Bar-HRM for Species Confirmation of Native Plants Used in Forest Restoration in Northern Thailand

Round 1

Reviewer 1 Report

Es necesario replantear la pregunta de investigación, dado que en una plantación forestal con especies como las seleccionadas en este trabajo, por la experiencia del personal técnico con semillas y plántulas en sus diferentes estados de crecimiento en vivero, su identificación es no es un problema.

En los resultados no se encontró información sobre Chukrasia velutina quizás porque la especie es sinónimo de Chukrasia tabularis . https://tropical.theferns.info/viewtropical.php?id=Chukrasia+tabularis&redir=Chukrasia+velutina

el mismo caso se aplica a Careya sphaerica que es un sinónimo de Careya arborea,

https://tropical.theferns.info/viewtropical.php?id=Careya+arborea&redir=Careya+sphaerica 

Ambos datos se pueden encontrar en la base de datos GenBank https://www.ncbi.nlm.nih.gov/nucore/?term=Careya+arb%C3%B3rea , https://www.ncbi.nlm.nih.gov/ nuccore/?term=Chukrasia+tabularis tal vez revisar eso puede ser útil

The discussion highlights the importance of identifying native plants for reforestation and its cost in time, assuming that a fast and reliable identification method is required. Also, it states that the method, despite being useful, is limited, expensive and time consuming (lines 179 & 180). This part should go to conclusions because the result was not discussed by anyone and neither costs nor time were contrasted.

Author Response

Reviewer 1

Comments and Suggestions for Authors

Es necesario replantear la pregunta de investigación, dado que en una plantación forestal con especies como las seleccionadas en este trabajo, por la experiencia del personal técnico con semillas y plántulas en sus diferentes estados de crecimiento en vivero, su identificación es no es un problema.

Translation: It is necessary to rethink the research question, since in a forest plantation with species such as those selected in this work, due to the experience of the technical staff with seeds and seedlings in their different stages of growth in the nursery, their identification is not a problem.

Response: Yes, it is so true what you said. However, in some cases seeds stocks were found to be a mix of several plant species (without intension). In addition, due to the decline of experts in plant identification or technical staffs who have been intensively trained, a quick and reliable method such HRM would be useful. Also, the method seems to be useful to apply with other species.

En los resultados no se encontró información sobre Chukrasia velutina quizás porque la especie es sinónimo de Chukrasia tabularis . https://tropical.theferns.info/viewtropical.php?id=Chukrasia+tabularis&redir=Chukrasia+velutina

Translation: No information on Chukrasia velutina was found in the results, perhaps because the species is synonymous with Chukrasia tabularis. https://tropical.theferns.info/viewtropical.php?id=Chukrasia+tabularis&redir=Chukrasia+velutina

Response: Really sorry for this mistake and thank you very much for pointing this out. You are the best.

el mismo caso se aplica a Careya sphaerica que es un sinónimo de Careya arborea,

https://tropical.theferns.info/viewtropical.php?id=Careya+arborea&redir=Careya+sphaerica 

Translation: the same case applies to Careya sphaerica which is a synonym of Careya arborea, https://tropical.theferns.info/viewtropical.php?id=Careya+arborea&redir=Careya+sphaerica

Response: Again, really sorry for this mistake and thank you very much for pointing this out. You are the best.

Ambos datos se pueden encontrar en la base de datos GenBank https://www.ncbi.nlm.nih.gov/nucore/?term=Careya+arb%C3%B3rea , https://www.ncbi.nlm.nih.gov/ nuccore/?term=Chukrasia+tabularis tal vez revisar eso puede ser útil

Translation: Both data can be found in the GenBank database https://www.ncbi.nlm.nih.gov/nucore/?term=Careya+arb%C3%B3rea, https://www.ncbi.nlm.nih. gov/ nuccore/?term=Chukrasia+tabularis maybe reviewing that can be useful

Response: Thank you so much for this. We did the analyses again (included the 2 species). Edited relevant texts, Table 3 and 4, and Figure 2.

The discussion highlights the importance of identifying native plants for reforestation and its cost in time, assuming that a fast and reliable identification method is required. Also, it states that the method, despite being useful, is limited, expensive and time consuming (lines 179 & 180). This part should go to conclusions because the result was not discussed by anyone and neither costs nor time were contrasted.

Response: Edited as suggestion.

Thank you very much for your time and comments. We really appreciate it.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript the authors investigate a hybrid method of DNA barcoding and High-Resolution Melting analysis (Bar-HRM), demonstrating its usefulness for identification of native plant species used in tropical forest restoration not only in Thailand but also in other areas.

 

Minor Comments

In figure 1 on the map the scale should appear and the north arrow is very small

Author Response

Reviewer 2

Comments and Suggestions for Authors

In this manuscript the authors investigate a hybrid method of DNA barcoding and High-Resolution Melting analysis (Bar-HRM), demonstrating its usefulness for identification of native plant species used in tropical forest restoration not only in Thailand but also in other areas.

 

Minor Comments

In figure 1 on the map the scale should appear, and the north arrow is very small

Response: Edited as suggestion. Thank you very much.

 

Thank you very much for your time and comments. We really appreciate it.

Author Response File: Author Response.pdf

Reviewer 3 Report

I think the importance of this paper would be enhanced if the following points are mentioned.

1. The field-collected samples were not sequenced in this study.  The possibility exists that the sequences may not be identical to those in the database.  In other words, do we need to consider intraspecific variation and regional differences?

2. It is known that the accuracy of species identification can be improved by appropriate phylogenetic analysis of BLAST search results.  Is it necessary to compare the results of this study with those of phylogenetic analysis?

3. In the relation to question 1, to what extent can the reproducibility of the Bar-HRM experiment be guaranteed for native plants collected from the field?  Is it necessary to present the results compared with multiple samples?

4. In the ms, you mention the cost of HRM.  Is it possible to show the actual cost of HRM and compare it with the cost of other methods?

Author Response

Reviewer 3

Comments and Suggestions for Authors

I think the importance of this paper would be enhanced if the following points are mentioned.

1. The field-collected samples were not sequenced in this study.  The possibility exists that the sequences may not be identical to those in the database.  In other words, do we need to consider intraspecific variation and regional differences?

Response: Yes, we do need to consider intraspecific variation and regional differences like you said. However, we only used the sequences for prediction of the melting curve of each species and thus to present differences between the tested species. In addition, sequencing free is the point we would like to prove here. We do agree on the point you raised here about important of field-collected samples but at the same time the successful of this method indicated that when using the HRM, sequencing is not necessary.

2. It is known that the accuracy of species identification can be improved by appropriate phylogenetic analysis of BLAST search results.  Is it necessary to compare the results of this study with those of phylogenetic analysis?

Response: Yes, it is. But again, we would like to prove here that this species identification/distinguish method is a sequencing free method and thus we did not include any analysis based on DNA sequencing which would be generated in this study. Thank you for the point.

3. In the relation to question 1, to what extent can the reproducibility of the Bar-HRM experiment be guaranteed for native plants collected from the field?  Is it necessary to present the results compared with multiple samples?

Response: Yes, it is reproducible without any doubt as we tested with 3 sets of samples collected from the field.

4. In the ms, you mention the cost of HRM.  Is it possible to show the actual cost of HRM and compare it with the cost of other methods?

Response: Yes, we can compare the costs among methods but based on specific chemicals and reagents we used in our lab. We did not included comparison of the actual cost as this would be a source of bias as it literally depends on the lab and country of the study. For example, in our lab the cost of HRM was around 10-20% lower than DNA sequencing, we need to send the samples to be sequenced outsource as we do not have sequencing facility in our lab. However, this may be or may be not the case in other labs with their own sequencing facility. Nevertheless, one thing can assure here is that the HRM is quicker than sequencing method.

 

Thank you very much for your time and comments. We really appreciate it.

 

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

A very good research which will give new insights to species identification, also, in forest conservation and restoration.

Author Response

Point 1: A very good research which will give new insights to species identification, also, in forest conservation and restoration.

 

Response 1:

We really appreciate the Reviewer’s input to review the revised version and give this positive comment. Thank you very much.

Reviewer 2 Report

The review on the manuscript titled "Bar-HRM for species confirmation of native plant used in forest restoration in Northern Thailand"

 

1. In abstract, Results from both simulation and in vitro analyses revealed that ITS region can be used as a primer set, which can clearly discriminate native plant species in this study. what is ITS region? These results can used in other study area?
2. In the introduction, the aim of this study should be clearly elaborated.
3. In Materials and methods section, more information on the stand and site characteristics should be given.
4. In results section, the comparisons of results need to be more quantitative e.g. how much higher or more?
5. In Discussion section, the structure of Discussion is differed from the Results. You have something to do with it.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors did not answer my questions in the response list.