Long-Read cDNA Sequencing Revealed Novel Expressed Genes and Dynamic Transcriptome Landscape of Triticale (x Triticosecale Wittmack) Seed at Different Developing Stages
, Anna Bolotina 1,2, Pavel Merkulov 1,2, Maxim Dudnikov 1,2, Alexander Soloviev 1,3 and Ilya Kirov 1,2,*Round 1
Reviewer 1 Report
In general, the manuscript is well-written, methodology is adequate to reach the work main goal. However, I suggest a careful review of the section: results, to present a detailed description of the results. There are many paragraphs which presented the methodology and strategy to analyze sequencing data. In addition, Discussion should present a comparison to previous works showing the novelty brought by the authors, as well as, hypothesis of the main mechanisms modulated during seed development.
Author Response
We thank the reviewer for such positive evaluation of our manuscript.
Q1: However, I suggest a careful review of the section: results, to present a detailed description of the results. There are many paragraphs which presented the methodology and strategy to analyze sequencing data.
A: We have modified the first section and move some information to M&M section.
Q2: Discussion should present a comparison to previous works showing the novelty brought by the authors, as well as, hypothesis of the main mechanisms modulated during seed development.
A: We modified a part of the discussion and provided more evidence from another grasses (rice, maize and wheat) about the role of RNA processing genes in seed development and particularly in proper control of transposons. The provided data further supported our hypothesis that endosperm DNA hypomethylation has a TE-protective role for the embryo.
Reviewer 2 Report
Dear Editorial Manager
The manuscript has been written well, but references must be checked again into the text and the end of manuscript.
Regards
Author Response
We thank the reviewer for such positive evaluation of our manuscript. The reference list was extended during the revision process. Also duplicated references were removed.
Reviewer 3 Report
The authors of the manuscript titled “Long-Read cDNA Sequencing Revealed Novel Expressed Genes and Dynamic Transcriptome Landscape of Triticale (x Triticosecale Wittmack) Seed at Different Developing Stages” identified a set of novel and lnc RNAs related to seed developmental stages in Triticale. The work is scientifically important and can be accepted. However, the authors need to address the comments for improving the present manuscript. The negative word for the manuscript is it lacks any experimental demonstration for the genes identified which could have been very interesting.
Comment
1. Line 385: Why was the RNA concentration and integrity estimated for DNA sample?
2. The sequencing details using replicates should be mentioned for each seed growth stage in the materials and method section. Line 121: Indicate the different samples as to how many biological/technical replicates were used for the sequencing initially.
3. The growth conditions of the samples used for sequencing should be provided in detail.
4. The authors do not report any experimental validation of the known and novel genes identified for strengthening their stance.
Author Response
We thank the reviewer for valuable comments that improved our manuscript.
Q1: Line 385: Why was the RNA concentration and integrity estimated for DNA sample?
A: We thank the reviewer for reporting this mistake. It was changed to DNA.
Q2: The sequencing details using replicates should be mentioned for each seed growth stage in the materials and method section.
A: Unfortunately, we did not have replicates per se but we used three different samples for 15 and 20 dpa stages (whole seed, dissected endosperm and dissected germ part). The information was added to the M&M. The lack of replicates was because we did not intend to perform differentially expression analysis, but rather we were interested to cover as much biological variation as possible during seed development. In addition, it is known that Nanopore reads are not the best choice for DEG analysis.
Q3: The growth conditions of the samples used for sequencing should be provided in detail.
A: The detailed information was added to the M&M section.
Q4: The authors do not report any experimental validation of the known and novel genes identified for strengthening their stance.
A: We agreed with the reviewer that experimental evaluation of the defined novel genes is a very interesting topic. But this is out of scope of the current manuscript and the work aimed to check the functional importance of the found novel genes is ongoing in our lab now.
Reviewer 4 Report
The article Long-Read cDNA Sequencing Revealed Novel Expressed 2 Genes and Dynamic Transcriptome Landscape of Triticale (x 3 Triticosecale Wittmack) Seed at Different Developing Stages revealed a large number of transcripts with important role in seed development. The article can be published with the following modifications:
1. Introduction part should be precise elaborating the back-ground, research gap, current status and obkective of the study.
2. Materials and methods: this part looks fine
3. Results: This part has included several sentences not relevent to this section. These part should be either included in materials or Discussion as suggested in the attached file.
4. Discussion part should include all the relevant information available for the role of lncRNA in seed development in different crops. This part needs to re-written with clear focus on the area of the work.
Comments for author File: 
Comments.pdf
Author Response
We thank the reviewer for valuable comments that improved our manuscript.
Q1: Introduction part should be precise elaborating the back-ground, research gap, current status and objective of the study.
A: The introduction part was modified.
Q2: Results: This part has included several sentences not relevent to this section. These parts should be either included in materials or Discussion as suggested in the attached file.
A: We modified the highlighted parts but we prefer to leave the explanation of the samples in the Results part because we think that it is important to understand the meaning of the rest of the section.
Q3: Discussion part should include all the relevant information available for the role of lncRNA in seed development in different crops. This part needs to re-written with clear focus on the area of the work.
A: We modified a part of the discussion and provided more evidence from another grasses (rice, maize and wheat) about the role of RNA processing genes in seed development and particularly in proper control of transposons. The provided data further supported our hypothesis that endosperm DNA hypomethylation has a TE-protective role for the embryo.
