RNA-Seq Revealed the Molecular Mechanism of Nutritional Quality Improvement in o16-wx Double-Mutation Maize

Round 1

Reviewer 1 Report

Introduction section:

Wang et al. (2019) examined the transcriptome on kernels (18th day after pollination) of the o2o2wxwx double mutant line QCL5013 and parent lines using RNA-Seq. The present study performed similar work in QCL8012_2, aline developed through marker assisted selection by crossing QCL8007_5 (o16wx) and CML 30. The selection of the study material is interesting as the new line has 94% genomic background of CML 30, making QCL8012_2 a near isogenic line of CML 30. A DEG abnalysis would thus identify the specific genes associated with these double mutations. It has been previously reported that o16 mutatnts does not affect key physiological and biochemical traits (Sarika et al., 2018). The objective of this study, therefore is to find out whether o16, in wxwx background influence key grain development processes in maize and how it is different from the effect of o2 in wx background. The introduction does not reflect on this aspect, rather presents a generalized view of wx and o16 interaction.

I would also suggest to avoid use of the phrase 'polymerization of o16 and wx'. Genes or alleles are not polymerized physically, they can be pyramided or accumulated in a genetic background.    

 

Materials and methodology

Section 2.4: Details on RNA seq experimentation needed.  As the researchers outsourced the experimentation, a validation check should be mentioned. Also please clarify whether the kernels remained at milk stage at 28 day and 38 day. Because milk stage generally refers to 18-25 days after silking. 

 

Section 2.5: Reference of SOAPnuke needed

 

Section 2.6: Give experimental details on qPCR. Wet lab experimental conditions should be mentioned instead of giving only reference.

 

Section 2.8: Bioproject PRJCA017345 available but mentions only one entry named CRA011345, as per record iti is 'GSA'. There is no mention of SRA or transcriptome database. Search with subPRO025789 did not return any entry in the database. Please recheck your submission details.

 

Results:

3.1 The difference between the wild type and the mutant is not clear form the SEM image or photograph. The photgraph of cob of CML530/WT is out of focus. Clear photographs are needed. As the mutant is related to starch content it is suggested that a histochemical test for starch staining should be performed to clarify the difference. Please refer to the light box test for opaqueness (Sarika et al., 2018, PLOS). 

 

3.2 The amino acid profile in figure 2 shows that the range of lysine in the wild type is around 0.25%, while in the improved line is around 0.37% (calculated by multiplying with 1.5048). The o2 mutant itself has a lysine content of 0.37-0.4%. Pyramiding o2 and o16, lysine content of >0.5% have been achieved. The authors also have given several instances of achieving >0.5% lysine content in waxy background. Hence, they should recheck their amino acid profile or explain why the improved line has less lysine content than the single gene o2 lines or other lines in waxy background. Also, the same group reported that in double mutant QCL8004_2 (o2o2wxwx) lysine content is 0.38%. 

Moreover, the graph shows significant reduction in glu, cys, leu and ile, physiological basis needs to be explained. Is any of the genes ivolved in biosynthesis of these amino acids reflected in DEG?  

3.6-3.7 Should also check for DEG for amino acids that were reduced. 

Some typographical/grammatical mistakes

 Section 2.3 heading is incomplete

Figure 1: In the last line, the authors have written '...the blue arrow is a matrix protein'. It should be 'the blue arrow indicates matrix protein'.

Grammatical and typographical mistakes are present.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

overall the manuscript is well written but I have the following questions/suggestions on this:

How the tryptophan (Trp) contents were measured. Please describe in detail.

Figure 8 is not clear, please increase the resolution.

Please provide the full names of the abbreviations at first appearance, please check the whole manuscript carefully.

Add the latest references in the particular directions.

The conclusion is not clear, please rewrite it.

 Minor editing of the English language is required.

Author Response

Please see the attachment.

Author Response File: Author Response.docx