Identification and Genetic Mapping of Potential QTLs Conferring Heat Tolerance in Cotton (Gossypium hirsutum L.) by Using Micro Satellite Marker’s Approach

Round 1

Reviewer 1 Report

In this study, authors used progeny of heat tolerant and heat sensitive varieties to map heat tolerance related QTLs. The manuscript is poorly written, lack clarity and incomplete figures with bad quality. Similarly, the results are poorly described and discussion is surprisingly short with no significance.

some other specific comments are;

• Figure 1 a b c lacks graph axes, even one of the graph missing the title.
• Figure 2 resolution is very bad
• As I said earlier, article is poorly written with repetition of ideas/sentences, including the sentences at inappropriate position. For example, lines 131-135 do not represent material and methods.
• The concept of heat stress measured in this study is not logical. Authors should have tested parameters that describe the level of heat stress. How about irrigation schedule? May be the affect they saw are also contributed by drought.  
• Selection of parental lines is somewhat strange; authors could have used other statistical analysis to better the judge the material.
• How many plants per replicates they used to asses the morphological parameters?
• Table 1. Authors should provide +- standard deviation for each parameter.
• Table of correlation analysis is hard to follow as lines are skewed due to inappropriate size.
• Results description should be improved, and discussion should be carefully organized. Especially discussion is one of the most important components of this type of study and authors seems to overlook this perspective.

Author Response

file uploaded in response to the reviewer comments

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, the authors identified the QTLs in cotton controlling heat tolerance. They used 94 F2 population and 175 SSR markers. In the end, they found 17 QTLs that contribute to heat tolerance phenotypes. This study could be useful for the future of cotton breeding, although I have some suggestions listed below.

Major:

1. The plots need a lot of improvement. For figure 1s, please use a density plot or boxplots (with individual dots). And absolutely no 3D histogram.
2. For Figure 1s, please choose 4-6 most interesting phenotypes from Fig1a, Fig1b, and Fig1c combined. Otherwise, too many figures are just distractions. Other figures can go to supplemental materials.
3. The tables need a lot of improvement too. Tables 1,2,3 are too big to read. They contain too much info. Please combine table2 and table3. Then please simplify Table1 and Table2+3. The full table can go to supp.
4. One line 307, the table below is not labeled as Table 4. Please use a correlation heatmap to show this data. If there are too many phenotypes, please choose the typical ones.
5. In general, about phenotypes, I’m not clear what are the major ones to focus on. The way it is now is to lay out everything, which makes me unclear on the major point. All the abbreviations make it even harder to understand.
6. On line 208, how does the 175 SSR number compare with other studies? Is 175 on the lower end or the normal size of SSR markers? In other words, are 175 SSR markers enough for QTL? If so, please discuss.
7. Figures 2a and 2b should be combined. Plus, the symbols are hard to link to SSRs.
8. Are there any interesting genes around the QTL? Was the range too large to locate genes?
9. Please discuss further how to use this QTL information in molecular breeding. If you need more space, please cut the length of the introduction. The introduction has some redundant information.
10. In table 4, the meaning of the header should be clearly written in the result section. And please include the calculation method in the method section. What is LOD value, Additive, Dominance, PV %age? How are they calculated? For now, they are not clear to the readers.

Minor:

1. The number of F2 individuals (94) should be included in the abstract and introduction.
2. On line 48, the data should be updated to a recent year, for example, 2019-2020 at least. “holds third position in the world as per records of 2012–13”.
3. On lines 102-105, not sure what this means. Does it mean very few studies on upland cotton? What is upland cotton? Why it's different from other types? Are other studies using varieties that are not upland cotton?
4. The first section of “Materials and Methods” has a lot of redundant information. Please combine with other sections. Or write it as Section 2.1.
5. On line 195, the first paragraph of 2.1 is redundant.
6. On line 233, the primer sequences should be included as a supplementary table.
7. In Table 4, the physical location of these QTLs should be included. To my knowledge, serval cotton genomes are available on Phytozome (https://phytozome-next.jgi.doe.gov/).

Author Response

File uploaded in response to the reviewer comments

Author Response File: Author Response.docx

Reviewer 3 Report

Rani et al. evaluated morphological and physical traits in 14 cotton cultivars under heat stress and selected 2 culticurs with contrasting phenotypes as parents for mapping population. Using SSR markers, QTLs responsible for heat tolerance were identified. The study of cotton heat resistance is important but the overall quality of the manuscript must be improved before publishing. 

Comments:

1) The phenotypic values of 23 morpho-physiological traits under non-heat stress condition need to be reported. Evidence needed before to draw conclusions that those 23 traits are related to heat tolerance. 

2) The introduction contains redundant information, such as, the impacts of heat appear several times in different paragraphs. Please combine the same information and be succinct.

3) Aim b 'estimate genetic diversity and phylogenetic relationship assessment by applying SSRs on 14 cultivars of cotton' proposed in the last paragraph of introduction wasn’t shown in the manuscript.

4) In the Material & Method section, the 14 cultivar names and 23 traits redundantly appeared twice. 

5) More details are required for the Material & Method section. Which year the experiment conducted? Information of the 175 SSR markers? What's the ploidy number of the 14 cultivars?

6) Line 327 and 328, chi-square tested need to support conclusions for 1:3 or 1:2:1 segregations.  

7) The presentation of tables and figures in the manuscript not read friendly. 

    a) Table1, the trait value need to be present as 'average ± sd' for each trait for each cultivar. 

    b) Table 1 and 2: keep consistency for the digit of decimals.

    c) Table2, isn’t the max and min values for F2 population incorrectly placed?

    d) Table 4 (correlation table) is better to be presented as heatmap instead of a table. And there's another 'Table 4' in the manuscript!

    e) Figure 2a and 2b are the same? why not combine panel a and b? 

Author Response

File uploaded in response to the reviewer comments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Author answered all the queries. 

Author Response

Response to Reviewer 1 Comments

Point 1:  Figure 1 a b c lacks graph axis, even one of the graph missing the title

Response 1: Graphs have been changed as per reviewer’s demand and graph axis has been demonstrated in figure 1 b and c

Point 2: Figure 2 resolution is very bad

Response 2: Figure 2 has been improvised and both a & b of it has been combined to avoid confusion

Point 3: As I said earlier, article is poorly written with repetition of ideas/sentences, including the sentences at inappropriate position. For example, lines 131-135 do not represent material and methods.

Response 3: Line 131-135 has been removed as it has already been discussed in introduction what it contained, article has been rephrased to the best of my efforts, repeating material has been removed from introduction, material and methods, grammar mistakes have been addressed.

Point 4: The concept of heat stress measured in this study is not logical. Authors should have tested parameters that describe the level of heat stress. How about irrigation schedule? May be the affect they saw are also contributed by drought.  

Response 4: Heat stress was observed on different morpho-physiological attributes of cotton from F2 population especially through cell injury, proline contents, relative water content, sympdial branch traits, boll number and other morphological traits which are strongly associated with heat stress as proved by previous studies and irrigation was kept standard according to agricultural practices therefore drought was when not even imposed it could not be the reason behind effects. Heat stress was obvious because of late sown cotton during severely harsh temperature up to 48̊̊̊̊̊ C as per meteorological data. So, the studies logically support the hypothesis and objectives of the experiment.

Point 5: Selection of parental lines is somewhat strange; authors could have used other statistical analysis to better the judge the material.

Response 5: Statistical analysis based graphs of selectively most prominent characters for parental line screening where two genotypes showed diverse affects, has been given in the paper on page 6 as figure 1a.

Point 6: How many plants per replicates they used to assess the morphological parameters?

Response 6: 94 individuals for each genotype (MNH-886 and MNH-814) were used to assess the parameters

Point 7: Table 1. Authors should provide +- standard deviation for each parameter.

Response 7: It has been written.

Point 8: Table of correlation analysis is hard to follow as lines are skewed due to inappropriate size.

Response 8: The table has been changed to Pearson correlation which is easy to understand and shortened.

Point 9: Results description should be improved, and discussion should be carefully organized. Especially discussion is one of the most important components of this type of study and authors seem to overlook this perspective.

Response 9: Discussion has been more precisely rewritten to elaborate all the measurements and how they were likely to be involved in the study aim.

Author Response File: Author Response.docx

Reviewer 3 Report

The quality of the manuscript improved after revision and most concerns have been addressed. But the manuscript is still not qualified for publication. A further revision is necessary.

Comments:

1. Figure 1a, the label for x-axis and y-axis was wrong.

2. Figure 1 a,b and c, y-axis should begin with 0 instead of negative value. And please try to keep each chart with the same style.

3. Figure 1 a,b, and c, the standard deviation of phenotypic values shouldn’t be same for all the genotypes. The deviation comes from measurements for a number of individuals of the same genotype for a specific trait. 

Author Response

Response to Reviewer 3 Comments

Point 1: The phenotypic values of 23 morpho-physiological traits under non-heat stress condition need to be reported. Evidence needed before to draw conclusions that those 23 traits are related to heat tolerance.

Response 1: Evidences have been given in introduction and discussion sections to draw conclusions that those 23 traits are related to heat tolerance. 

Point 2:  The introduction contains redundant information, such as; the impacts of heat appear several times in different paragraphs. Please combine the same information and be succinct.

Response 2: Done and the changes have been marked

Point 3:  Aim b 'estimate genetic diversity and phylogenetic relationship assessment by applying SSRs on 14 cultivars of cotton' proposed in the last paragraph of introduction wasn’t shown in the manuscript.

Response 3: Review has been addressed and the aim have been modified and marked

Point 4:  In the Material & Method section, the 14 cultivar names and 23 traits redundantly appeared twice. 

Response 4: Redundancy has been removed and marked in parental line screening section of material and methods

Point 5: More details are required for the Material & Method section. Which year the experiment conducted? Information of the 175 SSR markers? What's the ploidy number of the 14 cultivars?

Response 5:  Year and ploidy has been mentioned

Point 6:  Line 327 and 328, chi-square tested need to support conclusions for 1:3 or 1:2:1 segregations.  

Response 6: It has been mentioned in the section

Point 7:  The presentation of tables and figures in the manuscript not read friendly. 

1. a) Table1, the trait value need to be present as 'average ± sd' for each trait for each
2. b) Table 1 and 2: keep consistency for the digit of decimals.
3. c) Table2, isn’t the max and min values for F2 population incorrectly placed?
4. d) Table 4 (correlation table) is better to be presented as heat map instead of a table. And there's another 'Table 4' in the manuscript!
5.   e) Figure 2a and 2b are the same? Why not combine panel a and b?
   

Response 7: They have been modified

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Reviewer 2 Report

Authors identified very useful QTLs associated with various heat tolerance related  phenotypes. The study seems interesting and well designed although the manuscript need some improvements. I suggest acceptance of manuscript after the following major revisions.

Introduction

1. How the heat stress affects the development of cotton and how do the studied phenotypes account for heat tolerance? These basic questions should be addressed in the introduction which is missing.
2. Line 30-31: add a suitable reference
3. Line 32: “basics7” replace with “basic 7 genomes”
4. Line 56-57: Very old reference, please add the latest reference

Materials and Methods

1. Line 82-89: this paragraph seems to be the part of the conclusion or introduction so it should not be here in MM. you can add a summary of plant materials used in this study in this paragraph
2. What do you think? the statistical analysis should not be addressed in a separate heading, I think separately explained statistical analysis is a more suitable approach. On the other hand, you can also give a stepwise summary of the statistical analysis used in the study.

Results

1. How did you do the selection of parent lines? Is it based on just mean performance? It is not a reliable approach to screeningor selection of material. I would suggest you, to perform multivariate analysis such as PCA and Heiraricle cluster analysis which gives a clear picture of the best performing line and also exhibit the effect of stress as compared to control which is not well explained in the manuscript. You can follow the following articles to perform such analysis for screening experiments.
2. Mahmood, T.; Abdullah, M.; Ahmar, S.; Yasir, M.; Iqbal, M.S.; Yasir, M.; Rehman, S.U.; Ahmed, S.; Rana, R.M.; Ghafoor, A.; et al. Incredible role of osmotic adjustment in grain yield sustainability under water scarcity conditions in wheat (Triticum aestivum L.). Plants2020, 9, 1208.
3. Mahmood, T.; Wang, X.; Ahmar, S.; Abdullah, M.; Iqbal, M.S.; Rana, R.M.; Yasir, M.; Khalid, S.; Javed, T.; Mora-poblete, F.; et al. Genetic Potential and Inheritance Pattern of Phenological Growth and Drought Tolerance in Cotton ( Gossypium Hirsutum L .). Plant Sci.2021, 12, 705392.

• Mahmood, T.; Rana, R.M.; Ahmar, S.; Saeed, S.; Gulzar, A.; Khan, M.A.; Wattoo, F.M.; Mora‐Poblete, F.; Gabrielle Sousa Mafra, X. Du Effect of drought stress on capsaicin and antioxidant contents in pepper genotypes at reproductive stage. Plants2021, 10, 1286.

2. The quality of figures is too low which does not meet the journal standards, so please add good quality figures

Discussion

1. The discussion segment is too short, the author should know that it’s the most important portion of any study so it should be extensively and comprehensively explained. It should cover all the dynamics of the study. I want to see the discussion related to the performance of screening lines under heat stress and how the selected parent line is better than other material, it should be explained in the discussion separately.

Reviewer 3 Report

The title of this manuscript mentions the mapping of potential QTLs conferring heat tolerance in cotton. However, the phenotype characterization and mapping are oriented toward developmental aspects, and not heat tolerance. Better development and growth are a consequence of heat tolerance. Thus, to find QTLs conferring heat tolerance, I think that heat stress physiological parameters should be analyzed in heat-tolerant cultivar and heat-sensitive variety. The authors could then try to link QTLs to genes potentially involved in the heat tolerance phenotype by measuring gene expression; RT-qPCR could be used to check if differential gene expressions are measured in heat tolerant variety compared to heat sensitive variety.

Moreover, the manuscript writing is incomplete. The text of the “Results” section should be extended and results better explained. Also, the discussion is too short, lacks of content, and should be developed.

Figure 1: titles should be added to the graph axes.

Figure 2: image resolution should be increased.