Accurate Cultivar Authentication of Jujube Fruits Using Nano-Fluidic Genotyping of Single Nucleotide Polymorphism (SNP) Markers
Round 1
Reviewer 1 Report
This is an interesting study, aiming to develop a DNA-based barcoding method that can accurately verify the botanical authentication of jujube fruits. The resulting information, according to the authors, can serve as a scientific baseline for the implementation of the GIs regulations in the jujube industry. The manuscript is well written, bringing recent literature, with methodology and statistical analysis well described. Results are presented in a very clear way. Discussion is the only part of the manuscript that needs a little bit of improvement, as well as the references in the reference list. I am describing below some minor corrections/suggestions, and further details on the discussion and the references.
Abstract:
All seems fine in the abstract, very well written and presented.
Introduction:
It is well presented and informative. Only one correction is needed on page 2, line 67:
The word ‘’ratio” needs a correction, in the description of IRMS.
Materials and methods:
The methods and statistical analysis are well described. There is only a minor correction on page 3, line 118:
Instead of Song et al., (Song Lihua ….), please alter to: Song et al. (Song Lihua….), without the comma.
Results:
Results are also well presented, with minor corrections needed, such as:
Page 6, line 159:
Instead of 186among the …., please alter to: 186 among, with a space between them.
Page 9, line 221. I suppose the authors are referring to Table 2 and not Table S2.
Page 9, lines 224, 230, and last line of Table 5. Please use the same abbreviation for the probability of identity among siblings, which on page 6, line 138, is written as PIB-sib. Right now there are four different abbreviations. I suggest that the same abbreviation should be used in all four abbreviations.
Page 9. Also, on line 230, on Table 5´s title, the description of the abbreviation should be provided, as well as the one of PI. Ex: ….. PI = Probability of Identity; PIB-sid = probability of identity among siblings.
Table 5 is not well presented. The columns are too narrow. The landscape orientation should be more appropriate, please verify.
Page 9, line 234: Please correct the word ‘multilocus’. It is written as: mutilocus.
Discussion:
Discussion is OK, but I noticed the lack of references in some parts of it. The first three paragraphs are providing some data on jujuba cultivars, botany, industry, at least the two first ones, but no references are presented. Also, the results of this study were not well compared to other similar studies, of other species with SNP markers, or even other markers. I would like to see some improvement here. References 15, 16, 17 and 18 with similar objectives for four different crops were cited in introduction, and their results could be compared to those of the present study.
The subtitles within this item (Discussion) are incorrect. There are two “DNA quality extracted from jujube fruits”, as sub-item 3.3. The first one should be 4.1, and the second 4.2 with a different sub-title. Also, the last one needs to be 4.4, instead of 3.4.
Page 10, line 251: Instead of “… from both Ningxia, China.”, I suppose it should be “… from Ningxia, China.”.
Page 11, line 322: Instead of “…both fresh and dry and generates high quality”. I suggest altering to “and generated high quality”, since the sentence is in the past.
The last paragraph of Discussion is totally repeated on item 5 – Conclusions. Either exclude this paragraph from Discussion, or exclude item 5 (Conclusions).
References:
Several references need correction.
The titles of many references have the second and/or third word of the Journal title in small letters, while it should have capital letters in the first letters.
Ex: Ref. 2 - Nature communications 2014, 5, 1-12. It should be Nature Communications.
I counted 10 references with this correction needed.
Ref. 5 has the page number incomplete. It should be: 1683-1698.
I noticed other incomplete references, such as Ref. 9: 2019,1, e190015.
Please check Refs. 10, 21 and 28 as well.
The scientific name in Ref. 28 needs to be in italics.
Author Response
Aug. 21st, 2022
Manuscript ID: horticulturae-1871182
Title: Accurate cultivar authentication of jujube fruits using nano-fluidic genotyping of Single Nucleotide Polymorphism (SNP) markers
horticulturae
Dear Editor,
The authors appreciate the editor's and reviewer’s time in evaluating the manuscript and providing constructive comments. The suggestions have significantly helped us in improving the quality of the manuscript. I look forward to working with you and the reviewers to move this manuscript closer to publication in horticulturae.
We revised the manuscript and made appropriate changes following the reviewer's suggestions. Please find our response to all reviewer’s comments below.
Thank you for your time and consideration. I look forward to hearing from you.
Sincerely,
Yaping Ma
Ningxia University
Review Report Form 1
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English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
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Does the introduction provide sufficient background and include all relevant references? |
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Are all the cited references relevant to the research? |
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Is the research design appropriate? |
(x) |
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Are the methods adequately described? |
(x) |
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Are the results clearly presented? |
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( ) |
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Are the conclusions supported by the results? |
(x) |
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( ) |
( ) |
Comments and Suggestions for Authors
This is an interesting study, aiming to develop a DNA-based barcoding method that can accurately verify the botanical authentication of jujube fruits. The resulting information, according to the authors, can serve as a scientific baseline for the implementation of the GIs regulations in the jujube industry. The manuscript is well written, bringing recent literature, with methodology and statistical analysis well described. Results are presented in a very clear way. Discussion is the only part of the manuscript that needs a little bit of improvement, as well as the references in the reference list. I am describing below some minor corrections/suggestions, and further details on the discussion and the references.
The authors appreciate the reviewer’s time in evaluating the manuscript and providing constructive comments. We revised the manuscript and made appropriate changes following the reviewer's suggestions.
Abstract:
All seems fine in the abstract, very well written and presented.
Thank you.
Introduction:
It is well presented and informative. Only one correction is needed on page 2, line 67:
The word ‘’ratio” needs a correction, in the description of IRMS.
We have made revisions in line 111.
Materials and methods:
The methods and statistical analysis are well described. There is only a minor correction on page 3, line 118: Instead of Song et al., (Song Lihua ….), please alter to: Song et al. (Song Lihua….), without the comma.
Thank you. We have removed the comma.
Results:
Results are also well presented, with minor corrections needed, such as:
Page 6, line 159:
Instead of 186among the …., please alter to: 186 among, with a space between them.
Thank you. We have made revisions.
Page 9, line 221. I suppose the authors are referring to Table 2 and not Table S2.
Thank you. We have made revisions referring to Table 2.
Page 9, lines 224, 230, and last line of Table 5. Please use the same abbreviation for the probability of identity among siblings, which on page 6, line 138, is written as PIB-sib. Right now there are four different abbreviations. I suggest that the same abbreviation should be used in all four abbreviations.
We have made revisions throughout the manuscript of PID-sib.
Page 9. Also, on line 230, on Table 5´s title, the description of the abbreviation should be provided, as well as the one of PI. Ex: ….. PI = Probability of Identity; PIB-sid = probability of identity among siblings.
We have added the explanation of this abbreviation PID-sib.
Table 5 is not well presented. The columns are too narrow. The landscape orientation should be more appropriate, please verify.
I made proper adjustments to make it better displayed.
Page 9, line 234: Please correct the word ‘multilocus’. It is written as: mutilocus. Multilocus
Thank you. We have made revisions.
Discussion:
Discussion is OK, but I noticed the lack of references in some parts of it. The first three paragraphs are providing some data on jujuba cultivars, botany, industry, at least the two first ones, but no references are presented. Also, the results of this study were not well compared to other similar studies, of other species with SNP markers, or even other markers. I would like to see some improvement here. References 15, 16, 17 and 18 with similar objectives for four different crops were cited in introduction, and their results could be compared to those of the present study.
Thank you for your suggestions. We have supplemented the content in the discussion, adding the comparison of the literature in lines 402-411
The subtitles within this item (Discussion) are incorrect. There are two “DNA quality extracted from jujube fruits”, as sub-item 3.3. The first one should be 4.1, and the second 4.2 with a different sub-title. Also, the last one needs to be 4.4, instead of 3.4.
We have checked and corrected the subtitle.
Page 10, line 251: Instead of “… from both Ningxia, China.”, I suppose it should be “… from Ningxia, China.”.
We have made revisions.
Page 11, line 322: Instead of “…both fresh and dry and generates high quality”. I suggest altering to “and generated high quality”, since the sentence is in the past.
We have made revisions followed the comments.
The last paragraph of Discussion is totally repeated on item 5 – Conclusions. Either exclude this paragraph from Discussion, or exclude item 5 (Conclusions).
Thank you. We have checked and revised the conclusion section.
References:
Several references need correction.
The titles of many references have the second and/or third word of the Journal title in small letters, while it should have capital letters in the first letters.
Ex: Ref. 2 - Nature communications 2014, 5, 1-12. It should be Nature Communications.
I counted 10 references with this correction needed.
Ref. 5 has the page number incomplete. It should be: 1683-1698.
I noticed other incomplete references, such as Ref. 9: 2019,1, e190015.
Please check Refs. 10, 21 and 28 as well.
We have examined and changed all the references to match the journal format.
The scientific name in Ref. 28 needs to be in italics.
Thank you. We have checked and revised the References.
Submission Date
01 August 2022
Date of this review
07 Aug 2022 21:51:07
Review Report Form 2
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
( ) |
(x) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
( ) |
( ) |
(x) |
( ) |
|
Are the results clearly presented? |
( ) |
(x) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
( ) |
(x) |
( ) |
Comments and Suggestions for Authors
Dear Authors,
This manuscript proposes a SNP fingerprint method to detect adulteration, a major issue in the fruit products industry. The target of the fingerprint is jujube industry which comprises fresh and dried fruit products. I have a few comments. First, please clarify if the pulp tissue used was fresh or dried as there are contradictory statements in the manuscript. If both tissues were used, provide results and a discussion of the DNA yield/quality and SNP scores. Secondly, you mention a comparison of SNP scores from fruit and leaf tissues but leaf SNP scores data is missing. While the leaf DNA SNP scores are unimportant to support the claims of the manuscript, indicate why a comparison was necessary for both jujube tissues. Did you expect different DNA profiles from a leaf and a pericarp of the same jujube tree?
Thank you for your suggestions. First, DNA extracted from fresh and dried jujube fruits. Second, the leaf result was presented in our previous publication. We added literature citation here.
In addition to the above, chromosomal information and PIC used to select the final subset of 24 SNPs are missing from the manuscript and supplementary material. Both can be included for SNPs in Table 5 and Table S2. A table of genetic distances is also necessary, preferably in the manuscript as it was used to generate the PCoA and NJ tree. Finally, instead of random example SNP profiles in Table 4, the fingerprint or SNP profiles of the 24 SNPs subset could be presented. It is missing in the manuscript and supplementary tables.
Thank you. We have made revisions in the manuscript.
What is the level of out-crossing, selfing and/or heterozygosity in jujube cultivars? Please discuss whether they can impact application of the suggested SNP fingerprint, especially if cultivars are not exclusively clonally propagated.
We have supplemented the content in the manuscript in section 4.3.
Finally, the conclusion section needs to be rewritten as it is wholly identical to a paragraph from the discussion section.
We have removed the duplicates, and have re-examined and modified the conclusions
Below are my other comments.
Abstract
Line 22: It is not clear what authors mean by 'same products'. Fruit pulp? Commercial jujube products?
Thank you. We made revisions in line 22.
Line 25: 'large number of samples in a short time'. Please be specific here to make this clearer. How many samples per unit time?
This method can handle 100s to 1000s of samples per day (based on the platform used). We made revisions in line 24.
Introduction:
Line 39: 'The market value is ...' Indicate which market region (Global, China or Ningxia?).
We have made revisions in China in line 39.
Line 46: 'fully ripen' change to fully ripened.
We have made revisions.
Line 55: At first use, expand the acronym IP?
We have made revisions.
Materials and Methods
Line 96 and Table 1: 'For each cultivar, three individual fruits were independently sampled from three trees which were clonally propagated.' At first glance, this appears to be 3 fruits from every clone per cultivar. Please rewrite to indicate it was a single fruit independently sampled from each of the 3 clones per cultivar.
We have written this sentence in 148 lines, “A single fruit was independently sampled from each of the 3 clones per cultivar.”
Line 106: 2-β-mercaptoethanol is available in various concentrations. Please add the concentration of your stock βME.
We have corrected it.
Line 106: 'DNA presence and quantity were assessed..'. It is not clear what this phrase refers to. Perhaps DNA quality and quantity? Assessing quantity indicates DNA is present in the sample.
We have corrected the sentence in 153 line, “DNA quality were evaluated on 0.8% agarose gel stained with ethidium bromide then visualized with a UV trans-illuminator model M-20”.
Line 110: 'Maryland, the USA for genotyping.' Remove 'the'.
We have made revisions.
Line 112 and Table 3: '260∶280 ratio' change to 'A260/280 ratio' here and throughout the manuscript.
We have made revisions.
Line 117: Remove 'the' before SNP.
We have made revisions.
Line 131: 'markers was listed in Supplementary' change to 'markers is listed in Supplementary'.
We have made revisions.
Table 2. Column on SNPs and Flanking Sequences. I'd suggest having separate columns for SNPs, 5' flanking sequence and 3' flanking sequence.
We have made revisions.
Line 134: 'whether the multiple fruits of the same' Remove 'the' before 'multiple fruits'.
We have made revisions.
Line 136: 'were considered identical and were derived from' remove 'were' before 'derived'.
We have removed it.
Lines 144 and 149/150: Is this the same genetic distance as that in Line 149 or different? Why wasn't the same genetic distance used for both PCoA and NJ clustering? Please clarify in the manuscript and, if they were different, indicate the algorithm useo estimate genetic distance mentioned in Line 144. It might be useful to include a table of the genetic distances.
We have made revision. This is because the distance method used in GenAlEx is not implemented in the Microsatellite Analyzer. Therefore, a different distance was used. In a way, we consider the use of two different distances an advantage, which double checked the consistency of the two ordination
Line 159: missing space in '1.86among'
We have made revisions.
Results
Line 159/160: Analyzed fruit tissue was fresh (line 102) but the wording here suggests a 'Zhongning Yuanzao' fruit was not. Please edit accordingly.
We have made corrected.
Line 166: 'which enables' change to 'which enabled'
We have made revisions.
Line 169: 'if no call were scored (as 0)' Do you mean missing data? Please indicate 'excluding missing data'.
We have made revisions.
Line 170: 'Fully matched' do you mean were identical? Rephrase as it is not clear. Also mention why it was necessary to compare SNP scores across tissues. Do SNP genotypes vary across vegetative tissues of the same jujube tree?
We have made revision. The SNP profiles among vegetative tissues are the same. The purpose here was to show the consistency between fruits and leaves, which were assessed in different experiments. The result demonstrated the robustness of this protocol.
Line 174: Superscript in 10^-15.
We have made revisions.
Line 175: This statement should mention clones being from the same mother tree, rather than fruits.
We have made revisions.
Table 4: In the caption, indicate if these are condensed cultivar SNP profiles or individual fruit SNP profiles
We have made revisions.
Table 4/Line 190: 'DNA extracted from fresh and dried jujube fruits' this contradicts line 102 'For DNA extraction from fruits ......pulp tissue was taken from each fresh fruit'.
We have made revisions.
Figure 2 or Line 203/204: '(First axis....of total information'. Change to 'Variation according to Coordinate 1=xx%, Coordinate 2=xx% and so on.)
We have made revisions.
Figure 3: Did you bootstrap the NJ tree? It is not mentioned in materials and methods. Indicate branch support values on the tree branches and include a tree scale.
Thank you. We have supplemented the Figure 3.
Line 219 and 234: 'has high polymorphism informative content'. Provide the cut-off PIC value instead and add SNP PIC values to Table 5 and Table S2. Change 'informative' to 'information'.
Thank you. We have added the PIC in the table 5.
Line 223: '3/4' use a percentage (75% relative to the full 96 panel) instead of the fraction.
We have made revisions.
Figure 4 Line 232: 'PID and PID-sib'. Please use a consistent format for these throughout the manuscript text and table 5. Sometimes it's PIDSIB, PISI or PI-SIB. The same goes for PID.
We have made revisions throughout the manuscript.
Discussion
Line 243-248: Please provide citations for claims made in this section.
We have supplemented the content in the discussion, and added the references.
Line 251: 'both Ningxia, China' change to Ningxia and China
We have made revisions.
Line 260 and Line 275 are identical.
We have made revisions.
Line 289: 'authenticity of a given jujube fruit'. Please rewrite this statement. The SNP subset provides a fingerprint for 8 tested genotypes and do not necessarily represent a fingerprint for the entire jujube germplasm in the market. A separate SNP set may be required to authenticate jujube genotypes with different profiles.
We have made revisions.
Line 307: Results from the leaf SNP analysis are not presented in the manuscript.
The leaf result was presented in our previous publication. We added literature citation here.
Line 311-312: SSRs are highly reproducible. Please provide a citation because these lines indicates otherwise.
We have supplemented the content in lines 511-524.
Line 320: 'verified the authentication of jujube fruits and processed material'. According to materials and methods only fresh fruit tissue was tested.
We have made revisions
Line 322: Same comment as Line 320.
We have made revisions
Line 324: 'Fluidigm genotyping system' is this a trademark?
We have made revisions in line 543, “This protocol used the nano-fluidic chip for SNP genotyping and enabled rapid cultivar authentication of jujube fruits.”
Conclusion:
Line 331-341: The lines 319-329 in the discussion section have been copy-pasted here.
Thank you. We have checked and revised the conclusion section.
Submission Date
01 August 2022
Date of this review
05 Aug 2022 19:04:26
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Authors,
This manuscript proposes a SNP fingerprint method to detect adulteration, a major issue in the fruit products industry. The target of the fingerprint is jujube industry which comprises fresh and dried fruit products. I have a few comments. First, please clarify if the pulp tissue used was fresh or dried as there are contradictory statements in the manuscript. If both tissues were used, provide results and a discussion of the DNA yield/quality and SNP scores. Secondly, you mention a comparison of SNP scores from fruit and leaf tissues but leaf SNP scores data is missing. While the leaf DNA SNP scores are unimportant to support the claims of the manuscript, indicate why a comparison was necessary for both jujube tissues. Did you expect different DNA profiles from a leaf and a pericarp of the same jujube tree?
In addition to the above, chromosomal information and PIC used to select the final subset of 24 SNPs are missing from the manuscript and supplementary material. Both can be included for SNPs in Table 5 and Table S2. A table of genetic distances is also necessary, preferably in the manuscript as it was used to generate the PCoA and NJ tree. Finally, instead of random example SNP profiles in Table 4, the fingerprint or SNP profiles of the 24 SNPs subset could be presented. It is missing in the manuscript and supplementary tables.
What is the level of out-crossing, selfing and/or heterozygosity in jujube cultivars? Please discuss whether they can impact application of the suggested SNP fingerprint, especially if cultivars are not exclusively clonally propagated.
Finally, the conclusion section needs to be rewritten as it is wholly identical to a paragraph from the discussion section.
Below are my other comments.
Abstract
Line 22: It is not clear what authors mean by 'same products'. Fruit pulp? Commercial jujube products?
Line 25: 'large number of samples in a short time'. Please be specific here to make this clearer. How many samples per unit time?
Introduction:
Line 39: 'The market value is ...' Indicate which market region (Global, China or Ningxia?).
Line 46: 'fully ripen' change to fully ripened.
Line 55: At first use, expand the acronym IP?
Materials and Methods
Line 96 and Table 1: 'For each cultivar, three individual fruits were independently sampled from three trees which were clonally propagated.' At first glance, this appears to be 3 fruits from every clone per cultivar. Please rewrite to indicate it was a single fruit independently sampled from each of the 3 clones per cultivar.
Line 106: 2-β-mercaptoethanol is available in various concentrations. Please add the concentration of your stock βME.
Line 106: 'DNA presence and quantity were assessed..'. It is not clear what this phrase refers to. Perhaps DNA quality and quantity? Assessing quantity indicates DNA is present in the sample.
Line 110: 'Maryland, the USA for genotyping.' Remove 'the'.
Line 112 and Table 3: '260∶280 ratio' change to 'A260/280 ratio' here and throughout the manuscript.
Line 117: Remove 'the' before SNP.
Line 131: 'markers was listed in Supplementary' change to 'markers is listed in Supplementary'.
Table 2. Column on SNPs and Flanking Sequences. I'd suggest having separate columns for SNPs, 5' flanking sequence and 3' flanking sequence.
Line 134: 'whether the multiple fruits of the same' Remove 'the' before 'multiple fruits'.
Line 136: 'were considered identical and were derived from' remove 'were' before 'derived'.
Lines 144 and 149/150: Is this the same genetic distance as that in Line 149 or different? Why wasn't the same genetic distance used for both PCoA and NJ clustering? Please clarify in the manuscript and, if they were different, indicate the algorithm used to estimate genetic distance mentioned in Line 144. It might be useful to include a table of the genetic distances.
Line 159: missing space in '1.86among'
Results
Line 159/160: Analyzed fruit tissue was fresh (line 102) but the wording here suggests a 'Zhongning Yuanzao' fruit was not. Please edit accordingly.
Line 166: 'which enables' change to 'which enabled'
Line 169: 'if no call were scored (as 0)' Do you mean missing data? Please indicate 'excluding missing data'.
Line 170: 'Fully matched' do you mean were identical? Rephrase as it is not clear. Also mention why it was necessary to compare SNP scores across tissues. Do SNP genotypes vary across vegetative tissues of the same jujube tree?
Line 174: Superscript in 10^-15.
Line 175: This statement should mention clones being from the same mother tree, rather than fruits.
Table 4: In the caption, indicate if these are condensed cultivar SNP profiles or individual fruit SNP profiles
Table 4/Line 190: 'DNA extracted from fresh and dried jujube fruits' this contradicts line 102 'For DNA extraction from fruits ......pulp tissue was taken from each fresh fruit'.
Figure 2 or Line 203/204: '(First axis....of total information'. Change to 'Variation according to Coordinate 1=xx%, Coordinate 2=xx% and so on.)
Figure 3: Did you bootstrap the NJ tree? It is not mentioned in materials and methods. Indicate branch support values on the tree branches and include a tree scale.
Line 219 and 234: 'has high polymorphism informative content'. Provide the cut-off PIC value instead and add SNP PIC values to Table 5 and Table S2. Change 'informative' to 'information'.
Line 223: '3/4' use a percentage (75% relative to the full 96 panel) instead of the fraction.
Figure 4 Line 232: 'PID and PID-sib'. Please use a consistent format for these throughout the manuscript text and table 5. Sometimes it's PIDSIB, PISI or PI-SIB. The same goes for PID.
Discussion
Line 243-248: Please provide citations for claims made in this section.
Line 251: 'both Ningxia, China' change to Ningxia and China
Line 260 and Line 275 are identical.
Line 289: 'authenticity of a given jujube fruit'. Please rewrite this statement. The SNP subset provides a fingerprint for 8 tested genotypes and do not necessarily represent a fingerprint for the entire jujube germplasm in the market. A separate SNP set may be required to authenticate jujube genotypes with different profiles.
Line 307: Results from the leaf SNP analysis are not presented in the manuscript.
Line 311-312: SSRs are highly reproducible. Please provide a citation because these lines indicates otherwise.
Line 320: 'verified the authentication of jujube fruits and processed material'. According to materials and methods only fresh fruit tissue was tested.
Line 322: Same comment as Line 320.
Line 324: 'Fluidigm genotyping system' is this a trademark?
Conclusion:
Line 331-341: The lines 319-329 in the discussion section have been copy-pasted here.
Author Response
Aug. 21st, 2022
Manuscript ID: horticulturae-1871182
Title: Accurate cultivar authentication of jujube fruits using nano-fluidic genotyping of Single Nucleotide Polymorphism (SNP) markers
horticulturae
Dear Editor,
The authors appreciate the editor's and reviewer’s time in evaluating the manuscript and providing constructive comments. The suggestions have significantly helped us in improving the quality of the manuscript. I look forward to working with you and the reviewers to move this manuscript closer to publication in horticulturae.
We revised the manuscript and made appropriate changes following the reviewer's suggestions. Please find our response to all reviewer’s comments below.
Thank you for your time and consideration. I look forward to hearing from you.
Sincerely,
Yaping Ma
Ningxia University
Review Report Form 1
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
This is an interesting study, aiming to develop a DNA-based barcoding method that can accurately verify the botanical authentication of jujube fruits. The resulting information, according to the authors, can serve as a scientific baseline for the implementation of the GIs regulations in the jujube industry. The manuscript is well written, bringing recent literature, with methodology and statistical analysis well described. Results are presented in a very clear way. Discussion is the only part of the manuscript that needs a little bit of improvement, as well as the references in the reference list. I am describing below some minor corrections/suggestions, and further details on the discussion and the references.
The authors appreciate the reviewer’s time in evaluating the manuscript and providing constructive comments. We revised the manuscript and made appropriate changes following the reviewer's suggestions.
Abstract:
All seems fine in the abstract, very well written and presented.
Thank you.
Introduction:
It is well presented and informative. Only one correction is needed on page 2, line 67:
The word ‘’ratio” needs a correction, in the description of IRMS.
We have made revisions in line 111.
Materials and methods:
The methods and statistical analysis are well described. There is only a minor correction on page 3, line 118: Instead of Song et al., (Song Lihua ….), please alter to: Song et al. (Song Lihua….), without the comma.
Thank you. We have removed the comma.
Results:
Results are also well presented, with minor corrections needed, such as:
Page 6, line 159:
Instead of 186among the …., please alter to: 186 among, with a space between them.
Thank you. We have made revisions.
Page 9, line 221. I suppose the authors are referring to Table 2 and not Table S2.
Thank you. We have made revisions referring to Table 2.
Page 9, lines 224, 230, and last line of Table 5. Please use the same abbreviation for the probability of identity among siblings, which on page 6, line 138, is written as PIB-sib. Right now there are four different abbreviations. I suggest that the same abbreviation should be used in all four abbreviations.
We have made revisions throughout the manuscript of PID-sib.
Page 9. Also, on line 230, on Table 5´s title, the description of the abbreviation should be provided, as well as the one of PI. Ex: ….. PI = Probability of Identity; PIB-sid = probability of identity among siblings.
We have added the explanation of this abbreviation PID-sib.
Table 5 is not well presented. The columns are too narrow. The landscape orientation should be more appropriate, please verify.
I made proper adjustments to make it better displayed.
Page 9, line 234: Please correct the word ‘multilocus’. It is written as: mutilocus. Multilocus
Thank you. We have made revisions.
Discussion:
Discussion is OK, but I noticed the lack of references in some parts of it. The first three paragraphs are providing some data on jujuba cultivars, botany, industry, at least the two first ones, but no references are presented. Also, the results of this study were not well compared to other similar studies, of other species with SNP markers, or even other markers. I would like to see some improvement here. References 15, 16, 17 and 18 with similar objectives for four different crops were cited in introduction, and their results could be compared to those of the present study.
Thank you for your suggestions. We have supplemented the content in the discussion, adding the comparison of the literature in lines 402-411
The subtitles within this item (Discussion) are incorrect. There are two “DNA quality extracted from jujube fruits”, as sub-item 3.3. The first one should be 4.1, and the second 4.2 with a different sub-title. Also, the last one needs to be 4.4, instead of 3.4.
We have checked and corrected the subtitle.
Page 10, line 251: Instead of “… from both Ningxia, China.”, I suppose it should be “… from Ningxia, China.”.
We have made revisions.
Page 11, line 322: Instead of “…both fresh and dry and generates high quality”. I suggest altering to “and generated high quality”, since the sentence is in the past.
We have made revisions followed the comments.
The last paragraph of Discussion is totally repeated on item 5 – Conclusions. Either exclude this paragraph from Discussion, or exclude item 5 (Conclusions).
Thank you. We have checked and revised the conclusion section.
References:
Several references need correction.
The titles of many references have the second and/or third word of the Journal title in small letters, while it should have capital letters in the first letters.
Ex: Ref. 2 - Nature communications 2014, 5, 1-12. It should be Nature Communications.
I counted 10 references with this correction needed.
Ref. 5 has the page number incomplete. It should be: 1683-1698.
I noticed other incomplete references, such as Ref. 9: 2019,1, e190015.
Please check Refs. 10, 21 and 28 as well.
We have examined and changed all the references to match the journal format.
The scientific name in Ref. 28 needs to be in italics.
Thank you. We have checked and revised the References.
Submission Date
01 August 2022
Date of this review
07 Aug 2022 21:51:07
Review Report Form 2
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
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Yes |
Can be improved |
Must be improved |
Not applicable |
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Does the introduction provide sufficient background and include all relevant references? |
(x) |
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Are all the cited references relevant to the research? |
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(x) |
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Is the research design appropriate? |
(x) |
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Are the methods adequately described? |
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(x) |
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Are the results clearly presented? |
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(x) |
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Are the conclusions supported by the results? |
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(x) |
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Comments and Suggestions for Authors
Dear Authors,
This manuscript proposes a SNP fingerprint method to detect adulteration, a major issue in the fruit products industry. The target of the fingerprint is jujube industry which comprises fresh and dried fruit products. I have a few comments. First, please clarify if the pulp tissue used was fresh or dried as there are contradictory statements in the manuscript. If both tissues were used, provide results and a discussion of the DNA yield/quality and SNP scores. Secondly, you mention a comparison of SNP scores from fruit and leaf tissues but leaf SNP scores data is missing. While the leaf DNA SNP scores are unimportant to support the claims of the manuscript, indicate why a comparison was necessary for both jujube tissues. Did you expect different DNA profiles from a leaf and a pericarp of the same jujube tree?
Thank you for your suggestions. First, DNA extracted from fresh and dried jujube fruits. Second, the leaf result was presented in our previous publication. We added literature citation here.
In addition to the above, chromosomal information and PIC used to select the final subset of 24 SNPs are missing from the manuscript and supplementary material. Both can be included for SNPs in Table 5 and Table S2. A table of genetic distances is also necessary, preferably in the manuscript as it was used to generate the PCoA and NJ tree. Finally, instead of random example SNP profiles in Table 4, the fingerprint or SNP profiles of the 24 SNPs subset could be presented. It is missing in the manuscript and supplementary tables.
Thank you. We have made revisions in the manuscript.
What is the level of out-crossing, selfing and/or heterozygosity in jujube cultivars? Please discuss whether they can impact application of the suggested SNP fingerprint, especially if cultivars are not exclusively clonally propagated.
We have supplemented the content in the manuscript in section 4.3.
Finally, the conclusion section needs to be rewritten as it is wholly identical to a paragraph from the discussion section.
We have removed the duplicates, and have re-examined and modified the conclusions
Below are my other comments.
Abstract
Line 22: It is not clear what authors mean by 'same products'. Fruit pulp? Commercial jujube products?
Thank you. We made revisions in line 22.
Line 25: 'large number of samples in a short time'. Please be specific here to make this clearer. How many samples per unit time?
This method can handle 100s to 1000s of samples per day (based on the platform used). We made revisions in line 24.
Introduction:
Line 39: 'The market value is ...' Indicate which market region (Global, China or Ningxia?).
We have made revisions in China in line 39.
Line 46: 'fully ripen' change to fully ripened.
We have made revisions.
Line 55: At first use, expand the acronym IP?
We have made revisions.
Materials and Methods
Line 96 and Table 1: 'For each cultivar, three individual fruits were independently sampled from three trees which were clonally propagated.' At first glance, this appears to be 3 fruits from every clone per cultivar. Please rewrite to indicate it was a single fruit independently sampled from each of the 3 clones per cultivar.
We have written this sentence in 148 lines, “A single fruit was independently sampled from each of the 3 clones per cultivar.”
Line 106: 2-β-mercaptoethanol is available in various concentrations. Please add the concentration of your stock βME.
We have corrected it.
Line 106: 'DNA presence and quantity were assessed..'. It is not clear what this phrase refers to. Perhaps DNA quality and quantity? Assessing quantity indicates DNA is present in the sample.
We have corrected the sentence in 153 line, “DNA quality were evaluated on 0.8% agarose gel stained with ethidium bromide then visualized with a UV trans-illuminator model M-20”.
Line 110: 'Maryland, the USA for genotyping.' Remove 'the'.
We have made revisions.
Line 112 and Table 3: '260∶280 ratio' change to 'A260/280 ratio' here and throughout the manuscript.
We have made revisions.
Line 117: Remove 'the' before SNP.
We have made revisions.
Line 131: 'markers was listed in Supplementary' change to 'markers is listed in Supplementary'.
We have made revisions.
Table 2. Column on SNPs and Flanking Sequences. I'd suggest having separate columns for SNPs, 5' flanking sequence and 3' flanking sequence.
We have made revisions.
Line 134: 'whether the multiple fruits of the same' Remove 'the' before 'multiple fruits'.
We have made revisions.
Line 136: 'were considered identical and were derived from' remove 'were' before 'derived'.
We have removed it.
Lines 144 and 149/150: Is this the same genetic distance as that in Line 149 or different? Why wasn't the same genetic distance used for both PCoA and NJ clustering? Please clarify in the manuscript and, if they were different, indicate the algorithm useo estimate genetic distance mentioned in Line 144. It might be useful to include a table of the genetic distances.
We have made revision. This is because the distance method used in GenAlEx is not implemented in the Microsatellite Analyzer. Therefore, a different distance was used. In a way, we consider the use of two different distances an advantage, which double checked the consistency of the two ordination
Line 159: missing space in '1.86among'
We have made revisions.
Results
Line 159/160: Analyzed fruit tissue was fresh (line 102) but the wording here suggests a 'Zhongning Yuanzao' fruit was not. Please edit accordingly.
We have made corrected.
Line 166: 'which enables' change to 'which enabled'
We have made revisions.
Line 169: 'if no call were scored (as 0)' Do you mean missing data? Please indicate 'excluding missing data'.
We have made revisions.
Line 170: 'Fully matched' do you mean were identical? Rephrase as it is not clear. Also mention why it was necessary to compare SNP scores across tissues. Do SNP genotypes vary across vegetative tissues of the same jujube tree?
We have made revision. The SNP profiles among vegetative tissues are the same. The purpose here was to show the consistency between fruits and leaves, which were assessed in different experiments. The result demonstrated the robustness of this protocol.
Line 174: Superscript in 10^-15.
We have made revisions.
Line 175: This statement should mention clones being from the same mother tree, rather than fruits.
We have made revisions.
Table 4: In the caption, indicate if these are condensed cultivar SNP profiles or individual fruit SNP profiles
We have made revisions.
Table 4/Line 190: 'DNA extracted from fresh and dried jujube fruits' this contradicts line 102 'For DNA extraction from fruits ......pulp tissue was taken from each fresh fruit'.
We have made revisions.
Figure 2 or Line 203/204: '(First axis....of total information'. Change to 'Variation according to Coordinate 1=xx%, Coordinate 2=xx% and so on.)
We have made revisions.
Figure 3: Did you bootstrap the NJ tree? It is not mentioned in materials and methods. Indicate branch support values on the tree branches and include a tree scale.
Thank you. We have supplemented the Figure 3.
Line 219 and 234: 'has high polymorphism informative content'. Provide the cut-off PIC value instead and add SNP PIC values to Table 5 and Table S2. Change 'informative' to 'information'.
Thank you. We have added the PIC in the table 5.
Line 223: '3/4' use a percentage (75% relative to the full 96 panel) instead of the fraction.
We have made revisions.
Figure 4 Line 232: 'PID and PID-sib'. Please use a consistent format for these throughout the manuscript text and table 5. Sometimes it's PIDSIB, PISI or PI-SIB. The same goes for PID.
We have made revisions throughout the manuscript.
Discussion
Line 243-248: Please provide citations for claims made in this section.
We have supplemented the content in the discussion, and added the references.
Line 251: 'both Ningxia, China' change to Ningxia and China
We have made revisions.
Line 260 and Line 275 are identical.
We have made revisions.
Line 289: 'authenticity of a given jujube fruit'. Please rewrite this statement. The SNP subset provides a fingerprint for 8 tested genotypes and do not necessarily represent a fingerprint for the entire jujube germplasm in the market. A separate SNP set may be required to authenticate jujube genotypes with different profiles.
We have made revisions.
Line 307: Results from the leaf SNP analysis are not presented in the manuscript.
The leaf result was presented in our previous publication. We added literature citation here.
Line 311-312: SSRs are highly reproducible. Please provide a citation because these lines indicates otherwise.
We have supplemented the content in lines 511-524.
Line 320: 'verified the authentication of jujube fruits and processed material'. According to materials and methods only fresh fruit tissue was tested.
We have made revisions
Line 322: Same comment as Line 320.
We have made revisions
Line 324: 'Fluidigm genotyping system' is this a trademark?
We have made revisions in line 543, “This protocol used the nano-fluidic chip for SNP genotyping and enabled rapid cultivar authentication of jujube fruits.”
Conclusion:
Line 331-341: The lines 319-329 in the discussion section have been copy-pasted here.
Thank you. We have checked and revised the conclusion section.
Submission Date
01 August 2022
Date of this review
05 Aug 2022 19:04:26
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
This manuscript is important and has practical application in the Chinese Jujube industry and/or stakeholders globally seeking to limit mislabeled fruit products or adulteration. I have few minor comments outlined below.
Abstract
Please check these words and change accordingly.
Line 12: 'highly adaptability' to 'highly adapted'
Line 21: 'baes' to 'based'
Line 24: 'reliable' to 'reliability'
Materials and methods
Line 100: 'extraction' change to 'extracted'
Table 1, column with 'No. of analyzed'. Should be 'No of samples analyzed' or simply 'n'
Line 146: 'boost strapping' change to 'bootstraps'
Results
Line 172: Please confirm if Table S2 is in the supplementary material.
Discussion
If at all possible, please provide citations in section 306-314. This information is very useful for readers as it compares two marker systems (SNPs and SSRs) used routinely for DNA fingerprinting. SNPs have worked very well in your study.
Line 310: acronyms for Applied Biosystems and SEQ need to be written in full.
References
Check consistency in the formatting. Journal names are italicized except references 27-30.
Author Response
Aug. 23rd, 2022
Manuscript ID: horticulturae-1871182
Title: Accurate cultivar authentication of jujube fruits using nano-fluidic genotyping of Single Nucleotide Polymorphism (SNP) markers
horticulturae
Dear Editor,
The authors appreciate the editor's and reviewer’s time again in evaluating the manuscript and providing comments.
We revised the manuscript and made appropriate changes following the reviewer's suggestions. Please find our response to all reviewer’s comments below.
Thank you for your time and consideration. I look forward to hearing from you.
Sincerely,
Yaping Ma
Ningxia University
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
( ) |
(x) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
This manuscript is important and has practical application in the Chinese Jujube industry and/or stakeholders globally seeking to limit mislabeled fruit products or adulteration. I have few minor comments outlined below.
Abstract
Please check these words and change accordingly.
Line 12: 'highly adaptability' to 'highly adapted'
We have made revisions in line 13.
Line 21: 'baes' to 'based'
We have made revisions in line 21.
Line 24: 'reliable' to 'reliability'
We have made revisions in line 24.
Materials and methods
Line 100: 'extraction' change to 'extracted'
We have made revisions in line 104.
Table 1, column with 'No. of analyzed'. Should be 'No of samples analyzed' or simply 'n'
We have corrected it in Table 1.
Line 146: 'boost strapping' change to 'bootstraps'
We have corrected in line 150.
Results
Line 172: Please confirm if Table S2 is in the supplementary material.
Thank you. We have confirmed the Table S2 was in the supplementary.
Discussion
If at all possible, please provide citations in section 306-314. This information is very useful for readers as it compares two marker systems (SNPs and SSRs) used routinely for DNA fingerprinting. SNPs have worked very well in your study.
Thank you. We have added two references to support the description, in line 313.
Line 310: acronyms for Applied Biosystems and SEQ need to be written in full.
We have added the abbreviations and full names at 315 lines.
References
Check consistency in the formatting. Journal names are italicized except references 27-30.
Thank you. We have checked and made revisions for all References.
Submission Date
01 August 2022
Date of this review
22 Aug 2022 19:29:06
Author Response File: Author Response.docx
