Ethyl Methyl Sulfonate-Induced Mutagenesis and Its Effects on Peanut Agronomic, Yield and Quality Traits

Round 1

Reviewer 1 Report

Chen et al. describe the development and a forward genetic screen of two EMS-populations of cultivated peanuts. In the first part of the study, the impact of EMS dosage and duration on germination rates of each genotype was tested, which will provide helpful context for other mutant population development in this species. The authors further describe the variation in quality and yield traits in these populations, identifying several mutant lines that have potentially beneficial phenotypes for breeding and genetic analysis.

As the authors describe in the introduction, mutagenized populations have been widely applied in peanut research and breeding, so the study is not particularly novel. However, the mutant materials developed here could be used in genetic mapping studies to help characterize genetic variation in this crop, which in turn could help selection in breeding programs, so there is value for the research community in the results presented.

I have three major concerns that should be addressed, mainly relating to the data analysis and its presentation, followed by some more minor comments that I have listed below.

1. LD50 calculation – This calculation of LD50 is incorrect, since the authors have simply selected the dose that is closest to 50%. I believe the correct analysis here requires the calculation of LD50 values for each duration of treatment and for each genotype by plotting all five data points, then performing regression analysis to identify the dose at 50% mortality. The optimization of EMS dose is presented as an important justification for the value of the study, so this analysis should be performed correctly.
2. M2 population analysis – I do not understand the numbers in the data presented in tables 3-5. I think the measurements of traits should be made on M2 families, which each represent independent mutagenesis events, but the numbers here suggest that individual plants were measured, including multiple times from the same M2 population. Please clarify and justify these measurements.
3. Mutant phenotypes - Some of these mutant phenotypes described from line 156 onwards are quantitative, and must be presented quantitatively, as has been done for the whole population earlier in the study. This must be included for leaf morphology, seed size and height mutants described in this section and should be measured from multiple individuals from the M3 population. The current description and photo of an individual plant is insufficient.

 

Minor comments:

Line 13 and line 33 – What supporting evidence is there for low genetic variability in cultivated peanuts? No citation or other information is provided to support the statements in the abstract and introduction sections.

Line 37 – When introducing EMS, I think it would be helpful to include information on the mode-of-action of this mutagen, which is related to the types of mutations that can be expected in a population.

Line 41 – What physical and chemical mutagens?

Line 59 – I think it would be helpful to clarify that a control treatment was included for each, so 17 treatments. If these treatments are well-described in the text, Table 1 is unnecessary.

Line 64 – Please provide details of how seeds were germinated.

Line 71-74 – 341 and 377 M2 families from two genotypes were retained. Were these separated by treatment and replication for M3 and subsequent phenotyping? Were mutants treated as a singular block and might this impact the results from the phenotypic screen?

Line 78-79 – How is wild-type defined in this experiment? The untreated control?

Line 88 – Why were the results from the M2 and M3 generations not also analyzed statistically?

Line 95 – Again, it will be helpful to clarify what the wild-type is in this comparison, I assume this is untreated control? These results should be described in more detail e.g. were the differences in germination significant at all doses in both genotypes, to different extents with higher doses?

Table 2 – These differences are hard to interpret with upper and lower case letters and standard deviations. Perhaps a graph may be a simpler way for the reader to interpret this data?

Line 107 – Do I understand correctly that the subsequent mutant analyses are using all M2 populations (i.e. 341 for HY22 and 377 for YY45) regardless of their treatment status? The different plants and populations will contain differences in the dosage and duration of these mutants, making it hard to compare across populations? I think this experimental design is worth clarifying here.

Line 110 – Do you mean the average height from the entire population, or from non-mutagenized controls? Linked to my point above, the impact on overall population will depend on the selection of these mutant lines. For height here, but other traits in this section, on what basis is a measurement “considered normal”? Would this be based on the mean parental non-mutagenized line? This needs clarification and details of the measurements that were made to make this determination.

Data from tables 3-5 might be better represented in a histogram with smaller bin sizes, than as these broad classes, especially for those at the extremes where it is hard to interpret.

Line 183 – Replace “Figre” with “Figure”. Also, there is no figure 4 in this manuscript?

Line 201 – To place this study in the context of other EMS populations in peanut, it would be good to compare rates and durations of EMS treatment with other genotypes described in previous studies. One conclusion from this study is the difference between in genotypes - is there evidence for broader variation in EMS-resilience between genotypes?

Line 207 – Linked to my comment in the introduction to include details of EMS mode of action, I think possible mutations to confer these traits should be discussed in the context of the mutagen and the peanut genome. EMS introduces mainly point mutations and deletions, which are more likely to be loss-of-function genetic variants. In a tetraploid crop, how does the genetic redundancy affect the appearance and degree of phenotypic variation?

Line 249 – I think an expanded discussion of how these materials can be applied would be worthwhile, especially since this is one justification for the value of this study. The manuscript describes briefly that they can help identify genetic mechanisms and be applied directly in breeding, but further context of the next steps would be good to include. How would mapping studies be performed, and how would breeders make use of the mutant lines identified? How will the authors make this material available to breeders and researchers?

Line 258 – Replace “greed” with “agreed”.

Author Response

Comments ：

Reviewer 1

1. LD50 calculation – This calculation of LD50 is incorrect, since the authors have simply selected the dose that is closest to 50%. I believe the correct analysis here requires the calculation of LD50 values for each duration of treatment and for each genotype by plotting all five data points, then performing regression analysis to identify the dose at 50% mortality. The optimization of EMS dose is presented as an important justification for the value of the study, so this analysis should be performed correctly.

Responses:

Thanks for your comment. We have calculated the LD50 values for mutagenesis with different EMS concentrations for each duration through linear regression. Please review line 128-137 and Table 1.

2. M2 population analysis – I do not understand the numbers in the data presented in tables 3-5. I think the measurements of traits should be made on M2 families, which each represent independent mutagenesis events, but the numbers here suggest that individual plants were measured, including multiple times from the same M2 population. Please clarify and justify these measurements.

Responses:

Thanks for your comment. As mentioned above, less M1 plants were obtained from each replicate. Then, we mixed M2 families obtained with all different treatments for next analysis. So, there is no data statistics for results from the M2 and M3 generations.

3. Mutant phenotypes - Some of these mutant phenotypes described from line 156 onwards are quantitative, and must be presented quantitatively, as has been done for the whole population earlier in the study. This must be included for leaf morphology, seed size and height mutants described in this section and should be measured from multiple individuals from the M3 population. The current description and photo of an individual plant is insufficient.

 Responses:

Thanks for your comment. We have added the plant heights in line 240, 142 and Table 2. The seed size could be referred by a ruler in Figure 7. We are regret that leaf morphology was not to measure in the field.  

Minor comments:

Line 13 and line 33 – What supporting evidence is there for low genetic variability in cultivated peanuts? No citation or other information is provided to support the statements in the abstract and introduction sections.

Responses:

Thanks for your comment.  I have added the reference in line 34.

Line 37 – When introducing EMS, I think it would be helpful to include information on the mode-of-action of this mutagen, which is related to the types of mutations that can be expected in a population.

Responses:

Thanks for your comment. Some information was introduced in line 47-52.

Line 41 – What physical and chemical mutagens?

Responses:

Thanks for your comment. I have changed “chemical and physical mutagens” to “induced mutagenesis” in line 44.

Line 59 – I think it would be helpful to clarify that a control treatment was included for each, so 17 treatments. If these treatments are well-described in the text, Table 1 is unnecessary.

Responses:

Thanks for your comment. I have deleted the Table 1.

Line 64 – Please provide details of how seeds were germinated.

Responses:

Thanks for your comment. I have added the details in line 72-74.

Line 71-74 – 341 and 377 M2 families from two genotypes were retained. Were these separated by treatment and replication for M3 and subsequent phenotyping? Were mutants treated as a singular block and might this impact the results from the phenotypic screen?

Responses:

Thanks for your comment. Less M1 plants were obtained, because (1) replicates and samples in this experiment are limit, (2) low germination rate with different EMS treatments, even germination rates were 0% under high EMS concentration and long duration. So, we mixed 349 and 374 M2 families obtained from two genotypes with all different treatments for next analysis. It might be some value to analyze effect on agronomic, yield and quality traits under 16 same treatments for each genotype. In addition, several benefit mutants could be chosen for peanut breeding.

Line 78-79 – How is wild-type defined in this experiment? The untreated control?

Responses:

Thanks for your comment. I have revised the “wild-type” to “the control” in line 90-91, and below text.

Line 88 – Why were the results from the M2 and M3 generations not also analyzed statistically?

Responses:

Thanks for your comment. As mentioned above, less M1 plants were obtained from each replicate. Then, we mixed M2 families obtained with all different treatments for next analysis. So, there is no data statistics for results from the M2 and M3 generations.

Line 95 – Again, it will be helpful to clarify what the wild-type is in this comparison, I assume this is untreated control? These results should be described in more detail e.g. were the differences in germination significant at all doses in both genotypes, to different extents with higher doses?

Responses:

Thanks for your comment. I have revised the “wild-type” to “the control” and added more details to describe the differences in germination at all doses in both genotypes in line 109-118.

Table 2 – These differences are hard to interpret with upper and lower case letters and standard deviations. Perhaps a graph may be a simpler way for the reader to interpret this data?

Responses:

Thanks for your comment. I have changed Table 2 to Figure 1.

Line 107 – Do I understand correctly that the subsequent mutant analyses are using all M2 populations (i.e. 341 for HY22 and 377 for YY45) regardless of their treatment status? The different plants and populations will contain differences in the dosage and duration of these mutants, making it hard to compare across populations? I think this experimental design is worth clarifying here.

Responses:

Thanks for your comment. As mentioned above, less M1 plants were obtained from each replicate. Then, we mixed M2 families obtained with all different treatments for next analysis. So, we mixed all M2 families with different treatments for each genotype for next analysis. It might be valuable to analyze the effect on agronomic, yield and quality traits under 16 same treatments for each genotype. In addition, several benefit mutants could be chosen for peanut breeding.

Line 110 – Do you mean the average height from the entire population, or from non-mutagenized controls? Linked to my point above, the impact on overall population will depend on the selection of these mutant lines. For height here, but other traits in this section, on what basis is a measurement “considered normal”? Would this be based on the mean parental non-mutagenized line? This needs clarification and details of the measurements that were made to make this determination.

Responses:

Thanks for your comment. We have deleted the classification of phenotypic traits and divided with smaller interval in Figure 2. Then the details of phenotypic traits have been revised in the whole text.  

Data from tables 3-5 might be better represented in a histogram with smaller bin sizes, than as these broad classes, especially for those at the extremes where it is hard to interpret.

Responses:

Thanks for your comment. We have changed Table 3-5 to Figure 2-4, which is a histogram with smaller bins.

Line 183 – Replace. Also, there is no figure 4 in this manuscript?

Responses:

Thanks for your comment. We have changed “Figre” to “Figure”.

Line 201 – To place this study in the context of other EMS populations in peanut, it would be good to compare rates and durations of EMS treatment with other genotypes described in previous studies. One conclusion from this study is the difference between in genotypes - is there evidence for broader variation in EMS-resilience between genotypes?

Responses:

Thanks for your comment. We have discussed in line 288-292.  

Line 207 – Linked to my comment in the introduction to include details of EMS mode of action, I think possible mutations to confer these traits should be discussed in the context of the mutagen and the peanut genome. EMS introduces mainly point mutations and deletions, which are more likely to be loss-of-function genetic variants. In a tetraploid crop, how does the genetic redundancy affect the appearance and degree of phenotypic variation?

Responses:

Thanks for your comment. We have discussed in line 300-314.  

Line 249 – I think an expanded discussion of how these materials can be applied would be worthwhile, especially since this is one justification for the value of this study. The manuscript describes briefly that they can help identify genetic mechanisms and be applied directly in breeding, but further context of the next steps would be good to include. How would mapping studies be performed, and how would breeders make use of the mutant lines identified? How will the authors make this material available to breeders and researchers?

Responses:

Thanks for your comment.  We have discussed in line 357-364.  

 

Line 258 – Replace “greed” with “agreed”.

Responses:

Thanks for your comment. I have replace “greed” with “agreed”.

  

Sincerely,

Lei Zhang

 

Reviewer 2 Report

Dear Authors,

First sentence in Abstract and First sentence in the introduction is almost same. Please change it.

First paragraph of the introduction contains only one reference. Please add more reference to support the paragraph.

What is CK in the table?

Table 2 - Why germination rate is high in 0.3% EMS at 3h?No difference between 1h, 3h and 5h? Which kind of mean comparison test was used here?

L-101 - Where these values taken from? Table 2? Please add table or figure numbers.

L102- "YY45 were 53% and 56% at 0.3% EMS...."i cannot find such a values in Table 2.  

Is it possible to add clear figures for Plant height, branch number and pod number as well? In addition,graphs will be more informative. In your reference, Wang et al. 2015 has added clear photos to explain their data. 

Your objective is to find out the effect of EMS concentration and yeild on plants. Then how did you compare Table 3 data with EMS concentrations and duration? It is not clear that which treatment plants (M2) have grown.

L110- The average height HY22 - 43.4, YY45 - 38. But in Table 3 Normal height should be between 40-60 in YY45 as well. Therefore, Your description in the table is not correct. Its not the best way to explain your data clearly.

What is the significance of your study compared with Nadaf et al.? Variety difference? You said you are going to find optimum EMS treatment for peanut. Finally you have pooled all the treatments and try to characterize EMS induced mutants. I do not feel any novelty of this study except the 3.1 section. Which treatment is good for your cultivars? Why? How do you prove it?

You are trying to explain your data with tables. But Graphs will be very useful for readers. Please go through your refered papers and try to improve it.  

Thanks.

 

Author Response

Comments ：

Reviewer 2:

First sentence in Abstract and First sentence in the introduction is almost same. Please change it.

Responses:

Thanks for your comment. We have changed the first sentence in abstract of Lines 12-15.

First paragraph of the introduction contains only one reference. Please add more reference to support the paragraph.

 Responses:

Thanks for your comment. We have added two references in Lines 34 and 36.

What is CK in the table?

 Responses:

Thanks for your comment. We have changed the Table 2 to Figure 1. CK was changed to control (untreated HY22 or YY45 plants) in Figure 1.

Table 2 - Why germination rate is high in 0.3% EMS at 3h? No difference between 1h, 3h and 5h? Which kind of mean comparison test was used here?

 Responses:

Thanks for your comment. Although the germination rate is slightly higher in 0.3% EMS at 3 and 5h than 1h, there is no significant difference between 1h, 3h and 5h according to the ANVON analysis. The reason might be measurement deviation. The ANOVA analysis through SPSS software was used to analyze the data of germination rate, which showed in part of “Materials and methods”, Lines 100-102.

L-101 - Where these values taken from? Table 2? Please add table or figure numbers.

 Responses:

Thanks for your comment. We have added Table 1 to explain the LD50 value for mutagenesis of peanut with different EMS concentration for each duration, which was calculated through linear regression based on lethal rate. Please review it in Line 138.

L102- "YY45 were 53% and 56% at 0.3% EMS...."i cannot find such a values in Table 2.  

Responses:

Thanks for your comment. We have rewritten Lines 132-138 to clarify the LD50 value with different EMS treatments for each genotype. 

Is it possible to add clear figures for Plant height, branch number and pod number as well? In addition, graphs will be more informative. In your reference, Wang et al. 2015 has added clear photos to explain their data. 

 Responses:

Thanks for your comment. We have changed Table 3-5 to Figure 2-4 according to the reference of Wang et al. 2015.

Your objective is to find out the effect of EMS concentration and yeild on plants. Then how did you compare Table 3 data with EMS concentrations and duration? It is not clear that which treatment plants (M2) have grown.

 Responses:

Thanks for your comment. Less M1 plants were obtained, because (1) replicates and samples in this experiment are limit, (2) low germination rate with different EMS treatments, even germination rates were 0% under high EMS concentration and long duration. So, we mixed 349 and 374 M2 families obtained from HY22 and YY45 with all different treatments, respectively. It might be value to analyze effect on agronomic, yield and quality traits under 16 same treatments for each genotype. In addition, several benefit mutants could be chosen for peanut breeding.

L110- The average height HY22 - 43.4, YY45 - 38. But in Table 3 Normal height should be between 40-60 in YY45 as well. Therefore, Your description in the table is not correct. Its not the best way to explain your data clearly.

 Responses:

Thanks for your comment. The data from Tables 3 might be better represented in a histogram with smaller bin sizes. So We have changed it to Figure 2, which was explained in Line 146-166.

What is the significance of your study compared with Nadaf et al.? Variety difference? You said you are going to find optimum EMS treatment for peanut. Finally you have pooled all the treatments and try to characterize EMS induced mutants. I do not feel any novelty of this study except the 3.1 section. Which treatment is good for your cultivars? Why? How do you prove it?

 Responses:

Thanks for your comment. Compared with Nadaf et al, LD50 value was calculated to optimal the proper EMS concentration for each duration.

You are trying to explain your data with tables. But Graphs will be very useful for readers. Please go through your refered papers and try to improve it.  

 Responses:

Thanks for your comment. Data from Tables 3-5 might be better represented in a histogram with smaller bin sizes, than as these broad classes, especially for those at the extremes where it is hard to interpret. So, Tables 3-5 were changed to new Figure 2-4.

 

Sincerely,

Lei Zhang

 

 

Round 2

Reviewer 1 Report

My major concern remains in the analysis of M2 populations, which the numbers show are mixing multiple individuals from the same populations. Each M1 mutant is an independent biological replicate, equating to a unique genotype. The M2 family is segregating for this induced genetic variation and represent technical replicates of the same genetic variation. Therefore, the n of all the phenotypic measurements should be the number of M2 populations. With the numbers presented, it seems as if all individuals were treated in isolation, even if multpiple individuals from the same population were selected. If the source of the M2 population for each individual cannot be traced, a minimum is to alert the reader to this experimental design, so this can be understood when interpreting these results.

Line 12 – The original wording of this sentence was clearer, in my opinion.

Table 1, I am not sure it is necessary to display this table with all calculations in the main paper. This could be presented in an online supplementary file.

Line 151 – This conclusion is wrong, the EMS-treatment caused variation in plant height, with some lines taller and some shorter.

Author Response

Journal Name: agronomy

Manuscript Number: agronomy-770472

Manuscript Title:  Ethyl methyl Sulfonate-induced mutagenesis and its effects on peanut agronomic, yield and quality traits

 

 

 

 

Dear Editor,

Thank you for your letter and careful consideration of our manuscript agronomy-770472 entitled “Ethyl methyl Sulfonate-induced mutagenesis and its effects on peanut agronomic, yield and quality traits”. We appreciate the opportunity to revise and resubmit our manuscript. The comments were extremely valuable and have helped us to improve our manuscript greatly. We have modified the manuscript accordingly and hoped the revised manuscript would meet your requests. And then detailed corrections are listed below point by point.

 

 

Comments ：

Reviewer 1

My major concern remains in the analysis of M2 populations, which the numbers show are mixing multiple individuals from the same populations. Each M1 mutant is an independent biological replicate, equating to a unique genotype. The M2 family is segregating for this induced genetic variation and represent technical replicates of the same genetic variation. Therefore, the n of all the phenotypic measurements should be the number of M2 populations. With the numbers presented, it seems as if all individuals were treated in isolation, even if multpiple individuals from the same population were selected. If the source of the M2 population for each individual cannot be traced, a minimum is to alert the reader to this experimental design, so this can be understood when interpreting these results.

Responses:

Thanks for your comment.  We have revised to the original meaning.  We have analyzed the phenotypic data of M2 population on the basis of each M2 families from corresponding M1 plant. Please review the Figure 3-5 and the corresponding details in the text.

Line 12 – The original wording of this sentence was clearer, in my opinion.

Responses:

Thanks for your comment.  We have revised to the original meaning.

Table 1, I am not sure it is necessary to display this table with all calculations in the main paper. This could be presented in an online supplementary file.

Responses:

Thanks for your comment. We have changed Table 1 to Figure 2 in the main paper, and original table 1 was presented in a supplementary file.

Line 151 – This conclusion is wrong, the EMS-treatment caused variation in plant height, with some lines taller and some shorter.

Responses:

Thanks for your comment. We have deleted the wrong conclusion.

  

 

This revised manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All the authors have approved the manuscript and agree with submission to your esteemed journal. There are no conflicts of interest to declare. Thank you for your consideration. I look forward to hearing from you. 

Sincerely,

Lei Zhang

Department of Crop Science and Technology,

College of Agriculture, South China Agricultural University,

Guangzhou, 510642, PR China

Phone: (86)-20-85280203

Fax: (86)-20-85280203

E-mail: [email protected]

 

Reviewer 2 Report

Dear Authors,

Current form of the manuscript is acceptable.

Regards

Author Response

Journal Name: agronomy

Manuscript Number: agronomy-770472

Manuscript Title:  Ethyl methyl Sulfonate-induced mutagenesis and its effects on peanut agronomic, yield and quality traits

 

 

Dear Editor,

Thank you for your letter and careful consideration of our manuscript agronomy-770472 entitled “Ethyl methyl Sulfonate-induced mutagenesis and its effects on peanut agronomic, yield and quality traits”. We appreciate the opportunity to revise and resubmit our manuscript. The comments were extremely valuable and have helped us to improve our manuscript greatly. We have modified the manuscript accordingly and hoped the revised manuscript would meet your requests. And then detailed corrections are listed below point by point.

 

Comments ：

Current form of the manuscript is acceptable.

Thanks.  

 

 

This revised manuscript has not been published or presented elsewhere in part or in entirety, and is not under consideration by another journal. All the authors have approved the manuscript and agree with submission to your esteemed journal. There are no conflicts of interest to declare. Thank you for your consideration. I look forward to hearing from you. 

Sincerely,

Lei Zhang

Department of Crop Science and Technology,

College of Agriculture, South China Agricultural University,

Guangzhou, 510642, PR China

Phone: (86)-20-85280203

Fax: (86)-20-85280203

E-mail: [email protected]