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Review
Peer-Review Record

Quantitative Epigenetics: A New Avenue for Crop Improvement

Reviewer 1: Anonymous
Reviewer 2: Anonymous
Received: 14 September 2020 / Revised: 24 October 2020 / Accepted: 4 November 2020 / Published: 7 November 2020
(This article belongs to the Special Issue Advances in Plant Epigenetics and Epigenomics)

Round 1

Reviewer 1 Report

In this study, Gahlaut and colleauges provide an overview of how our developing understanding of genome-wide epigenetic marks in model plants can be applied for agricultural biotechnology. This is an important emerging area of general interest to scientists working in the field and the Figures provide a useful summary.

I particularly like the Tables, which summarise many studies of possible relevance and highlight the phenotypes that can be affected. These will be a useful resource for pre-breeding research.

Generally the scope and writing is accurate and interesting although the authors must take care to distinguish between what has already been proven to be possible, and things which are potentially very interesting but still hypothetical. I have made some suggestions of where these changes should be made but the Editors may want to suggest others.

One other important thing needs to be added is a summary of legislative issues - CRISPr-Cas9 modificaitons at the DNA level cause crops to be regulated as GMOs but would use of epi-alleles get around this? I recommend to describe the specific routes to using this variation in Section 9, the Conclusions.

Some specific comments:

Abstract: I think that EWAS and epi-GBS should both briefly be mentioned in the Abstract as these are the most important ways of identifying which epigenetic mechanisms are causal for phenotypic variation.

General: use the name 'Arabidopsis' consistently - sometimes it is given as a taxonomic name, sometimes a common one; I suggest to give its full name (Arabidopsis thaliana (L.) Heynh.) at first mention and 'Arabidopsis' after that.

line 15: remove word 'the'

line 21: change this sentence to say 'can potentially facilitate' as this is unproven.

line 47 and throughout: I think now 'next-generation sequencing (NGS)' is an obsolete term and the techniques should just be called RNA-seq, bisulfite-seq etc.

lines 51 and 53: remove word 'the' from before WGBS

line 56: should say 'has been observed'.

line 61: when discussing the variation in DNA methylation levels, explicit mention should be made of its roles in controlling TEs - the way the paper is written implies that mC is mostly about controlling gene expression which is not so. Table 1 should be adapted to give the approximate proportion of each genome that consists of repetitive elements as this is important context for understanding the variation reported.

Line 71: better definitions are needed here. It is important to emphasise that most chromatin configurations and DNA methylation patterns vary with the environment and are reset during reproduction, with stable inheritance of epi-alleles being rare.

The authors could cite papers such as:

Wang, G., & Köhler, C. (2017). Epigenetic processes in flowering plant reproduction. Journal of experimental botany, 68(4), 797-807.   Chow, H. T., Chakraborty, T., & Mosher, R. A. (2020). RNA-directed DNA methylation and sexual reproduction: Expanding beyond the seed. Current opinion in plant biology, 54, 11-17.

Similarly, at line 80 the text should read 'some epiQTLs are stably inherited'. Although 'up to 90%' may be true, many are much less stable than this. Ensuring that there is stable inheritance will be vital for using epigenetic variation commercially.

The issue of missing heritably also needs to be treated with caution - the authors address this by citing quite an old paper, reference [27], but not all researchers agree with this interpretation. Conventional QTLs of polygenic nature could be the main players. This section should be expanded and made more cautious.

Table 2: this would be more useful if the phenotypes were briefly described e.g. instead of 'starch metabolism' describe what actually happens

Section 3. This is moslty fine, but epiRILs have paticular applications in novel mechanisms for heterosis which - a paragraph describing this with papers making specific suggestions supporting this e.g.

Chen, Z. J. (2013). Genomic and epigenetic insights into the molecular bases of heterosis. Nature Reviews Genetics, 14(7), 471-482.  

McKeown, P. C., Fort, A., Duszynska, D., Sulpice, R., & Spillane, C. (2013). Emerging molecular mechanisms for biotechnological harnessing of heterosis in crops. Trends in biotechnology, 31(10), 549-551.  

Lauss, K., Wardenaar, R., Oka, R., van Hulten, M. H., Guryev, V., Keurentjes, J. J., ... & Johannes, F. (2018). Parental DNA methylation states are associated with heterosis in epigenetic hybrids. Plant Physiology, 176(2), 1627-1645.  

Line 142: 'have' not 'has'  

Line 143: again, very important to say that epigenetic modifications can be heritable, not are - the great majority simply are not.  

Line 145: 'at least two different epiRIL populations' - I know of others which are not yet published; important also to clarify that these are populations, not characterised lines.  

Line 151: 'the' DDM locus; which 'encodes...' (not 'harbors...')        

Lines 173-4: citation [66] (Li et al.) is indeed an important paper in the field. Its key conclusions should be summarised at this point.

Line 209: supply of 'methyl groups'.

Section 8: this section needs to briefly mention epi-GBS as means of discovering valuable epigenetic variation where the genome sequence tools needed for EWAS may not be available. See:

van Gurp, T. P., Wagemaker, N. C., Wouters, B., Vergeer, P., Ouborg, J. N., & Verhoeven, K. J. (2016). epiGBS: reference-free reduced representation bisulfite sequencing. Nature Methods, 13(4), 322-324.  

Werner, O., Prudencio, Á. S., de la Cruz-Martínez, E., Nieto-Lugilde, M., Martínez-Gómez, P., & Ros, R. M. (2020). A cost reduced variant of epi-genotyping by sequencing for studying DNA methylation in non-model organisms. Frontiers in plant science, 11, 694.  

The authors could also cite the following as an example of use in non-model crops:  

Prudencio, Á. S., Werner, O., Martínez-García, P. J., Dicenta, F., Ros, R. M., & Martínez-Gómez, P. (2018). DNA methylation analysis of dormancy release in almond (Prunus dulcis) flower buds using epi-genotyping by sequencing. International journal of molecular sciences, 19(11), 3542.  

Lines 211-213: is this gap supposed to be here?  

Line 237: what does 'deprived' mean here? I don't think this is the right word. 

Line 240: 'published' instead of 'done'  

Line 258: suggest 'the most suitable material' instead of 'commendable'

Line 330: FWA in italics

Line 333: this sentence is unclear, please rewrite.

 

Author Response

Reviewer 1

Comments and Suggestions for Authors

In this study, Gahlaut and colleauges provide an overview of how our developing understanding of genome-wide epigenetic marks in model plants can be applied for agricultural biotechnology. This is an important emerging area of general interest to scientists working in the field and the Figures provide a useful summary.

I particularly like the Tables, which summarise many studies of possible relevance and highlight the phenotypes that can be affected. These will be a useful resource for pre-breeding research.

Generally, the scope and writing is accurate and interesting although the authors must take care to distinguish between what has already been proven to be possible, and things which are potentially very interesting but still hypothetical. I have made some suggestions of where these changes should be made but the Editors may want to suggest others.

One other important thing needs to be added is a summary of legislative issues - CRISPr-Cas9 modifications at the DNA level cause crops to be regulated as GMOs but would use of epi-alleles get around this? I recommend to describe the specific routes to using this variation in Section 9, the Conclusions.

Responses: Thank you for your suggestion. Accordingly, we have inserted a paragraph describing the legislative issues on CRISPR-Cas9 modifications in section 8. Please see Line No. 934-941

Some specific comments:

Abstract: I think that EWAS and epi-GBS should both briefly be mentioned in the Abstract as these are the most important ways of identifying which epigenetic mechanisms are causal for phenotypic variation.

Response: We agree with the reviewer's point, and in the revised manuscript, EWAS and epi-GBS have been briefly described in the Abstract. Please see Line No. 18-19

General: use the name 'Arabidopsis' consistently - sometimes it is given as a taxonomic name, sometimes a common one; I suggest to give its full name (Arabidopsis thaliana (L.) Heynh.) at first mention and 'Arabidopsis' after that.

Response: We corrected this error throughout the manuscript. For example, changes have been made in Line 46, 51,53, 56, 66, 149

line 15: remove word 'the'

Response: We removed word “the”.

line 21: change this sentence to say 'can potentially facilitate' as this is unproven.

Response: We changed the sentence as suggested by the reviewer in the revised manuscript. Please see Line No. 24

line 47 and throughout: I think now 'next-generation sequencing (NGS)' is an obsolete term and the techniques should just be called RNA-seq, bisulfite-seq etc.

Response: We corrected this and revised the manuscript as per reviewers suggestions. For example, changes have been made in Line 55-56

lines 51 and 53: remove word 'the' from before WGBS

Response: The word “the” has been removed at both places.

line 56: should say 'has been observed'.

Response: We corrected this error in the revised manuscript.

line 61: when discussing the variation in DNA methylation levels, explicit mention should be made of its roles in controlling TEs - the way the paper is written implies that mC is mostly about controlling gene expression which is not so. Table 1 should be adapted to give the approximate proportion of each genome that consists of repetitive elements as this is important context for understanding the variation reported.

Response: We agree with the reviewer's comments, and in the revised manuscript we now mention the role of DNA methylation in controlling TEs. Please see Line No. 76-78. We also added data of repetitive elements of each species in Table 1.

Line 71: better definitions are needed here. It is important to emphasise that most chromatin configurations and DNA methylation patterns vary with the environment and are reset during reproduction, with stable inheritance of epi-alleles being rare.

The authors could cite papers such as:

Wang, G., & Köhler, C. (2017). Epigenetic processes in flowering plant reproduction. Journal of experimental botany68(4), 797-807.   Chow, H. T., Chakraborty, T., & Mosher, R. A. (2020). RNA-directed DNA methylation and sexual reproduction: Expanding beyond the seed. Current opinion in plant biology54, 11-17.

Response: We revised the epi-allele definition as suggested by the reviewer and cited the suggested citations of Wang and Kohler (2017) and Chow et al. (2020). Please see Line No. 79-82.

Similarly, at line 80 the text should read 'some epiQTLs are stably inherited'. Although 'up to 90%' may be true, many are much less stable than this. Ensuring that there is stable inheritance will be vital for using epigenetic variation commercially.

Response: We rephrased this statement in the revised manuscript. Please see Line No. 92

The issue of missing heritably also needs to be treated with caution - the authors address this by citing quite an old paper, reference [27]. Still, not all researchers agree with this interpretation. Conventional QTLs of polygenic nature could be the main players. This section should be expanded and made more cautious.

Response: We agree with the points made by the Reviewer’s and modified the text. Please see Line No. 104

Table 2: this would be more useful if the phenotypes were briefly described e.g. instead of 'starch metabolism' describe what actually happens

Response: Thanks for the suggestion, we briefly described the epiallele related phenotypes in Table 2.

Section 3. This is mostly fine, but epiRILs have paticular applications in novel mechanisms for heterosis which - a paragraph describing this with papers making specific suggestions supporting this e.g.

Chen, Z. J. (2013). Genomic and epigenetic insights into the molecular bases of heterosis. Nature Reviews Genetics14(7), 471-482.  

McKeown, P. C., Fort, A., Duszynska, D., Sulpice, R., & Spillane, C. (2013). Emerging molecular mechanisms for biotechnological harnessing of heterosis in crops. Trends in biotechnology31(10), 549-551.  

Lauss, K., Wardenaar, R., Oka, R., van Hulten, M. H., Guryev, V., Keurentjes, J. J., ... & Johannes, F. (2018). Parental DNA methylation states are associated with heterosis in epigenetic hybrids. Plant Physiology176(2), 1627-1645.  

Response: We added a new section “3.3.Epigenetics andbasis of Heterosis” as suggested by the reviewer and also added the above citations in the revised manuscript. Please see Line No.213-234

Line 142: 'have' not 'has'  

Response: We have revised it.

Line 143: again, very important to say that epigenetic modifications can be heritable, not are - the great majority simply are not.  

Response: We corrected it in the revised manuscript. Please see Line No. 156

Line 145: 'at least two different epiRIL populations' - I know of others which are not yet published; important also to clarify that these are populations, not characterised lines.  

Response: We added the “populations” after epi-RIL in the revised manuscript. Please see Line No. 158

Line 151: 'the' DDM locus; which 'encodes...' (not 'harbors...')

Response: We corrected this error in the revised manuscript. Please see Line No. 164

Lines 173-4: citation [66] (Li et al.) is indeed an important paper in the field. Its key conclusions should be summarised at this point.

Response: We took reviewer’s suggestions into consideration and highlighted the key conclusions of the suggested paper in the revised manuscript. Please see Line No. 188-190

Line 209: supply of 'methyl groups'. 

Response: We corrected it in the revised manuscript. Please see Line No. 248

Section 8: this section needs to briefly mention epi-GBS as means of discovering valuable epigenetic variation where the genome sequence tools needed for EWAS may not be available. See:

van Gurp, T. P., Wagemaker, N. C., Wouters, B., Vergeer, P., Ouborg, J. N., & Verhoeven, K. J. (2016). epiGBS: reference-free reduced representation bisulfite sequencing. Nature Methods13(4), 322-324.  

Werner, O., Prudencio, Á. S., de la Cruz-Martínez, E., Nieto-Lugilde, M., Martínez-Gómez, P., & Ros, R. M. (2020). A cost reduced variant of epi-genotyping by sequencing for studying DNA methylation in non-model organisms. Frontiers in plant science11, 694.  

The authors could also cite the following as an example of use in non-model crops:  

Prudencio, Á. S., Werner, O., Martínez-García, P. J., Dicenta, F., Ros, R. M., & Martínez-Gómez, P. (2018). DNA methylation analysis of dormancy release in almond (Prunus dulcis) flower buds using epi-genotyping by sequencing. International journal of molecular sciences19(11), 3542.  

Response: We added a paragraph in Section 7, which briefly describes epi-GBS and its utilization in plants. Please see Line No. 355-371

Lines 211-213: is this gap supposed to be here?  

Response: We deleted the gap/space in the revised manuscript.

Line 237: what does 'deprived' mean here? I don't think this is the right word. 

Response: We replaced “deprived” with “less”. Please see Line No. 276

Line 240: 'published' instead of 'done'  

Response: We corrected it in the revised manuscript. Please see Line No. 279

Line 258: suggest 'the most suitable material' instead of 'commendable'

Response: We corrected it in the revised manuscript. Please see Line No. 297

Line 330: FWA in italics

Response: We corrected it in the revised manuscript. Please see Line No. 390

Line 333: this sentence is unclear, please rewrite.

Response: We rewrote the sentence to make it readable. Please see Line No. 390-391

Reviewer 2 Report

In this manuscript, Gahlaut et al., review the application of epigenetics for crop improvement. They introduced the current status of epigenetic studies and suggested future prospect in crop sciences. Overall, this manuscript well summarized the published data on epigenetics in crop plants. The followings are my comments:

  1. Overall, this manuscript simply describes the published results. It will be better to summarize the published results and provide the conclusive sentences in the last paragraphs of each sections.
  2. Focus on the DNA methylation: Although the authors introduced that epigenetic modifications involve DNA methylation, histone modification, and small RNAs, the majority of this review focuses on the DNA methylation. Please indicate the limit of this review and change the title of this manuscript.
  3. Table 1: The authors summarized the DNA methylation levels in plant species in the table 1. It is not clear whether methylation percentage is proportion of total methylation or per-site methylation. For instance, the sum of the percentages of three methylations in Arabidopsis is 100% while the others are not. Please provide the legend how the methylation percentages were obtained.
  4. Minors: 1) The position of several abbreviation is inappropriate such as line 217. 2) Remove the wavy lines in figure 1. 3) Please provide the reference of figure 2 if the statistical analysis was obtained from other papers. 4) Please check the typos, especially the references such as line 375 (Genome boil à Genome biology)

Author Response

Thank you for giving us the opportunity to submit a revised draft of the manuscript. We appreciate the time and effort that the reviewer dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated most of the suggestions made by the reviewer. Those changes are highlighted within the manuscript (track changes).

Comments and Suggestions for Authors

In this manuscript, Gahlaut et al., review the application of epigenetics for crop improvement. They introduced the current status of epigenetic studies and suggested future prospect in crop sciences. Overall, this manuscript well summarized the published data on epigenetics in crop plants. The followings are my comments:

  1. Overall, this manuscript simply describes the published results. It will be better to summarize the published results and provide the conclusive sentences in the last paragraphs of each sections.

Response: We have revised the whole manuscript as per the reviewer's comment. Also, we now clearly wrote conclusive sentences in the last paragraphs of each section, where-ever we don't give it earlier. Please see Line No. 231-234, 263-264, 288-292, 408-411.

2. Focus on the DNA methylation: Although the authors introduced that epigenetic modifications involve DNA methylation, histone modification, and small RNAs, the majority of this review focuses on the DNA methylation. Please indicate the limit of this review and change the title of this manuscript.

Response: We agree with the reviewer's comments that we discussed various aspects of epigenetics in the Introduction to set the background, and then primarily focus on DNA methylation as literature exists in this aspect only (for Quatitative epigenetics). We wish to keep the title more general, allowing researchers to explore this area of epigenetics further.

Table 1: The authors summarized the DNA methylation levels in plant species in the table 1. It is not clear whether methylation percentage is proportion of total methylation or per-site methylation. For instance, the sum of the percentages of three methylations in Arabidopsis is 100% while the others are not. Please provide the legend how the methylation percentages were obtained.

Response: We thank Reviewer for raising this important point. Previously, in Table 1, the methylation percentage was presented as a proportion of per-site methylation for all species except Arabidopsis. The Table 1 has been revised and presented data as per-site methylation for all species. Table 1. Legend has also been revised accordingly.

Minors: 1) The position of several abbreviation is inappropriate such as line 217. 2) Remove the wavy lines in figure 1. 3) Please provide the reference of figure 2 if the statistical analysis was obtained from other papers. 4) Please check the typos, especially the references such as line 375 (Genome boil à Genome biology)

Responses: 1. As suggested by the reviewer, we have corrected the abbreviation thought-out the manuscript. For example, please see Line No. 240, Line No. 255 and Line No. 256, 332 etc.

  1. The wavy line has been removed from Figure 1.
  2. In Figure 2, we used arbitrary data to demonstrate the association between epialleles and trait, hence no reference is required.
  3. The formatting errors of references has also been take care.

Round 2

Reviewer 2 Report

In this revised manuscript, the authors tried to do their best to address my comments. 

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