Comparative Genetic Diversity Assessment and Marker–Trait Association Using Two DNA Marker Systems in Rice (Oryza sativa L.)
, Mohamed M. El-Malky 3, Tarek A. Shalaby 4,5 and Mohamed Ismail 6
Round 1
Reviewer 1 Report
The manuscript by Mohammed Al-daej et al. studied the genetic diversity of 12 rice varieties using 12 ISSR and 5 SSR markers. In addition, the rice varieties were evaluated for 11 agro-morphological traits in a two-year trial. They analyzed the association between the DNA markers and agro-morphological traits. I can hardly support publishing this paper in its current form. The main reasons are as follows.
The number of tested varieties is very small and cannot represent a type of rice germplasm. It is better to increase the number of varieties or use a mini-core collection for such research.
They used 12 ISSR and five SSR markers. Molecular markers evenly distributed on 12 chromosomes should be suitable for analyzing the association with agricultural morphological traits.
Author Response
Responses for reviewers
Reviewer 1
- Comment: The number of tested varieties is very small and cannot represent a type of rice germplasm. It is better to increase the number of varieties or use a mini-core collection for such research.
Response: The selected varieties are the most common varieties cultivated in Egypt and the kingdom of Saudi Arabia, as such; the currents study aimed to investigate if the local varieties, not all types of rice germplasm, harbour any allele(s) that is associated with agro-morphological traits of interest. A statement regarding the significance of selected varieties to local rice breeders and farmers is included (Line 106-109).
The use of a mini-core collection has constraints associated with our study. Although using more rice genotypes would significantly generalize the outcomes, many previous studies have already used the mini-core collection in the genetic association study in rice.
Many researchers use a large selection of genotypes to capture most of the genetic variation within the rice genome. However, other researchers use a small collection of genotypes with special interests/characters for local breeders.
For instance, using 14 basmati rice genotypes along with 10 SSRs located on seven chromosomes, Zulfiqar et al. 2023 were able to determine the genetic association between seed components and SSRs (Zulfiqar et al. 2023. Genetic diversity studies for grain iron and zinc content analysis for elite rice (Oryza sativa L.) genotype by using SSR markers J. Food Compost. Anal. 115: 104816). Also, Fazal et al., 2022, used 12 rice genotypes in addition to five SSRs located on three different chromosomes to assess genetic diversity in Pakistani rice germplasm (Fazal et al., 2022)
The use of the mini-core collection in the field trials evaluation and laboratory work requires significant fund that is challenging and not currently accessible in the existing circumstances across most of third-world countries, including Egypt.
Future proposals were set to include more genotypes once collaboration and significant funds are available.
- Comments: They used 12 ISSRs and five SSRs markers. Molecular markers evenly distributed on 12 chromosomes should be suitable for analyzing the association with agricultural morphological traits.
Response: Not clear at this point if the reviewer advising of using markers evenly distributed across the 12 chromosomes of rice. The selected SSRs located on five different chromosomes; 1, 2, 6, 11, and 12. Additionally, ISSRs used herein is randomly spread across the rice genome, which, in part, would complement the limited number of SSRs.
While selecting SSRs across the 12 rice chromosomes is more comprehensive, in some circumstances, it is not feasible to adopt such approach.
In many studies, researchers use SSRs located on specific chromosomes, and the selection of these markers is not necessarily spread across the entire rice 12 linkage groups. Many researchers adopt such approach, especially when markers previously showed strong relationships with the specific trait(s) of interest.
For instance, using 10 SSRs located on seven chromosomes, Zulfiqar et al. 2023 were able to determine the genetic association between seed components and SSRs (Zulfiqar et al. 2023. Genetic diversity studies for grain iron and zinc content analysis for elite rice (Oryza sativa L.) genotype by using SSR markers J. Food Compost. Anal. 115: 104816). Also, Fazal et al., 2022, used five SSRs located on three different chromosomes to assess genetic diversity in Pakistani rice germplasm (Fazal et al., 2022)
The selected SSRs previously reported being associated with biotic and abiotic stresses, nutritional contents, and physiological traits using quantitative trait loci (QTL) approach and/or segregation analysis (e.g., heat tolerance; RM 209, blast resistance; RM 217, RM 224, RM 1216, nutritional content; RM 211, fertility restoration; RM 3873, rice cooking characteristics; RM 29, plant height characteristics and grain quality; RM 225, antioxidant traits; RM 247).
We believe that we were able to utilize contemporary mythologies and statistical analyses comparable to other literature in order to address critical questions related to rice breeding without affecting the confidence of the findings.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper presents a well organized experiment on genetic diversity about different rice genotypes. Appart from minor English style and language check that is required, the overall presentation is very good. But the discussion section is too short and I believe that more references should be added and the results should be discussed and not only presented again.
Lines 120-122: Please correct ''ha-1 and m2'' to "ha-1 and m2"
Line 137-140: Please explain more about the extraction procedure.
Lines 157-159: Please correct " oC" to "oC"
Author Response
Responses of reviewers
Reviewer 2
- Comment: Minor English style and language check is required.
Response: English style and language style have been modified as necessary.
- Comment: The discussion section is too short, and I believe that more references should be added, and the results should be discussed and not only presented again.
Response: The discussion section has been extended with more references included. Results have been discussed comprehensively.
- Comments: Lines 120-122: Please correct ''ha-1 and m2'' to "ha-1and m2"
Line 137-140: Please explain more about the extraction procedure.
Lines 157-159: Please correct " oC" to "oC"
Response: Correction has been made for ha-1 and m2 to ha-1 and m2 (line xxx-line xxx). PCA extraction procedure has been updated (line 134-138).
Correction has been made for oC" to "°C"(line 159-160)
Author Response File: Author Response.pdf
Reviewer 3 Report
The study presents an interesting effort to apply association mapping for marker-trait associations in rice. The authors used current methods and presented their results concisely. Although for association mapping the number of tested genotypes and molecular markers are rather low, the study has focus and could possibly be of interest to scientists of the relative scientific field.
I think that the article could benefit if a dendrogram for the genetic relationships of the varieties was presented, based on the genetic diversity matrix. Also, as the authors state, population structure and similarities among genotypes could result in false positive associations. This could be checked from marker data using a suitable tool (e.g. Pritchard, J., Stephens, M., and Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics 155, 945–959. ). According the results the authors should discuss strengths and weaknesses of their approach.
Author Response
Responses of reviewers
Reviewer 3
- Comment: I think that the article could benefit if a dendrogram for the genetic relationships of the varieties was presented, based on the genetic diversity matrix.
Response: The article primarily focuses on genetic diversity assessment and marker-trait associations using two DNA marker systems. However, to highlight the genetic relationship among the studied varieties, a dendrogram based on Nei’s genetic distance of the combined datasets (SSR and ISSR) is provided as a Supplementary material in the revised manuscript. Line 174-178, Supplementary Figure S1.
- Comment: The authors state, population structure and similarities among genotypes could result in false positive associations. This could be checked from marker data using a suitable tool (e.g., Pritchard, J., Stephens, M., and Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics 155, 945–959).
Response: Indeed, population structure was already inferred as mentioned in the material and method using STRUCTURE software (Pritchard et al. 2000), line 182-184. Also, kinship among genotypes was also inferred using SpaGeDi software (Hardy et al. 2002) which also included in the association analysis model.
To avoid any additional false positive that might arise after population structure and kinship correction, a false discovery rate method for multiple comparisons was include which would eliminate any additional false positive associations.
- Comment: According the results the authors should discuss strengths and weaknesses of their approach.
Response: Strengthen and weakness of the applied methodologies discussed thoroughly in the revised manuscript. (Line 367-369, Line 404-412)
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Regarding my comment on using STRUCTURE and SpaGeDi you mention the softwares in M&M but you do not report your findings in Results and the consequences to your study. Please describe if population structure was observed and how this could affect marker-trait associations.
Author Response
Comment,
Regarding my comment on using STRUCTURE and SpaGeDi you mention the softwares in M&M but you do not report your findings in Results and the consequences to your study. Please describe if population structure was observed and how this could affect marker-trait associations.
Response,
- This point was described in M&M part lines from 187 to 190 and Result part lines 243-246.
- One reference was added Evanno et al 2005 [34].
- Two sublimantry figures were added S2 and S3.
- The changed parts are colored with yellow and track changes
Author Response File: Author Response.pdf
