Chemical Composition and Genome Pattern as a Means of Identifying the Origin of Preserved Wild Garlic (Allium ursinum L.) in Poland
, Piotr GÄ™bczyÅ„ski 1, KatarÃna Ražná 2 and Jana Žiarovská 2
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the manuscript entitled “Chemical composition and genome pattern as a means of identifying the origin of preserved wild garlic (Allium ursinum L.) in Poland”, the authors compared the genome and chemical composition of the wild garlic leaves collected in four locations in southern Poland. I highlighted below some points that can be taken into consideration by the authors to improve the work.
1. Lines 20-24: The results and conclusion presented in the abstract should be improved. Authors need to be more specific about the key findings identified in the study. For example: “The profiles of the analysed components differed in the material from a different site”. What components?
2. Line 25: The keywords “wild garlic” and “genome pattern” are already included in the manuscript title, please, replace with others. Furthermore, it does not make sense to include “freezing”, “air-drying”, and “freeze-drying” as keywords in this study.
3. Lines 29-38: It is necessary to include citations in the first paragraph of the introduction.
4. Line 41: Please, include the meaning of the acronym FAO (Food and Agriculture Organization).
5. Lines 48-53: In total, the genus Allium groups how many species? Also comment on the Amaryllidaceae family.
6. Lines 54 and 58: The genus Allium should be in italics.
7. Lines 244, 263, and 362: The scientific names Allium vienale and Allium ursinum should be in italics.
8. Authors should include more information about the chemical composition and genomic analysis of Allium ursinum in the manuscript introduction.
9. Line 85: In what year were the plants collected?
10. Lines 128-129: The extract preparation method must be detailed. How many grams of plant material were used? How many mL of solvent? What was the extraction method? Was the crude extract obtained concentrated? What was the final yield of the extract? Was the extract stored at room temperature or under refrigeration?
11. What were the concentrations of the ethanolic extract used to quantify total polyphenols and antioxidant activity? Were the analysis performed in triplicate or quadruplicate? This information needs to be very clear in the methodology.
12. The writing of the methodology on antioxidant activity must be improved. Please, describe in detail each method used to carry out antioxidant activity (ABTS, DPPH, and FRAP). The mathematical formulas used to analyze the data obtained must be included for each method.
13. Lines 207-220: This paragraph does not report the results and discussions of the present study. In fact, it is just bibliographic information that does not fit in the “Results and discussion” section. Therefore, I suggest it be deleted.
14. Lines 252-255: Statistical analysis must be described in the methodology.
15. In the conclusion, the authors must report future perspectives in relation to the results obtained in this study.
Author Response
ANSWERS TO THE COMMENTS OF THE REFEREE 1
Firstly, we wish to thank the referee whose useful comments have contributed to the improvement of our work.
Changes made in the text and tables as a result of a referee's suggestions are typed in highlight.
- Lines 20-24: The results and conclusion presented in the abstract should be improved. Authors need to be more specific about the key findings identified in the study. For example: “The profiles of the analysed components differed in the material from a different site”. What components?
- the sentence was replaced by: The content of the analysed components differed in leaves from individual locations, but no leaves were obtained from any location with the highest content of all components.
- Line 25: The keywords “wild garlic” and “genome pattern” are already included in the manuscript title, please, replace with others. Furthermore, it does not make sense to include “freezing”, “air-drying”, and “freeze-drying” as keywords in this study.
- Keywords have been replaced by: vitamin C, chlorophylls, β-carotene
- Lines 29-38: It is necessary to include citations in the first paragraph of the introduction.
- Citations have been added
- Line 41: Please, include the meaning of the acronym FAO (Food and Agriculture Organization).
- The full name is provided next to the acronym
- Lines 48-53: In total, the genus Alliumgroups how many species? Also comment on the Amaryllidaceae family.
- Information about the Allium groups and the Amaryllidaceae family has been added.
- Lines 54 and 58: The genus Alliums hould be in italics.
- Corrected
- Lines 244, 263, and 362: The scientific names Allium vienale and Allium ursinum should be in italics.
- Corrected
- Authors should include more information about the chemical composition and genomic analysis of Allium ursinumin the manuscript introduction.
- Some information about garlic was added to introduction. Lines 60-88.
- Line 85: In what year were the plants collected?
- The harvest year has been added
- Lines 128-129: The extract preparation method must be detailed. How many grams of plant material were used? How many mL of solvent? What was the extraction method? Was the crude extract obtained concentrated? What was the final yield of the extract? Was the extract stored at room temperature or under refrigeration?
- Detailed information provided
- What were the concentrations of the ethanolic extract used to quantify total polyphenols and antioxidant activity? Were the analysis performed in triplicate or quadruplicate? This information needs to be very clear in the methodology.
- Detailed information provided
- The writing of the methodology on antioxidant activity must be improved. Please, describe in detail each method used to carry out antioxidant activity (ABTS, DPPH, and FRAP). The mathematical formulas used to analyze the data obtained must be included for each method.
- Mathematical formulas used to analyse the obtained data are given.
- Lines 207-220: This paragraph does not report the results and discussions of the present study. In fact, it is just bibliographic information that does not fit in the “Results and discussion” section. Therefore, I suggest it be deleted.
The paragraph has been deleted.
- Lines 252-255: Statistical analysis must be described in the methodology.
- Description of statistical analysis was added.
- In the conclusion, the authors must report future perspectives in relation to the results obtained in this study.
Conclusions 5 and 6 were rebuild, Conclusion added: Taking into account the content of the analyzed ingredients in the products, each of the preservation methods can be successfully used to process wild garlic. Only in the case of air-dried leaves can a significant reduction in the vitamin C content be expected.
Reviewer 2 Report
Comments and Suggestions for AuthorsA very interesting paper. The author focuses on wild garlic in Poland. Attempting to use chemical composition and genomic pattern methods as a basis for identifying the origin. In fact, the most reliable way is to perform DNA resequencing analysis on the collected samples, verifying their origin at the molecular level. In view of this study, the author conducted experiments from the perspectives of chemical composition and MMA(microsatellite markers assays), and achieved good results. I hope the author can pay attention to the ecological characteristics of plants, supplement pictures or measurement data from the appearance and morphology of plants, so that other non- horticultural readers can better understand the state of wild garlic.
Author Response
ANSWERS TO THE COMMENTS OF THE REFEREE 2
Firstly, we wish to thank the referee whose useful comments have contributed to the improvement of our work.
Following the referee’s suggestion we have added an explanation to the material and methods section (lines 84-85):
The leaves were collected once, early in the morning, and were fully developed. The size of the leaves was approximately 10 to 20 cm in length.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Dear Editors,
Presented MS describes variability phytochemical and genetic characteristics of Allium ursinum from several origins in Southern Poland. The research approach is comprehensive, allowing the assessment of valuable natural resources. I have several comments that should be taken to account so to make the paper publishable:
The Introduction poses several statements that are not substantiated - see file with track with these and other comments and suggestions.
M&M - The plant material origin is quite vaguely described. A map or coordinates would be helpful. The ownership of the forest areas are mentioned but not the type of forests that would provide information for the growing conditions. Moreover, it remain uncertain if these populations are harvested or they are in some part/tourist area as pressure on the population should be taken into account. Statistical methods are not explained.
R&D - the title implies that origin (elevation and genetics) are explored. However, the data presentation does not give clear view how these factors influence different variables. Origins are not grouped, correlations and/or upmg analyses are not presented. This makes the discussion underwhelming and especially these "conclusions" rather inconclusive:
Marker analysis of coding region-based CDDP and PBA techniques showed sequence variability in the samples from the mountainous areas – Ropa, Limanowa, and Sucha Beskidzka – compared to the sample from Krakow. One of the reasons for this variability might be environmental conditions.
5. Taking into account the level of total polyphenols and antioxidant activity, the best preservation method was usually air drying; in the case of vitamin C and chlorophylls, it was freezing; and in the case of β-carotene, freeze-drying was best.
6. The leaves collected in the Limanowa area were characterised by the highest level of total polyphenols, chlorophylls, β-carotene, and antioxidant activity (FRAP), and those picked in the Sucha Beskidzka area had the highest levels of vitamin C.
Comments for author File: 
Comments.pdf
English is Ok, minor editing should be done by the Authors when cheking the text.
Author Response
ANSWERS TO THE COMMENTS OF THE REFEREE 3
Firstly, we wish to thank the referee whose useful comments have contributed to the improvement of our work.
Changes made in the text and tables as a result of a referee's suggestions are typed in highlight.
R&D - the title implies that origin (elevation and genetics) are explored. However, the data presentation does not give clear view how these factors influence different variables. Origins are not grouped, correlations and/or upmg analyses are not presented. This makes the discussion underwhelming and especially these "conclusions" rather inconclusive:
- In Tables 1-3, the authors presented the influence of two factors (harvesting place and preservation method) on the chemical composition of wild garlic. Data taking into account factor I - the place of collection, also include the influence of the plant genome (DNA) and the location of the place of collection above sea level. Therefore, in order not to excessively expand the already extensive work and not to duplicate data, the authors did not decide to introduce additional statistical calculations.
Line 15: bear was replaced by ramson - as suggested by the Reviewer
Line 17: The analytical method was added.
Line 22-23: the sentence was rephrased.
Line 54: Allium was italicized. (the same line 393)
Line 67: camembert was deleted.
Lines 74-77 georeferences were added.
Lines 79-84: the sentence was deleted.
Line 121: the sentence was rewritten.
Lines 190-194: The description of the statistical analysis has been added:
All samples were analysed in three independent replications (n = 3). Results were analysed statistically using a double-factor analysis of variance (ANOVA) on the basis of Duncan's range test (P < 0.05 The independent variables were the place of origin (altitude above sea level) and the method of preservation. Calculations were performed using Statistica 13.0 Pl (TIBCO Software Inc., Palo Alto, CA, USA).
Table 1. Titles of the tables were rewritten. A number of repetitions was added. Factor analysis to mark (significant) factors (place of origin, processing type) and their interaction was added.
Line 268: L-ascorbic acid, due to its reducing properties, can be transformed into L-dehydroascorbic acid. Due to this, it can create a redox system. It is an oxidized form of ascorbic acid. Both forms - reduced and oxidized - are biologically active molecules having the same vitamin activity. In plant material, L-ascorbic acid is most often determined directly and, after reducing L-dehydroascorbic acid, L-ascorbic acid is again determined as the sum of ascorbic acid and dehydroascorbic acid, i.e. vitamin C. Many researchers are interested in the extent to which ascorbic acid has been oxidized to dehydroascorbic acid, therefore we decided to discuss the results when expressed in this form, i.e. in % ascorbic acid in the total vitamin C content.
An explanation of Vitamin C has been added in Table 3 (sum of AA and DHAA)
Line 394: preserving replaced by: preventing chlorophyll degradation.
Conclusions: Conclusions 1 and 2 were combined.
Line 339: Please, avoid comparisons of means in % - the sentence was rewritten: In both air-dried and freeze-dried leaves, a significant increase in this indicator was found; in the case of air-dried leaves from Limanowa it was even twofold, compared to frozen leaves.
-conclusions 5 and 6 were connected and A new conclusion has been added.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have answered my points mentioned completely. The manuscript is much stronger and acceptable.
