Epigenetic and Genetic Contribution for Expression Bias of Homologous Alleles in Polyploid Sugarcane

Round 1

Reviewer 1 Report

1.      Author should mention that, what is expression of methylated gene (weather it is increased or decreased) through real-time PCR?

2.      What is the effect of methylation on sugar content (increased or decreased) of sugarcane.

Author Response

Response to Reviewer 1 Comments

Point 1: Author should mention that, what is expression of methylated gene (weather it is increased or decreased) through real-time PCR?

Response 1: This is a good suggestion, we have performed RT-PCR from randomly selected four DNA methylase genes, and we could observe that Sspon.02G0023350-1P (MET) and Sspon.01G0015710-2B (CMT3) were most expressed at root tissues compared with leaf and rind, but Sspon.08G0012870-2B (DDM) and Sspon.01G0000660-1A (DRM2) were expressed at the lowest levels of root tissues (Supplementary Figure 1A).

Point 2: What is the effect of methylation on sugar content (increased or decreased) of sugarcane.

Response 2: This is a good question, but we can’t simply correlate the amount of sugar with increased or decreased methylation. As we know, high sucrose accumulation is a characteristic feature of sugarcane. Sugarcane has a universal source-sink system, sucrose in leaves is mainly derived from the SPS-mediated sucrose synthesis pathway. Except for consumption during leaf growth, the sucrose synthesized in leaves is exported to sink tissues and used for consumption and storage. Our previous research found that the methylation level increased gradually from leaves to roots, and stem (rind and pith) [1]. In addition, we found that genes involved in sucrose synthesis and accumulation pathways are regulated by DNA methylation [1]. For example, the genes involved in the sucrose synthesis pathway, including FBPase, PGI, SPS, and SPP, were highly expressed in the leaves; SWEETs involved in sucrose transport were highly expressed in the leaves and stems; invertases (INVs) involved in sucrose hydrolysis had a lower expression in stem tissue (rind and pith) than that in leaf and root tissue. We suggest that sucrose synthesis and accumulation is a complex network, and hypomethylation in leaves is very important for sucrose synthesis in sugarcane. Taken together, DNA methylation plays an important role in sucrose accumulation in sugarcane.

1. Xue, Y.; Zou, C.; Zhang, C.; Yu, H.; Chen, B.; Wang, H. Dynamic DNA methylation changes reveal tissue-specific gene expression in sugarcane. Front Plant Sci 2022, 13, 1036764. https://doi.org/10.3389/fpls.2022.1036764.

Reviewer 2 Report

It has been understood that DNA methylation regulates gene expression in eukaryotes. However, the role of methylation in gene expression changes in polyploids is poorly understood. In this manuscript, authors have comparatively analyzed DNA methylation profiles and transcriptome maps in four different tissues (leaf, rind, pith and root) in autopolyploid sugarcane. Higher DNA methylation levels were found in non-balanced alleles compared to balanced alleles, as well as higher CG to TG substitution rates, suggesting epigenetic and genetic variation associated with transcription divergence.

I offer following comments for further improvement of the manuscript,

1.       Authors need to explain in the introduction section what made them to think that methylation occurs in polyploid genome like Sugarcane leading to gene expression bias.

2.       Authors are supposed to base their results on Methylation ratio to show the very occurrence of methylation in sugarcane.

3.       There is a need for some more data to show differentially expressed homologous alleles have strong association with genetic substitution and DNA methylation (Sub section 3.3)

4.       There is not much to show in the manuscript that non-balanced alleles, which were associated with DNA methylation changes, were involved in processes relevant to agronomic traits (Sub section 3.4)

5.       Authors may please be advised to improve the language and spelling mistakes at many places in the manuscript (For instance pages 63-64, 131, 144-145, 149, 227, 253).

 This manuscript may be accepted for publication after revision.

Author Response

Response to Reviewer 2 Comments

It has been understood that DNA methylation regulates gene expression in eukaryotes. However, the role of methylation in gene expression changes in polyploids is poorly understood. In this manuscript, authors have comparatively analyzed DNA methylation profiles and transcriptome maps in four different tissues (leaf, rind, pith and root) in autopolyploid sugarcane. Higher DNA methylation levels were found in non-balanced alleles compared to balanced alleles, as well as higher CG to TG substitution rates, suggesting epigenetic and genetic variation associated with transcription divergence.

I offer following comments for further improvement of the manuscript,

We thank the reviewer for this positive suggestions and comments. We have addressed all the concerns as below:

Point 1: Authors need to explain in the introduction section what made them to think that methylation occurs in polyploid genome like Sugarcane leading to gene expression bias.

Response 1: This is a good point. We have added several sentences to explain this accordingly.

Point 2: Authors are supposed to base their results on Methylation ratio to show the very occurrence of methylation in sugarcane.

Response 2: Thank you for your suggestion. We have performed systematic analysis of sugarcane methylation in our previous manuscript [1], such as methylation ratio, methylation landscape, and distribution.

Point 3: There is a need for some more data to show differentially expressed homologous alleles have strong association with genetic substitution and DNA methylation (Sub section 3.3)

Response 3: Thank you for your suggestion. We have calculated the relationship between genetic variation and changes in DNA methylation in differentially expressed homologous alleles. We found that genetic variation in homologous alleles was positive with DNA methylation changes (Figure 4I). We have added this analysis in this part.

Point 4: There is not much to show in the manuscript that non-balanced alleles, which were associated with DNA methylation changes, were involved in processes relevant to agronomic traits (Sub section 3.4)

Response 4: Sorry for this misunderstanding caused to the reviewer. We have correlated DNA methylation changes with several agronomic traits, such as dominant and suppressed alleles, which were represent as non-balanced alleles in this part.

Point 5: Authors may please be advised to improve the language and spelling mistakes at many places in the manuscript (For instance pages 63-64, 131, 144-145, 149, 227, 253).

Response 5: Thank you for your suggestion. We have corrected them accordingly.

1. Xue, Y.; Zou, C.; Zhang, C.; Yu, H.; Chen, B.; Wang, H. Dynamic DNA methylation changes reveal tissue-specific gene expression in sugarcane. Front Plant Sci 2022, 13, 1036764. https://doi.org/10.3389/fpls.2022.1036764.

Reviewer 3 Report

In the manuscript titled “Epigenetic and genetic contribution for expression bias of homologous alleles in polyploid sugarcane” Shi et al suggested that transcriptional regulation of homologous alleles by epigenetic contribute to key agronomic traits in sugarcane.

In general, the paper is well written and the results are clearly present.  However, in my opinion, the methods section has to be improved by explain with more detailed all the methods used.  Also, some spelling problems have been detected in the different sections.

Author Response

Response to Reviewer 3 Comments

In the manuscript titled “Epigenetic and genetic contribution for expression bias of homologous alleles in polyploid sugarcane” Shi et al suggested that transcriptional regulation of homologous alleles by epigenetic contribute to key agronomic traits in sugarcane. 

In general, the paper is well written and the results are clearly present.  However, in my opinion, the methods section has to be improved by explain with more detailed all the methods used.  Also, some spelling problems have been detected in the different sections.

Thank you for these positive comments. We have added more details in method part and revised some spelling problems accordingly.

Reviewer 4 Report

• The study has good and in-depth coverage. However I  suggest  th following corrections to authors to improve the MS 
•  
• 1. Add latest reference related to study like meena et al., 2022 DOI: 10.3389/fgene.2022.854936 in line no 36 or some where.

2. In Line 39, after behind "than the" is missing

3. In lIne No 40-50 , author should reframe the sentence for proper understadning of the test

4. In Line No  77 author mentioned only leaf tissue However he used four different type tissues in the study

5. line no 88, how low quality reads are filtered , tool used should be mentioned?

6. In Line 115 reference of paper published using Omics share tools should be given

7. Author should justify why gene content bias was not significant in line no 128-129

8. It would be better if lab validation (RT-PCR) of work need to be presented thorugh gel picture at specific places

Author Response

Response to Reviewer 4 Comments

The study has good and in-depth coverage. However I suggest  the following corrections to authors to improve the MS  

We thank the reviewer for this positive suggestions and comments.

Point 1: Add latest reference related to study like meena et al., 2022 DOI: 10.3389/fgene.2022.854936 in line no 36 or some where.

Response 1: This is a good suggestion. We have added this reference in line no 37.

Point 2: In Line 39, after behind "than the" is missing

Response 2: Corrected.

Point 3: In line No 40-50 , author should reframe the sentence for proper understanding of the test

Response 3: Thank you for your suggestion. We have reframed this part.

Point 4: In Line No  77 author mentioned only leaf tissue However he used four different type tissues in the study

Response 4: Thank you for this point. We have added the other three tissues (root, rind, and pith) in line no 81.

Point 5: line no 88, how low quality reads are filtered , tool used should be mentioned?

Response 5: Thank you for this suggestion. We filtered low-quality reads by using FASTP with default parameters.

Point 6: In Line 115 reference of paper published using Omics share tools should be given

Response 6: Omicshare tool is an online analysis tool that has no published paper. We have added the URL in this part when using it in our research.

Point 7: Author should justify why gene content bias was not significant in line no 128-129

Response 7: Sorry to bring this misunderstanding. We did not find genome-wide gene content, structure and composition between different sub-genomes by checking the annotation and assembly from published sugarcane genome paper [1].

Point 8: It would be better if lab validation (RT-PCR) of work need to be presented thorugh gel picture at specific places

Response 8: This is a good suggestion, we have performed RT-PCR of several randomly selected DNA methyltransferase genes, and found that most of them were expressed at the same trend among three tissues, and this phenomenon is correlated between RT-PCR and RNA-seq data.

1. Zhang, J.; Zhang, X.; Tang, H.; Zhang, Q.; Hua, X.; Ma, X.; Zhu, F.; Jones, T.; Zhu, X.; Bowers, J.; Wai, C.M.; Zheng, C.; Shi, Y.; Chen, S.; Xu, X.; Yue, J.; Nelson, D.R.; Huang, L.; Li, Z.; Xu, H.; Zhou, D.; Wang, Y.; Hu, W.; Lin, J.; Deng, Y.; Pandey, N.; Mancini, M.; Zerpa, D.; Nguyen, J.K.; Wang, L.; Yu, L.; Xin, Y.; Ge, L.; Arro, J.; Han, J.O.; Chakrabarty, S.; Pushko, M.; Zhang, W.; Ma, Y.; Ma, P.; Lv, M.; Chen, F.; Zheng, G.; Xu, J.; Yang, Z.; Deng, F.; Chen, X.; Liao, Z.; Zhang, X.; Lin, Z.; Lin, H.; Yan, H.; Kuang, Z.; Zhong, W.; Liang, P.; Wang, G.; Yuan, Y.; Shi, J.; Hou, J.; Lin, J.; Jin, J.; Cao, P.; Shen, Q.; Jiang, Q.; Zhou, P.; Ma, Y.; Zhang, X.; Xu, R.; Liu, J.; Zhou, Y.; Jia, H.; Ma, Q.; Qi, R.; Zhang, Z.; Fang, J.; Fang, H.; Song, J.; Wang, M.; Dong, G.; Wang, G.; Chen, Z.; Ma, T.; Liu, H.; Dhungana, S.R.; Huss, S.E.; Yang, X.; Sharma, A.; Trujillo, J.H.; Martinez, M.C.; Hudson, M.; Riascos, J.J.; Schuler, M.; Chen, L.Q.; Braun, D.M.; Li, L.; Yu, Q.; Wang, J.; Wang, K.; Schatz, M.C.; Heckerman, D.; Van Sluys, M.A.; Souza, G.M.; Moore, P.H.; Sankoff, D.; VanBuren, R.; Paterson, A.H.; Nagai, C.; Ming, R. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L. Nat Genet 2018, 50 (11), 1565-1573. https://doi.org/10.1038/s41588-018-0237-2.