AFLP-Based Analysis of Variation and Population Structure in Mutagenesis Induced Faba Bean

Round 1

Reviewer 1 Report

1) In the Introduction is not so clear what is the research question? or why induce mutations?, because it seems obvious that any induced mutation generate genetic variability or changes in the base sequences/AND but what for?.

2) Also in the Introduction there a clear tendency to remark the generation of genetic variation but what is the goal or objective to induce such variation, and there is not information about of the context of the mutations as strategy of plant breeding or any other aspect. All this generate doubts on the objective stated because is obvious that any induced mutation generate genetic variation but is good, bad or what for and also is not so clear what is the contribution of the paper.

3) In materials and methods not present details about form of use of the agent mutagenic. For example, what plant material was irradiated or treated was seed, seedlings or what propagule was used, what is technical basis to decide the doses and agent mutagenic because is no clear what traits were the study object to address the genetic analysis. In the same section indicate that 116 mutant families where chosen for the analysis but no what criteria used to discriminate. What was the objective/purpose to incorporate two landraces and one cultivar?.

4) The sections of results and discussion the authors remark the genetic variability and differences between doses and mutagenic agents but what is the contribution or probable relationships with trait of agronomic interest or any other interest.

Author Response

Reviewer  1

1) In the Introduction is not so clear what is the research question? or why induce mutations?, because it seems obvious that any induced mutation generate genetic variability or changes in the base sequences/AND but what for?

Thank you very much for your questions. This study was conducted based on our previous study that assesses genetic diversity at the morphological level. Our study found various mutant types that were different from the control plant. The present study aimed to assess genetic diversity at a molecular level that gives a comprehensive picture of genetic diversity induced by mutation at selected doses of mutagens and compare with phenotypic data.  

2) Also in the Introduction there a clear tendency to remark the generation of genetic variation but what is the goal or objective to induce such variation, and there is no information about the context of the mutations as a strategy of plant breeding or any other aspect. All this generates doubts on the objective stated because is obvious that any induced mutation generates genetic variation but is good, bad or what for and also is not so clear what is the contribution of the paper.

Thank you very much for the comments. We improve our introduction as well as the objectives of the study.

3) In materials and methods not present details about form of use of the agent mutagenic. For example, what plant material was irradiated or treated was seed, seedlings or what propagule was used, what is technical basis to decide the doses and agent mutagenic because is no clear what traits were the study object to address the genetic analysis. In the same section indicate that 116 mutant families where chosen for the analysis but no what criteria used to discriminate. What was the objective/purpose to incorporate two landraces and one cultivar?

Thank you very much for the questions.

1. The plant material was dry seed. The additional explanation has been incorporated in section Materials and Methods.
2. A technical basis of deciding the dose and kinds of mutagen was based on our previous study that gives high phenotypic variation. Our publications found that gamma radiation and DES at selected doses generated variations at the morphological level. Please kindly find our publications below.
3. Selection criteria were based on phenotypic differences of mutant plants. The additional explanation has been incorporated in section Materials and Methods.
4. These three genotypes are well adapted to Saudi Arabia environment. Based on our previous study, the three genotypes displayed high genetic variability. Moreover, the four reference genotypes represented three different geographical origins than can be beneficial for studying population structure and we can measure the genetic distance of the mutant population relative to these reference genotypes.

• Nurmansyah, Alghamdi,S.,Migdadi, H.M., and Farooq, M. 2019.Novel inflorescence architecture in gamma radiation-induced faba bean mutant populations. International Journal of Radiation Biology 95(12): 1744-1751.
• Nurmansyah, Alghamdi,S.,and Migdadi, H.M.. 2020. Morphological diversity of faba bean (Vicia faba L.) M₂ mutant populations induced by gamma radiation and diethyl sulfate (DES). Journal of King Saud University-Science 32(2): 1647-1658.

 

 4) The sections of results and discussion the authors remark the genetic variability and differences between doses and mutagenic agents but what is the contribution or probable relationships with trait of agronomic interest or any other interest.

Thank you very much for your suggestion. The additional subsection (3.6 Relationship between molecular and phenotypic data) has been incorporated to cover these questions.

Reviewer 2 Report

１st-peer review
The manuscript is deal with genetic diversity and population
structure by mutagen treatments of faba bean. Some of revisions are
needed for the publication of the Journal.
1. In general, LD50 for gamma ray radiation is 200Gy to 250Gy for
soybean and faba bean. Also, 250Gy in rice (Lines 339-342). The
radiation intensity is designed for 25Gy to 50Gy in this study. Would
you explain the reason, please?
2.　The selected sample numbers are lack of uniformity, such as, 13
(0.01%, DES), 5 (0.05%, DES), 61 (25Gy, gamma ray) and 37 plants
(50Gy, gamma ray), respectively. It is very important to basal concept
in the experiment design. Actually, polymorphic loci % (% P) is
increased by the number of samples’ increase rather than kinds of
mutagen and radiation/treatment intensity. Total plant numbers of
DES treated are 18 and the numbers of gamma ray treated are 98
plants.
3. Lines 99~100: What is the selection criteria　when the
‘representing 116 plants’ were collected from 1,324 M1 mutant plants?
Phenotype, survival rate or any other factors of the plants? The lack
of uniformity of the sample numbers are related with the selection
criteria?
4. Line 186, Line 330: All　of the primer　combinations exhibited　
100% polymorphism. Is there any mono－morphic band？ Or you
count only polymorphic bands?
Line 190 (Table 1): Change the pont on Table 1, from ‘EcoR1 and
Mse1’ to ‘Eco R1 and Mse 1’.
5. Lines 201~203: Sentences (or Explanation) of Table 2 may be
deleted and skipped. Would you add more description for Table 2?
6. Line 208 (Table 2): Table 2 have to include data of the reference
plants as control. Data for control (the reference plants) are skipped
in Table 2.
7. Lines 219~220: Sentence revision may be need.
‘The mean number of different alleles (Na) and the number of
effective alleles of mutants and the reference plants were~．’？
8. Lines 225-226 (Table 3): Would you change ‘No. of mutants’ to
‘No. of samples’ on Table 3.
9. Lines 311-312: Would you change ‘~by four mutagen treatments’
to like ‘~by four treatments (two kinds of mutagen and two kinds of
intensity)’.
10. Lines 352-354: It was mentioned that chemical mutagen treatment
showed higher genetic diversity than that of physical mutagens.
However, it is very hard to compare with chemical mutagens vs
physical mutagens. Because kind of mutagen and intensity of
mutagen are more important to yield genetic diversity. And, so many
kinds of chemical and physical mutagens, and radiation intensity (or
treatment concentration) are used at the mutation fields. Therefore,
it is natural that describe range of radiation intensity and kinds of
radiation when the genetic diversity are compared by kinds of
mutagens.　
11. There are differences among the populations (four treatments) at
the molecular based diversity. How about the relation among the
populations at the phenotype level?

Comments for author File: Comments.zip

Author Response

The manuscript is deal with genetic diversity and population structure by mutagen treatments of faba bean. Some of revisions are needed for the publication of the Journal.

1. 
   In general, LD50 for gamma ray radiation is 200Gy to 250Gy for soybean and faba bean. Also, 250Gy in rice (Lines 339-342). The radiation intensity is designed for 25Gy to 50Gy in this study. Would you explain the reason, please?

Thank you very much for the question. The reason for using 25 Gy and 50 Gy gamma radiation was based on our previous studies. Previously, we tried a wide range of gamma intensity on faba bean from 25 Gy to 500 Gy and only worked well up to 50 Gy. Our previous publication (https://www.tandfonline.com/doi/abs/10.1080/09553002.2018.1409913) showed that 50 Gy gamma radiation caused 69% reduction of seedling emergence at M1 generation. Therefore, we use 25 Gy and 50 Gy based on this consideration.

2.　The selected sample numbers are lack of uniformity, such as, 13 (0.01%, DES), 5 (0.05%, DES), 61 (25Gy, gamma ray) and 37 plants (50Gy, gamma ray), respectively. It is very important to basal concept in the experiment design. Actually, polymorphic loci % (% P) is increased by the number of samples’ increase rather than kinds of mutagen and radiation/treatment intensity. Total plant numbers of DES treated are 18 and the numbers of gamma ray treated are 98 plants.

Thank you very much for the question. The lack of sample uniformity was due to the survival rate of different doses of mutagen. Previously, we generated M1 generation from separate 120 dry seeds that were treated to the four treatments. Then the survive M1 plants were individually harvested and sown as M2 families. After that, we select mutant from M2 families which had phenotypical differences with control plant.

3. 
   Lines 99~100: What is the selection criteria　when the ‘representing 116 plants’ were collected from 1,324 M1 mutant plants? Phenotype, survival rate or any other factors of the plants? The lack of uniformity of the sample numbers are related with the selection criteria?

Thank you very much for the questions. Yes, the lack of uniformity was based on the selection criteria. The selection criteria of selected mutants were based on survival rates at the M₁ and M₂ generations followed by phenotypic differences. Additional explanation has been incorporated in section Materials and Methods.

4. 
   Line 186, Line 330: All　of the primer　combinations exhibited 100% polymorphism. Is there any mono－morphic band？ Or you count only polymorphic bands? Line 190 (Table 1): Change the pont on Table 1, from ‘EcoR1 and Mse1’ to ‘Eco R1 and Mse 1’.

Thank you very much for the question and suggestion. As our observation of banding pattern generated by GeneMapper, we did not find any monomorphic band. The suggestion has been incorporated.

5. 
   Lines 201~203: Sentences (or Explanation) of Table 2 may be deleted and skipped. Would you add more description for Table 2?

Thank you very much for your suggestion. The suggestion has been incorporated.

6. 
   Line 208 (Table 2): Table 2 has to include data of the reference plants as control. Data for control (the reference plants) are skipped in Table 2.

Thank you very much for your suggestion. The suggestion has been incorporated.

7. 
   Lines 219~220: Sentence revision may be need. ‘The mean number of different alleles (Na) and the number of effective alleles of mutants and the reference plants were~．’？

Thank you very much for your suggestion. The suggestion has been incorporated.

8. 
   Lines 225-226 (Table 3): Would you change ‘No. of mutants’ to ‘No. of samples’ on Table 3.

Thank you very much for your suggestion. The suggestion has been incorporated.

9. Lines 311-312: Would you change ‘~by four mutagen treatments’ to like ‘~by four treatments (two kinds of mutagen and two kinds of intensity)’.

Thank you very much for your suggestion. The suggestion has been incorporated.

10. 
    Lines 352-354: It was mentioned that chemical mutagen treatment showed higher genetic diversity than that of physical mutagens. However, it is very hard to compare with chemical mutagens vs physical mutagens. Because kind of mutagen and intensity of mutagen are more important to yield genetic diversity. And, so many kinds of chemical and physical mutagens, and radiation intensity (or treatment concentration) are used at the mutation fields. Therefore, it is natural that describe range of radiation intensity and kinds of radiation when the genetic diversity are compared by kinds of mutagens.　

Thank you very much for your valuable comments and suggestions. The additional explanations have been incorporated.

11. 
    There are differences among the populations (four treatments) at the molecular based diversity. How about the relation among the populations at the phenotype level?

Thank you very much for your question and suggestion. The additional subsection (3.6 Relationship between molecular and phenotypic data) has been incorporated.

Round 2

Reviewer 1 Report

Two considerations:

a) Include the references used for define the doses and agent mutagenic.

b) Define why include the controls ILB4347, Hassawi 2, 112 Hassawi 3, and Misr 3, no as geographic representation, much more as references of genetic diversity versus mutants and homogenous population.

Author Response

Dear Prof.

With greetings

Thank you for your valuable comments. Please find our corrections made on the manuscript.

Reviewer  1

Two considerations:

1. Include the references used for defining the doses and agent mutagenic.

Thank you very much for your suggestion. We have incorporated additional reference in section Materials and Methods.

1. b) Define why include the controls ILB4347, Hassawi 2, Hassawi 3, and Misr 3, not as geographic representation, much more than references of genetic diversity versus mutants and homogenous population.

We based the deciding of reference genotypes on our previous studies when we screened various faba bean genotypes using ISSR and SRAP markers. Each of deciding reference genotypes represented different clusters as described by these molecular markers.  Please kindly find our publications below.

Alghamdi, S.S., Al-Faifi, S.A., Migdadi, H.M., Ammar, M.H., and Siddique, K.M. (2011). Inter-simple sequence repeat (ISSR)-based diversity assessment among faba bean genotypes. Crop Pasture Science 62(9), 755–760. https://doi.org/10.1071/CP11045

Alghamdi, S.S.,  Al-Faifi, S.A., Migdadi, H.M., Khan, M.A., EL-Harty, E.H., and Ammar, M.H. (2012b). Molecular diversity assessment using Sequence Related Amplified Polymorphism (SRAP) markers in Vicia faba L. International Journal of Molecular Sciences 1(12) 3, 16457-16471. https://doi.org/10.3390/ijms131216457

Reviewer 2 Report

Most of revisions are excuted. The lack of uniformity of the sample numbers still remain weak point. Lines 281 to 284: I think the sentence is correct. 

Author Response

Dear Prof.

With greetings

Thank you very much for your valuable comments. Please find our correction on the manuscript.

Most of the revisions executed. The lack of uniformity of the sample numbers remain weak point. Lines 281 to 284: I think the sentence is correct. 

Thank you very much for your comments. However, the lack of sample uniformity was because of the survival rate of different doses of mutagen where increased doses reduced the survival rate. We based the comparison between phenotypic and molecular data on the relative number of mutants within the dose of mutagen treatment.