Association Mapping for Quantitative Trait Loci Controlling Superoxide Dismutase, Flavonoids, Anthocyanins, Carotenoids, γ-Oryzanol and Antioxidant Activity in Rice
, Elssa Pandit 1,3,†, Priyadarsini Sanghamitra 1, Saumya Ranjan Barik 1, Deepak Kumar Nayak 1, Auromira Sahoo 1, Arpita Moharana 1, Jitendriya Meher 1, Prasanta K. Dash 4, Reshmi Raj 1, Binod Kumar Jena 5, Kartik Chandra Pradhan 2, Devraj Lenka 2, Nabaneeta Basak 1, Srikanta Lenka 1 and Sharat Kumar Pradhan 1,*Round 1
Reviewer 1 Report
In this work, the authors use association mapping to identify a number of QLT controlling antioxidant activity in rice. Overall, it is an interesting work, however the following questions are needed attentions:
1. What are the conditions of the selection of the panel population (120) out of the 270 genotypes, please provide more information. As for rice, a well-studied model plant, the population size (270, and panel population of 120) and the number of biomarkers (136) in this AM study maybe less than ideal. I would like to see the authors’ options to that.
2. Please describe more clearly the selection of 136 SSR from online database, e.g. filtering criterial and include database version number.
3. Is there any reason carotenoids is in your keywords and results, but not mentioned in your conclusion? Please discuss this issue.
4. In Section 3.1 and figure 2, what are the standards of “very high” to “very low” of antioxidant content? Please be more specific.
5. Please discuss the selection of K value in the discussion section.
6. The ∆K peak value in 3.5 is not 264.2 at K = 2 among the assumed K, but around 350 ?
7. In section 3.2, Figure S1 is not a graph of principal component analysis.
8. In section 3.2 the 6th line, how does figure 3 show one compound contributed more than the others？I don’t think fig3 provides enough information to show that.
9. Please label all the axes on all the graphs.
10. Please fix the missing space and tables overlapping issues.
Author Response
Point by point response to the queries of Reviewer 1
Reviewer 1:
Comments and Suggestions for Authors:
In this work, the authors use association mapping to identify a number of QLT controlling antioxidant activity in rice. Overall, it is an interesting work, however the following questions are needed attentions:
Query 1: What are the conditions of the selection of the panel population (120) out of the 270 genotypes, please provide more information. As for rice, a well-studied model plant, the population size (270, and panel population of 120) and the number of biomarkers (136) in this AM study maybe less than ideal. I would like to see the authors’ options to that.
Response:
About the panel constitution:
The initial population shortlisted was 270 based on(i) maturity duration (upto 135 days) and (ii) kernel color (red, black, purple and white) from about 1000 germplasm lines. Seeds of these germplasm were collected from Gene bank, ICAR-National Rice Research Institute, Cuttack and were grown in the experimental plot of the Institute during wet season, 2019.
The initial shortlisted 270 germplasm lines (121 white and 149 colored rice grains) were raised in the field in an augmented block design and seeds were stored for 3 months for dormancy break after harvest, the germplasm lines were phenotyped for Carotenoids (mg g -1), super oxide dismutase (unit g-1), total anthocyanin content (mg 100g-1), gamma-oryzanol (mg 100 g-1), total flavonoids content (mg catechine or CEt 100 g-1) and 2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The genotypes were classified into 5 groups based on the phenotyping results of each compounds seeing the frequency distribution groups (Fig. 1). A panel population was prepared by selecting genotypes represented from each 5 groups from each of the 6 antioxidant traits and the total genotypes in the panel become 120 which were taken from the initial population of 270 germplasm lines.
About SSR markers selection:
A total of 136 SSR (simple sequence repeat) markers were selected from the database (http://gramene.org/) available in the public domain and used for genotyping of the panel population (Supplementary Table 5). The markers were selected based on positions covering all the chromosomes to illustrate the diversity and to identify the polymorphic loci among the rice germplasm lines present in the panel.
Query 2. Please describe more clearly the selection of 136 SSR from online database, e.g. filtering criteria and include database version number.
Response:
The 136 SSR markers were selected across all the 12 rice chromosomes on the basis of their location on each chromosome so as to represent all the chromosomes and maximum genome coverage. On an average 12 markers per chromosome having a range of 6-17 were taken for the study. The genome coverage was more than 90% on average. The details of the selection of markers are given in the following table. This was performed manually by selecting markers having around 3-4 Mb distance from each other in ascending manner so that individual chromosomes could be covered from beginning to the end point. No software was used for this selection of markers.
|
Chromosome No. |
No of Markers Used |
Genome Coverage (%) |
|
1 |
17 |
94.63 |
|
2 |
13 |
95.13 |
|
3 |
12 |
96.50 |
|
4 |
12 |
94.07 |
|
5 |
10 |
88.75 |
|
6 |
11 |
97.67 |
|
7 |
10 |
90.31 |
|
8 |
13 |
84.80 |
|
9 |
11 |
70.93 |
|
10 |
9 |
61.18 |
|
11 |
12 |
85.55 |
|
12 |
6 |
83.26 |
Query 3. Is there any reason carotenoids is in your keywords and results, but not mentioned in your conclusion? Please discuss this issue.
Response: Our target was to map the QTL regulating carotinoids in the germplasm lines. We obtain a moderate level of diversity for the trait in the panel population. We also obtained marker-trait associations for carotenoids by individual models i.e., either by GLM or by MLM. We did not get the genomic region controlling the carotinoids content detected by both the models of GLM and MLM for considering robust association. Therefore, we did not conclude the control of carotinoid content in the present set of germplasm lines.
Query 4. In Section 3.1 and figure 2, what are the standards of “very high” to “very low” of antioxidant content? Please be more specific.
Response: The estimated values of each trait for the germplasm lines are grouped into 5 groups for this study i.e., very low, low, medium, high and very high. We also obtained the frequency distribution of germplasm lines in each group. Therefore, the cut of value for each group is determined.
Query 5. Please discuss the selection of K value in the discussion section.
Response: Thanks. We have now includedthe selection of K in the revised manuscript.
Query 6. The ∆K peak value in 3.5 is not 264.2 at K = 2 among the assumed K, but around 350 ?
Response: Thanks a lot. The delta K value at assumed K=2 is 362.4 but not 264.2. Sorry for the mistake. We have corrected this in the revised manuscript.
Query 7. In section 3.2, Figure S1 is not a graph of principal component analysis.
Response: Thanks. This is not PCA but Scree plotgenerated by plotting the eigen values on the y-axis and the number of factors on the x-axis. It always displays a downward curve. The point where the slope of the curve is clearly leveling off (the “elbow) indicates the number of factors that should be generated by the analysis.
Query 8. In section 3.2 the 6th line, how does figure 3 show one compound contributed more than the others？I don’t think fig3 provides enough information to show that.
Response: PCA diagram is generated based on the variance-covariance of the traits from which length of the line representing each trait can be assessed. So, we are now replacing the PCA diagram containing the diversity of each trait.
Query 9. Please label all the axes on all the graphs.
Response: All the axes in the figures are now labeled.
Query 10. Please fix the missing space and tables overlapping issues.
Response: We have now removed the missing spaces and tables overlapping issues.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments for author File: 
Comments.pdf
Author Response
Point by point response to the queries of Reviewer 2
Reviewer 2
Query: The screen shots (peer-review-22329392.v1.pdf) were supplied for correction of few misspelling.
Response: Thanks a lot for improving our manuscript. We corrected the spelling mistakes in the revised manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
Please check the attached file.
Comments for author File: Comments.pdf
Author Response
Point by point response to the Reviewers queries
Reviewer 3
The piece of work is interesting and generated some useful information, Overall, the manuscript was written well to explain the objectives. However, I have some major issues that are to be addressed before the article being accepted.
These are as follows:
Query 1. The abstract is descriptive and can not summarize the results. For example, “…A total of 14 significant marker-trait associations were detected for the studied antioxidant traits…Antioxidant hot spot locations for the QTLs namely OZ11.1, TFC11.1 and AC11.1 regulating γ-oryzanol, flavonoid and anthocyanin content, respectively were detected. Additional co-localization of PAC12.2 for anthocyanin content with TFC12.1 for flavonoid content was observed.” Therefore, this part must be rewritten.
Response: Thanks for the suggestion. These above lines were rewritten and a modified version of these suggested lines are included in the abstract.
Query 2. What does the number of Y axis in Figure 1 and Figure 2B indicate?
Response: Y-axis in Fig.1 and Fig. 2B indicate the germplasm numbers. Revised Figure 1 and Figure 2B containing the Y-axis scale is submitted in the revised manuscript.
Query 3. How to get the SOD content in Figure 2?
Response: The Spider graph in the Figure 2 indicates the comparative variation of antioxidant traits among the panel germplasm lines. The exact value is presented in the Table 1.
Query 4. The references are not well prepared and kept uniformity according to the journal instruction. Ref.69? 2014 appears in two places, and the title is different from Ref.68.etc
Response: Thanks. The reference in proper format of the journal is now presented.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The author has carefully answered all the questions I asked and has made the appropriate changes in the manuscript. I will recommend the editor to accept this article if the following question is addressed.
1 Remove carotenoids from the keywords.
Author Response
Point by point response to Reviewer 1
MS No. agronomy-1905652
Title: Association mapping for quantitative trait loci controlling superoxide dismutase, flavonoids, anthocyanins, carotenoids, γ-oryzanol and antioxidant activity in rice.
Suggestions by the Reviewer 1
The author has carefully answered all the questions I asked and has made the appropriate changes in the manuscript. I will recommend the editor to accept this article if the following question is addressed.
Query1: Remove carotenoids from the keywords.
Response: Thank you very much for your sugestion. I have deleted the keyword, carotenoid from the keywords.
Author Response File: Author Response.docx
