Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques

Round 1

Reviewer 1 Report

The manuscript entitled “Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques” demonstrated that the wheat plants were mutated by NaN3 and were checked by iPBS and CRED-iPBS for identification of genome instability and cytosine methylation. The results showed that the mutation ratios are determined by action time and concentration of NaN3. This research could provide a good method to identify genomic mutation by chemical materials for plant breeding.

This story is simple and fair, but it is useful in plant germplasm development.

 

Concerns for revision:

1.     The bands were disappeared compared to the control, while the new bands had to be appeared because of the primer amplification products.

2.     The lots of genomic mutation loci were taken place, the phenotypes need to be shown in this research.

3.     The genomic mutation including cytosine methylation detected by iPBS and CRED-iPBS need to be further verified by other methods by selection of represent loci.

 

English editing was morderatedly needed.

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 Agriculture journal. All revised parts have been highlighted in blue.

 

Sincerely,

Dr. Aras Turkoglu

Comments

Comment 1# The manuscript entitled “Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques” demonstrated that the wheat plants were mutated by NaN3 and were checked by iPBS and CRED-iPBS for identification of genome instability and cytosine methylation. The results showed that the mutation ratios are determined by action time and concentration of NaN3. This research could provide a good method to identify genomic mutation by chemical materials for plant breeding.

This story is simple and fair, but it is useful in plant germplasm development.  The bands were disappeared compared to the control, while the new bands had to be appeared because of the primer amplification products.

 

Response to Comment 1#

Many thanks for the valuable insights of Review 1

 

Comment 2# The lots of genomic mutation loci were taken place, the phenotypes need to be shown in this research.

Response to Comment 2#

This part added the main text.

 

Comment 3# The genomic mutation including cytosine methylation detected by iPBS and CRED-iPBS need to be further verified by other methods by selection of represent loci.

Response to Comment 3#

The sentences " The iPBS and CRED-iPBS methodologies have been proficiently employed for genetic scrutiny in wheat that has been subjected to mutagenesis. It is advisable to conduct additional research to elucidate the various facets of phytotoxicity induced by mutagenicity in plants." were added to the Conclusions section of the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Wheat is one of key field crop in the world. The use of molecular mutation breeding as one of key techniques for crop improvement. In this study, authors use the conventional chemical mutagenesis technique via sodium azide (NaN3) for germination in wheat. The results showed that treatments with NaN3 had an effect on the level of cytosine methylation and the stability of the genomic template in wheat plants in germination stage and produced some significant results. In general , this manuscript will well-written and experimental design is good and had some novel data, but the presentation of data are too simple, only two tables, no figure. Here are my comments:

 

Commnets:

1.     The data are only two tables, it is too simple and there is no figure in the paper, at least you should make the figure to present part of your data.

2.     There is no confirmation data to support your results

3.     Table 1 should keep the same digital number;

4.     Table 2 and 3 are very difficult to see, it should make it more concise.

Author Response

Responses to Comments of Reviewer 2

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 Agriculture journal. All revised parts have been highlighted in green.

Sincerely,

Dr. Aras Turkoglu

 

Comments

Comments 1# The data are only two tables, it is too simple and there is no figure in the paper, at least you should make the figure to present part of your data.

Response to Comment 1#:

We added several figures (figure 1,2,3 and 4) related to table in the main text.

 

Comments 2# Table 1 should keep the same digital number.

Response to Comment 2#:

Digital numbers were corrected in Table1 considering the reviewer's suggestion.

 

Comments 4# Table 2 and 3 are very difficult to see, it should make it more concise.

Response to Comment 4#:

We added the Figure 1-2-3 and 4 in main text for tables 2 and 3 for better understanding.

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Türkoğlu et al. presented a work to estimate genomic instability and DNA methylation due to NaN3 treatment in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS markers. In general, I found some novel point in this paper, but I have three major concerns as listed below:

1) A similar work (from same group) has been reported the effect of aluminum (Al) treatment in wheat by these two markers (iPBS and CRED-iPBS).  Please see: POUR, ARASH HOSSEIN; ÖZKAN, GÜLLER; NALCI, ÖZGE; and HALİLOĞLU, KAMİL (2019) "Estimation of genomic instability and DNA methylation due to aluminum (Al) stress in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS analyses," Turkish Journal of Botany: Vol. 43: No. 1, Article 2. https://doi.org/10.3906/bot-1804-23

Thus, it reminds me whether this job is of great novelty.

And even, whether CRED-iPBS is an effective method to identify DNA methylation level before and after treatment by NaN3, as there may have many other DNA methylation detecting methods (for example, Bisulfite sequencing).

2) To more attractive ilustration of the diverse bands of iPBS and CRED-iPBS, it is needed to proved pic after agrose gel electrophoresis.

3) Is there any phenotype change before and after treatment by NaN3?

I am worried aboutwhether this job is of great novelty as a strong candidate.

Author Response

Responses to Comments of Reviewer 3

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 Agriculture journal. All changes are highlighted in yellow.

 

Sincerely,

Dr. Aras Turkoglu

 

Comments

Comment 1# Türkoğlu et al. presented a work to estimate genomic instability and DNA methylation due to NaN₃ treatment in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS markers. In general, I found some novel point in this paper, but I have three major concerns as listed below: A similar work (from same group) has been reported the effect of aluminum (Al) treatment in wheat by these two markers (iPBS and CRED-iPBS).  Please see: POUR, ARASH HOSSEIN; ÖZKAN, GÜLLER; NALCI, ÖZGE; and HALİLOĞLU, KAMİL (2019) "Estimation of genomic instability and DNA methylation due to aluminum (Al) stress in wheat (Triticum aestivum L.) using iPBS and CRED-iPBS analyses," Turkish Journal of Botany: Vol. 43: No. 1, Article 2. https://doi.org/10.3906/bot-1804-23. Thus, it reminds me whether this job is of great novelty. And even, whether CRED-iPBS is an effective method to identify DNA methylation level before and after treatment by NaN3, as there may have many other DNA methylation detecting methods (for example, Bisulfite sequencing).

Response to Comment 1#

The article the reviewer pointed to differs from this manuscript presented. In the article examining the results of aluminum (Al) treatment, only the number of bands is presented in the tables instead of the molecular size of the bands. Sodium azide therapy, which has a different molecular structure and effect from Al in the current manuscript, also has different application times 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. The iPBS amplification technique has been demonstrated to be a notable DNA fingerprinting technology not requiring sequence data. The use of the iPBS marker is an easy and rapid method for monitoring changes in the DNA profile of plants. 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. There are numerous methods for investigating epigenetic modifications. One of these methods involves combining PCR with enzymes that are sensitive to cytosine methylation in order to detect DNA modifications. Enzymes sensitive to cytosine methylation make high-throughput analysis of this epigenetic control mechanism feasible, and the procedure is straightforward and cost-effective.

Comment 2# To more attractive ilustration of the diverse bands of iPBS and CRED-iPBS, it is needed to proved picture after agrose gel electrophoresis.

Response to Comment 2#

We added agarose gel gel electrophoresis photo for iPBS and CRED-iPBS.

Comment 3# Is there any phenotype change before and after treatment by NaN3?

Response to Comment 3#

This part added the main text.

 

 

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

In the manuscript “Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques” Aras Türkoğlu and co-authors described experiments with NaN3 mutagenesis. Experiment was employed to assesment potential applay of chemical mutagenesis technique for germination in wheat. Congratulation for this work where an exhaustive DNA fragment analysis were done.

This is well written and clear, and with a good experimental design. However, the paper is moderately innovative. Authors have previously published a complementary work. The manuscript is suitable for publication in the Genotype evaluation and selection section. However, some issues should be revised.

-        In introduction section, authors should introduce clearly the effect of azide and reported results (genotypic and phynotypic)

-        Section 2.3 : 40 cycles for PCR using Taq is not suitable because DNA changes could be produced !!!?...please use numbers for PCR step temperatures.

-        Table 1 : did you use one primer or pair of primers !? if yes, please, forward and reverse primer should be metioned.

-        Line 186,  GTS should be identified and explaind for the first use !

-        Table 2, control is plant whithout sodium azide treatment ? bands in (-) are not synthetized ? It was not clear !!!! the same with table 3 !

-        Discussion section, did authors check other kind of mutations ? if not discussion should be done.

-        We don’t know if there was some problems with plant growth etc !!? germination rates.

-        What is the real purpose of the study, given that the same experiments have been carried out before?

-        In addition, we know that this type of technique is expensive in plant breeding programs, and that they are avoided.

what does this technique offer?

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 Agriculture journal. All changes are highlighted in red.

 

Sincerely,

Dr. Aras Turkoglu

 

Comments

Comment 1# In the manuscript “Sodium Azide as a Chemical Mutagen in Wheat (Triticum aestivum L.): Patterns of the Genetic and Epigenetic Effects with iPBS and CRED-iPBS Techniques” Aras Türkoğlu and co-authors described experiments with NaN3 mutagenesis. Experiment was employed to assesment potential applay of chemical mutagenesis technique for germination in wheat. Congratulation for this work where an exhaustive DNA fragment analysis were done. This is well written and clear, and with a good experimental design. However, the paper is moderately innovative. Authors have previously published a complementary work. The manuscript is suitable for publication in the Genotype evaluation and selection section. However, some issues should be revised.

In introduction section, authors should introduce clearly the effect of azide and reported results (genotypic and phenotypic).

Response to Comment 1#

In line with the reviewer's suggestion, effect of sodium azide was written in the "introduction" part.

 

Comment 2# Table 1: did you use one primer or pair of primers !? if yes, please, forward and reverse primer should be metioned.

Response to Comment 2#

We used one primer (5’-3’). Primer list was showed in Table 1.

 

Comment 3# Line 186, GTS should be identified and explaind for the first use!

Response to Comment 3#

In line with the reviewer's suggestion, a description of "GTS" was added where it was first used.

 

Comment 4# Table 2, control is plant whithout sodium azide treatment? bands in (-) are not synthetized? It was not clear !!!! the same with table 3 !

Response to Comment 4#

The sentence "The (-) a sign in Tables 2 and Table 3 indicates that there was no difference between the control and experimental groups in terms of band increase or band decrease" was added to the manuscript in method section.

 

Comment 5# Discussion section, did authors check other kind of mutations? if not discussion should be done.

Response to Comment 5#

Yes, we checked mutation such as EMS. In manuscript, it was showed highlighted in red.

 

Comment 6#   We don’t know if there were some problems with plant growth etc !!? germination rates.

Response to Comment 6#

This part added the main text.

 

Comment 7# What is the real purpose of the study, given that the same experiments have been carried out before?

Response to Comment 7#

The objective of this research was to investigate the impact of sodium azide, on genomic instability and cytosine methylation in the Kirik variety of bread wheat (Triticum aestivum L.) in the seedling growth stage. In this study not only we applying the CRED-iPBS techniques for detection changes in cytosine methylation but also, we identified the polymorphism rate. In the previous study, only study on iPBS marker techniques for detection polymorphism.

 

Comment 8# In addition, we know that this type of technique is expensive in plant breeding programs, and that they are avoided. what does this technique offer?

Response to Comment 8#

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. The iPBS amplification technique has been demonstrated to be a notable DNA fingerprinting technology not requiring sequence data. The use of the iPBS marker is an easy and rapid method for monitoring changes in the DNA profile of plants. 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. There are numerous methods for investigating epigenetic modifications. One of these methods involves combining PCR with enzymes that are sensitive to cytosine methylation in order to detect DNA modifications. Enzymes sensitive to cytosine methylation make high-throughput analysis of this epigenetic control mechanism feasible, and the procedure is straightforward and cost-effective.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The reversion is in a better form. I have no further quesitons

Reviewer 3 Report

Authors have addressed all my concerns, I suggest to publish this paper.

Reviewer 4 Report

Dear authors,

congratulations;

Best regards