Comparative Study of Isoflavone Synthesis Genes in Two Wild Soybean Varieties Using Transcriptomic Analysis

Round 1

Reviewer 1 Report

Isoflavone composition varies across different soybean seeds due to the expression of multiple genes. RNA-seq technology was employed to identify potential differentially expressed genes (DEGs) and to compare the differences of isoflavone content between only two wild soybean varieties, ZYD7068 (high iso-flavone) and ZYD7194 (low isoflavone). Differentially expressed metabolic DEGs were identified involved in Isoflavonoid biosynthesis and Flavone and flavonol biosynthesis: 4 genes were found to positively regulate isoflavone synthesis and 2 were involved in negative regulation.

The studied soybean varieties ZYD7068 and ZYD7194 were grown by the Institute of Tillage and Cultivation, Heilongjiang Academy of Agricultural Sciences, and in the pot farm of Hei-longjiang Academy of Agricultural Sciences.

The material is not sufficiently described at all. RNA was sampled and extracted from the seeds at R5 and R8 stages (sometimes R6 is mentioned); the phenotypic stages need to be clearly defined. The genetic status of the varieties should also be mentioned, e.g. whether the material was provided from a gene bank or multiplied (several times) in the institutions mentioned before.

Figure 1 (A) shows seed phenotypes of the 2 wild soybean varieties; this figure does not seem to show anything relevant, so that it is obsolete and must be omitted.

Table 1 should show the Summary of transcriptome data of soybean seeds. However, the presented data is difficult to understand; it would deserve better presentation and clear explanation.

 

Conclusion: This paper presents on the results of sophisticated metabolic analyses applied to only two wild soybean accessions which are very little described phenotypically.

It would have been advised to better identifiy suitable experimental material and describe this meterial in detail phenotypically as well as genotypically. 

Author Response

Dear Editor:

Re: View Decision: major revisions .

We truly appreciate your efforts in handling this manuscript, and herein submit a revised version of our manuscript entitled " Comparative Study of Isoflavone Synthesis Genes in Two Wild Soybean Varieties using Transcriptomic Analysis.

We would like to thank the Reviewer for voicing concerns, suggesting amendments and providing cogent advice as to how to address certain deficiencies in the manuscript. We have carefully studied and addressed the critiques of the reviewers to clarify and improve the paper. We respond to the Reviewers’ Comments in order below:

Q1: The material is not sufficiently described at all. RNA was sampled and extracted from the seeds at R5 and R8 stages (sometimes R6 is mentioned); the phenotypic stages need to be clearly defined. The genetic status of the varieties should also be mentioned, e.g. whether the material was provided from a gene bank or multiplied (several times) in the institutions mentioned before.

Response: Thank you for pointing out the problem. Based on your suggested changes, we have added the source of the material to the method, and we have also specified the sampling period. The seeds of R6 and R8 stages were used for RNA sequencing (RNA-seq) and the seeds of R5 to R8 stages were used for quantitative real-time polymerase chain reaction (qRT-PCR) experiments.

Q2: Figure 1 (A) shows seed phenotypes of the 2 wild soybean varieties; this figure does not seem to show anything relevant, so that it is obsolete and must be omitted. 

Response: Thank you for pointing out this. We added a detailed phenotype about the isoflavone fraction in the corresponding results section as well as modified Figure 1(A).

Q3: Table 1 should show the Summary of transcriptome data of soybean seeds. However, the presented data is difficult to understand; it would deserve better presentation and clear explanation.

Response: Thank you for pointing out this. We modified the data names in Table 1, added more detailed descriptions, and gave a more detailed introduction in the article.

Conclusion: This paper presents on the results of sophisticated metabolic analyses applied to only two wild soybean accessions which are very little described phenotypically. It would have been advised to better identifiy suitable experimental material and describe this meterial in detail phenotypically as well as genotypically.

Response: We gratefully appreciate for your valuable comment. We have made detailed changes to your suggestion.

Reviewer 2 Report

Major:

1) I recommend that the authors should use some help of a native English speaker or send the manuscript (Ms) to an English Editing Service that proofreads scientific writing.

 

2) In general, authors should improve all used figures and tables, e.g. increase font size, image quality, explain all used abbreviations in legends, etc. It is very difficult to understand the results of the Ms in this form.

 

3) Authors should explain in detail the origin and features of the plants used in experiments (ZYD7068 and ZYD7194). Also, explain R5 and R8 stages (maybe give photos as additional material?).

 

4) In my opinion, authors should avoid clear unambiguous expressions like: “Four genes (Glyma.13G173300, 27 Glyma.13G173600, Glyma.14G103100, and Glyma.17G158900) were found to positively regulate isoflavone synthesis, whereas two genes (Glyma.04G036700 and Glyma.19G030500) showed negative regulation.”

Because with the help of the chosen methods (real-time PCR and next generation sequencing), we can only talk about the possible

 

5) Is it possible to provide data on individual isoflavones?

 

Minor:

6) Fig. 1: include the bar (1 cm).

I recommend that the authors should use some help of a native English speaker or send the manuscript (Ms) to an English Editing Service that proofreads scientific writing.

Author Response

Dear Editor:

Re: View Decision: major

We truly appreciate your efforts in handling this manuscript, and herein submit a revised version of our manuscript entitled " Comparative Study of Isoflavone Synthesis Genes in Two Wild Soybean Varieties using Transcriptomic Analysis.

We would like to thank the Reviewer for voicing concerns, suggesting amendments and providing cogent advice as to how to address certain deficiencies in the manuscript. We have carefully studied and addressed the critiques of the reviewers to clarify and improve the paper. We respond to the Reviewers’ Comments in order below:

Q1:I recommend that the authors should use some help of a native English speaker or send the manuscript (Ms) to an English Editing Service that proofreads scientific writing.

Response: We gratefully appreciate for your valuable comment. We have checked and revised the article according to your opinion.

Q2:In general, authors should improve all used figures and tables, e.g. increase font size, image quality, explain all used abbreviations in legends, etc. It is very difficult to understand the results of the Ms in this form. 

Response: Thank you for pointing out this. In response to your suggestion, we have revised the pictures and tables.

Q3: Authors should explain in detail the origin and features of the plants used in experiments (ZYD7068 and ZYD7194). Also, explain R5 and R8 stages (maybe give photos as additional material?). 

Response: We gratefully appreciate for your valuable comment. We supplemented the source of plants and introduced the isoflavone content in more detail. In Figure 1A, the photos of seeds from R5 to R8 stages are added, and the references of the standard of soybean growth period division are also added in the methods and materials.

Q4: In my opinion, authors should avoid clear unambiguous expressions like: “Four genes (Glyma.13G173300, 27 Glyma.13G173600, Glyma.14G103100, and Glyma.17G158900) were found to positively regulate isoflavone synthesis, whereas two genes (Glyma.04G036700 and Glyma.19G030500) showed negative regulation.”

Because with the help of the chosen methods (real-time PCR and next generation sequencing), we can only talk about the possible

Response: We gratefully appreciate for your valuable comment. According to your suggestion, corresponding changes have been made.

Q5: Is it possible to provide data on individual isoflavones?

Response: Thank you for the nice suggestion. We have added an introduction to the isoflavone content of different components in Figure 1B and the results.

Minor:

Q6: Fig. 1: include the bar (1 cm).

Response: Thank you so much for your careful check. For your suggestion, we have modified Figure 1.

Round 2

Reviewer 1 Report

Dear authors, thank you for carefully revising your paper. It will now be subject to decision by the chief editor.

Author Response

Dear Editor:

We sincerely thank you for your efforts in handling this manuscript, and thank you very much for your opinions.

Reviewer 2 Report

1) authors should improve all used figures and tables in manuscript (Ms) text, e.g.

a) Fig. 1a: increase figure quality and present on the white background, not black. Also, include the bar (1 cm).

b) Fig. 1b: enhance the font size.

c) Fig. 1b: “The isoflavone content in the mature seeds of ZYD7068 and ZYD7194”. please, explain “mature seeds”. Is this the R8 stage? Сan the authors submit isoflavone content data at stage R5 or R6? To show the dynamics of isoflavone accumulation.

d) Legend for Table 1: explain A1, B1, A3, B3, Q30.

e) Fig. 2b: enhance the font size and I do not understand this figure.

f) Fig 3: enhance the font size.

g) Fig 4: enhance the font size.

h) Fig 5: explain A1, A3, B1, B3 in the legend for figure.

i) Fig 6: explain A1, A3, B1, B3 in the legend for figure.

j) Fig 7: correct “in 4 seed developmental stages” to “in 4 seed developmental stages (R5, R6, R7, and R8)”.

 

2) How many biological repeats were there when measuring the isoflavone content (Fig. 1b) and in qRT-PCR analysis (Fig. 7)? Why such small values of error values? Authors used SEM or SD? Explain in the legends.

 

3) “Authors should explain in detail the origin and features of the plants used in experiments (ZYD7068 and ZYD7194).

Response: We gratefully appreciate for your valuable comment. We supplemented the source of plants and introduced the isoflavone content in more detail.”

I did not find this information in Ms text. Please, include in response the line number.

 

4) Line 139-140: “The calculation of gene expression level followed the 2-ΔCT method described by Livak & Schmittgen[27].”

Correct to

“The calculation of gene expression level followed the 2-ΔΔCT method described by Livak & Schmittgen [27].”

 

5) Usually in Livak & Schmittgen (2001) calculation there is a sample with a value of about 1.0. I don't find such samples in the Fig.7a, b, c, d, e, and f.

English: I recommend that the authors should use some help of a native English speaker or send the manuscript (Ms) to an English Editing Service. I still find a lot of small typos and inaccuracies. Maybe it will just be enough for the authors to carefully re-read the Ms text.

Author Response

Dear Editor:

Re: View Decision: major revisions .

We truly appreciate your efforts in handling this manuscript, and herein submit a revised version of our manuscript entitled "Comparative Study of Isoflavone Synthesis Genes in Two Wild Soybean Varieties using Transcriptomic Analysis".

We would like to thank the Reviewer for voicing concerns, suggesting amendments and providing cogent advice as to how to address certain deficiencies in the manuscript. We have carefully studied and addressed the critiques of the reviewers to clarify and improve the paper. We respond to the Reviewers’ Comments in order below:

1. a) Fig. 1a: increase figure quality and present on the white background, not black. Also, include the bar (1 cm). 

Response: Thank you for your rigorous consideration advice. We have modified Figure 1A as you suggested.

 

1. b) Fig. 1b: enhance the font size. 

Response: We gratefully appreciate for your valuable suggestion. We have enhanced the font size.

 

1. c) Fig. 1b: “The isoflavone content in the mature seeds of ZYD7068 and ZYD7194”. please, explain “mature seeds”. Is this the R8 stage? Сan the authors submit isoflavone content data at stage R5 or R6? To show the dynamics of isoflavone accumulation.

Response: We gratefully appreciate for your valuable suggestion. In this study mature seeds are those at R8 stage, which we have labeled in the article.

 

30. d) Legend for Table 1: explain A1, B1, A3, B3, Q30. 

Response: Thank you for your questions. We have explained A1, B1, A3, B3 and Q30 in the note. A1:ZYD7068 seeds at R6 stage. B1:ZYD7194 seeds at R6 stage. A3:ZYD7068 seeds at R8 stage. B3:ZYD7194 seeds at R8 stage. Q30: The base mass value is 30. Raw reads Q30 (%): The number of bases with Phred values greater than 30 in the raw data as a percentage of the total number of bases. Clean reads Q30 (%): The number of bases with Phred values greater than 30 in the clean data as a percentage of the total number of bases.

 

1. e) Fig. 2b: enhance the font size and I do not understand this figure.

Response: Thank you for pointing out the problem. We have enhance d the font of Figure 1B. The four colors respectively represent DEGs of different combinations of A1 vs A3, A1 vs B1, A3 vs B3 and B1 vs B3, and the number of identical DEGs among the four combinations.

 

1. f) Fig 3: enhance the font size.

Response: Thank you for your questions. We have enhanced the font as you suggested.

 

1. g) Fig 4: enhance the font size.

Response: Thank you for pointing out the problem. We have enhanced the font of Figure 4.

 

1. h) Fig 5: explain A1, A3, B1, B3 in the legend for figure. 

Response: Thank you for your questions. We have explained A1, B1, A3 and B3 in the legend.

 

i)Fig 6: explain A1, A3, B1, B3 in the legend for figure.

Response: Thank you so much for your careful check. We have supplemented the legend according to your suggestion.

 

1. j) Fig 7: correct “in 4 seed developmental stages” to “in 4 seed developmental stages (R5, R6, R7, and R8)”. 

Response: Thank you for pointing out this. We have revised it according to your suggestion.

 

2) How many biological repeats were there when measuring the isoflavone content (Fig. 1b) and in qRT-PCR analysis (Fig. 7)? Why such small values of error values? Authors used SEM or SD? Explain in the legends. 

Response: Thank you for your questions. Three biological replicates of each sample. The error of this study accords with the experiment, but the error line is not obvious because of the large experimental value. Error bars represent the standard deviations of data.

 

3) “Authors should explain in detail the origin and features of the plants used in experiments (ZYD7068 and ZYD7194). 

Response: We gratefully appreciate for your valuable comment. We supplemented the source of plants and introduced the isoflavone content in more detail.”

I did not find this information in Ms text. Please, include in response the line number. 

Response: Thank you for pointing out this. The origin and characteristics of plants (ZYD7068 and ZYD7194) are in line 78 to line 81. 

 

4) Line 139-140: “The calculation of gene expression level followed the 2-ΔCT method described by Livak & Schmittgen[27].”

Correct to “The calculation of gene expression level followed the 2-ΔΔCT method described by Livak & Schmittgen [27].”

Response: Thank you for pointing out this. In this study, the method of  in Livak & Schmittgen[27] is adopted, which is also usually written as 2^-ΔCT.

 

 

5) Usually in Livak & Schmittgen (2001) calculation there is a sample with a value of about 1.0. I don't find such samples in the Fig.7a, b, c, d, e, and f.

Response: We gratefully appreciate for your valuable comment. In this study, the method of  in Livak & Schmittgen[27] was adopted, which was suitable for determining the influence of experimental treatment on the expression of candidate internal control genes. In this study, it was to determine whether soybean resources with different isoflavone contents could affect the expression of candidate genes.

 

Comments on the Quality of English Language

English: I recommend that the authors should use some help of a native English speaker or send the manuscript (Ms) to an English Editing Service. I still find a lot of small typos and inaccuracies. Maybe it will just be enough for the authors to carefully re-read the Ms text.

Response: Thank you for pointing out this. We have corrected and checked for spelling issues.