Genome-Wide Identification and Expression Analysis of Cytosine DNA Methyltransferase Genes Related to Somaclonal Variation in Pineapple (Ananas comosus L.)
Round 1
Reviewer 1 Report
DNA methylation is a critical process because it responses to the surrounding changes and regulates the cellular metabolism. The authors evaluated the methylation in the total DNA of somaclonal pineapple variants. They also searched the pineapple genome and identified six methyltransferases. Their phylogenetic analysis suggested the enzymes present conserved motifs and are highly homologous with the identified family members. Their analysis of the cis-elements and the transcription evaluation indicated the enzymes potentially response to either different environment or different growth stages. The results are interesting and extended the knowledge in this area. However, the manuscript requires dramatic revisions especially in the language. The followings are my suggestions.
- Please find a native English speaker to help in the language. A lot of descriptions are misleading and need professional revision. As a Chinese, I understand what the authors were talking. However, other readers may find it is difficult to follow the manuscript.
- Please be careful with the scientific terms such as ORF and expression of genes. It looks like it is CDS instead of ORF in the table2 according to the length. ORF contains introns while CDS does not. Also, RT-PCR measures the amount of a specific range of nucleic acid. In the manuscript, it is RNA. Therefore, the Y-axles should be the level of transcription instead of expression since expression contains both transcription and translation.
- Are the numbers in Figure1 obtained from the averages from replicates? Please add the deviations and statistic analysis for the conclusion of difference detected.
- Please remake the panel B in Figure5 since both axles are missing.
- I don't following the explanation in the section 3.7 and Figure 7. What is the methylation level? How does it corelated with the RNA levels of the enzymes?
Author Response
Response to Reviewer 1 Comments
DNA methylation is a critical process because it responses to the surrounding changes and regulates the cellular metabolism. The authors evaluated the methylation in the total DNA of somaclonal pineapple variants. They also searched the pineapple genome and identified six methyltransferases. Their phylogenetic analysis suggested the enzymes present conserved motifs and are highly homologous with the identified family members. Their analysis of the cis-elements and the transcription evaluation indicated the enzymes potentially response to either different environment or different growth stages. The results are interesting and extended the knowledge in this area. However, the manuscript requires dramatic revisions especially in the language. The followings are my suggestions.
- Please find a native English speaker to help in the language. A lot of descriptions are misleading and need professional revision. As a Chinese, I understand what the authors were talking. However, other readers may find it is difficult to follow the manuscript.
Answer : Thank you very much for your comments, which are very helpful for us to improve the manuscript. And we have invited a native English speaker to help correct grammar and sentences, and we hope the revised paper will be more clear and accurate on expressions.
- Please be careful with the scientific terms such as ORF and expression of genes. It looks like it is CDS instead of ORF in the table2 according to the length. ORF contains introns while CDS does not. Also, RT-PCR measures the amount of a specific range of nucleic acid. In the manuscript, it is RNA. Therefore, the Y-axles should be the level of transcription instead of expression since expression contains both transcription and translation.
Answer : Thanks for your comments , after carefully check, the ORF length have amended to CDS length in table2. Furthermore, the Y-axles have amended to transcription level in Figure6.
- Are the numbers in Figure1 obtained from the averages from replicates? Please add the deviations and statistic analysis for the conclusion of difference detected.
Answer : We have used the MSAP for analyzing the rate of mehtylation site on individual plant, so deviations and statistic analysis was not performed.
- Please remake the panel B in Figure5 since both axles are missing.
Answer : Figure5 has been amendment.
- I don't following the explanation in the section 3.7 and Figure 7. What is the methylation level? How does it corelated with the RNA levels of the enzymes?
Answer : Thanks very much for your comments. The section 3.7 and Figure 7 don't relate to the methylation level, and the contents have been corrected.
Author Response File: 
Author Response.docx
Reviewer 2 Report
1) line 22: need to standardize the font size
2) line 159: what are the reference gene(s) used in this analysis?
3) line 292: what is the control sample (to normalize with) for this experiment? Normally, the relative expression value for the control sample should be 1.
4) line 392: There is redundant numbering in the reference list.
Author Response
Response to Reviewer 2 Comments
- line 22: need to standardize the font size
Answer :The font has been standardize in the same size.
- line 159: what are the reference gene(s) used in this analysis?
Answer : AcACTIN was used as the reference gene in this study and we have been added it in section 2.6. The sequence of AcACTIN primer see Supplementary Table S2.
- line 292: what is the control sample (to normalize with) for this experiment? Normally, the relative expression value for the control sample should be 1.
Answer : The mixed samples of different tissue such as leaf, fruit and root from pineapple which were used as control, and the relative expression were carried out intra-gene standardization and calculate Zscore.
- line 392: There is redundant numbering in the reference list.
Answer : The redundant numbers have been deleted.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
My concerns have been addressed except for the following one.
Please check the caption of Figure 7 again. According to the authors' response, it should be the heatmap of expression instead of methylation and expression.
