DNA-Based Molecular Markers and Antioxidant Properties to Study Genetic Diversity and Relationship Assessment in Blueberries
Round 1
Reviewer 1 Report (New Reviewer)
see my suggestions in attached pdf
Comments for author File: 
Comments.pdf
Author Response
Page 1. Introduction is very weak. write all about the blue berries, its area of cultivation, breeding behave, germplasm across world, major problem etc.
Response: Thanks for your thoughtful and valuable comments. The Intrtoduction has been revised based on your suggestions.
Page 2. Give values of each group. write their importance, fruit weight, yield, important traits, prominent cultivating areas. etc.
Response: Have been done.
Page 4. Which species is more valued for medicinal point of view. . WHich phyrochemial is thers for anti-cancer capacity or anti-diabitic capacity?
Response: These are inluded in detrail under heading 5.
Page 6. How one can exploit chemicals present in leaves?
Can author add diversity at phenotypic level in this species?
Response: Included in lines 215-225; 498-507.
Page 8. No need to give the name of markers not deployed or reported in blue berry.
Response:.Have been revised. Thank you.
Page 9. Who reported SSRs in blueberries, how many SSRs which method-tool was used to report SSRs.
Response: Please see lines 437-451.
L392-412: do not about SNP based history. better to write about SNP in blue berry on which cultivar it was developed how these SNPs were identified.
Response: The manuscript has been revised as per your nice suggestions (much shortened).
Page 10. Table 2 is meaningless and not related to marker appliocation in blueberries.
Response: Table 2 has been revised based on the comments of Reviewer 4.
Page 16. Can author add some information on metabolomicsm RNA seq and proteomics. this will add more strength in review article.
Response: Has been included in lines 681-685
Reviewer 2 Report (New Reviewer)
I sugget you use genetic diversity, not biodiversity.
SNP marker are mentioned in the text, so the use of this type of marker in the genetic diversity of blueberries also needs to be involved or used as a perspective on how it can be used in corresponding work. You also need to compare the advantages and disadvantages of using these different markers when performing genetic diversity analysis, or the situations in which they are adopted.
I have to remind you that although the antioxidants can be used for cluster analysis, or to classify different blueberries varieties, there are still many differences between it and genetic diversity analysis, because it only contains a subset of traits and cannot assess the whole genetic background. Similarly, a small number of DNA markers cannot assess the genetic background of different blueberries varieties, and they are mainly used for the clustering analysis based on these markers or discrimination of different blueberries varieties. Of course, due to technical limitations, we used to do approximate genetic analysis with a small number of molecular markers. Therefore, it is recommended to use molecular markers (such as SNPs) that cover the whole genome as much as possible to obtain more realistic results for genetic analysis.
Due to the DNA markers used, the genes associated with them are not necessarily linked to the synthesis or accumulation of antioxidants, so clustering based on the two kinds of markers is not likely to be consistent.
English is ok.
Author Response
I sugget you use genetic diversity, not biodiversity.
Response: As per the comments of the reviewers, the title has been changed. Biodiversity has been deleted from the title.
SNP marker are mentioned in the text, so the use of this type of marker in the genetic diversity of blueberries also needs to be involved or used as a perspective on how it can be used in corresponding work. You also need to compare the advantages and disadvantages of using these different markers when performing genetic diversity analysis, or the situations in which they are adopted.
I have to remind you that although the antioxidants can be used for cluster analysis, or to classify different blueberries varieties, there are still many differences between it and genetic diversity analysis, because it only contains a subset of traits and cannot assess the whole genetic background. Similarly, a small number of DNA markers cannot assess the genetic background of different blueberries varieties, and they are mainly used for the clustering analysis based on these markers or discrimination of different blueberries varieties. Of course, due to technical limitations, we used to do approximate genetic analysis with a small number of molecular markers. Therefore, it is recommended to use molecular markers (such as SNPs) that cover the whole genome as much as possible to obtain more realistic results for genetic analysis.
Due to the DNA markers used, the genes associated with them are not necessarily linked to the synthesis or accumulation of antioxidants, so clustering based on the two kinds of markers is not likely to be consistent.
Response: Thanks for your thoughtful and valuable suggestions. The manuscript has been revised based on your suggestions (yellow highlighted).
Reviewer 3 Report (New Reviewer)
DNA-based Molecular Markers and Antioxidant Properties to Study Biodiversity and Relationship Assessment in Blueberries
Abstract
economic and medicinal importance of blueberries, application of molecular markers, and biochemical estimation in berry improvement and conservation, filling the gap in the literature.
Write the finding/results of economic increase or decrease/which cultivar has more chemicals/which molecular marker more helpful etc.
Introduction
Blueberry Production and Economic Importance
Give year wise economic if possible -fresh data will be better.
Reference:
Remove the references more than, 15 years
General
Add work plan model, some pic possible to insure landscape of your publication
Write references in one format. Also improve some English.
Also improve some English.
Author Response
Comments and Suggestions for Authors
DNA-based Molecular Markers and Antioxidant Properties to Study Biodiversity and Relationship Assessment in Blueberries
Abstract
economic and medicinal importance of blueberries, application of molecular markers, and biochemical estimation in berry improvement and conservation, filling the gap in the literature.
Write the finding/results of economic increase or decrease/which cultivar has more chemicals/which molecular marker more helpful etc.
Response: Thanks for your comments. The manuscript has been revised and related findings have been added in text of the manuscript.
Introduction
Blueberry Production and Economic Importance
Give year wise economic if possible -fresh data will be better.
Response: Has been revised as per your suggestions.
Reference:
Remove the references more than, 15 years
Response: Removal of more than 15-year references in the text has been done. Few old references are kept in Table 3 as they contain important results.
General
Add work plan model, some pic possible to insure landscape of your publication
Write references in one format. Also improve some English.
Comments on the Quality of English Language
Also improve some English.
Response: Thanks for your suggestions. The manuscript has been revised as per your thoughtful suggestions.
Reviewer 4 Report (New Reviewer)
Please see the attachment.
Comments for author File: Comments.pdf
Author Response
I have read several times manuscript entitled “DNA-based Molecular Markers and Antioxidant Properties to Study Biodiversity and Relationship Assessment in Blueberries” and it left ambiguous impression. In one way the review contains a lot of information about important chemical compounds found in blueberries and studies of genetic diversity or (QTL) mapping. On the other way some parts of text provide detailed explanation of very general topics like what is genetic diversity or what is molecular marker. It raised the question for what readers is the review intended? The title and larger portion of the manuscript indicate specialized topics for informed reader, but the parts “Genetic Diversity”, “Estimation of Genetic Diversity” and “Molecular Markers and Genetic Diversity” appear to be introductory text for students of Bachelor or Master programme Genetics/Plant breeding. The authors should decide what is their audience because both approaches won’t work together. Somebody with interest for general review about diversity and molecular markers or historical window to gene techniques development won’t search for review about blueberries. Therefore, I suppose the former is more probable and following suggestions are aligned with this assumption.
Graphs versus “text embedded numbers”
Why did you decide to make graph only for percentage share of farm gate value of fresh and processed fruits (Line 128)? Generally, it is easier for people to get idea about amount, differences or pattern when they see a picture instead of bunch of numbers and mainly the part about phytochemistry include many numerical indicators like ratios or concentrations. I think it could be useful to transform such data into graphs too. Anyway, I am able to understand and respect that you prefer numerical output or maybe you do not want to make a set of graphs. It is all right, but then you should remove Figure 1 because it is not clear why this and not other information was so important to deserve a graph.
Long citations
I have found unusually long citation in several cases (Line 199-200; Line 216) and I think you should change it to standard citation style like Prior et al. [59] etc.
Substantially reduce general parts of the text Primarily parts 6. Genetic Diversity (Line 243 - 274), 6.1 Estimation of Genetic Diversity (Line 275-321) and 7.1 DNA-based Markers (Line 322 - 412). Despite extensive citation, if I am not mistaken 96 i.e. 42% of all citations (!), only few lines of text is somehow connected to blueberries breeding or diversity studies.
Check logical connectivity between the sentences.
Here is example of what I mean:
“The ability to make billions of copies of short segments of desired DNA in no time using polymerase chain reaction [120] has enabled scientists to use many sophisticated techniques for population genetic studies and diversity analysis [121]. One of the first use of molecular markers was reported by Botstein et al. [122]. DNA polymorphism was detected using restriction fragment length polymorphism (RFLP) by constructing genetic maps. These markers have their advantages and disadvantages. To achieve the best genetic diversity results within a population, the marker system and statistical tool selection are the key factors after the generation of the dataset [102].” (Lines 316-321)
The first sentence informs reader about PCR based techniques and then follows information about early application of molecular markers (construction of RFLP based linkage map). It gives incorrect impression that RFLP is PCR based technique. Further, the sentence starts with “These markers have…” refers to RFLP markers as imply order of sentences or more generally to all DNAbased markers as imply the last sentence starts with “To achieve the best genetic…”? Moreover, this sentence and other sentences can stand by themselves, but they are poorly linked together.
Information in Table 2
This table deserves some improvement. I know and checked the Table 2 is based on information in two reviews from 2009 and 2017, but still. Table 2 is the part of explanatory text about molecular markers. Despite this assumption definition of “Marker index” or “Level of polymorphism” is missing. Just for my curiosity, why the level of polymorphism for the AFLP markers is “Very high”? It does not make a sense. AFLP marker is typically scored as binary (present/absent i.e. 1 or 0), it means only two options in contrast to SSR marker with even ten times more alleles. It mismatches polymorphism per locus (low for AFLP or SNP to name a few) and number of studied loci. For example, SNP marker has low level polymorphism, but the number of studied loci i.e. SNP could be extremely high (10E5 – 10E6).
Current status of research in blueberries?
I made own search for current (2020-2023) articles about genetics of blueberries. To my surprise, there were many articles using NGS techniques (like GBS) for study of genetics diversity, QTL mapping and genome sequencing (for example - Kulkarni et al., 2020; Manzanero et al., 2023;
Miao et al. 2021; Nagasaka et al., 2022; Nishiyama et al., 2021; Wu et al., 2022; Yu et al., 2021
etc.). Unfortunately, only few of them are mentioned when compared to broadly covered studies
working with older EST-SSR or RAPD markers. Moreover, I do not understand why was not mentioned any whole genome assembly in berries despite its usefulness for marker development among the other things. I believe the reduction or even removing of general parts and including more current studies would be greater benefit for the reader.
References
Cui F, Ye X, Li X, Yang Y, Hu Z, Overmyer K, Brosché M, Yu H, Salojärvi J. Chromosome-level
genome assembly of the diploid blueberry Vaccinium darrowii provides insights into its subtropical
adaptation and cuticle synthesis. Plant Commun. 2022 Jul 11;3(4):100307. doi:
10.1016/j.xplc.2022.100307.
Kulkarni KP, Vorsa N, Natarajan P, Elavarthi S, Iorizzo M, Reddy UK, Melmaiee K. Admixture
Analysis Using Genotyping-by-Sequencing Reveals Genetic Relatedness and Parental Lineage
Distribution in Highbush Blueberry Genotypes and Cross Derivatives. Int J Mol Sci. 2020 Dec
26;22(1):163. doi: 10.3390/ijms22010163.
Manzanero BR, Kulkarni KP, Vorsa N, Reddy UK, Natarajan P, Elavarthi S, Iorizzo M, Melmaiee
- Genomic and evolutionary relationships among wild and cultivated blueberry species. BMC Plant Biol. 2023 Mar 6;23(1):126. doi: 10.1186/s12870-023-04124-y.
Miao XR, Chen QX, Niu JQ, Guo YP. The complete chloroplast genome of highbush blueberry (Vaccinium corymbosum). Mitochondrial DNA B Resour. 2021 Dec 20;7(1):87-88. doi: 10.1080/23802359.2021.2009384.
Nagasaka K, Nishiyama S, Fujikawa M, Yamane H, Shirasawa K, Babiker E, Tao R. Genome-Wide Identification of Loci Associated With Phenology-Related Traits and Their Adaptive Variations in a Highbush Blueberry Collection. Front Plant Sci. 2022 Jan 21;12:793679. doi: 10.3389/fpls.2021.793679.
Nishiyama S, Fujikawa M, Yamane H, Shirasawa K, Babiker E, Tao R. Genomic insight into the developmental history of southern highbush blueberry populations. Heredity (Edinb). 2021 Jan;126(1):194-205. doi: 10.1038/s41437-020-00362-0.
Wu C, Deng C, Hilario E, Albert NW, Lafferty D, Grierson ERP, Plunkett BJ, Elborough C, Saei A, Günther CS, Ireland H, Yocca A, Edger PP, Jaakola L, Karppinen K, Grande A, Kylli R, Lehtola VP, Allan AC, Espley RV, Chagné D. A chromosome-scale assembly of the bilberry genome identifies a complex locus controlling berry anthocyanin composition. Mol Ecol Resour. 2022 Jan;22(1):345-360. doi: 10.1111/1755-0998.13467.
Yu J, Hulse-Kemp AM, Babiker E, Staton M. High-quality reference genome and annotation aids understanding of berry development for evergreen blueberry (Vaccinium darrowii). Hortic Res. 2021 Nov 1;8(1):228. doi: 10.1038/s41438-021-00641-9.
Response: Thanks for your thoughtful and valuale comments and suggestion. Figure 1 has been removed as was suggested keeping the findings in text. Other suggestions has been taken into consideration and the manuscript has been revised based on your suggestions (yellow highlighted). Suggested references have also been added.
Round 2
Reviewer 2 Report (New Reviewer)
line 246-249, what do clones mean? The anthocyanin content may also be varied when the same cultivar or variety is planted in different places. Please check other parts for the use of "clone".
In part 8, You have to also mention that there will be differences between the two clusterings because the DNA markers they use are not related to anthocyanin synthesis.
English is ok.
Author Response
Comments and Suggestions for Authors
line 246-249, what do clones mean? The anthocyanin content may also be varied when the same cultivar or variety is planted in different places. Please check other parts for the use of "clone".
Response: Thanks for your comments and suggestions. “Clone” has been defined (line 215).
In part 8, You have to also mention that there will be differences between the two clusterings because the DNA markers they use are not related to anthocyanin synthesis.
Response: Thanks. Differences between the two clusterings (genetic vs. biochemical) are already available in detail in the text (lines 533- 537).
Reviewer 4 Report (New Reviewer)
Second revision
I received new version of manuscript and the authors obviously followed at least some of my suggestions. I appreciate removing Figure 1 and extending section about current advances in the field of genetics and genomics with regards to evolution and breeding of (blue)berries. I have noticed some very general parts of the text are still there, but they were improved. Still, I have to say that mainly the section "6. Genetic diversity" is about "everything and nothing" and it has very low informative value. Last thing, I attached the list of minor mistakes, mostly typos.
Line 461 and in an LB -> and in a LB
Line 480 EST-PCR and EST-PCR markers were used -> 2x EST-PCR
Line 505 to characterize a set of blueberry wild blueberry clones, cultivars, and hybrids -> to characterize a set of blueberry including wild blueberry clones, cultivars, and hybrids
Line 507 woked -> worked
Line 514 Rodríguez-Peña1 -> Rodríguez-Peña
Line 528 "...closeness with diploid V. tenellum These findings provided..." ->
"...closeness with diploid V. tenellum. These findings provided..." (missing dot)
Line 571 unify shortcut "G-SSR" vs "g-SSR"
Line 578 by [75] -> by Debnath and Sion [75]
Line 659 pocanalysis -> PCA analysis (I suppose)
Line 743 conversation -> conservation
Author Response
Comments and Suggestions for Authors
Second revision
I received new version of manuscript and the authors obviously followed at least some of my suggestions. I appreciate removing Figure 1 and extending section about current advances in the field of genetics and genomics with regards to evolution and breeding of (blue)berries. I have noticed some very general parts of the text are still there, but they were improved. Still, I have to say that mainly the section "6. Genetic diversity" is about "everything and nothing" and it has very low informative value. Last thing, I attached the list of minor mistakes, mostly typos.
Line 461 and in an LB -> and in a LB
Line 480 EST-PCR and EST-PCR markers were used -> 2x EST-PCR
Line 505 to characterize a set of blueberry wild blueberry clones, cultivars, and hybrids -> to characterize a set of blueberry including wild blueberry clones, cultivars, and hybrids
Line 507 woked -> worked
Line 514 Rodríguez-Peña1 -> Rodríguez-Peña
Line 528 "...closeness with diploid V. tenellum These findings provided..." ->
"...closeness with diploid V. tenellum. These findings provided..." (missing dot)
Line 571 unify shortcut "G-SSR" vs "g-SSR"
Line 578 by [75] -> by Debnath and Sion [75]
Line 659 pocanalysis -> PCA analysis (I suppose)
Line 743 conversation -> conservation
Response: Thanks for your review and valuable suggestions. Section 6 has again been shortened and some basic ideas have been retained in the text that may be of useful to some readers. All minor corrections have also been taken into consideration and the manuscript has been revised following your suggestions (yellow highlighted).
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
The review article “Molecular Markers and Antioxidant Properties to Study Biodiversity in
Blueberries” by Debnath and Bhatt, who have presented a comprehensive insight to the role of
molecular markers and different antioxidants for biodiversity studies in Blueberries.
The manuscript is well organized and thoroughly described. The authors have covered almost
every aspect of the respective subject in order to achieve their developed hypothesis. To sum up,
this MS can find an interest for the specialists in this field. However, I have a few suggestions
that I hope the authors will find the following specific comments helpful in the revision.
The title is too comprehensive and I feel it should be a little more elaborative in terms of the
objectives of the MS.
Keywords must be different from title words.
All the sections are well explained and authors have almost covered all the aspects to support
their specific objectives of the study.
Groups of Blueberries: I think it could be more effective if the authors mention this information
in a tabular form. This can make the readers understand and compare the characteristics of
different groups of blueberries more easily.
Is there any phylogenetic relationship description of these groups of blueberries?
Figures: is there any latest data for the mentioned figures after 2019?
I can understand the authors have put a lot of efforts in this MS however I feel many sections of
the MS are too lengthy which is a good thing but on the same note it looks a bit exaggerated and
draws down the importance of the MS. You can be elaborative in a thesis but not for the
manuscript, for example heading 4 “Antioxidant Activity of Blueberry Phenolics” heading 6,
“molecular markers and genetic diversity” sections needs to be brief. Moreover, there are some
irrelevant citations as well in these sections.
In the conclusions section the authors should include and suggest the future prospects of the
proposed information and how researchers/farming community can benefit from it.
Best regards!
Reviewer 2 Report
I was reading this review with great interest. It is easy to read and give an extensive overview of both antioxidant properties of American blueberries and studies regarding biodiversity. I noticed latin words not in italic some places in the text.
Firstly: This manuscript is well written and the English is fine (taking into account that English is not my native language). It can be published, but need some revision.
I would like to correct the number of stars on novelty to 2-3 stars. The reason for this is that many of the techniques described for molecular markers and biodiversity studies are old-fashion. The table 1.1 page 12 is not interesting as it give an overview of the methods (this is common knowledge and should not be in focus in this review paper). I would like to see a table that give an overview of the different biodiversity studies and markers used for blueberries instead. Now this knowledge in hidden in the different chapters on molecular marker methods. It doesn't make sense to concentrate so much of the text describing the different molecular marker methods as such.
What is the rationale to bring in Vaccinium vitis idea, lingonberries, and not anything about Vaccinium myrtillus, the European wild blueberry?
I recommend to add work on Vaccinium myrtillus in the review as there are plenty of new publications on this species showing both the environmental effects on anthocyanin production and the difference between wild populations from different regions. It is a clear genotype X environmental effect. There are also publications on regulation mechanisms on the anthocyanins.
The influence of the environment on the polyphenol content is large. In that respect you can ask if the rationale to breed for higher content of healthy compounds is feasible. And what is the point as the consumer could have the same effect by eating more berries... ? The discussion and conclusion should bring this into account.
In chapter 7 " Relationship between Biochemical and Molecular Analysis" there is a need to discuss more about the effect of the environment on the biochemical composition. The text explain the discrepancy between antioxidant properties/content of phenolic compounds and the differences shown using molecular markers with flaws in the methods. This could of cause be the case, as this is very complex pathways controlled by a vast number of genes, but the environment will have a big impact. This should be discussed.
Sentence 138: theirbenefical - split into two words.
Finally, as I mentioned in my short report in the electronic form, the whole text could be shortened. Authors are advised to read through and avoid repeating information in different chapters.
Reviewer 3 Report
Review of the manuscript: Molecular markers and antioxidant properties to study biodiversity in blueberries
After reading the manuscript carefully I consider that needs significant changes to be able to contribute to the literature. In general, the manuscript tries to cover too much, and this results in areas being reviewed either being too shallow or outdated or containing misconceptions about vaccinium species. I hope these notes below can help to make a better manuscript:
- Tittle is deceiving as there is no way to figure out this is a review of the literature. An explicit title in this sense will be preferred
- The manuscript is mostly based in North America and thus does not include other Vacciniums species that are collected from the wild in Europe for example
- Vaccinium has between 150-450 species depending on the taxonomist you choose. And most of the species you list under blueberry in the introduction have their own common names and do not fall under the “blueberry” category
- Most V. angustifolium are not cultivars but patches of land that have been cleared and thus very few plantations are actually performed. So this species is mostly collected from wild patches.
- Use metric system consistently (ex: line 72)
- SHB is grown through all the south part of the states.
- More than 50% of HB are sold as fresh. So, Where is the reference for the 50% you use?
- The percentage of the global distribution of blueberry production has been very dynamic lately and thus you should use the IBO reports to report this. The numbers you mention in Fig 1.2 are not true anymore
- Part 4 is nicely written, and I see it as a contribution
- The genetic diversity is poorly reviewed, incomplete, and misleading. Probably thinking from a low bush perspective but lacks more info about HB and RE. The same is the case for estimation of the genetic diversity.
- The section about Mol Markers is elementary. There is no contribution to the literature from this area. Most of the text is part of most textbooks. Additionally, the literature is outdated and incomplete. There are significant newer contributions that are completely missed.
