A Useful Technical Application of the Identification of Nucleotide Sequence Polymorphisms and Gene Resources for Cinnamomum osmophloeum Kaneh. (Lauraceae)

Round  1

Reviewer 1 Report

The authors of the manuscript "A Useful Technical Application of The Identification of Nucleotide Sequence Polymorphisms and Gene Resources for tree Cinnamomum osmophloeum Kaneh. (Lauraceae)" describe their efforts to compute a phylogenetic tree across multiple sampled cinnamon trees. They especially focus on the previous used trnL vs. ITS2 genes and compare the trees using these genes and NJ and parsimony tree methods. Overall the manuscript is well written and easy to follow providing enough details to follow their steps and conclusion.

 My only concern or question about this study is on the pyhlogentic trees that they are showing.

It appears that the bootstrap values are very low in both cases. This is clearly due to the sequence similarity between the samples. However, this makes conclusions about their relatedness difficult. I don’t know if the resources are there but I would be interested in a multi gene tree and if this increases the resolution between the samples. In the simplest form this can be a combination of ITS + trnL.

 In addition, I am missing a bit of a conclusion. What is it that we have learned from this comparison. How does the phylogenetic clustering relate to the geographical clustering of the samples? The presented conclusion that ITS2 might be a better region to identify the clustering is interesting. However, what are the implications?

Author Response

Dear Reviewer,

Thank you very much for the useful comments.

We attached our edited manuscript here.

Our answers are as follows.

Comments 1. My only concern or question about this study is on the pyhlogentic trees that they are showing. It appears that the bootstrap values are very low in both cases. This is clearly due to the sequence similarity between the samples. However, this makes conclusions about their relatedness difficult. I don’t know if the resources are there but I would be interested in a multi gene tree and if this increases the resolution between the samples. In the simplest form this can be a combination of ITS + trnL.

Response: Thank you for these valuable comments. The bootstrap value is low due to the fact that the experimental samples analyzed are all the same cinnamon species (these are from the "species", but different "strains"), therefore, lower values are reasonable results. For this type research, a multiple gene tree is totally far away from the DNA barcoding goal to do the genetics resource identification. Moreover, ITS+trnL cannot be combined the dataset for this plant species study. Through the results of this experiment, support the use of ITS sequences and the import the concept of authentication and DNA barcoding, this method can classified well and achieve fast identification goal for the preservation of those research center long term collection Cinnamomum osmophloeum strains, will be better than the decision tree grouping method. Our analysis is enough to derive optimal result (Line 343-345; Line 402-408; Line 410-417）

Comments 2. In addition, I am missing a bit of a conclusion. What is it that we have learned from this comparison. How does the phylogenetic clustering relate to the geographical clustering of the samples? The presented conclusion that ITS2 might be a better region to identify the clustering is interesting. However, what are the implications?

Response: We demostrate the ITS2 rDNA local sequence database established in the present study could be used in gene resource identification and the selection of desirable Cinnamomum osmophloeum strains. Our study results can be used further for correctly and fast identifying the true Cinnamomum osmophloeum in the first line, and avoid fake Cinnamomum osmophloeum in the markets. This implication is useful for the forestry management for many countries in reality (Line 12-17; Line 30-33; please see our conclusion Line 410-417).

Reviewer 2 Report

  This MS contains sufficient information and could be warrant for the publication. Besides some minor errors, my primary concern is the discussion section which lacks depth. Please see my comments below.

Line 46: please designate the full name of CO before use abbreviations.

Line 95 to 97: If the sequence of pITS2 is identical among strains,  why conclude that it is sufficient to barcode CO?

Line 154: “350 L” must be wrong. Please also check the followings to make the corrections.

Line 202-216: this part should be re-organized and incorporated into M&M part.

Table 3: Under “LHC+HL” by “trnL-trnF IGS” category, the length of product should be 356-361 bp.

The discussion part should be rewritten. It just rephased the results and introduction. Authors should refer other studies to thoroughly reason your results.  

Author Response

Dear Reviewer,

Thank you very much for your useful and positive comments.

We edited this manuscript carefully according to your comments.

Comments 1: Line 46: please designate the full name of CO before use abbreviations.

Response: We have added the definition of CO. Cinnamomum osmophloeum Kaneh. (CO). (Line 13 and Line 45)

Comments 2: Line 95 to 97: If the sequence of pITS2 is identical among strains,  why conclude that it is sufficient to barcode CO?

Response: We have corrected this error and revised into "Our results indicated that the pITS2 nucleotide sequences for all seven of the geographical strains are not correlated with essential oil composition." (Line 96-98)

Comments 3: Line 154: “350 L” must be wrong. Please also check the followings to make the corrections.

Response: We have revised the paragraph for the unit mL accordingly (Line 150-187).

Comments 4: Line 202-216: this part should be re-organized and incorporated into M&M part.

Response: We have deleted the original Line 202-216 and incorporated this part into the methods (Line 107-114).

Comments 5: Table 3: Under “LHC+HL” by “trnL-trnF IGS” category, the length of product should be 356-361 bp.

Response: We have corrected the error (Line233-235).

Comments 6: The discussion part should be rewritten. It just rephased the results and introduction. Authors should refer other studies to thoroughly reason your results.

Response: We have revised the discussion accordingly (Line 326-408).

Reviewer 3 Report

Overall, the paper was well done. There are several areas of editing in the Materials and Methods. All the figures and tables need more complete information.

Comments for author File: Comments.docx

Author Response

Dear Reviewer,

We really appreciate your useful and careful suggestions and editing.

Comments 1: revised the typo error.

Response: We revise “The” into “the”. (Line 2)

 Comments 2: edited the title of this manuscript.

Response: We delete “tree”.( Line 4)

 Comments 3: revised the typo error.

Response: We revised into The plant genus Cinnamomum “contains” economically important evergreen”,” and…(Line 11)

 Comments 4: Please define with first use.

Response: We revised into “Our study tree species Cinnamomum osmophloeum Kaneh. (CO)” (Line 12-13).

 Comments 5: Please define with first use.

Response: We revised into the definition. “IGS is intergenic spacer.”(Line 25)

 Comments 6: The ending –aceae = family. You use the phrase correctly in the abstract.

Response: We delete “family” according to the reviewer’s suggestion.( Line 38)

 Comments 7: What do you mean by this?

Response: We revised into “and potential use as a medicinal material for decreasing high uric acid and high blood sugar [17,18]” (Line 49-50).

 Comments 8: rewrite “CO is an endemic species of Taiwan.”

Response: We revised the sentence into “CO is a species endemic to Taiwan.”acording to the reviewer’s suggestion (Line 53)

 Comments 9: edit 2 into two.

Response: We revised 2 into “Two” the acording to the reviewer’s suggestion (Line 70).

 Comments 10: edit grammar error.

Response: We revised lengthe”s”the acording to the reviewer’s suggestion. (Line 71)

 Comments 11: edit grammar error.

Response: We have added the “variation in” acording to the reviewer’s suggestion.( Line 72)

 Comments 12: Citation for this statement?

Response: We have cited the above paper. Please see Reference [28]. ( Line 86)

 Comments 13: You should include the author information for these all species in the paper at first mention. Afterward, you do not need to include the author.

Response: We revised the species name and put them into correct format.( Line 89-90)

 Comments 14: The second set of quotation marks are pointing in the wrong direction. They are opening, not closing.

Response: We have corrected this point. (Line 107)

 Comments 15: Table headings should include enough information for the table to stand alone. What Does the A stand for, why are some letters in bold and others not? Please add more detail to this heading.

Response: We have revised all tables and figures headings. For the table 1, “The List of the tested plant species names with three amplified partial nuclear regions pITS2, chloroplast regions trnL intron and trnL-trnF IGS, and GenBank Accession number indicated with base pair lengths. Letter in bold is amplified by this study.”(Line 123-125)

 Comments 16: The degree symbol is not placed correctly. You need to fix all these notations. I’m sure you didn’t use liters of these materials. There’s some sort of hidden character like a tab or something that is replacing your correct volume.

Response: We have revised all mistakes in the grammar in this paragraphy, such as ml and the degree symbol. (Line 147-184)

 Comments 17: How did you visualize the bands?

Response: We have added this method into “and visualized on an ABI3730XL capillary-based DNA sequencer (Applied Biosystems).”(Line 179)

 Comments 18: Citation?

Response: We have cited the above paper. Please see Reference [36]. (Line 182).

 Comments 19: These sentences have a lot of double spaces.

Response: We have corrected those mistakes according. (Line 183-187)

 Comments 20: This citation should be with the first mention.

Response: We have added the citation into the first mention in this manuscript. Please see Reference [36]. (Line 198)

 Comments 21: This is already mentioned in the paragraph before the figure.

Response: We have deleted this paragraph because of the reviewer’s suggestions, and thus this current revised version had been corrected.( Line 206-211)

 Comments 22: This line has a lot of double spaces.

Response: We have revised them into correct format (Line 226-227).

 Comments 23: What do you mean by this? This sentence doesn’t make sense.

Response: We have revised this paragraph based on the reviewer’s question into “The variation of sequence identity matrix was the greatest for pITS2 among 14 geographical strains of CO collected from LHC. The same specimens of these 14 geographical stains collected from LHC, they are identical for nucleotide sequences in the trnL-trnF IGS and trnL intron regions. Sequence analysis data of PCR products of the pITS2, trnL intron and trnL-trnF IGS in the present study are shown in Table 3.” (Line 228-232)

 Comments 24: See comment on Table 1. Please include more detail.

Response: We have added more details in this headline into Table 3, “The Size Distributions of PCR Products based on one partial nuclear region pITS2, and two chloroplast regions trnL intron and trnL-trnF IGS from the two study sites used in the present study.”. (Line 233)

 Comments 25: Please edited those grammar errors.

Response: We have corrected the grammar errors accordingly. (Line 244-247)

 Comments 26: rewrite this paragraph.

Response: We have corrected the errors and rewrote into “The length of pITS2 region is relatively short, when compared to those of trnL intron and trnL-trnF IGS for strains in the present study. However, there is a large intra-specific variation was observed. After sequence alimented alignment and compared comparison, only 30 out of 180 positions were conserved among 38 representative strain’s sequences.” “Ten insertions of nucleotides were observed at positions in the 4th, 22th, 55th, 56th, 78th and 79th bp. The deletion of nucleotides were observed at positions in the 6–11th, 32th, 78th, 79th, 117–122th, 146th and 147th bp. Total there were 123 substituted positions, which represented 68.3% of the total sequence length (Figure 4).”(Line 251-257)

 Comments 27: What do you mean by this? Do you mean that insertions of 1-5 bp were found in 10 locations? This is very confusing.

Response: We have revised this sentence into Figure 2. Nucleotide Sequence Polymorphism of C. osmophloem Kaneh. Sequence alignments of the trnL intronic region among the different C. osmophloeum Kaneh. varieties used in this study.( Line 260-261)

 Comments 28: See comment on table 1. Please include more detail.

Response: We have added more details into the headings. Our answer: “Figure 4. Nucleotide Sequence Polymorphism of C. osmophloem Kaneh. The Cinnamomum osmophloeum Kaneh. Varieties are shown according to the sequence alignments of the nuclear non-coding region pITS2 used in this study.”(Line 269-271)

 Comments 29: You should look for more recent information.

Response: We update our reference to the recently data as (data included was as of January 2019, see Table 1). ( Line 275)

 Comments 30:  

Response: We have corrected the grammar errors.( Line 276-277).

 Comments 31: Include what the scale represents since you have it on the figure.

Response: We have added the scale into the figure 5. Please see the Figure 5. (Line 290)

 Comments 32: You should include the author information for these all species in the paper at first mention. Afterward, you do not need to include the author.

Response: We have corrected it into “Cinnamon species in Taiwan has been used heavily for hundreds years. However, in the markets, a serious problem is about the fake or misidentified cinnamon materials. They are very difficult to be identified by the morphology. There are four indigenous cinnamon species in Taiwan: Cinnamomum osmophloeum, Cinnamomum insulari-montanum Hayata, Cinnamomum pedunculatum Nees, and Cinnamomum macrostemon Hayata [2].” (Line 327-331)

 Comments 33: What do you mean by this? I think you can end the sentence at greatest as you are only looking at members of Cinnamomum. This sentence reads as if you are comparing Cinnamomum to other genera.

Response: We have deleted the unnecessary sentences according to the reviewer’s suggestion. (Line 362-363)

 Comments 34: What do you mean by this? Did you have one individual of cassia and one of loureiroi that could not be distinguished, or did loureiroi match with all the cassia? Please clarify.

Response: We have rewritten this paragraph into “However, the two species C. loureiroi and C. cassia could not be separated successfully using the trnL-trnF IGS sequences. The IDs found in the trnL intron were less variable (between 0.993-1) among the Cinnamomum spp. Many different species have identical trnL intron sequences, such as C. cassia, C. insulari-montanum, C. burmannii and C. wilsonii Sarg. Based on our results, the trnL intron is not a good marker for identifying species, and instead, we recommend ITS2 because it provides better resolution for species identification among Cinnamomum spp.” According to the reviewer’s suggestion (Line 365-371)

 Comments 35: This is hyphenated in the first use. Is it supposed to be or was that because of the line break?

Response: We have corrected this into C. insulari-montanum. (Line 368)

 Comments 36: grammar error.

Response: We have corrected those errors in the conclusion (Line 409-417).

Round  2

Reviewer 2 Report

Authors have addressed my questions. I do not have other questions to raise.

Author Response

Dear Reviewer,

Thank you very much for those careful comments and support our study.

Best regards,

Pei-Luen Lu