Assessment of Simple Sequence Repeat (SSR) Markers Derived from Whole-Genome Sequence (WGS) of Mungbean (Vigna radiata L. Wilczek): Cross-Species Transferability and Population Genetic Studies in Vigna Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See my report review in attachment

Comments for author File: Comments.pdf

Comments on the Quality of English Language

In all manuscripts, there are some words that run into each other. I suggest reading them carefully and correcting them.

 

Author Response

Response to Reviewers Comments

Manuscript ID: Horticulturae-2748740

Title: “Assessment of Simple Sequence Repeat (SSR) Markers Derived from Whole Genome Sequence (WGS) of Mungbean (Vigna radiata L. Wilczek): Cross-Species Transferability and Population Genetics Studies in Vigna Species”

 

Dear Editor, Authors are thankful to the reviewers for their valuable comments. The valuable and insightful comments of the reviewers have led to possible improvements in the current version. We have carefully considered the comments and tried our best to address every one of them and added the required information as suggested by the reviewers. We hope the manuscript after careful revisions meets your high standards. All modifications in the manuscript have been highlighted in yellow and have been addressed in the following sections.

 

Reviewer(s)' Comments to Author:

 

Reviewer: 1

In this article the authors provided information about a study focused on the genus Vigna, specifically on the validation of in-silico polymorphism of whole-genome sequence-developed mungbean-specific SSR markers and their transferability among different Vigna species.

1) The introduction part is too exhaustive, shows too much detail about the Vigna species. I recommend that only those paragraphs that focus on the goals of the work and its importance for the genetic study, diversity and resources, remain. For example: the taxonomy of the Vigna sp. does not necessarily need to be presented in so many details (from line 56-61).

From 75-88 - The passage highlights various beneficial aspects of mungbean, emphasizing its nutritional value, suitability for animal husbandry, absence of anti-nutritional factors, and its positive impact on soil fertility through nitrogen fixation, but is not so relevant for this study.

And from 89 to 104, also is not so relevant for this paper.

Here “This process is very cumbersome and expensive” I suggest using "laborious" instead of “cumbersome.

 

Author response (AR): As suggested by the reviewer the information about taxonomy (Line 56-61), beneficial aspects of mungbean (Line 75-88) and information from Line 89-104 has been deleted. Instead of cumbersome; the word laborious is used.

 

2) The objectives are well presented. The study aims to test the in-silico polymorphism of the WGS-developed SSR markers. This involves computationally assessing the genetic variations represented by these markers.

AR: Thank you

3) In all manuscripts, there are some words that run into each other. I suggest reading them carefully and correcting them.

AR: As suggested the manuscript is carefully read and corrected wherever necessary and highlighted.

4) The material and method parts are well presented. - But I have a question…. You wrote that “The amplified PCR product was run on 2.5% agarose gel.” But it is known that for migration of the amplified PCR product, PAGE is used. Why did you migrate into agarose gel? - you can mention studies that have used the migration of SSR into agarose gels.

AR: Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Our study resolved all the SSR markers used. Few studies are cited in this research paper (Satinder Kaur et al. (2017), Simranjit Kaur et al. (2019), Choudhary et al. (2022)) have used agarose gel electrophoresis for migration of SSR into agarose gel. PAGE (Polyacrylamide gel electrophoresis) is used for separating the molecules of DNA where agarose gel cannot separate and has high resolving power.

 

5) I suggest using “unequally” instead “unevenly” see line 276.

AR: Corrected as unequally

 

6) Missing all supplementary materials (table S1, S2, S3).

AR: All the supplementary materials (table S1, S2, S3) are submitted along with the original manuscript.

 

7) “The number of alleles amplified by WGS-SSRs were ranged as 7 (SSR 274) to 24 (SSR 271)”. In figure 2 shows other SSR markers that have only one allele per sample (from 1 to 25), amplified. - Maybe is better to show as imagines with this primers SSR 274, or SSR 271 or SSR 271 (24 alleles), SSR 123 (23 alleles), SSR 208, SSR 262, SSR 273, SSR 287 and SSR 289 (21 alleles) amplified more than 20 alleles…. - More, in the title and explanations of figure 2 are presented the 25 samples you worked with, but what are the other samples, from number 26 to 33?.

AR: The gel picture of SSR 274 (7 alleles) and SSR 271 (24 alleles) is included in the figure 2 along with 26 to 33 samples.

 

8) In 3.4, the study applied a specific clustering method (UPGMA neighbor joining) to group Vigna accessions based on their genetic dissimilarity, and the outcome of this analysis resulted in the identification of three main clusters. This kind of analysis is valuable for understanding the genetic diversity and relationships among different accessions within the Vigna genus. How do you explain why V. trilobata (GP9) is in sub-cluster 1a while the rest of V. trilobata (GP8, 6 and 7) are in sub-cluster 3a?

AR: This may be attributed to the genetic composition of the accessions which resulted in the grouping of accessions of the same species in different clusters/groups.

9) GeetaKumari et al. [85] – correct is Gita Kumari –

see https://pubmed.ncbi.nlm.nih.gov/34611751/

AR: Corrected as suggested

10) The conclusions are supported by the results

AR: - Yes

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study focused on validating mungbean-specific markers within the Vigna genus. Results indicated strong transferability of these markers (more than 90%) across diverse Vigna species. These findings highlight their promising application in genetic studies and breeding programs aimed at enhancing species within the Vigna genus. In general, I think the methods employed are likely adequate, and these markers will facilitate additional research in the field. However, the manuscript could benefit greatly from a thorough and careful revision.

 

1. The manuscript consistently exhibits spacing problems, with words frequently merging together in various sections. Please correct them carefully. Line 39, “two sub-populationsbased”;  Line 163, “750 85’ E)with”; line 173, “20 ng/μland”; line 196, “A series of250”; line 230, “each10 independent”; line 328, “andrelationship”; line 336, “GP 24”; line 425, “52.40 per cent”; line 453, “90 per cent”; line 490, “types.Thus,”

2. Some sentences require revision to enhance the overall readability and flow. For example, line 25, verb "are" is missing before "very much useful”; line 34, verb “were” is missing before “grouped into three clusters”. Sentences need to be revised in lines 444-445; line 447, “is higher than between different”. Line 527, “observed our study”

3. Line 62, it should be (2n=4x=44, tetraploid) instead of (2n=2x=44, tetraploid).

4. Abbreviations should be defined at first mention. Please revise line 81, “N-fixing ability”; Lines 108-109, “RFLP, RAPD, STS”;

5. In table 1, what does GP represent?

6. Please update all figures to higher resolutions.

7. In figure 2, please indicate the size of maker. Could the authors clarify the meaning of numbers 1-33? Kindly include a description in the figure legend.

8. In Figure 6, some of the annotations are overlapped together and difficult to read. Can author revise the figure to reduce the overlap?

9. Lines 434-436, “However, the chances of error like DNA slippage during PCR amplification by polymerase enzyme machinery cannot be ruled out.” In fact, High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest. I suggest to include High-fidelity PCR in the discussion.

10. Section 2.4, “annealing at 55°C which comprised of 35 cycles and followed by extension at 72°C for 10 minutes.” The extension time of taq polymerase is usually 1 minute/kb target length. Why the authors use 10 minutes here?

Comments on the Quality of English Language

Extensive editing of English language required. 

Author Response

Response to Reviewers Comments

Manuscript ID: Horticulturae-2748740

Title: “Assessment of Simple Sequence Repeat (SSR) Markers Derived from Whole Genome Sequence (WGS) of Mungbean (Vigna radiata L. Wilczek): Cross-Species Transferability and Population Genetics Studies in Vigna Species”

 

Dear Editor, Authors are thankful to the reviewers for their valuable comments. The valuable and insightful comments of the reviewers have led to possible improvements in the current version. We have carefully considered the comments and tried our best to address every one of them and added the required information as suggested by the reviewers. We hope the manuscript after careful revisions meets your high standards. All modifications in the manuscript have been highlighted in yellow and have been addressed in the following sections.

 

Reviewer(s)' Comments to Author:

 

Reviewer: 2

This study focused on validating mungbean-specific markers within the Vigna genus. Results indicated strong transferability of these markers (more than 90%) across diverse Vigna species. These findings highlight their promising application in genetic studies and breeding programs aimed at enhancing species within the Vigna genus. In general, I think the methods employed are likely adequate, and these markers will facilitate additional research in the field. However, the manuscript could benefit greatly from a thorough and careful revision.

 

Comments

 

1) The manuscript consistently exhibits spacing problems, with words frequently merging together in various sections. Please correct them carefully. Line 39, “two sub-populationsbased”; Line 163, “750 85’ E)with”; line 173, “20 ng/μland”; line 196, “A series of250”; line 230, “each10 independent”; line 328, “andrelationship”; line 336, “GP 24”; line 425, “52.40 per cent”; line 453, “90 per cent”; line 490, “types.Thus,”:

AR: As suggested by the reviewer, the merging of words into each other is corrected.

 

2) Some sentences require revision to enhance the overall readability and flow. For example, line 25, verb "are" is missing before "very much useful”; line 34, verb “were” is missing before “grouped into three clusters”. Sentences need to be revised in lines 444-445; line 447, “is higher than between different”. Line 527, “observed our study”.

AR: As per the suggestions the sentences have been revised and highlighted in the text.

3) Line 62, it should be (2n=4x=44, tetraploid) instead of (2n=2x=44, tetraploid). 

AR: As suggested by Reviewer 1; the taxonomy details have been deleted from the Introduction.

 

4) Abbreviations should be defined at first mention. Please revise line 81, “N-fixing ability”; Lines 108-109, “RFLP, RAPD, STS”.

AR: Abbreviations have been defined for RFLP, RAPD, STS, SSR. The N-fixing ability has been deleted as per Reviewer 1 suggestion in the introduction.

 

5) In table 1, what does GP represent?          

AR: GP denotes genotype

 

6) Please update all figures to higher resolutions.

AR: All the original figures with higher resolutions are updated.

 

7) In figure 2, please indicate the size of marker. Could the authors clarify the meaning of numbers 1-33? Kindly include a description in the figure legend.

AR: The size of the marker is 100 bp. 1-25 are Vigna species accessions.  26 to 33, pigeonpea and soybean accessions are not shown in the legend and were used to check the polymorphism and cross-genera transferability.

 

8) In Figure 6, some of the annotations are overlapped together and difficult to read. Can author revise the figure to reduce the overlap?

AR: Figure 6 is generated through software and there is no editing possible in the JPEG format of Figure 6.

9) Lines 434-436, “However, the chances of error like DNA slippage during PCR amplification by polymerase enzyme machinery cannot be ruled out.” In fact, High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest. I suggest to include High-fidelity PCR in the discussion. see https://pubmed.ncbi.nlm.nih.gov/34611751/

AR: Suggestion noted and high-fidelity PCR included in discussion.

10) Section 2.4, “annealing at 55°C which comprised of 35 cycles and followed by extension at 72°C for 10 minutes.” The extension time of Taq polymerase is usually 1 minute/kb target length. Why the authors use 10 minutes here?

AR: Yes, the extension time of Taq polymerase is usually 1 minute/kb target length. PCR reactions using more than 30 cycles, longer extensions are required. Therefore for 35 cycles, 10 minutes extension time is given in present study.

 

11) Comments on the Quality of English Language: Extensive editing of English language required.

AR: Extensive editing of English has been done with proper grammar check and reviewed by an experienced English speaking colleague.

 

 

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The majority of the comments have been addressed in the revised manuscript. I just have two remaining comments.

1. In Figure 2, please lable the the size of the marker band. See the example in Figure 1C of https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00090 or Figure 2b of https://www.nature.com/articles/s41587-022-01585-8. 

2. The description of PCR steps in 2.4 is still not intact and confused. A standard PCR steps should include: 1) An initial step of DNA denaturation at 94°C to 98°C for 3 to 5 min depending on the optimal temperature of the DNA polymerase; 2) Denaturation temperature (the same used in the step before) for 30 seconds; 3) Annealing temperature taking into account the Tm of the primers for 30 seconds; 4) Extension temperature at 72°C and taking into account the size of the fragment to be amplified. Step 2-4 should be repeated for 25 to 35 rounds (cycles). A final step of extension is required to allow all the PCR products to be correctly synthesized, usually at 72°C for 10 min. Finally, the temperature should be reduced to 4°C to store the PCR product. Please revised the PCR steps correctly.

Author Response

Reviewer Report pf Referee 2 for Round 2:

Comment 1: In Figure 2, please label the the size of the marker band. See the example in Figure 1C of https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00090 or Figure 2b of https://www.nature.com/articles/s41587-022-01585-8. 

AR: As per suggestion, the Figure 1C of https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00090 have been refereed and the size of the marker band labeled (100 bp, 200 bp, 500 bp, 700 bp, 1000 bp and 1100 bp)

 

Comment 2: The description of PCR steps in 2.4 is still not intact and confused. A standard PCR steps should include: 1) An initial step of DNA denaturation at 94°C to 98°C for 3 to 5 min depending on the optimal temperature of the DNA polymerase; 2) Denaturation temperature (the same used in the step before) for 30 seconds; 3) Annealing temperature taking into account the Tm of the primers for 30 seconds; 4) Extension temperature at 72°C and taking into account the size of the fragment to be amplified. Step 2-4 should be repeated for 25 to 35 rounds (cycles). A final step of extension is required to allow all the PCR products to be correctly synthesized, usually at 72°C for 10 min. Finally, the temperature should be reduced to 4°C to store the PCR product. Please revised the PCR steps correctly.

AR: As suggested, the PCR profile for the amplification of DNA was revised.

The PCR profile for the amplification of DNA was set as an initial denaturation at 94°C for 03 minutes, followed by denaturation at 94°C for 01 minute, annealing at 55°C for 01 minute which comprised 35 cycles, extension at 72°C for 01 minute and final extension at 72°C for 10 minutes and hold at 4°C.