GGE Biplot-Based Transcriptional Analysis of 7 Genes Involved in Steroidal Glycoalkaloid Biosynthesis in Potato (Solanum tuberosum L.)

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

The quality of the english is fine.

Author Response

Reviewers' comments:

Reviewer #1:

The author should include the primers sequences used in the study, either as supplemental data or in a table. If they can also state if these primers span intronic regions that would enhance the experimental approach.

Response: We have included the primer sequences used in the study as supplementary data. Please see supplementary table S1.

The PCR analysis was based on a ΔΔCt. Was a reference gene such as act2 to ef1-alpha used in this analysis?

Response: The internal reference gene was Actin I, please see lines 111.

Table 1 What is the significance of the lower-case letters following gene expression levels, for example, cultivar D-3 has hmg-1 measured at 0.44±0.09d. What does the “d” signify? There are other letters attached to other entries on this table.

Response: Duncan multiple comparison method was used and different lower-case letters represented significantly difference in genotype or environment. Duncan's (1955) multiple range test is widely accepted by research workers, especially those in the agricultural sciences (Chew, 1977).

Aleong, J., and D. Howard. "Extensions of the Duncan's Multiple Range Test for unbalanced data." Journal of Applied Statistics 12.1 (1985): 83-90.

Duncan, David B. "Multiple range and multiple F tests." biometrics 11.1 (1955): 1-42.

The authors state “It should be noted that this study only examined the changes in SGAs content in 301 potato tubers under red light…”. This needs to be rewritten as the authors never measured SGA content in the manuscript. They do state two sentences later “However, it is important to acknowledge that we did not investigate the SGAs content level in potato stems, leaves and other parts under red light.”

Response: It has been revised. Please see lines 283-289.

The manuscript does demonstrate a potentially novel approach to analysis. Figures 2 and 3 are the same data in a different format. I suggest the authors merge these into one figure with a, b, c and d subgraphs.

Response: The figures were redrawn as suggesting. Please see lines 157.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

I appreciate your efforts and time in preparing the manuscript. However, the whole manuscript needs to be organized prior to acceptance. Please find below my comments and suggestions:

Comment 1: Figure 1 can be removed or shifted to the supplementary.

Comment 2: The introduction is really well-written. I appreciate the authors for this concise and clear introduction. However, there are some minor grammatical errors in this section, which need to be corrected.

Comment 3: Please mention the RNA concentration, used for cDNA synthesis.

Comment 4: Please use a more updated reference for the ΔΔCT method (line 125).

Comment 5: Please italicize all the gene names.

Comment 6: most of the references are outdated. Please replace them with recent references (preferably from the last 10-15 years).

Comment 7: Without enzymatic assays or other validation experiments, the reliability of any methods become sometimes tricky. I suggest the authors to consider this point. For example, in line 324, the authors claimed “These markers can be applied to investigate the 324 association between gene expression levels and SGAs accumulation.” But without any validation.

Comment 8: No reference or housekeeping genes are mentioned in the methodology section.

 

Comment 9: Result sections are unnecessarily long. I recommend the authors to wisely choose the lines and points which can be shifted to the discussion section.

Extensive editing of the English language is required.

Author Response

Reviewers' comments:

Reviewer #2:

Comment 1: Figure 1 can be removed or shifted to the supplementary.

Response: Figure 1 was migrated to supplementary. Please see Figure S1.

Comment 2: The introduction is really well-written. I appreciate the authors for this concise and clear introduction. However, there are some minor grammatical errors in this section, which need to be corrected.

Response: Thanks for your suggestions. The introduction was rewritten and edited and we try our best to avoid grammar errors.

Comment 3: Please mention the RNA concentration, used for cDNA synthesis.

Response: The RNA concentration is 600-800 ng/ng/μL. The cDNA working concentration was uniformly diluted to 200 ng/μL.

Comment 4: Please use a more updated reference for the ΔΔCT method (line 125).

Response: Thanks for your mention. The relational references for ΔΔCT method were added. Please see lines 112.

Comment 5: Please italicize all the gene names.

Response: All gene names were italicized.

Comment 6: Most of the references are outdated. Please replace them with recent references (preferably from the last 10-15 years).

Response: The relevant references were replaced including published recent years.

Comment 7: Without enzymatic assays or other validation experiments, the reliability of any methods become sometimes tricky. I suggest the authors to consider this point. For example, in line 324, the authors claimed “These markers can be applied to investigate the 324 association between gene expression levels and SGAs accumulation.” But without any validation.

Response: Line 324 was deleted.

Comment 8: No reference or housekeeping genes are mentioned in the methodology section.

Response: It was clarified and explained in Lines 111.

Comment 9: Result sections are unnecessarily long. I recommend the authors to wisely choose the lines and points which can be shifted to the discussion section.

Response: Appreciate for your comments. The structure of results and discussion was rearranged and edited.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Even though the manuscript is improved, however, the choice of the reference gene still seems superficial. The reference provided for choosing Actin as a reference gene is also irrelevant because it is just a general reference on actin and its regulating factors. I recommend carefully choosing the condition-specific reference gene, based on ranking via commonly used reference gene software.

Minor editing of the English language is required.

Author Response

Reviewers' comments:

Reviewer #2:

Comment : Even though the manuscript is improved, however, the choice of the reference gene still seems superficial. The reference provided for choosing Actin as a reference gene is also irrelevant because it is just a general reference on actin and its regulating factors. I recommend carefully choosing the condition-specific reference gene, based on ranking via commonly used reference gene software.

Response: We have provided new references for choosing Actin as a reference gene . please see lines 111. Upadhyaya et al. choosing the actin as a reference gene in their study on enhanced α-tocopherols and abiotic stress tolerance by genetic engineering of potato. Qin T et al. choosing the actin as a reference gene in their study on analyses reveal cultivar-specific molecular signatures associated with different rooting depth responses to drought stress in potato.

 

Upadhyaya D C, Bagri D S, Upadhyaya C P, et al. Genetic engineering of potato (Solanum tuberosum L.) for enhanced α‐tocopherols and abiotic stress tolerance[J]. Physiologia Plantarum, 2021, 173(1): 116-128.

Qin T, Ali K, Wang Y, et al. Global transcriptome and coexpression network analyses reveal cultivar-specific molecular signatures associated with different rooting depth responses to drought stress in potato[J]. Frontiers in Plant Science, 2022, 13: 1007866.

Comment : Minor editing of the English language is required.

Response: The language of the manuscript has been corrected and evidence of the corrections is provided.

Author Response File: Author Response.docx