Starch Bio-Synthetic Pathway Genes Contribute to Resistant Starch Content Differentiation in Bread Wheat
, Zhengqing Wu 1,2, Zhensheng Lei 1,2,3,4,* and Zhengfu Zhou 1,2,4,*
Round 1
Reviewer 1 Report
The manuscript investigates an interesting and important topic on the genetic control and sources of resistant starch, which has beneficial effects on human health.
1. Most noticeable drawback of the manuscript is a poor command of English. There are numerous grammar and spelling errors, and awkward sentences throughout the text. I strongly recommend it undergoes professional English editing.
Introduction
2. The introduction should include more information about the previous research on resistant starch (RS) in wheat. What types of RS are present in wheat flour? The authors listed the genes involved in starch biosynthesis in rice and Arabidopsis [cited literature 28-31], but it would be good to also include some previous findings on wheat genes controlling RS biosynthesis. The authors should elaborate more on how RS content in wheat is related to amylose content and the dysfunction of the amylopectin (lines 78-81).
3. The last paragraph in the introduction contains information on the materials and methods and the results. This should not be in the introduction. Instead, the end of the introduction ought to provide readers with the information on what it is that has not been investigated so far that this study aims to investigate/elucidate, i.e. the aim of the study.
Materials and methods
4. In the Materials and methods, it is stated that the population of 207 analysed accessions were collected from China and 14 other countries. What are these countries? Please, add an additional column in the Table S1 and specify the country for each accession.
It is confusing how this same population of 207 accessions, which is used before for dissecting quality and nutritional traits, in the cited papers [38-41] contains accessions that:
“were from China while the rest were originated from 7 other countries [in 38 and 39]“,
“from the Henan Province Crop Germplasm Bank and The International Maize and Wheat Improvement Center (CIMMYT)” [in 40],
„from the Yellow and Huai Valley and Southwestern Wheat Region of China, including Henan, Hebei, Shaanxi, Shanxi, Shandong, Jiangsu, Sichuan, and Yunnan provinces” [in 41].
Are the accessions from China only, or from China and 7 other countries, or from China and 14 other countries? Which ones?
5. In the lines 118-119, it is stated “The authors declare the total permissions to use the collections.” Is there any entity that holds rights to the collections? Who granted the permission to the authors? Could the authors, please, clarify their statement?
6. The author should explain what specific agronomic practices local management involves in the statement “The field nursery was according to the local management” (lines 123-124).
6. A big shortcoming of this paper is the low significance of results due to only one experimental year, despite three locations.
7. I suggest testing and presenting the results of Shapiro-Wilk test for normality distribution of RS values.
8. Please, provide a scientific reference that can support the formula from the RS assay kit.
Results
9. How many replications per accession per location were used for measuring RS? Please present ANOVA table with sources of variance, sum of squares, degrees of freedom, and p values in Table S3.
10. Discuss why all accessions (total average 5.13%) had much higher RS content compared to the usual RS content in wheat flour (as referred to in the introduction - usually 1-2%, even <1%).
11. The description of methodology in the results should be moved to the MM section (lines 216-219; lines 286-288). The method of gene expression should be described in mnore detail in MM. The results section should not contain citations.
Conclusion
The conclusion section is missing. It should be separated from the Discussion. The varieties with highest RS and superior alleles, such as variety Xi’nong979, should be highlighted here.
Author Response
The manuscript investigates an interesting and important topic on the genetic control and sources of resistant starch, which has beneficial effects on human health.
- Most noticeable drawback of the manuscript is a poor command of English. There are numerous grammar and spelling errors, and awkward sentences throughout the text. I strongly recommend it undergoes professional English editing.
Response: Thank you for the comments and suggestion for our manuscript. The manuscript has been edited by profession language editing service provided by MDPI.
Introduction
2.The introduction should include more information about the previous research on resistant starch (RS) in wheat. What types of RS are present in wheat flour? The authors listed the genes involved in starch biosynthesis in rice and Arabidopsis [cited literature 28-31], but it would be good to also include some previous findings on wheat genes controlling RS biosynthesis. The authors should elaborate more on how RS content in wheat is related to amylose content and the dysfunction of the amylopectin (lines 78-81).
Response: The previous research on RS was described in the introduction. The kit can measure the sum of all the types of RS, in the present study we used whole-meal flour for RS measurement, when referencing the classification of RS, type 2 was the main RS in wheat. Corresponding information was added in the revision. Also the genes controlling RS biosynthesis in wheat and how RS content in wheat is related to amylose content and the dysfunction of the amylopectin were mentioned in the new revision.
3.The last paragraph in the introduction contains information on the materials and methods and the results. This should not be in the introduction. Instead, the end of the introduction ought to provide readers with the information on what it is that has not been investigated so far that this study aims to investigate/elucidate, i.e. the aim of the study.
Response: Thank you very much for the suggestion. The last paragraph has been re-wrote. The objective of the study was illustrated instead of the materials and results.
Materials and methods
- In the Materials and methods, it is stated that the population of 207 analysed accessions were collected from China and 14 other countries. What are these countries? Please, add an additional column in the Table S1 and specify the country for each accession.
It is confusing how this same population of 207 accessions, which is used before for dissecting quality and nutritional traits, in the cited papers [38-41] contains accessions that:
“were from China while the rest were originated from 7 other countries [in 38 and 39]“,
“from the Henan Province Crop Germplasm Bank and The International Maize and Wheat Improvement Center (CIMMYT)” [in 40],
„from the Yellow and Huai Valley and Southwestern Wheat Region of China, including Henan, Hebei, Shaanxi, Shanxi, Shandong, Jiangsu, Sichuan, and Yunnan provinces” [in 41].
Are the accessions from China only, or from China and 7 other countries, or from China and 14 other countries? Which ones?
Response: Your feedback was appreciated. The varieties collected from the main planting region of China and other 7 countries. The corresponding description was revised in the new revision. Additionally, following your suggestion, the information has been provided in Table S1.
- In the lines 118-119, it is stated “The authors declare the total permissions to use the collections.” Is there any entity that holds rights to the collections? Who granted the permission to the authors? Could the authors, please, clarify their statement?
Response: That is a nice suggestion. Actually, the Chinese varieties were collected and provided by Henan Province Crop Germplasm Bank which is constructed and managed by Henan Academy of Agricultural Sciences (HAAS) (affiliation of most authors of the manuscript), and the varieties from other countries were provided by International Maize and Wheat Improvement Center (CIMMYT) through a project of a Joint Laboratory of HAAS and CIMMYT. Therefore, our institute and the researches has the total permission to used the collection for scientific research.
- The author should explain what specific agronomic practices local management involves in the statement “The field nursery was according to the local management” (lines 123-124).
Response: The management has been described in plant materials section in the revision to make the method much more clarified.
- A big shortcoming of this paper is the low significance of results due to only one experimental year, despite three locations.
Response: Thank you very much for the concern. The journal Agronomy does not have clarified requirement of replicate for field experiment, as most scientific journals asked for at least three replicates. Although our present study has only one experimental year, the plant materials were planted in three locations. As mentioned in discussion, three locations spanning E113°97′ to E115°65′ in longitude and N34°45 to ′N35°5′ in latitude. The environment are divergent especially during the crucial developmental stage for starch accumulation. The common genetic loci detected under different locations can represent stable genetic loci for RS control.
- I suggest testing and presenting the results of Shapiro-Wilk test for normality distribution of RS values.
Response: Thank you for the comments. The the results of Shapiro-Wilk test was added in Table S2.
- 9. Please, provide a scientific reference that can support the formula from the RS assay kit.
Response: The link of the website for the RS assay kit was provided, at the same time, as you suggested the reference was added.
Results
- How many replications per accession per location were used for measuring RS? Please present ANOVA table with sources of variance, sum of squares, degrees of freedom, and p values in Table S3.
Response: Each variety was planted 42 individuals within one plot. The grains from each plot were harvest and milled mixed together. For measuring the RS content, each sample was measured with 3 replicates. The sources of variance, sum of squares, degrees of freedom, and p values of ANOVA was presented in Table S3.
- Discuss why all accessions (total average 5.13%) had much higher RS content compared to the usual RS content in wheat flour (as referred to in the introduction - usually 1-2%, even <1%).
Response: The RS content of fine milled flour in wheat varieties are usually 1-2%. There was also studies reported RS comprised on average 6.6% of ground grain and 3.9% of wheat flour. The RS content of varieties in the present study are 3% - 5% with whole-meal flour, consistent with previous study. The corresponding details and reference were added in discussion.
- 12. The description of methodology in the results should be moved to the MM section (lines 216-219; lines 286-288). The method of gene expression should be described in mnore detail in MM. The results section should not contain citations.
Response: Thank you for pointing out this. The methodological description in the result part was moved to the materials and method part. Moreover, we provided more detail for gene expression and candidate gene prediction in materials and methods section.
Conclusion
13.The conclusion section is missing. It should be separated from the Discussion. The varieties with highest RS and superior alleles, such as variety Xi’nong979, should be highlighted here.
Response: The conclusion section has been added following the discussion. As you suggested, the variety Xi’nong979 which contained the most superior RS alleles was highlighted in conclusion. The variety would be a elite material for high RS wheat breeding.
Reviewer 2 Report
The manuscript provides a fairly robust dataset on exploring genetic resources for highly resistant starch in bread wheat. A large number of wheat accessions from different countries were phenotyped in three locations as well as genotyped using 660K SNPs and KASP. I suggest accepting the manuscript following minor revision.
Specific comments
The manuscript needs major English editing.
The introduction needs to be improved and the importance of exploring the genetic resources for highly resistant starch in wheat should be clarified. The hypothesis, rationale, and objectives should be improved. The presented findings in lines 95-107 should be moved to the discussion section.
The used plant materials are diverse accessions collected from different countries. The authors used different terms such as varieties, genotypes, and accessions throughout the manuscript. There is a difference between the three terms, thus the authors should use just the appropriate term for their study.
The scientific name of bread wheat should be presented in the section of plant material.
Figure 2 and Table S4 have not been cited in the text.
Cluster analysis could be applied to classify the assessed genotypes based on their resistant starch content.
The obtained findings should be discussed better and associated with the previously published results.
A conclusion should be added
The journals are abbreviated in some references while not in others. The reference style should be revised and the same style should be followed in all references.
The scientific names should be in italic throughout the manuscript (as in lines 478, 511)
Author Response
Comments and Suggestions for Authors：
The manuscript provides a fairly robust dataset on exploring genetic resources for highly resistant starch in bread wheat. A large number of wheat accessions from different countries were phenotyped in three locations as well as genotyped using 660K SNPs and KASP. I suggest accepting the manuscript following minor revision.
Specific comments
The manuscript needs major English editing.
Response: The manuscript has been edited by profession language editing service provided by MDPI.
- The introduction needs to be improved and the importance of exploring the genetic resources for highly resistant starch in wheat should be clarified. The hypothesis, rationale, and objectives should be improved. The presented findings in lines 95-107 should be moved to the discussion section.
Response: Thank you very much for your comments. The introduction section has been re-wrote. Especially, we added a new paragraph to describe of the rationale and objectives of the present study.
- The used plant materials are diverse accessions collected from different countries. The authors used different terms such as varieties, genotypes, and accessions throughout the manuscript. There is a difference between the three terms, thus the authors should use just the appropriate term for their study.
Response: Thank you very much for pointing this negligence. All the materials used in the present study are varieties. The term has been revised and keep consistence throughout the manuscript.
- The scientific name of bread wheat should be presented in the section of plant material.
Response: The scientific name of the bread wheat was added in the first sentence of the materials and methods section.
- Figure 2 and Table S4 have not been cited in the text.
Response: Figure 2 and Table S4 were cited appropriately in the main text in the new revision.
- Cluster analysis could be applied to classify the assessed genotypes based on their resistant starch content.
Response: Thank you very much for the suggestion. The cluster analysis is a routine process for GWAS and is vital to detect the genetic loci correctly. The cluster analysis was published by our previous publication. The reference ‘Liu, C.; Zhou, Z.; Wu, Z.; Qin, M.; Shi, X.; Wang, Y.; Li, W.; Yao, W.; Lei, Z.; Zhao, R. In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level. Genomics 2020, 112, 4690-4700, doi:10.1016/j.ygeno.2020.08.019.’ Therefore, the cluster analysis was not present in the RS manuscript.
- The obtained findings should be discussed better and associated with the previously published results.
Response: Most of the starch biosynthesis genes revealed by studies in the model plants, Arabidopsis and rice. There are few studies reported genetic locus for RS content. As you suggested, we compared previous studies with our present result in the discussion.
- A conclusion should be added
Response: The conclusion section was added.
8.The journals are abbreviated in some references while not in others. The reference style should be revised and the same style should be followed in all references.
Response: The format of references was checked and revised according to the guide from the journal’s submission web.
9.The scientific names should be in italic throughout the manuscript (as in lines 478, 511)
Response: The scientific names in lines 478 and 511 were in italic, and we have check and revised similar mistake throughout the manuscript.
Reviewer 3 Report
It is an interesting and well planned and nicely written research article which addresses up to date topic.
In general English is fine, but in some parts plural or signal form are use not incorrectly.
Author Response
Comments and Suggestions for Authors:
It is an interesting and well planned and nicely written research article which addresses up to date topic.
In general English is fine, but in some parts plural or signal form are use not incorrectly.
Response: We are appreciate your comments on our manuscript. The manuscript has been edited by a professional language editing service.
Round 2
Reviewer 1 Report
The authors addressed all the comments and considerably improved the manuscript.
