Genome-Wide Scan for Genetic Signatures Based on the Whole-Genome Resequencing of Salt- and Drought-Tolerant Rice Varieties

Round 1

Reviewer 1 Report

 

General: 

The following article reviews candidate genes for salt tolerance in rice: Fan, X.; Jiang, H.; Meng, L.; Chen, J. Gene Mapping, Cloning and Association Analysis for Salt Tolerance in Rice. Int. J. Mol. Sci. 2021, 22, 11674. https://doi.org/10.3390/ijms222111674 

Have the authors checked their gene list to for any overlap with those identified by any of the articles reviewed Chen et al.?  

 

The Methods section needs revising to ensure all relevant methods are included. Full details of all experiments and materials (including details of breeders and release dates and locations for all elite lines). Full details of phenotyping measurements and growth stages are needed for the field experiment and explain fully how selections were made – on what criteria did you select the VSDT? 

 

The Results section needs revising to remove methods and focus on explaining results only. 

 

Introduce the two acronyms NGR and VSDT in the Abstract or early in the Introduction. 

 

Title: “genetic signatures” is a vague term – title should be more focused 

 

Abstract:  

line 22 specify which phenotypes were analysed 

line 25 – screened or identified? “Natural variation analysis” is vague. Does this mean sequence or SNP and InDel variation? 

 

 

Introduction 

Line 49 – Should this state “Most previously identified genes related to abiotic stresses…”?  

Line 81 – A recent study has been done on salt tolerant genotypes and this should be considered here. Higgins et al., 2021 Evolutionary Applications 15: 1141-1161 DOI: 10.1111/eva.13433 

 

Lines 86- 87 give more details about what trait(s) were controlled by the allele(s) identified and validated. Delete ‘tag’ and replace with ‘tagged’. 

 

109 Which country? Need to mention a table and give a list with full details of all 200 accessions. 

 

Line 92 – identified or selected? 

 

Line 101 delete ‘the natural’. 

 

Methods 

109 which country? 

110. How was preliminary selection done? Give full details of stresses were imposed and at what growth stages. 

 

111 – Delete “Among them” and replace with “They were”. What were the conditions of light, temperature in the growth chamber? Were any stresses imposed? How was screening done? 

 

Section 2.2 

Describe each separate experimental trial in a separate paragraph. Were all 10 selected varieties grown in all three experiments (i.e. five salt stress and five drought stress cultivars?). For each experiment state what controls were used? How many replicates were used for each variety? What was the experimental design for each experiment? Were randomised layouts used for the experiments? What statistical tests were used  to compare phenotypic differences? 

 

122 – what is underground water? What was it mixed with? 

 

124 – write in past not present tense. Give precise details of what was measured for “survival rate” and “yield”. 

 

128-130 Re-write this sentence because it is unclear as to what was done – were statistical analyses used – if so which ones? What traits were recorded? What is meant by “using fields”? Which indicators were used for what? 

 

137-138 Should this state “salinity and drought”? 

140-140 Need to be re-written as a complete sentence (first clause needs a verb). 

149 Give full name of reference genome (NIP is an abbreviation that needs defining) 

 

 

Line 173-174 Clarify definition of genotypes 0/0 and 1/1, it is unclear what they relate to. 

 

Section 2.7 

Not clear why this has been described an additional set of accessions from published sequencing data in lines 102-105. 

 

Results 

Avoid repetition of methods in results. 

 

213 not sure why this is referred to “deep”-sequencing analysis.  

219-220 Were the SNPs and InDels found between each variety and Nipponbare or between pairs of the 10 test varieties? 

 

Figure 1 – Why was T-test used? Why not ANOVA? 

 

Section 3.2 

This section seems to be in the wrong place, it does not clearly relate to the prevous and subsequent sections.  

Need to clarify what was added to what (data from 116 rice materials with available subgroup information were added to what exactly? Were these data added to the data from the sequencing described in the previous section?  

 

259-261 – this sentence should be moved to the appropriate section of Methods. 

 

 

Section 3.4 

Please say if this section reports on findings from the genome wide scan for loci related to drought and salt tolerance described in section 2.6, otherwise it is not clear which methods were used for this part. 

When describing genes in the text please indicate the chromosome location and give the LOC identifier as well as its abbreviation so it can be found on Fig 4 A. 

 

Line 238 – genes without detailed functional annotations – does this mean they had no functional annotations on AgriGO? Were any other databases checked? (e.g. http://rice.uga.edu; https://www.ebi.ac.uk/ols/ontologies/agro; https://plants.ensembl.org/) 

 

Figure 4 A must be made bigger to be readable. 

 

292 – Pok is an abbreviation that must be defined. The sentence in lines 291 -293 should should be moved to the appropriate section of Methods. Give the full web link for the relevant part of the NCBI database. 

 

The GO terms are listed in a different order to the order presented in fig 4b. Please re-order to match the figure (top to bottom). 

 

Fig 4 C legend does not explan what CK and Salt represetn in terms of treatments and a reference is needed in the figure legend to the relevant study from which these data were taken. 

 

Section 3.6 

317 – Rephase the opening sentence of section 3.5 to start “Ecotypes of Asian cultivated rice classified as upland and lowland often differ...”. These two sentences should be in the Introduction not the results. The second two should be in the Methods section. No need to say “finally” in line 323. Remove ‘the’ from line 325. 

 

The first four sentences starting on line 338 to 346 should be moved to the Introduction and details should be cited about the contribution or effect (R2) on root growth of the QTL which the AIM1 gene underlies. Explain in section 3.5 and figure 5 A why the AIM1 gene is shown in red in this figure. What is the horizontal black dotted line in Fig 5 A? 

 

Line 347 should be re-worded to state that the 17 variations were found either between elite varieties (pairwise) or between each elite variety and the Nipponbare reference genome, whichever of these is correct. (I can’t tell from the information in the methods section). 

 

Figure 5 A is does not include “analysis of AIM1 Haplotypes” therefore remove the mention of this in the main ledgend for Figure 5. 

 

Explain in the main text what is meant by ‘differential sites’ in Fig 5A. 

 

Figure 5 C is a table and a graph and both need separate legends and full explanations. What are the values in the last two columns of the table? Are the positions in the promoter given in bp and are they for Nipponbare? Define CDS in the legend. Define Hap. Give the number of individuals for which phenotype data are show for each haplotype shown in the graph. Was the total number of elite varieties 10? From which experiment were the root data obtained? (This should be described in Methods unless they are from another study, and if these phenotypes are from another study it must be cited in the figure legend for Figure 5C). Are the central bars in the plots means or medians? Are the top and bottom of coloured designs and the outer two bars representing the quartiles?  

 

Line 369-371 This explanation of Fig 6 A does not help me interpret the figure – please re-phrase it. How are the highly divergent regions represented in the figure? What is the red dotted line top 5% of? Include more information for full interpretation in the figure legend. 

371-374 – check that Fig 6 B is fully explained here and the legend. 

 

Fig 6 C Each pannel needs a full description in the legend including chromosomes, position and LOC ID information. What are X axes? Define VSDT and NGR in the legend so the figure can stand alone. 

 

Discussion 

Revise the whole discussion to be more focused and less repetitive.  

 

Lines 394-396 might be country specific so please give some more context for these statements. 

 

Line 398 – After reading this far I am still not sure how you identified these 10 varieties. What was the full list from which they were selected? The phenotypic selection experiment was not explained well in the methods section. What were the selection criteria used in that experiment? 

 

Line 410 “A few previously reported salt stress-related genes were identified...” Re-write this to say how many were identified, which genes they are and what is known about their functions in relation to salt tolerance. 

 

Line 417 – four genes are mentioned that were studied for overexpression – were any of them identified in this study? 

 

Line 419 – Can you say more about what is known of the functionality of the gene product of AIM1? Which root traits has it been associated with? Can you advise which genotypes for specific haplotypes should be selected by breeders to improve drought? 

 

What is meant by “A genome wide selection analysis”? (line 439) 

 

Line 445 – what is the population mentioned here?

Some minor improvements would help readability and flow.

Author Response

Respected reviewer:

We sincerely thank you for their valuable comments. All the suggested revisions and aroused questions have been considered and addressed individually. Additionally, we corrected the minor mistakes in the previous manuscript by self-checking. All the changes can be found in the revised manuscript with tracked changes. I hope that the revised manuscript will fulfill the quality of the journal.

Author Response File: Author Response.pdf

Reviewer 2 Report

This research aims to identify five typical salt-tolerant varieties and five typical drought-tolerant materials and constructed variation maps of the resistance. Anyway considering also the field of research of this journal the work is very valuable.

The introduction section is very well written, but I think that can be added more information.

The experimental part is accurately done and very complex, and the results are well-presented bringing solid discussions.

 

Overall I think this is a very good article, easy to read, and I have no other comments.

Author Response

Respected reviewer:

Thank you for your recognition of our research. According to the comments of the other reviewers, we have made revisions to the manuscript. Additionally, we corrected the minor mistakes in the previous manuscript by self-checking. All the changes can be found in the revised manuscript with tracked changes.

Reviewer 3 Report

I read with interest the manuscript.

Kindly see the attached word table with all improvement need,

All suggestions are meant to highlight the quality of the research.

Comments for author File: Comments.pdf

Minor edits

Author Response

Respected reviewer:

We sincerely thank you for their valuable comments. All the suggested revisions and aroused questions have been considered and addressed individually. Additionally, we corrected the minor mistakes in the previous manuscript by self-checking. All the changes can be found in the revised manuscript with tracked changes. I hope that the revised manuscript will fulfill the quality of the journal. 

Author Response File: Author Response.pdf