PONE-D-22-14636A comparison of drying methods on the quality for bryophyte molecular specimens collected in the fieldPLOS ONE

Dear Dr. Shuo Shi

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Additional Editor Comments:

REVIEWER 1.

Title: 1. The title of manuscript is little inappropriate and incomplete.

Introduction

The text should be more inclined towards the problems associated with the sampling and DNA isolation from bryophytes species. What are the methods traditionally used for the collection of bryophytes sample? Why different treatment is required and what are their drawbacks?

Materials and Methods

1. Line 76-77: What is the location or collection site of the studied bryophytes specimen?

2. Line 79-80: All four samples were kept fresh at the beginning of the experiment…??? Means stored in -80oC

3. Line 86: The collected bryophytes samples from field contains mud, dirt and microscopic organisms which may cause undesirable PCR amplification. What are the steps taken to clean the collected samples?

4. Line 94-100: Some parts should be reconsidered and rewritten. Experiment design is not clear from the text. From the text it appears that samples are dried in oven at 150°C, 80°C, and 40°C, respectively and then kept in a paper bag in a cool, well-ventilated place, second in a sealed plastic bag with excess silica gel, and third in a sealed plastic bag in a –80°C refrigerator and then again dried at 150°C, 80°C, and 40°C. That is bit confusing.

5. Line 121: Please specify the sequence and Tm value of PCR primers ITS-P5 and ITS-U4 used in present study.

6. Line 124-127: Please specify the method used for evaluation of effect of different drying method on the morphological characteristics of bryophytes.

7. What concentration of gel is used for the gel electrophoresis (DNA isolation and PCR products)??

Results:

1. Line 131: The ratio of the spectrophotometric absorbance of sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid contaminated by phenol or proteins and organic acids respectively. The present study includes DNA purity result based on only the 260/230 ratio.

2. S1 Table: Title should be completed, must be well understandable without the text. What is the difference between DNA purity and DNA concentration (Table 1& 2)? Please provide details of abbreviations (C, P, H, M, N, S, F) in the footnote of table.

3. Figure (1-9): Legends should be completed and well understandable without text.

4. Line 133-138 (Table 1): Spectrophotometer, nanodrops and gel electrophoretogram are used to determine the concentration of DNA. The extracted DNA having a ratio of absorbance A260/280- 1.8 and A260/230- 2.0-2.2 are considered as pure. The A260/230 values of three samples are less than 2.0. The contamination of organic acids might be present in sample. Please provide the justification of statement in your manuscript

5. Instead of long fragment DNA author can use “high-molecular weight genomic DNA”.

6. Provide details (name, collection site, specimen no. family, etc.,)in tabular form about the bryophytes specimens used in present study.

Discussion

Please cite the literature associated with problems arises during collection storage and DNA isolation from bryophyte specimens and how this study provides solutions to those problems. Please give conclusion and future prospects of your study.

REVIEWER 2.

The manuscript brings a simple but important technical advance in the area of molecular studies of bryophytes. Considering the findings presented here, it is possible to carry out the field collection more easily and, at the same time, guarantee the obtaining of quality DNA samples for molecular investigations.

Some minor revisions are necessary:

1- Page 5, line 102: write only "DNA extraction"

2- Page 8, lines 147 to 152: for H. calcicola, the results for hot-air drying at 40°C was not commented. The results obtained for M. polymorpha was not commented. Need to reference that the paragraph is the analysis of Fig. 2.

3- Page 8, lines 153 to 155: consider reviewing the comparison between the results for the fresh freezing control group and hot-air drying at 80°C for M. polymorpha; in table S2 it is clear that, for this species, the only treatment that showed a significant difference (p > 0.05) was drying with hot air at 150°C.

4- Page 8, lines 161 to 163: consider reviewing the comparison of the success rate of PCR amplification after hot-air drying at 80°C and the other treatments; in Fig. 4 (and supplementary figures as well) it is not possible to detect a difference between this treatment and others in some cases.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title:

1. The title of manuscript is little inappropriate and incomplete.

Introduction

The text should be more inclined towards the problems associated with the sampling and DNA isolation from bryophytes species. What are the methods traditionally used for the collection of bryophytes sample? Why different treatment is required and what are their drawbacks?

Materials and Methods

1. Line 76-77: What is the location or collection site of the studied bryophytes specimen?

2. Line 79-80: All four samples were kept fresh at the beginning of the experiment…??? Means stored in -80oC

3. Line 86: The collected bryophytes samples from field contains mud, dirt and microscopic organisms which may cause undesirable PCR amplification. What are the steps taken to clean the collected samples?

4. Line 94-100: Some parts should be reconsidered and rewritten. Experiment design is not clear from the text. From the text it appears that samples are dried in oven at 150°C, 80°C, and 40°C, respectively and then kept in a paper bag in a cool, well-ventilated place, second in a sealed plastic bag with excess silica gel, and third in a sealed plastic bag in a –80°C refrigerator and then again dried at 150°C, 80°C, and 40°C. That is bit confusing.

5. Line 121: Please specify the sequence and Tm value of PCR primers ITS-P5 and ITS-U4 used in present study.

6. Line 124-127: Please specify the method used for evaluation of effect of different drying method on the morphological characteristics of bryophytes.

7. What concentration of gel is used for the gel electrophoresis (DNA isolation and PCR products)??

Results:

1. Line 131: The ratio of the spectrophotometric absorbance of sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid contaminated by phenol or proteins and organic acids respectively. The present study includes DNA purity result based on only the 260/230 ratio.

2. S1 Table: Title should be completed, must be well understandable without the text. What is the difference between DNA purity and DNA concentration (Table 1& 2)? Please provide details of abbreviations (C, P, H, M, N, S, F) in the footnote of table.

3. Figure (1-9): Legends should be completed and well understandable without text.

4. Line 133-138 (Table 1): Spectrophotometer, nanodrops and gel electrophoretogram are used to determine the concentration of DNA. The extracted DNA having a ratio of absorbance A260/280- 1.8 and A260/230- 2.0-2.2 are considered as pure. The A260/230 values of three samples are less than 2.0. The contamination of organic acids might be present in sample. Please provide the justification of statement in your manuscript

5. Instead of long fragment DNA author can use “high-molecular weight genomic DNA”.

6. Provide details (name, collection site, specimen no. family, etc.,)in tabular form about the bryophytes specimens used in present study.

Discussion

Please cite the literature associated with problems arises during collection storage and DNA isolation from bryophyte specimens and how this study provides solutions to those problems. Please give conclusion and future prospects of your study.

Reviewer #2: The manuscript brings a simple but important technical advance in the area of molecular studies of bryophytes. Considering the findings presented here, it is possible to carry out the field collection more easily and, at the same time, guarantee the obtaining of quality DNA samples for molecular investigations.

Some minor revisions are necessary:

1- Page 5, line 102: write only "DNA extraction"

2- Page 8, lines 147 to 152: for H. calcicola, the results for hot-air drying at 40°C was not commented. The results obtained for M. polymorpha was not commented. Need to reference that the paragraph is the analysis of Fig. 2.

3- Page 8, lines 153 to 155: consider reviewing the comparison between the results for the fresh freezing control group and hot-air drying at 80°C for M. polymorpha; in table S2 it is clear that, for this species, the only treatment that showed a significant difference (p > 0.05) was drying with hot air at 150°C.

4- Page 8, lines 161 to 163: consider reviewing the comparison of the success rate of PCR amplification after hot-air drying at 80°C and the other treatments; in Fig. 4 (and supplementary figures as well) it is not possible to detect a difference between this treatment and others in some cases.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field

PONE-D-22-14636R1

Dear Dr. Shi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Rosani do Carmo de Oliveira Arruda

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript contains information relevant to the study of DNA in Bryophytes and will certainly have a positive impact on research on this topic. All suggestions made by the reviewers of the manuscript were accepted and carried out by the authors.All queries were kindly explained and resolved. The methodological issues raised were resolved and also answered by the authors. Thus, I consider that the answers were sufficient, and that the article is ready to be accepted for publication.

Reviewers' comments:

The manuscript contains information relevant to the study of DNA in Bryophytes and will certainly have a positive impact on research on this topic. All suggestions made by the reviewers of the manuscript were accepted and carried out by the authors.All queries were kindly explained and resolved. The methodological issues raised were resolved and also answered by the authors. Thus, I consider that the answers were sufficient, and that the article is ready to be accepted for publication.

Attachments

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Submitted filename: Revised Manuscript with Track Changes (1).docx

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Submitted filename: Response to Reviewers (1).docx