In Silico Apple Genome-Encoded MicroRNA Target Binding Sites Targeting Apple Chlorotic Leaf Spot Virus
Round 1
Reviewer 1 Report
Please refer to the attached file.
Comments for author File: 
Comments.docx
Extensive English language edit is required.
Author Response
RESPECTED REVIEWER 1 (GENERAL COMMENTS):
The read the manuscript entitled “In Silico Identification of Apple Genome-Encoded MicroRNA Target Binding Sites Potentially Targeting the ACLSV” submitted to Horticulturae. Firstly, I reviewed the manuscript and pointed out my major and minor comments for the improvement of the manuscript. However, I found that the manuscript is already online and published in the below link (1st comment).
General Comment-1: The paper is already online in the below link. (https://www.preprints.org/manuscript/202305.0517/v1).
Response- Respected Sir, thank you very much for your kind comment. After submission, Respected Editor has approved the manuscript suitability for publication in the MDPI Preprint Server.
General Comment-2: The title should be clear and concise without abbreviations.
Response- Respected Sir, Correction has been made. Please see line 2-3.
General Comment-3: The abstract is written complicated and does not provide clear message, findings and contribution.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see lines 16-40.
General Comment-4: Once the scientific name (M. domestica) is mentioned in the text, there is no need to rewrite it several times, such as in lines 18, 23, and 80.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Please see lines 24, 27, 30, 46.
General Comment-5: The writing flow of introduction section is not appropriate. The authors should focus on the research gaps, and clearly point out, what have done from the published articles.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. The introduction has revised extensively. Correction has been made as per reviewer suggestion. Please see lines 50-54 and 79-92.
General Comment-6: Line 45 – 47: not providing the flow of the sentence.
Response- Respected Sir. Correction has been made as per reviewer suggestion.
General Comment-7: The main objective and aim of the study is written complicate. The authors should rephrase the sentence and clearly present the aim of the study.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion in the abstract and introduction. Please see lines 38-40 and 92 and 98.
General Comment-8: Table 1 has several links which needs citation.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Citation has already added in the text. But I also added in the table 1 as per reviewer recommendation. Please see lines 115-116 in table 1.
General Comment-9: The first cited abbreviation needs explanation.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-10: Figure 1 is not cited in the text.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Please see line 170.
General Comment-11: The resolution of Figure 2 and 4 is very low and is not readable.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Resolution figure 2 and 4 are greatly increased. Please see lines 203-205 and lines 274-275.
General Comment-12: The result section is boring and is not written interestingly.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. All the result section greatly revised as per reviewer recommendation
General Comment-13: I recommend that the authors should carefully check the manuscript. Present their work clearly and concisely. Select the tables and figures which are very important and give values to their text.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. The manuscript was greatly revised.
General Comment-14: The discussion section is weak. Try to improve it and discuss the relevant topics.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Discussion section extensively revised.
Reviewer 2 Report
The manuscript entitled “In silico identification of Apple genome-encoded microRNA target binding sites potentially targeting the ACLSV” authored by Ashraf et al is about the in silico identification of microRNA that could bind to the apple chlorotic leaf spot virus. They use four different algorithms to identify miRNAs and to in silico validate their possible function. The experimental procedure is identical to their previously published work of M. A. Ashraf (https://doi.org/10.1371/journal.pone.0261807), just different plant and different virus. After reading the manuscript, I have some major and minor points to be addressed.
Major points
Results section
1.- Results section looks like a data summary. The whole results section is not described properly and some sections like 3.2, 3.3 and 3.4 are just a list of names. It is necessary to improve the wording and perhaps to include these information (sections 3.1, 3.2 and 3.3) in a table.
2.- The union plot (Figure 3) show the entire set of in silico identified miRNAs and even it is possible to observe the results of each algorithm in this figure. So, considering that the manuscript has five figures and four tables, I suggest that figure 2 changes to a supplementary figure. It is not necessary to show the same results twice. I have a question about Figure 3 units, because in this manuscript, in the y-axis is shown minimum free energy-kcal/mol, ratio and expectations. But in the previously published work (https://doi.org/10.1371/journal.pone.0261807), the unit for the same figure are minimum free energy-kcal/mol. So, it is not clear how you joined the different data obtained from the four different algorithms.
3.- You should clarify why are you using a two-steps filtering process to identify miRNAs. First you choose those miRNAs identified by the four algorithms (nine miRNAs) and then you repeat the process but now selecting those identified by at least two algorithms (58 miRNAs). The other main problem is that in Table 2 is shown that only three miRNAs [mdm-7121 (a, b, c)] were identified by the four algorithms. Where are the other five miRNAs?
4.- Please clarify how can you construct a regulatory network based on the in silico predicted binding of miRNAs and their putative targets. Where is the term regulatory network involved? Also, it should be clarified the term “validation”. Validation considering what features. Since my point of view, the validation of your putative identified miRNAs should be the experimental data.
5.- The section 3.8 is called “Secondary structures of the consensual RNA”, but the aforementioned secondary structures are not shown, at least as it was shown in the previously published work (https://doi.org/10.1371/journal.pone.0261807).
6.- Would it be possible to experimentally validate your in silico results?
Discussion section
7.- It is not explained/discussed why you get so different results in each algorithm. Based on Table 2, I just see three miRNAs [mdm-7121 (a, b, c)] that were identified by the four algorithms. So what about the confidence of the in silico identification of miRNAs.
8.- If it is not possible to experimentally validate your in silico results, please validate the in silico approach by mentioning some references about miRNAs identification based on the used algorithms where the in silico approach was followed by experimental data.
9.- There is no conclusion section.
Minor points
10.- Please, homogenize the name of the miRNAs. It is shown different names in the different figures and tables for the same miRNA. This is so confusing.
11.- Please, revise the grammar and the acronyms.
Please find some extra comments in the attached file.
Comments for author File: 
Comments.pdf
Please, revise the grammar and the acronyms.
Author Response
RESPECTED REVIEWER 2 COMMENTS:
The manuscript entitled “In silico identification of Apple genome-encoded microRNA target binding sites potentially targeting the ACLSV” authored by Ashraf et al is about the in silico identification of microRNA that could bind to the apple chlorotic leaf spot virus. They use four different algorithms to identify miRNAs and to in silico validate their possible function. The experimental procedure is identical to their previously published work of M. A. Ashraf (https://doi.org/10.1371/journal.pone.0261807), just different plant and different virus. After reading the manuscript, I have some major and minor points to be addressed.
General Comment-1: Results section looks like a data summary. The whole results section is not described properly and some sections like 3.2, 3.3 and 3.4 are just a list of names. It is necessary to improve the wording and perhaps to include these information (sections 3.1, 3.2 and 3.3) in a table.
Response- Respected Sir, thank you very much for your kind comment. Supplementary table 2 and 3 mentioned and provided. All the results greatly revised as per reviewer recommendation.
General Comment-2: The union plot (Figure 3) show the entire set of in silico identified miRNAs and even it is possible to observe the results of each algorithm in this figure. So, considering that the manuscript has five figures and four tables, I suggest that figure 2 changes to a supplementary figure. It is not necessary to show the same results twice. I have a question about Figure 3 units, because in this manuscript, in the y-axis is shown minimum free energy-kcal/mol, ratio and expectations. But in the previously published work (https://doi.org/10.1371/journal.pone.0261807), the unit for the same figure is minimum free energy-kcal/mol. So, it is not clear how you joined the different data obtained from the four different algorithms.
Response- Respected Sir, we are highly thankful to for the useful comments to improve the manuscript. It has given new direction to improve the manuscript title. Correction has been made in the figure 3 legend. Please see line 276-280. Figure 2 is the most important graphs; we have supplemented with table S2 and S3. Secondly, resolution of figure 2 is greatly increased.
General Comment-3: You should clarify why are you using a two-steps filtering process to identify miRNAs. First you choose those miRNAs identified by the four algorithms (nine miRNAs) and then you repeat the process but now selecting those identified by at least two algorithms (58 miRNAs). The other main problem is that in Table 2 is shown that only three miRNAs [mdm-7121 (a, b, c)] were identified by the four algorithms. Where are the other five miRNAs?
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see line 281-286. The target binding sites are mentioned in the table 2. We have evaluated results on the basis of consensus genomic binding sites of apple miRNAs. Please see lines 29 to 33 in the abstract. We also explained in the section 3.6, lines 281-286.
General Comment-4: Please clarify how can you construct a regulatory network based on the in silico predicted binding of miRNAs and their putative targets. Where is the term regulatory network involved? Also, it should be clarified the term “validation”. Validation considering what features. Since my point of view, the validation of your putative identified miRNAs should be the experimental data.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see line 319-326 in the results section 3.7.
General Comment-5: The section 3.8 is called “Secondary structures of the consensual RNA”, but the aforementioned secondary structures are not shown, at least as it was shown in the previously published work (https://doi.org/10.1371/journal.pone.0261807).
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see line 338-347. We have added figure 6.
General Comment-6: Would it be possible to experimentally validate your in silico results?
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see lines 438-447.
General Comment-7: It is not explained/discussed why you get so different results in each algorithm. Based on Table 2, I just see three miRNAs [mdm-7121 (a, b, c)] that were identified by the four algorithms. So what about the confidence of the in silico identification of miRNAs.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. We greatly revised the abstract, results discussion and conclusion to illustrate the confidence level of predicted data.
General Comment-8: If it is not possible to experimentally validate your in silico results, please validate the in silico approach by mentioning some references about miRNAs identification based on the used algorithms where the in silico approach was followed by experimental data.
Table 1 has several links which needs citation.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Citation added in the table 1.
General Comment-9: There is no conclusion section.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion Conclusion section has been added. Please see lines 449-460
General Comment-10: Please, homogenize the name of the miRNAs. It is shown different names in the different figures and tables for the same miRNA. This is so confusing.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion in the whole manuscript.
General Comment-11: Please, revise the grammar and the acronyms.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. The text is greatly revised.
General Comment-12: Please find some extra comments in the attached file.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
Reviewer 3 Report
In this work, Asraf and collaborators use a bioinformatic approach to predict apple locus-derived microRNAs and their potential targets in the mRNAs derived from ACLSV genome. The main objective of this research is clear, but the manuscript has to be improved before it be considered for publication. My main concerns are: 1) Why only 9 mdm-miRNAs were common using the 4 algorithms? Please, compare and discuss the differences found in the 4 algorithms. Why was the best algorithm for the research purposes? 2) Results: The last section of the results (3.7-3.9) are poorly described. 3) Discussion. Plenty of paragraphs describe just results again; a robust discussion still needs to be included in the manuscript.
Minor issues:
Line 13. Please, provide the full scientific name of Malus domestica; the descriptor is missing.
Lines 18 and 23. Split Malus.domestica, please.
As made for other abbreviations than were declared in the abstract and following sections, v.g. ACLSV (lines 12 and 41), please, declare the mdm-mirRNAs in the introduction section.
Please, make double-check the English grammar throughout the document. Here are only examples where I found some mistakes:
Line 46. Check the concordance of verb tenses used throughout the manuscript. In some paragraphs, passive voice in the past and present simple are mixed, v.g. lines 44-46.
Lines 161, In this work, Asraf and collaborators use a bioinformatic approach to predict apple locus-derived microRNAs and their potential targets in the mRNAs derived from ACLSV genome. The main objective of this research is clear, but the manuscript have to be improved before to be considered for publication. My main concerns are: 1) Why only 9 mdm-miRNAs were common using the 4 algorithms? Please, compare and discuss the differences found the 4 algorithms. Why was the best algorithm for the research purposes? 2) Results: The last section of the results (3.7-3.9) are poorly described. 3) Discussion. There is still a plenty of paragraphs describing just results again; a robust discussion still needs to be included in the manuscript.
Minor issues:
Line 13. Please, provide the full scientific name of Malus domestica; the descriptor is missing.
Lines 18 and 23. Split Malus.domestica, please.
As made for other abbreviations than were declared in the abstract and following sections, v.g. ACLSV (lines 12 and 41), please, declare the mdm-mirRNAs in the introduction section.
Please, make a double check of the English grammar throughout the document. Here are few examples where I found some mistakes:
Line 46. Check the concordance of verb tenses used throughout the manuscript. In some paragraphs passive voice in past and present simple are used mixed.
Line 248. …consensus between all four algorithms. Between? Or …of the four…?
Line 285. Was visualized or were?
3.7, 3.8, and 3.9. May you describe more the Circos map of mdm-mirRNAs and their targets? Same thing with Table 3: the description made needs to be longer. What do you could say about the values found for MFE, AMFE, MFEI, and GC%, for example?
Please, check the References section format.. Check grammar in these titles (…that encodes… (a/an is missing before noun).
Line 248. …consensus between all four algorithms. Between? Or …of the four…?
Line 285. Was visualized, or itwas?
3.7, 3.8, and 3.9. Please describe more the Circos map of mdm-mirRNAs and their targets. Same thing with Table 3: the description made needs to be longer. What could you say about the values found for MFE, AMFE, MFEI, and GC%, for example?
Please, check the References section format.
Extensive editing of English language required
Author Response
RESPECTED REVIEWER 3 COMMENTS:
In this work, Ashraf and collaborators use a bioinformatic approach to predict apple locus-derived microRNAs and their potential targets in the mRNAs derived from ACLSV genome. The main objective of this research is clear, but the manuscript has to be improved before it be considered for publication. My main concerns are: 1) Why only 9 mdm-miRNAs were common using the 4 algorithms? Please, compare and discuss the differences found in the 4 algorithms. Why was the best algorithm for the research purposes? 2) Results: The last section of the results (3.7-3.9) is poorly described. 3) Discussion. Plenty of paragraphs describe just results again; a robust discussion still needs to be included in the manuscript.
General Comment-1: Why only 9 mdm-miRNAs were common using the 4 algorithms? Please, compare and discuss the differences found in the 4 algorithms. Why was the best algorithm for the research purposes?
Response- Respected Sir, thank you very much for your kind comment. We have already explained different features of algorithms in the table. Please see lines 380-392 in the discussion.
General Comment-2: Results: The last section of the results (3.7-3.9) is poorly described.
Response- Respected Sir, we are highly thankful to for the useful comments to improve the manuscript title. It has given new direction to improve the manuscript title. Correction has been made. Last section has been greatly revised. Please see lines 319-326, 338-347. Figure 6 has been added.
General Comment-3: Discussion. Plenty of paragraphs describe just results again; a robust discussion still needs to be included in the manuscript.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Discussion section greatly revised.
General Comment-4: Line 13. Please, provide the full scientific name of Malus domestica; the descriptor is missing.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-5: Lines 18 and 23. Split Malus.domestica, please.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-6: As made for other abbreviations than were declared in the abstract and following sections, v.g. ACLSV (lines 12 and 41), please, declare the mdm-miRNAs in the introduction section.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-7: Please, make double-check the English grammar throughout the document. Here are only examples where I found some mistakes:
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-8: Line 46. Check the concordance of verb tenses used throughout the manuscript. In some paragraphs, passive voice in the past and present simple are mixed, v.g. lines 44-46.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion in the introduction. Please see lines 47-53.
General Comment-9: Line 248. …Consensus between all four algorithms. Between? Or …of the four…?
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see line 273.
General Comment-10: Line 285. Was visualized or were?
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see line 315.
General Comment-11: 3.7, 3.8, and 3.9. May you describe more the Circos map of mdm-miRNAs and their targets?
Response- Respected Sir, we are highly thankful to for the useful comments to improve the manuscript title. It has given new direction to improve the manuscript title. Correction has been made. Last section has been greatly revised. Please see lines 319-326, 338-347.
General Comment-12: Same thing with Table 3: the description made needs to be longer. What do you could say about the values found for MFE, AMFE, MFEI, and GC%, for example?
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see lines 319-326, 338-347. Figure 6 has been added.
General Comment-13: Please, check the References section format.. Check grammar in these titles (…that encodes… (a/an is missing before noun).
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-14: Extensive editing of English language required.
Response- Thank you very much for your valuable comments for the improvement of our manuscript. We have taken reviewer’s comment in full consideration and it will be well reflected by the revised version of manuscript. All the comments have been added and manuscript was scientifically edited with highest standard of English editing and writing. Please see the Tracked Changes version.
Round 2
Reviewer 1 Report
The authors revised the manuscript and made a reply sheet. However, they used track changes in the manuscript and did not clearly point out the changes in the reply sheet, therefore, it is difficult to understand. Thus, I recommend that the authors revised the manuscript clearly without track changes and make all the changes with red color. In addition, the English quality become more worse.
Require more attention.
Author Response
Dear Editor
We are grateful to the reviewers and respected editor for their insightful comments on my original research manuscript (manuscript ID# horticulturae-2407353) which is currently under major revision in the Horticulturae journal. Coauthors and I very much appreciated the encouraging, critical and constructive comments on this manuscript by the respected editor and reviewers. The comments have been very thorough and useful in improving the manuscript. We strongly believe that the comments and suggestions have increased the scientific value of revised manuscript by many folds. We have taken them fully into account in this major revision. We are submitting the revised manuscript with the suggestion incorporated the manuscript. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript (red color). Here is a point-by-point response to the reviewers' comments and concerns. The manuscript has been revised as per the comments given by the respected reviewers and our responses to all the comments are as follows:
RESPECTED REVIEWER 1 (GENERAL COMMENTS):
The authors revised the manuscript and made a reply sheet. However, they used track changes in the manuscript and did not clearly point out the changes in the reply sheet, therefore, it is difficult to understand. Thus, I recommend that the authors revised the manuscript clearly without track changes and make all the changes with red color. In addition, the English quality becomes more worse.
Response: Respected Sir, track changes removed and we highly manuscript with red color (all changes previously done)
General Comment-1: The paper is already online in the below link. (https://www.preprints.org/manuscript/202305.0517/v1).
Response- Respected Sir, thank you very much for your kind comment. After submission, Respected Editor has approved the manuscript suitability for publication in the MDPI Preprint Server.
General Comment-2: The title should be clear and concise without abbreviations.
Response- Respected Sir, Correction has been made. Please see line 2-3.
General Comment-3: The abstract is written complicated and does not provide clear message, findings and contribution.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Please see lines 14-39.
General Comment-4: Once the scientific name (M. domestica) is mentioned in the text, there is no need to rewrite it several times, such as in lines 18, 23, and 80.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Please see lines 23, 26, 28 in the abstract. We have only given apple description in the introduction line 43. In the whole manuscript apple is used.
General Comment-5: The writing flow of introduction section is not appropriate. The authors should focus on the research gaps, and clearly point out, what have done from the published articles.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. The introduction has revised extensively. Correction has been made as per reviewer suggestion. Please see lines 44-45, 46-52. In addition, text revised with correct English language scientifically. We also added lines 80-95 new lines to revise introduction.
General Comment-6: Line 45 – 47: not providing the flow of the sentence.
Response- Respected Sir. Correction has been made as per reviewer suggestion. Please see lines 49-53.
General Comment-7: The main objective and aim of the study is written complicate. The authors should rephrase the sentence and clearly present the aim of the study.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion in the abstract and introduction. Please see lines 28-39 and 80 and 99.
General Comment-8: Table 1 has several links which needs citation.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Citation has already added in the table 1. Please see lines 116-117 in table 1.
General Comment-9: The first cited abbreviation needs explanation.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion.
General Comment-10: Figure 1 is not cited in the text.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Please see line 171.
General Comment-11: The resolution of Figure 2 and 4 is very low and is not readable.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. Resolution figure 2 and 4 are greatly increased. Please see lines 203-205 and lines 307-308.
General Comment-12: The result section is boring and is not written interestingly.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. All the result section greatly revised as per reviewer recommendation
General Comment-13: I recommend that the authors should carefully check the manuscript. Present their work clearly and concisely. Select the tables and figures which are very important and give values to their text.
Response- Respected Sir, thank you very much for your kind comment. It has given us new direction to improve the manuscript. Correction has been made as per reviewer suggestion. The manuscript was greatly revised. Supplementary tables S2-S3 added.
General Comment-14: The discussion section is weak. Try to improve it and discuss the relevant topics.
Response- Respected Sir, thank you very much for your kind comment. Correction has been made as per reviewer suggestion. Discussion section extensively revised. Please see lines 366-396, 438-441 and 447-453
Author Response File: Author Response.pdf
Reviewer 2 Report
Authors revised and made important changes on their manuscript. I do not have any additional comments
Author Response
Respected Sir
I am highly thankful to you for the improvement of the manuscript.
Reviewer 3 Report
The authors have made considerable changes to the original submitted manuscript as requested by the reviewers. The weakest section of the manuscript was the discussion. The current version looks better. At this stage, I don't have any more suggestions. Only one thing: there is plenty of typos in the document. Please, make double-check before submitting any manuscript.
Minor editing of English language required.
Author Response
Respected Sir
Thanks for your kind comment. The manuscript is greatly revised.
Author Response File: Author Response.pdf
