L 53. Please add reference
Response: Reference information has been added to line 53, as recommended.
Genetic diversity analysis of male date palms (Pollinizers) offers multifaceted benefits for the advancement of date palm cultivation(Bourguiba et al., 2023).
Comment 3: Line 62. It should the genetic diversity and differentiation
Response: Thank you for bringing this to our attention. We have revised line 62 as recommended.
In order to overcome this limitation, molecular markers have been employed to effectively reveal and characterize the genetic diversity differentiation present in date palm.
Comment 4: L 69. The ideal molecular marker must be polymorphic, reproducible, and most importantly co-dominant.
Response: Done, the sentence is revised.
Indeed, an ideal molecular marker for genetic analysis must be polymorphic, reproducible, should possess significant heritability, be applicable to any part of the genome, and exhibit sufficient polymorphism to distinguish closely related genotypes, and preferably be a co-dominant marker.
Comment 5: MM were well described.
Response: Thank you for taking the time to review our work. We appreciate your feedback.
Comment 6: L 238 – 241. This part is already mentioned in MM section. It can be deleted from the results section and please rephrase the rest of the paragraph accordingly.
Response: The necessary revisions have done.
In this study, we observed high genetic diversity in male date palm genotypes. The values obtained for the observed number of alleles (Na), effective number of alleles (Ne), Shannon's information index (I), expected heterozygosity (He), and unbiased expected heterozygosity (uHe) for male date palm genotypes were as follows: 1.820, 1.30, 0.322, 0.199, and 0.200, respectively. In contrast, females exhibited comparatively lower values, with respective figures of 1.318, 1.272, 0.271, 0.172, and 0.176 (Table 7).
Comment 7: In tables, I think (,) must changed by (.) to be uniform with the text.
Response: We appreciate your input and have considered your recommendation carefully. We have made the change throughout our article. Please see the manuscript.
Comment 8: In table 9, what are M and F?
Response: Thank you for your inquiry. In Table 9, 'M' represents males, and 'F' represents females. The necessary change has been made.
Comment 9: Figure 5 can be removed since the results are already available in table 8.
Response: Thank you for your suggestion. we have removed Figure 5 as recommended.
Comment 10: In table 10, the author showed the genetic distance based on the three markers. As both dominant and co-dominant markers were used, the authors must analyze each maker separately.
The same for PCoA, it is better to check the patterns of differentiation based on each marker separately and then make a comparison
Later, a combination between all markers can be used and discussed
Response: Thank you for your feedback. We appreciate your suggestion. in our study we found that the results for each marker exhibited a similar trend to the overall analysis. Therefore, we opted for the global results to provide a comprehensive and deep evaluation of diversity, considering the combined information from all three markers.
Comment 11: In the PCoA an cluster analysis, I see the that the populations are well differentiated so I expect higher value of PhPiT. In the results I see this value is relatively small (6%)
Response: Thank you for your observation. In the PCoA results, we found that the total variation explained by PCoA was 19.66%. Breaking this down, the first three axes (1, 2, and 3) corresponded to 8.38%, 6.26%, and 5.02%, respectively. While these values may seem relatively small, they collectively contribute to the overall differentiation observed in the populations. The small value (6%) for PhPiT, as mentioned in the results, reflects the proportion of total genetic diversity among populations, which aligns with the modest variation explained by the PCoA axes. We appreciate your comment.
Comment 12: L 371 – L 374. The authors repeated the results. These should not be shown in the discussion section. They should be discussed.
Response: We have made the change throughout our article.
Comment 13: L 377. The authors must discuss why markers were good enough
Response: The necessary revisions have done.
Comment 14: L 404. Please use the past. Please check the whole text
Response: The necessary revisions have done.
Comment 15: L 409. Tunis should be Tunisia
Response: Done, the sentence is revised.
Comment 16: English should revised in the whole text
Response: Thank you, we have considered your recommendation.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis research provides valuable insights into Moroccan male date palm diversity, the following points should be addressed to substantiate the conclusions more strongly in my opinion.
Major Suggestions:
- Expand the discussion to provide more insights into how the findings can inform breeding programs and conservation efforts for male date palms specifically.
- Validate the results using additional samples or molecular marker systems. This would strengthen conclusions.
- Complement the molecular data with phenotypic diversity assessments of the male genotypes. This allows checking consistency with field observations.
Potential Issues:
- The sampling sizes are imbalanced across locations. Small sample numbers for some populations (e.g. Tata) could bias results.
- Human-mediated movement of male palms between oases for cultivation may confuse geographical structure. The authors should account for this possibility.
- Relatedness between individual palms is not considered. This could inflate diversity estimates if close relatives are sampled.
- There is no confirmation that the molecular markers accurately reflect adaptive genetic diversity as opposed to neutral diversity.
- Standardization of DNA quality, PCR conditions etc. across sample genotypes should be demonstrated clearly. Variation in these parameters could alter outputs.
- Appropriate controls, replications and validations need to be included during genotyping work to support robustness of the diversity analyses.
Author Response
Dear Reviewer 2,
We are grateful to you, for your careful review and the valuable feedback provided. Your insightful comments have been considered to improve current version. Please find below our responses to your comments:
Comment 1: Expand the discussion to provide more insights into how the findings can inform breeding programs and conservation efforts for male date palms specifically.
Response: We appreciate your input and have considered your recommendation carefully. We have made the change throughout our article.
Comment 2: Validate the results using additional samples or molecular marker systems. This would strengthen conclusions.
Response: We appreciate the suggestion. Our study built upon earlier research in the assessment of male date palm diversity, such as Asma Chaouch Khouane et al (using microsatellites, 50 accessions)(Chaouch Khouane et al., 2020), Hedia Bourguiba (15 microsatellite loci, 72 accessions)(Bourguiba et al., 2023), and Nabila El Kadri et al (eight SSR loci, 24 genotypes)(El Kadri et al., 2019). In our study, we adopted a comprehensive approach, incorporating three types of molecular markers: SSR, ISSR, and DAMD. We believe this diverse set of markers will provide robust and reliable information, enhancing the depth of our results. In terms of sample size, we chose 79 accessions as a representative number. This decision is rooted in the unique dynamics of oasis environments, where the number of male palms is inherently limited as they primarily serve for pollination, contrasting with the larger population of female trees responsible for fruit production.
Comment 3: Complement the molecular data with phenotypic diversity assessments of the male genotypes. This allows checking consistency with field observations.
Response: We value the suggestion to include phenotypic diversity assessments in our study. Our three-year study primarily focused on molecular analysis, and we recognize the importance of the phenotypic aspect. Currently, colleagues in our research unit are dedicated to studying the phenotypic diversity of male genotypes.
Comment 4: The sampling sizes are imbalanced across locations. Small sample numbers for some populations (e.g. Tata) could bias results.
Response: Thank you for highlighting this concern. The sampling sizes were varied based on the number of plants and total density in each region. Our goal was to collect plants strategically to encompass the greatest possible genetic diversity in the studied regions.
Comment 5: Human-mediated movement of male palms between oases for cultivation may confuse geographical structure. The authors should account for this possibility.
Response: Thank you for raising this point. In our analysis, we did observe genotypes clustering according to geographical origin, suggesting that the influence of human-mediated movement might be relatively limited in our dataset. However, we appreciate the importance of considering this possibility more explicitly in our discussion and will ensure to address it comprehensively in the revised manuscript.
Comment 6: Relatedness between individual palms is not considered. This could inflate diversity estimates if close relatives are sampled.
Response: Thank you for highlighting the concern. During the sampling process, we indeed took into account that individuals were distant, and representativeness in space was considered. However, it's essential to note that we also paid attention to minimizing the inclusion of close relatives in the sampled palms to mitigate any potential inflation of diversity estimates.
Comment 7: There is no confirmation that the molecular markers accurately reflect adaptive genetic diversity as opposed to neutral diversity.
Response: Thank you for your valuable feedback. I appreciate your concern regarding the accuracy of molecular markers in reflecting adaptive genetic diversity compared to neutral diversity. In our study on the genetic diversity of male date palm, we carefully selected SSR, ISSR, and DAMD markers based on their documented sensitivity to various aspects of genetic variation.
The choice of these markers was informed by their demonstrated efficacy in similar studies and their ability to capture different facets of genetic diversity.
Comment 8: Standardization of DNA quality, PCR conditions etc. across sample genotypes should be demonstrated clearly. Variation in these parameters could alter outputs.
Response: Rest assured, our experimental protocols include rigorous standardization procedures for DNA quality assessment and PCR conditions.
-For DNA quality: To determine the DNA concentration and ensure its purity, the extracted DNA was analyzed using a spectrophotometer, specifically the Nano-100 micro spectrophotometer (XianYima Opto-electrical Technology, Shaanxi, China) (Figure 1). This device enables simultaneous measurement of optical densities at three wavelengths, providing results that encompass DNA concentration, optical densities (at 260 nm, 230 nm, and 280 nm), the 260/230 ratio, and the 260/280 ratio. These ratio values offer insights into potential DNA contaminations. Proteins absorb at 280 nm, while substances such as carbohydrates, peptides, phenols, or aromatic compounds absorb at 230 nm.
DNA purity is confirmed when the 260/280 ratio falls within the range of 1.8 to 2, and the 260/230 ratio is between 1.8 and 2.2. Only DNA meeting these criteria, demonstrating good quality and adhering to the specified intervals, is selected for PCR analysis (Figure 2).
Figure 1: DNA Quantification by Nano-100 Spectrophotometer
Photo: Property of the Biotechnology Unit at INRA
Figure 2: Example of DNA Sample Quantification Results Using a Spectrophotometer
-For PCR conditions: In our experimental design, we have relied on insights from previous articles, and we have carefully considered the composition of the primers. To further ensure the robustness of our PCR protocols, we conducted extensive testing before the actual applications. This involved running multiple tests to optimize and validate each PCR program, considering factors such as annealing temperatures, extension times, and other relevant parameters.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript was greatly improved, and I recommend its publication in its current form.
Comments on the Quality of English LanguageThe manuscript was greatly improved, and I recommend its publication in its current form.
Author Response
thank you for your comments
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for the revised version.
