Study on the Extraction and Identification of DNA from Ten Dalbergia Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript Study on Extraction and Identification of DNA from Ten Species of Dalbergia by Changtao Gan, Haishan He and Jian Qiu, is focus on the identification of Dalbergia species by using molecular tools (ITS2 barcoding fragment).

The traditional morphological identification of the species of the genus was defined by authors as problematic, for many reasons. The upcoming of barcoding methods to species identification was then assumed to be the way to solve this issue, thus it was applied in this study. As many wooden species present problems for DNA extraction due to the presence of polysaccharids, polyfhenols, resins, gums or other, authors propose the optimization of the extraction DNA method as a complementary aim of the research.

I think this kind of works are useful for the scientific community, since it is very common to face methodological constraints when working with molecular techniques, even more with species that have complex tissues. Moreover, to optimize barcoding assessment is also so relevant in species complexes that present taxonomic doubts. However, my main concern about this article is the lack of consistency in the story to tell and the difficulty in the language.

Abstract: I think that the main story to tell and general ideas of the work are included in the abstract, but I would suggest to fully improve language to communicate it properly. Some sentences are really confusing.

55-63; 68-78: please divide the ideas in main sentences, is not possible to follow entire paragraphs without periods.

Line 79: 10 species of wood? or 10 wooden species from the genus Dalbergia?

Line 126-154: You should translate this into a readable material and methods paragraph, in which you tell the public what did you do in your experiments based on this protocol. A copy and paste of the protocol you used does not seem to be a proper text to an article to be published.

Line 167: You should specify the test you run since the proposed extraction methods are more than one and with modifications i.e. different concentrations of ammonium acetate solution on the purity and yield of wood DNA, so I imagine that you tried to inspect the differences in performance among all of them.

Lines 182-192: Please organize this section better.

Lines 209-215: I would suggest to start with the general trend and then describe the results of each method. It would be nicer if you invert the order of these two sentences within the paragraph.

Lines 225-230: Please try to shorten the sentence and/or split it into more than one to improve reading and give clearer ideas.

Line 267- 275: try to rewrite this paragraph using shorter sentences and clearer ideas.

Line 273: this is discussion, not a result itself.

Figure 2: I would suggest to move this to a supplemental, besides letters cannot be fully read at first sight. The differences between species are already shown in your fig4.

Figure 3: I can’t really understand it. At the intra-species, level how do explain full divergence with almost null genetic distance? Are colors green and orange correctly labeled in the reference box? Could you please give a more detail explanation in the figure legend?

Line 313- 318; 322-327; 335-342: It is very difficult to reach to an idea/message with such long sentences. Please re-write.

Discussion: I would suggest starting the discussion with a phrase that indicates at a global level what this work contributes to general knowledge, for example:

In some plant species, tissues can contain complex molecules, which become inhibitory compounds in fragment amplification reactions (PCR). By testing different DNA extraction protocols, we were able to optimize one that offers good performance/yield and has no problems in obtaining barcode sequences (i.e. ITS). As a whole, this is a good achievement in this complex of species difficult to identify that are economically relevant for this and that (complete) …and you can continue arguing your results..

 Line 355-360: I couldn’t understand this long sentences. Please re-write.

Suggestion for supplementary material: consider to add the models and raw results of the statistical analysis done where you compared for the different extraction methods, yields and purification variables.

Comments on the Quality of English Language

 

Although I’m not a native English speaker, I believe that the manuscript should be revised entirely to improve language consistently. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study contributes to DNA extraction methodology. There are a lot of typos in the article. It was difficult to read.

I have comments on the manuscript:

1)       The abstract should be a total of about 200 words maximum. Make it more concise.

2)       Please, check spaces (“vines[1, 2]”, “species [1, 3] ,some” and so on).

3)       Lines 44 – Traditional wood identification mainly relies on wood…

4)       Line 48 – specie -> species.

5)       Line 171-172 – Add primers names.

6)       Line 269 – sequence sequencing comparison.

7)       Line 270 – variant or variable?

8)       Line 289 – interspecific species?

9)       Figure 4 – Please, make the colors less bright. Make description about the triangles?

10)   Line 300 – zone -> helix?

11)   Lines 293-304 – Since you are modeling the secondary structures of the ITS2 region, clarify the differences between the different species. To say that the structure of helices was different is not enough.

12)   Lines 360-362 – So many “species” in one sentence.

The article presents a methodology. I don't really understand why the authors add phylogeny and modeling of secondary structures here. It has long been shown that the ITS2 region is a good marker for species identification, even in this genus there are several publications using this region. In general, I did not see anything essentially new in the article, except the methodology. Try to revise the article and add relevance and novelty.

Comments on the Quality of English Language

There are a lot of typos in the article.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript Study on the Extraction and Identification of DNA from Ten Dalbergia Species by Changtao Gan et al. focus on the identification of Dalbergia species by barcoding, and aims in optimizing a DNA extraction protocol from wood tissue.

This is the second revision round, and I think that this new version significantly improved from the original one.

I would suggest minor changes this time, but after the final acceptance I would suggest to think about adding a more powerful message to the audience. Much of your results are scientific knowledge already reported, like the discrimination power of ITS region, or its secondary structure. The optimization of the DNA extraction method is fine, and if you think that this is helpful for processing species material and identify them, as well as been a tool to promote the conservation of these species, go deeply on this direction to give the audience a novel knowledge around these ideas. That could be an option among others to strenght your paper.

 

Pag 3/17: it is empty, please check edition.

Line 114-116: what does it mean when you said ‘ammonium acetate was added to a solution containing ammonium acetate’? To what do you refer with nuclear isolate*? Please clarify this two points.

*I did not realize that this was mentioned in the original version, I guess that it is a mistake, since the kind of protocol you are describing in your mauscript does not allow to isolate cell nucleus but total DNA (nuclear and organelle DNA). Cell nucleus extraction methods are much more laborious than this, specific for NGS methods, and if that would be the case you should explain in detail and that would be another paper. So please, clarify this point.  

Line 129: I guess that after this pre-treatment procedure you used the milled and rinsed samples to performed all 3 different extraction methods: normal CTAB and the two kits. I suggest to mentioned this specifically in the text.

Line 149-151: I’m afraid you are repeating the same description twice

Line 131: section 2.1.1. This section had considerably improved from the original manuscript version.

Line 193. Section 2.5. In your study you generated sequences for 3 species, but then you included 7 extra species in your phylogenetic tree. You should declare in your manuscript the source of this information, the repository from where they were taken, bibliographic references, ecc.

Line 270-272: It is contradictory to start with however, and then express the successful amplification. Please check.

Figure 2: I still think that this figure should be part of a supplementary material. That the barcode has a large number of mutation sites between the species is expected, that is the purpose of selecting ITS for distinguishing species.

Lines 329-333. This first paragraph of the discussion is a more proper way to start the section. I think it correctly sum up your main results and it is what expected for introducing your arguments with respect to the available background scientific knowledge.

Line 382: I’m not sure about this affirmation, in which way do you think that second generation sequencing technology improved it? What is the difference with respect to first generation sequencing in the particular case of barcoding regions?

Lines 389-393: In relation to my previous comment, I think this point cannot be fully understanding since the ITS region has been known to differentiate species for a long time, and with a sequence generated by the Sanger method it is easily achieved, without the need to recur to NGS

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for heeding most of my advice. However, there are some unresolved issues.

* Page 3 is blank.

* Lines 180-181 Add references to past research.

* Figure 4 - Colors are still too saturated and some are similar to each other. Why do some numbers have ".1" and others do not?

* I don't change my opinion regarding the chapter about the secondary structure of the ITS2 region. It provides little to no information. It is briefly described in the results, and is missing from the discussion altogether. In Figure 5, the authors do not label the differences in structures in any way.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf