Ethyl Methanesulfonate (EMS) Mutagen Toxicity-Induced DNA Damage, Cytosine Methylation Alteration, and iPBS-Retrotransposon Polymorphisms in Wheat (Triticum aestivum L.)
, Kamil Haliloğlu 2,*, Metin Tosun 2, Henryk Bujak 3,4, Barış Eren 5, Fatih Demirel 5, Piotr Szulc 6, Halit Karagöz 7, Marek Selwet 8, Güller Özkan 9 and Gniewko Niedbała 10,*Round 1
Reviewer 1 Report
line 37: ...delete...
line 104: ...is used to better understand...,
line 120: ..study we investigate/was investigated...?
line 133: ...delete...
line 149: ? ...µL/ or only 50 ng/L ...? ...10x ?
line 160: ...10x buffer...?
line 161: ? ...µL/ or only 50 ng/L ...?
Comments for author File: 
Comments.pdf
Author Response
Responses to Comments of Reviewer 1
General Response:
First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Agronomy journal. All revised parts have been highlighted in blue.
Sincerely,
Dr. Aras Turkoglu
Comments
Comment 1# line 37: ...delete...
Response to Comment 1# This sentence was removed.
Comment 2# line 104: ...is used to better understand...,
Response to Comment 2# This sentence was removed.
Comment 3# line 120: ...study we investigate/was investigated...?
Response to Comment 3# This sentence was rewritten.
Comment 4# line 133: ...delete.
Response to Comment 4# This sentences was necessary in mutation study.
Comment 5# line 149: ? ...µL/ or only 50 ng/L ...? ...10x ?
Response to Comment 5# This sentence was rewritten.
Comment 6# line 160: ...10x buffer...?
Response to Comment 6# This sentence was rewritten.
Comment 7# line 161: ? ...µL/ or only 50 ng/L ...?
Response to Comment 7# This sentence was rewritten.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Based on molecular marker technologies iPBS and CRED-iPBS, genetic polymorphism and methylation of wheat seedlings that derived from EMS treatment were studied. The results showed that EMS treatment had considerable polymorphisms on cytosine methylation and genomic instability of wheat. However, on the whole, the current research lacks innovation, the content is too simple, the result part is too few, the discussion part is too descriptive and not in-depth, and there are many unreasonable descriptions in the manuscript. In this situation, it requires reconsideration after major revision. The following are the details:
1. It is suggested to provide phenotypic and physiological change data of each treatment group, so as to reflect how epigenetic changes affect plant response to EMS treatment, and help readers better understand the impact of EMS treatment on wheat.
2. Electrophoretic gel imaging picture is more intuitive, it is recommended to supplement in the manuscript.
3. How to understand the genetic polymorphism bands of the control?
4. As we know that, the M1 generation is highly chimeric, so please describe more detail of how do you harvest the samples, by pooling several plants or by individual plant? How many samples in each treatment?
5. Line 119 “EMSs”?
6. Line 37 and 133, there are three “0.2%”;
7. Line 138, “25 oC” should be “25℃”;
8. Line 144, do you mean “-20℃”?
9. It was unbelievable as the volume was 1 L for DNA. Please check the PCR system.
10. Line 168, dose the formula is wrong according to the reference https://doi.org/10.1007/s11240-022-02440-z
11. Line 191-192, “the number of polymorphic bands” should not be presented by “%”;
12. Line 234 Table, in the last column should the “29,4” be “29.4”, 70,6” be “70.6”?
13. Line 235 and 288, what dose “without hormone” mean?
Minor editing of English language is required.
Author Response
Responses to Comments of Reviewer 2
General Response:
First of all, we thank the potential reviewer for her/his valuable time and also raised helpful comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Agronomy journal. All revised parts have been highlighted in red.
Sincerely,
Dr. Aras Turkoglu
Comments
Comment 1# It is suggested to provide phenotypic and physiological change data of each treatment group, so as to reflect how epigenetic changes affect plant response to EMS treatment, and help readers better understand the impact of EMS treatment on wheat
Response to Comment 1# Germination and seedling characteristics of each treatment group used in the research were presented in these papers [19].
Comment 2# Electrophoretic gel imaging picture is more intuitive, it is recommended to supplement in the manuscript.
Response to Comment 2# This gel imaging picture was added.
Comment 3# How to understand the genetic polymorphism bands of the control?
Response to Comment 3# By comparing the iPBS profile to the control, polymorphisms are found, either in the form of a new band that is absent in the control or a band that is absent in the control. Each experimental group's means were computed, and changes in each group's means relative to the control were calculated as a percentage. The formula 100 a/n was used to determine the mean polymorphism values for the CRED-iPBS investigation [15, 16, 18].
Comment 4# As we know that, the M1 generation is highly chimeric, so please describe more detail of how do you harvest the samples, by pooling several plants or by individual plant? How many samples in each treatment?
Response to Comment 4# Dear reviewer, after treatment (M1 plant), the seeds were washed in tap water for 4 hours to remove the mutagen. In this study, 25 seeds from each treatment were germinated on two layers of filter paper in 9-cm petri dishes for 4 replicates. Each petri dish was filled with 14 ml of distilled water. The germination process was carried out in a germination cabinet with the temperature adjusted to 25 OC for 14 days under the conditions of 16 hours light and 8 hours dark period. Germination and seedling characteristics of each treatment group used in the research were presented in these papers [Hossein pour et al, 2021; Dear reviewer, this article belong me (Aras TürkoÄŸlu), my name and surname was changed in 2021 year after i take the Türkiye citizenship; my prior name was Arash Hosseinpou ]. To determine the genomic DNA isolation all of leaves for each treatment was mixed and used. Dear reviewer you know better the chimeric is observed after seed harvest (M2 Plant). We perform this experiment 14 days after mutagen treatment and ended the experiment.
Comment 5# Line 119 “EMSs”?
Response to Comment 5# It is corrected.
Comment 6# Line 37 and 133, there are three “0.2%”;
Response to Comment 6# It is corrected.
Comment 7# Line 138, “25 oC” should be “25℃”;
Response to Comment 7# It is corrected.
Comment 8# Line 144, do you mean “-20℃”?
Response to Comment 8# It is corrected.
Comment 9# It was unbelievable as the volume was 1 L for DNA. Please check the PCR system.
Response to Comment 9# It is corrected.
Comment 10# Line 168, dose the formula is wrong according to the reference https://doi.org/10.1007/s11240-022-02440-z
Response to Comment 10# The formula was rewritten.
Comment 11# Line 191-192, “the number of polymorphic bands” should not be presented by “%”;
Response to Comment 11# They are corrected.
Comment 12# Line 234 Table, in the last column should the “29,4” be “29.4”, 70,6” be “70.6”?
Response to Comment 12# They are changed with figure.
Comment 13# Line 235 and 288, what dose “without hormone” mean?
Response to Comment 13# They are corrected.
Author Response File: Author Response.docx
Reviewer 3 Report
Line 114: Please delete “Coupled Restriction Enzyme Digestion” here, as Line101 has commented on this abbreviation CRED
Introduction: Please elaborate more clearly in the introduction the advantages and differences of the three methods IPBS, CRED and CRED-iPBS, especially what does IPBS refer to?
Line 138: 25 oC should be 25°C
Materials and Methods: In addition to the concentration, different solution volumes also affect the mutagenesis rate. Please specify the amount of seed samples used and the volume of EMS solution; also, after the scheduled mutagenesis time is up, is the termination method used to terminate further mutagenesis?
Line 150: MgCl2 should be MgCl2
What are the usual EMS concentrations for other tests? How long is the treatment time? Compared to the concentrations in this study, it seems to be generally higher? Then what is the special application scenario of this study? Why not use the conventional concentration for the test?
Line 114: Please delete “Coupled Restriction Enzyme Digestion” here, as Line101 has commented on this abbreviation CRED
Introduction: Please elaborate more clearly in the introduction the advantages and differences of the three methods IPBS, CRED and CRED-iPBS, especially what does IPBS refer to?
Line 138: 25 oC should be 25°C
Materials and Methods: In addition to the concentration, different solution volumes also affect the mutagenesis rate. Please specify the amount of seed samples used and the volume of EMS solution; also, after the scheduled mutagenesis time is up, is the termination method used to terminate further mutagenesis?
Line 150: MgCl2 should be MgCl2
What are the usual EMS concentrations for other tests? How long is the treatment time? Compared to the concentrations in this study, it seems to be generally higher? Then what is the special application scenario of this study? Why not use the conventional concentration for the test?
Author Response
Responses to Comments of Reviewer 3
General Response:
First of all, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Agronomy journal. All revised parts have been highlighted in green.
Sincerely,
Dr. Aras Turkoglu
Comments
Comments 1# Line 114: Please delete “Coupled Restriction Enzyme Digestion” here, as Line101 has commented on this abbreviation CRED
Response to Comment 1#: The sentence was removed.
Comments 2# Introduction: Please elaborate more clearly in the introduction the advantages and differences of the three methods IPBS, CRED and CRED-iPBS, especially what does IPBS refer to?
Response to Comment 2#: The sentence was added in introduction and highlighted.
Comments 3# Line 138: 25 oC should be 25°C
Response to Comment 3#: It was corrected.
Comments 4# Materials and Methods: In addition to the concentration, different solution volumes also affect the mutagenesis rate. Please specify the amount of seed samples used and the volume of EMS solution; also, after the scheduled mutagenesis time is up, is the termination method used to terminate further mutagenesis?
Response to Comment 4#: This sentence was rewritten.
Comments 5# Line 150: MgCl2 should be MgCl2
Response to Comment 5#: It was corrected.
Comments 6# What are the usual EMS concentrations for other tests? How long is the treatment time? Compared to the concentrations in this study, it seems to be generally higher? Then what is the special application scenario of this study? Why not use the conventional concentration for the test?
Response to Comment 6#: In our study, the experimental procedure for induced EMS mutagenesis of Bahar and Akkaya (2009) [Bahar, B., & Akkaya, M. S. 2009. Effects of EMS treatment on the seed germination in wheat. Journal of applied biological sciences, 3(1), 59-64.] was followed and triple technical repeats of three different percentages of EMS (Ethylmethane Sulphonate) doses (0.10%, 0.20%, 0.30%) and a control dose (0% EMS) were applied.
Author Response File: Author Response.docx
Reviewer 4 Report
The manuscript entitled “Ethyl Methanesulfonate (EMS) Mutagen Toxicity-Induced DNA Damage, Cytosine Methylation Alteration, and iPBS-Retrotransposon Polymorphisms in Wheat (Triticum aestivum L.)” provided two types of analysis methods, iPBS and CRED-iPBS, to monitor the DNA Damage and Cytosine Methylation Alteration in wheat plants treated with EMS. The results are useful to wheat gene function identification and breeding. However, there are something concerns to be needed to explain.
1. The two result sections focused on the two analyses, it is not appear to present the meaningful conclusions.
2. The discussion appeared to be over discussed because the result information is too less.
3. If the two methods, iPBS and CRED-iPBS, are highlights, authors had reported in their selves paper, ”Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques”. The advance has missed.
English Language quality is good in this manuscript.
Author Response
Responses to Comments of Reviewer 4
We thank the potential reviewer her/his valuable time and consider our work. In the revised text we have addressed all comments and suggestion by point-to-point. Moreover, we respond to all queries as follows. We hope the revised manuscript will merit to publication in Agronomy journal. All changes are highlighted in yellow.
Sincerely,
Dr. Aras Turkoglu
Comments
Comment 1# The manuscript entitled “Ethyl Methanesulfonate (EMS) Mutagen Toxicity-Induced DNA Damage, Cytosine Methylation Alteration, and iPBS-Retrotransposon Polymorphisms in Wheat (Triticum aestivum L.)” provided two types of analysis methods, iPBS and CRED-iPBS, to monitor the DNA Damage and Cytosine Methylation Alteration in wheat plants treated with EMS. The results are useful to wheat gene function identification and breeding. However, there are something concerns to be needed to explain.
The two result sections focused on the two analyses; it is not appeared to present the meaningful conclusions.
Response to Comment 1# Dear reviewer based on valuable comment we added different table and figure in main text.
Comment 2# The discussion appeared to be over discussed because the result information is too less.
Response to Comment 2# This section was rewritten.
Comment 3# If the two methods, iPBS and CRED-iPBS, are highlights, authors had reported in their self’s paper,” Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques”. The advance has missed.
Response to Comment 3#
Dear reviewer; sodium azide mutagen, which has a different molecular structure and effect from EMS mutagen in the current manuscript, also has different application times and concentration in trial design. Plants exposed to such mutagens can represent a wide variety of mutants at very high frequencies. In the current study, we aimed to determine the methylation changes in the variations that may occur. There are numerous methods for investigating epigenetic modifications. One of them is iPBS and CRED approaches. The iPBS amplification technique has been demonstrated to be a notable DNA fingerprinting technology not requiring sequence data. CRED involving the profiling of DNA with molecular markers is used to determine the changes in DNA methylation in plant genome. This technique has been effective in detecting changes in cytosine methylation due to various abiotic stresses, such as chromium nitrate, zinc, arsenic, and lead sulfate stress/toxicity as well as sodium azide. In conclusion we can use these approaches to different experimental group related to different stress.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
This revised version has modified and responded the suggestions.
Reviewer 4 Report
The authors had revised the manuscript and provided the response to each comment. There are no any new question for this manuscript.
minor revision on this manuscript in this manuscript.
