Development, Identification and Validation of a Novel SSR Molecular Marker for Heat Resistance of Grapes Based on miRNA

Round 1

Reviewer 1 Report

The manuscript entitled “Development, identification, and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA. The present paper, the author identified a key SSR marker to distinguish heat tolerance from sensitive germplasm, which will be useful for the grapevine breeder community. However, the clarity and presentation of data given in the manuscript are limited, preventing the audience's interest. 

Comments:

1.       IF the VMIRSSR167c3 marker plays a role in heat tolerance, the author should verify molecular mechanisms governed by the tolerance. For instance, the author should analyze the VMIRSSR167c3 marker-controlled list of candidate genes can be analyzed.

2.       The author should elaborate more molecular mechanisms VMIRSSR167c3 marker.

3.       Manuscript looks like wordiness and the repetition of sentences in material, methods, results, and discussion. The main concern is that the discussion section should be rewritten clearly with current results rather than already published research. I would suggest that the results and discussion should merge for better clarity and follow.   

Manuscript looks like wordiness and the repetition of sentences in material, methods, results, and discussion. The main concern is that the discussion section should be rewritten clearly with current results rather than already published research. I would suggest that the results and discussion should merge for better clarity and follow.   

Author Response

Dear Editor and Reviewers,

Subject: Submission of a revised manuscript “Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA”.

Thank you for the time and effort that you have put into the manuscript review of the last version. Your useful comments and suggestions are helpful for improving our work. According to the comments, we have revised the manuscript thoroughly and completely. If still there are still some mistakes and shortcomings, we will happily address them. Please scroll down for the details of our response to your comments.

Appended to this letter is our point-by-point response to the comments raised by the reviewers. Our responses are given directly afterward (the Reviewer's comments are reproduced and represented in blue).

Best regards

Sincerely yours

Chao Ma

Reviewer #1

Comments and Suggestions for Authors

The manuscript entitled “Development, identification, and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA. The present paper, the author identified a key SSR marker to distinguish heat tolerance from sensitive germplasm, which will be useful for the grapevine breeder community. However, the clarity and presentation of data given in the manuscript are limited, preventing the audience's interest.

Response

Thank you for your review. Your affirmation and recognition of “Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA” research paper will be of great help to our follow-up scientific research. Based on your suggestion, we have reviewed and revised the full text.

Comments

1. IF the VMIRSSR167c3 marker plays a role in heat tolerance, the author should verify molecular mechanisms governed by the tolerance. For instance, the author should analyze the VMIRSSR167c3 marker-controlled list of candidate genes can be analyzed.

Response

SSRs could respond to environmental changes to produce mature RNA isomers by influencing alternative splicing. The VMIRSSR167c3 is in the position of vvi-MIR167c, as shown below. Previous studies reported that vvi-miR167 was involved in the regulation of the heat resistance of grapes. The vv-MIR167 gene can be cleaved and processed into vvi-miR167. Target gene prediction of vvi-miR167 showed that vvi-miR167 could be cleaved with vvARF6, vvARF6-like and vvARF8 (as shown in the following figure A). Based on instantaneous leaf transformation and RT-qPCR results, miR167 was able to cleaved target genes vvARF6, vvARF6-like and vvARF8 (as shown in the following figure B, C). These partial results are cited from previous studies (Zhang et al., 2023). The above results are only described here and do not appear in the manuscript.

>Vvi-MIR167c sequence (pre-miR167c ± 500bp)

GAGAAAGAGAGAGAGAGATGCCTTGCTTTCCTTAGTCCTACATCTAAGAAGCCACTGAAATGGTCCCCCTATGAACTCCCAGTGTATTACACCACTTTTGGGTTCATGAGAGACACAGATGGATGAGAGTGGCCTCTCACCTAAAGTTAATAAAGTCTCTGGGGAAGTTTTAGAAACTCTCACAAGCTTGTCTGCATGGTGGTGGCCTGGGTTCGATAATATTGAATTTGTCTCTCATTGGACCACTTCTTTCTCTCTCTCTCTTCCCCTTGTATAATTTATTATGTTGCTTAAGCTTTTTCAGTCCCTAAAGCAATAATAAAATGGCCAAGAAGACTTTAAACAAAAGCAGATGACATCTTCTTGGCCTCTATTTATACCCCCTCCCCTCTCTTCTTCTCTTGCATCAATGCATATTATGATAGAGCAGATGAAACTCTCTCTCTCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGGGCTTACTTGCTTTACGTGCACCAGTAGCAGTTGAAGCTGCCAGCATGATCTCAACTTCCCTATACAAGTCAAGGAAAGATCAGATCATGTGGTAGCCTCACCTGTTGATGGGGTCACGAAAAACCCTAATTATCATAAAACCCTATTCCACCTTGCTCATTAGGGTTTCTCAAAACCCCCCCATATTAAGGTTTGTTCATGATCATTTCCTTACAACTTGATTTCTGCTCATCTGGGTACCCACAATTTGCTCTTCTCTCCCTATGTTTTCACAGTATAATTCCTCCTATTAATTTCTCAAGTATTAATATTCTTCTGTTGCCCTTTTTTTATTTTATTTTTCTGTGTGTCAAGTTTTGACAGGGAATCTTAACAACAAGAAGATGTCATTGTTAGGGTTTTAAGTGACAGAAATCATCTCTAATCCTGATTGGTGCTTTTGAGATCTCATCTTATCTCTTCTCCTCATGTCTGTAGAGATCTCTTTACATTCCCATTTTTTGGTATTATTTATTACCATTTTTCTTGCCTTTGTGACACATACTTTAATCCTGGCATCAACCATACAAGAATATAAAACTAGTAAAAGGGACAAAAGAAAAAAAAAAAAAGGAAGGAAAAA (notes: Green represents the SSR locus, yellow represents pre-miR167c, and red represents miR167c)

Zhang, L.; Fan, D.; Li, H.; Chen, Q.; Zhang, Z.; Liu, M.; Liu, J.; Song, Y.; He, J.; Xu, W.; Song, S.; Liu, H.; Ren, Y.; Ma, C., Characterization and identification of grapevine heat stress-responsive microRNAs revealed the positive regulated function of vvi-miR167 in thermostability. Plant Sci 2023, 329, 111623.

2. The author should elaborate more molecular mechanisms VMIRSSR167c3 marker.

Response

According to your suggestion, after agroinfiltration of the vector pHB into leaves from tissue culture plantlets of Thompson Seedless grapevines. Heat stress treatment at 45 ℃ for 2 h showed that the heat resistance of OE-vvi-miR167c increased compared with the CK group. MDA content confirmed this phenotypic change (as shown in the following figure). The above results are only described here and do not appear in the manuscript.

Figure legend: Transient transformation of vvi-miR167c in grapevine leaves. (A) Phenotypic appearance of grape tissue culture plantlets leaves (WT and OE-vvi-miR167c) during heat stress. (B) Relative expression level of vvi-miR167 in Thompson Seedless leaves infiltrated with vvi-miR167c were measured by RT-qPCR. (C) Analysis of malondialdehyde (MDA) contents in Thompson Seedless leaves infiltrated with vvi-miR167c under heat stress and CK.

3. Manuscript looks like wordiness and the repetition of sentences in material, methods, results, and discussion. The main concern is that the discussion section should be rewritten clearly with current results rather than already published research. I would suggest that the results and discussion should merge for better clarity and follow.

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised, including material, methods, results, and discussion. Please refer to the revised manuscript with changes marked.

Comments on the Quality of English Language

Manuscript looks like wordiness and the repetition of sentences in material, methods, results, and discussion. The main concern is that the discussion section should be rewritten clearly with current results rather than already published research. I would suggest that the results and discussion should merge for better clarity and follow.

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised, including material, methods, results, and discussion. Please refer to the revised manuscript with changes marked.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors

The following modifications are required:

Abstract

ü  This section is generally poorly written. It is written in a straightforward manner. This section should include the study's most important findings. As a result, this section should be strengthened. There are no novelties in this manuscript.

ü  For this section, the authors should stick to the title. The authors should adhere to the sequence and order of writing in this section as specified in the title.

ü  L16-18 (Therefore, miRNA-based 16 molecular marker development for high temperature resistance identification is an innovation strategy in molecular marker assisted breeding in grapevine): This sentence should be deleted

ü  L25-26 (On the other hand, pri-miRNAs responding to heat stress were identified by RT-qPCR (Real-time quantitative PCR): This sentence should be removed

ü  The authors must define the issue in a single line before describing the goal and explain why they chose this approach to study this research.

ü  No information about the type of experimental design and its component is available in this manuscript.

ü  The authors should be provided some measured data

ü  The authors should present a decisive conclusion derived from the research in the final line of the abstract and provide a single line of future prospects..

Keywords

ü  The keywords did not accurately reflect the manuscript's content, and the words that comprised the title should not be used as keywords. As a result, the keyword structure should be changed.

Introduction

ü  The authors should follow the title in the introduction section. This content of this section should follow the sequence of writing in the materials and method and results

ü  No information about the heat stress genes is available

ü  Along with the hypothesis statement, the authors should include some lines about the knowledge gap addressed by their research.

ü  The authors should also include a novelty statement at the end. In this study, what new things have the authors done or correlated in comparison to previous ones?

ü  The general and specific aim should be inserted

Materials and methods

ü  The age of seedlings should be added in the section 2.1

ü  The detailed of statistical data analysis should be inserted

Results and discussion

ü  In general, this section is not clearly written and should be modified

ü  Why did not the authors measure the quantitative data

ü  The captions of all figures should be detailed

ü  The captions of all tables should be detailed

ü  The bootstrap values should be added to the phylogenetic tree

ü  The authors should calculate the maximum composite likelihood

ü  The discussion is weak and should be improved because most of the sentences are written as the literature review rather than interpretation of results

Conclusion

ü  The authors should summarize the most important findings from this study. Furthermore, additional works pertaining to this research should be included as future works.

Extensive correction is needed

Author Response

Reviewer #2

Comments and Suggestions for Authors

The following modifications are required:

Abstract

1. This section is generally poorly written. It is written in a straightforward manner. This section should include the study's most important findings. As a result, this section should be strengthened. There are no novelties in this manuscript.
2. For this section, the authors should stick to the title. The authors should adhere to the sequence and order of writing in this section as specified in the title.

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised. Please refer to the revised manuscript with changes marked.

3. L16-18 (Therefore, miRNA-based 16 molecular marker development for high temperature resistance identification is an innovation strategy in molecular marker assisted breeding in grapevine): This sentence should be deleted
4. L25-26 (On the other hand, pri-miRNAs responding to heat stress were identified by RT-qPCR (Real-time quantitative PCR): This sentence should be removed.

Response

According to your suggestion, we have decided to delete the sentence.

5. The authors must define the issue in a single line before describing the goal and explain why they chose this approach to study this research.

Response

According to your suggestion, the revised manuscript (page 1, line 28-29) adds the goal and explains. As follows,

On the other hand, there are differences in heat resistance among grape varieties.

6. No information about the type of experimental design and its component is available in this manuscript.
7. The authors should be provided some measured data.

Response

According to your suggestion, the revised manuscript (page 1, lines 30-31, 37-38) adds the content. As follows,

Based on the results of leaf Fv / Fm images, values, and phenotypic changes under high temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 sensitive (such as, Shine Muscat and Jumeigui) grape varieties were identified.

The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties is 90%.

8. The authors should present a decisive conclusion derived from the research in the final line of the abstract and provide a single line of future prospects.

Response

According to your suggestion, we have made the corresponding modification (page 1, line 41-44).

In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and was beneficial to the breeding of heat-resistant varieties in the future.

Keywords

1. The keywords did not accurately reflect the manuscript's content, and the words that comprised the title should not be used as keywords. As a result, the keyword structure should be changed.

Response

According to your suggestion, we have made the keyword structure modification (page 1, line 45).

Keywords: miRNA; SSR; molecular markers; heat stress; grape; germplasm

Introduction

1. The authors should follow the title in the introduction section. This content of this section should follow the sequence of writing in the materials and method and results.

Response

According to your suggestion, we have made the Introduction structure modification (pages 2-4, lines 50-168). Please refer to the revised manuscript with changes marked.

2. No information about the heat stress genes is available.

Response

According to your suggestion, the revised manuscript (page 3, lines 101 - 126) perfect miRNA and target genes associated with heat stress. As follows,

    Under high temperature stress, miR398 enhanced heat resistance by decreasing the expression of target genes (COPPER/ZINC SUPEROXIDE DISMUTASE 1 (CSD1), CSD2, and COPPER CHAPERONE FOR SOD1 (CCS)) in Arabidopsis. The transgenic Arabidopsis thaliana that further overexpressed CSD1, CSD2 and CCS transcripts was more sensitive to heat stress than the wild type [21]. Overexpression of MIR398b and MIR398c activates NAT398b and NAT398c, thereby inhibiting the biogenesis of miR398 and reducing the heat tolerance of plants [22]. In addition, the production of tocopherol (vitamin E) under heat stress could promote the accumulation of miR398, thus improving heat resistance [23]. The results highlight the significance of miR398 ex-pression in plant thermotolerance. Other microRNAs, such as miR156, regulate memory in response to heat stress by targeting SQUAMOSA-PROMOTER BIND-ING-LIKE (SPL) transcription factors [24]. MiR160-ARFs (AUXIN RESPONSE FACTOR) gene module mediates thermostability.

3. Along with the hypothesis statement, the authors should include some lines about the knowledge gap addressed by their research.

Response

According to your suggestion, we have made the corresponding modification (page 3, lines 127 and 131).

Recent studies have shown that miR167 positively regulates heat resistance in grapes [8]. There were an increasing number of reports on the involvement of miRNA in grape development and response to stress [26-28]. However, large-scale development of molecular markers based on miRNA-SSR was still lacking in grapes.

4. The authors should also include a novelty statement at the end. In this study, what new things have the authors done or correlated in comparison to previous ones?
5. The general and specific aim should be inserted.

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised. Based on the complete genome sequences of grapes has important potential for developing novel functional molecular markers. In this study, we analyzed a large number of pri-miRNAs and pre-miRNAs SSR loci in the grape genome. Then, combined with RT-qPCR, we screened the differential genes related to heat resistance and carried out SSR design. We reported the development of miRNA-SSRs in grapes, which were validated in combination with PCR-Sanger sequencing. In conclusion, we developed a miRNA-SSR molecular marker (VSSRMIRNA) combining the comparison results of heat resistance of different varieties, which will greatly strengthen the molecular marker breeding research of grapes. This will lay a certain foundation for the breeding of heat-resistant and high-quality fruit varieties in the future.

Materials and methods

1. The age of seedlings should be added in the section 2.1.

Response

According to your suggestion, the manuscript has been revised (page 4, lines 175 and 177).

The experimental materials were selected from 100 days of growing cuttings of grapes with 7–10 leaves that could meet the needs of the experiment.

2. The detailed of statistical data analysis should be inserted.

Response

According to your suggestion, we have made the 2.7 Statistical analysis structure modification (page 5, lines 248 and 250).

The plant phenotype data and physiological indexes were statistically analyzed by Microsoft Excel (2016). Use TBtools software to make diagrams. The statistical software package IBM SPSS Statistics (Version 20.0) for Window was used.

Results and discussion

1. In general, this section is not clearly written and should be modified.

Response

According to your suggestion, the Results and discussion has been revised. Please refer to the revised manuscript with changes marked.

2. Why did not the authors measure the quantitative data？

Response

Thank you for your reviews. About measure the quantitative data, in order to further explore the high-temperature response of pri-miRNA, the primary transcript of miRNA, in grape.

3. The captions of all figures should be detailed.
4. The captions of all tables should be detailed.

Response

Thank you for your reviews. According to your suggestion, the captions of figures and tables have been perfected. Please refer to the revised manuscript with changes marked.

5. The bootstrap values should be added to the phylogenetic tree.

Response

According to your suggestion, the bootstrap values (Bootstrap = 1000) have been perfected.

6. The authors should calculate the maximum composite likelihood.

Response

Thank you for your reviews. Since the data for the phylogenetic tree is selected from different species, the construction of the NJ phylogenetic tree is more suitable than that of the ML phylogenetic tree.

7. The discussion is weak and should be improved because most of the sentences are written as the literature review rather than interpretation of results.

Thank you for your reviews. According to your suggestion, the discussion has been perfected. Please refer to the revised manuscript with changes marked.

Conclusion

The authors should summarize the most important findings from this study. Furthermore, additional works pertaining to this research should be included as future works.

Thank you for your reviews. According to your suggestion, the conclusion has been revised. As follows,

A total of 192 vvi-pri-miRNAs and 192 vvi-pre-miRNAs sequences were analyzed in the grape genome. 391 SSR markers were identified in vvi-pri-miRNA, and 12 SSR markers were found in vvi-pre-miRNA. In this study, the accuracy of the presence of miRNA-SSR was verified by PCR amplification combined with Sanger sequencing, and a molecular marker VMIRSSR167c3 was developed to distinguish heat-resistant and sensitive types. Notably, this was the genome-wide identification of miRNA-SSRs in grapes. A novel approach to developing molecular markers has been proposed and validated, which provides a reference for identifying more sequences from the genomes non-coding library. The development of heat-resistant molecular markers was beneficial to the genetic improvement of grape germplasm resources and the identification and application of heat-resistant high-quality varieties.

Comments on the Quality of English Language

Extensive correction is needed

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised. Please refer to the revised manuscript with changes marked.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors, I have read the MS entitled Development, identification and validation of a novel SSR mo- 2 lecular marker for heat resistance of grapes based on miRNA. I have some observations:

-the research studies adverse effects of high temperatures on fruit quality and yield of grapevines, where MiRNAs play an important role in plant growth and development and in the molecular mechanism of regulating the high temperature response. 

-please use passive voice in the whole MS. Do not use "we" or "our" all over text

-please format with italics when using Latin denomminations of plants all over text (e.g. line 412, 413 but check eveywhere)

-italics are not used for common plant names (line 496). Please format accordingly

-please add some more info on the grape varieties taken into study in the plant material section

-please add a subsection on reagents and equipements used and their origin

-please develop more the statistical analysis paragraph

-please add measuring units in graphs (fig. 3)

-please shortly describe the analysis methods used and after add the bibliographical refferences.

Dear Editors, I have read the MS entitled Development, identification and validation of a novel SSR mo- 2 lecular marker for heat resistance of grapes based on miRNA. 

The English language is fine, there are some minor corrections that need to be made, like from active to passive voice.

Author Response

Reviewer #3

Comments and Suggestions for Authors

Dear Authors, I have read the MS entitled Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA. I have some observations:

1. -the research studies adverse effects of high temperatures on fruit quality and yield of grapevines, where MiRNAs play an important role in plant growth and development and in the molecular mechanism of regulating the high temperature response.

Response

Thank you for your reviewing. Your affirmation and recognition of“Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA” research paper will be of great help to our follow-up scientific research.

2. -please use passive voice in the whole MS. Do not use "we" or "our" all over text.
3. -please format with italics when using Latin denomminations of plants all over text (e.g. line 412, 413 but check everywhere).
4. -italics are not used for common plant names (line 496). Please format accordingly.

Response

Thank you for your reviews. According to your suggestion, the manuscript has been revised, including grammar improvement and content perfected. Please refer to the revised manuscript with changes marked.

5. -please add some more info on the grape varieties taken into study in the plant material section.

Response

According to your suggestion, the manuscript has been revised. Please refer to Table S1 for grape information

6. -please add a subsection on reagents and equipment used and their origin.
7. -please develop more the statistical analysis paragraph.

Response

Thank you for your reviews. The reagents and equipment content have been perfected. In addition, according to your suggestion, we have made the 2.7 Statistical analysis structure modification (page 5, line 248 and 250).

The plant phenotype data and physiological indexes were statistically analyzed by Microsoft Excel (2016). Use TBtools software to make diagrams. The statistical software package IBM SPSS Statistics (Version 20.0) for Windows was used.

8. -please add measuring units in graphs (fig. 3).

Response

According to your suggestion, we have made the scale label modification (Figure 1 and Figure 2).

*Bar=1cm

9. -please shortly describe the analysis methods used and after add the bibliographical references.

Response

Thank you for your reviews. The analysis methods contents have been perfected. The main references were [37-39]. Please refer to the revised manuscript with changes marked.

Comments on the Quality of English Language

Dear Editors, I have read the MS entitled Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA.

The English language is fine, there are some minor corrections that need to be made, like from active to passive voice.

Response

Thanks for your comment on the article, we have corrected the English language. In addition, your affirmation and recognition of “Development, identification and validation of a novel SSR molecular marker for heat resistance of grapes based on miRNA” research paper will be of great help to our follow-up scientific research.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have addressed my questions.

Reviewer 2 Report

The authors have been addressed all comments

Moderate correction is needed