Genome-Wide Association Analysis Identified Newly Natural Variation for Photosynthesis-Related Traits in a Large Maize Panel

Round 1

Reviewer 1 Report

This work analizes a large panel of maize inbred lines in search for natural variation associated to photosynthesis and water use efficiency traits. The methods and the statistical analysis are correct. Findings, including QTLs, SNPs and potential genes involved set up interesting bases for breeding programmes, although the potential improvement in the studied traits associated to each variant are rather small.

 

I only have one questions regarding the SNP S8_165286201. In this paper, the gene associated in the +/-5 kbp proximity is Zm00001d005348 (https://www.maizegdb.org/gbrowse/maize_v4) or GRMZM2G134788 (v3), which code for the ctr3 (constitutive triple response3); serine/threonine protein kinase, and whose Arabidopsis homolog is AT5G03730. I do not understand the relation with the SNP found in the manuscript by Sekhon (2019) lying in the gene Zm00001d012159, which codes for the b-glucosidase orthologous to Arabidopsis BGLU42 (AT5G36890). Can you explain this?

Author Response

Dear Reviewer,

Please find enclosed the revised version of the manuscript Title: " Genome-wide association analysis identified newly natural variation of photosynthesis-related traits in a large maize panel " submitted to Agronomy

Below you can find a point by point answer

 

Comments and Suggestions for Authors

This work analizes a large panel of maize inbred lines in search for natural variation associated to photosynthesis and water use efficiency traits. The methods and the statistical analysis are correct. Findings, including QTLs, SNPs and potential genes involved set up interesting bases for breeding programmes, although the potential improvement in the studied traits associated to each variant are rather small.

 

I only have one questions regarding the SNP S8_165286201. In this paper, the gene associated in the +/-5 kbp proximity is Zm00001d005348 (https://www.maizegdb.org/gbrowse/maize_v4) or GRMZM2G134788 (v3), which code for the ctr3 (constitutive triple response3); serine/threonine protein kinase, and whose Arabidopsis homolog is AT5G03730. I do not understand the relation with the SNP found in the manuscript by Sekhon (2019) lying in the gene Zm00001d012159, which codes for the b-glucosidase orthologous to Arabidopsis BGLU42 (AT5G36890). Can you explain this?

In the present manuscript, we have addressed that the significant SNP S8_165286201 simultaneously affects gS and E and was close to the loci associated with Fv/Fm for stay-green (around ~1.3Mbp, Sekhon et al. 2019). It means that physical distance between Zm00001d012159 and GRMZM2G134788 is around 1.3Mbp. Besides, Sekhon et al. (2019) searched all annotated genes within these 200-kb windows surrounding the identified SNPs for photosynthetic traits. Though the gene associated in the +/-5 kbp proximity is Zm00001d005348 based on our previous report, the number of inbred lines of this maize panel varied from three different environments. So, we suppose that they have specific and potential connections.

Sekhon, R.S.; Saski, C.; Kumar, R.; Flinn, B.S.; Luo, F.; Beissinger, T.M.; Ackerman, A.J.; Breitzman, M.W.; Bridges, W.C.; Leon, N. De; et al. Integrated Genome-Scale Analysis Identi Fi Es Novel Genes and Networks Underlying Senescence in Maize [ OPEN ]. 2019, 31, 1968–1989, doi:10.1105/tpc.18.00930.

 

We would like to thank your comments; I hope all your inquiries are properly addressed.

We consider the manuscript has improved substantially with your input.

 

Sincerely,

 

Dr. Ana López Malvar

 

Author Response File: Author Response.docx

Reviewer 2 Report

General comments

This original paper presents a well-conducted study addressed to investigating the characterize the loci and candidate genes for five photosynthetic traits in maize. The results are well presented with all necessary statistical analyses performed.  Nevertheless, the paper's central question is fragile like a leap in the dark. Moreover, the ms is written in a frustratingly way to read which understates certain methods or findings. Therefore, I highly recommend English grammar and writing proofreading for this manuscript. 

Specific comments

Line 14: Check "has" or "have"?

Lines 35-36: That's a little bit confusing

Author Response

Dear Reviewer,

Please find enclosed the revised version of the manuscript " Genome-wide association analysis identified newly natural variation of photosynthesis-related traits in a large maize panel " submitted to Agronomy

 

 

Below you can find a point by point answer

 

Comments and Suggestions for Authors

General comments

 

This original paper presents a well-conducted study addressed to investigating the characterize the loci and candidate genes for five photosynthetic traits in maize. The results are well presented with all necessary statistical analyses performed.  Nevertheless, the paper's central question is fragile like a leap in the dark. Moreover, the ms is written in a frustratingly way to read which understates certain methods or findings. Therefore, I highly recommend English grammar and writing proofreading for this manuscript.

As suggested by the reviewer, the manuscript has been modified and updated in order to simplify the way the results were presented. Apart from that, the grammar and writing of the manuscript have been reviewed by an English speaking colleague that is not co-author of the paper

Specific comments

 

Line 14: Check "has" or "have"?

Since it refers to “any comprehensive study” it should be “has”

Lines 35-36: That's a little bit confusing

We have rephrase it

We would like to thank your comments; I hope all your inquiries are properly addressed.

We consider the manuscript has improved substantially with your input.

 

Sincerely,

 

Dr. Ana López Malvar

 

Author Response File: Author Response.docx

Reviewer 3 Report

Title: Genome-wide association analysis identified newly natural variation of photosynthesis-related traits in a large maize panel

Main comments

The manuscript is focused on the study of the genetic regulation of photosynthesis based on gas exchange-related parameters in maize. Several QTL were detected and some of them are indicated as new QTLs, associated with the theory of source-to-sink translocation. The results are interesting in the area and give new information for the Maize research community. However due the number of SNP used (152-670) and the unbalanced phenotyping described by authors, the strength of GWAS is limited.

 

Line 137: Please re-check the data set to be sure that there is no mistakes in this point or provide us the data set to corroborate this contradictorily correlation. Authors could use a similar approach than in Supplementary Fig.S2 to evaluate the alleles´s pyramidalization in the other traits, or mention if not are useful for MAS. The Supplementary material is not complete, the Tables S2, S3 and S4 are missing.

How long is the range in days to silking ? It was the correlation with photosynthesis parameters significant?. Although the plot correspond with a single line, authors was measured the relationship between plot yield and the parameters?  Is there any indication of effect on performance?

It is mentioned in M&M section a different number of lines evaluated each year (248 in 2017 and 581 and 565 in 2018 and 2019), how the BLUEs were estimated, which number of line was used?. Also the number of lines is confused (836 or 731).

 

Minor comments

Line 33: uppercase number 1 is a footnote? Or is the reference 1. Please fix it.

Line 53-59: The English is not fluent in some parts of the text, for ex line 53-59, 177-180,

Line 136, 196: The order of supplementary material is not sequential. Please modify this point.

The text ” The x-axis indicates groups 1, 2, 3, 4, and 5 with inbreds carrying 1, 2, 3, 4, and 5 favorable alleles in a large maize panel, respectively.” in the supplementary Fig 2 legend, could be improved.

Line 191: Please remove the uppercase in "The" and change "Among four SNPs, The" by  … Among these four SNPs, the

Line 447. The name of the reference has mistaken. Please revise all references to avoid this type of mistakes.

Reference that could be considered

Yannam, V.R.R.; Caicedo, M.; Malvar, R.A.; Ordás, B. Genome-Wide Association Analysis of Senescence-Related Traits in Maize. Int. J. Mol. Sci. 2022, 23, 15897. https://doi.org/10.3390/ijms232415897

Genome-wide Association Study of Chlorophyll Content in Maize Leaves TENG Shou-zhen, WANG Hai, LIANG Hai-sheng, XIN Hong-jia, LI Sheng-yan, LANG Zhi-hong. Biotechnology Bulletin . 2017, (4): 98 -107 .  DOI: 10.13560/j.cnki.biotech.bull.1985.2017.04.013 (in Chinese-Abstract in english)

Genome-wide association identifies impacts of chlorophyll levels on reproductive maturity and architecture in maize.Rajdeep S. Khangura,  View ORCID ProfileGurmukh S. Johal,  View ORCID ProfileBrian P. Dilkes. doi: https://doi.org/10.1101/2022.11.07.515492

 

Author Response

Dear Reviewer,

Please find enclosed the revised version of the manuscript " Genome-wide association analysis identified newly natural variation of photosynthesis-related traits in a large maize panel " submitted to Agronomy

We have corrected all the deficiencies highlighted by the reviewer

Below you can find a point by point answer

 

Comments and Suggestions for Authors

 

Main comments

The manuscript is focused on the study of the genetic regulation of photosynthesis based on gas exchange-related parameters in maize. Several QTL were detected and some of them are indicated as new QTLs, associated with the theory of source-to-sink translocation. The results are interesting in the area and give new information for the Maize research community. However due the number of SNP used (152-670) and the unbalanced phenotyping described by authors, the strength of GWAS is limited.

 

Line 137: Please re-check the data set to be sure that there is no mistakes in this point or provide us the data set to corroborate this contradictorily correlation. Authors could use a similar approach than in Supplementary Fig.S2 to evaluate the alleles´s pyramidalization in the other traits, or mention if not are useful for MAS. The Supplementary material is not complete, the Tables S2, S3 and S4 are missing.

Checking the data set we have not found any incongruency

In supplementary Figure S2 we chose that trait because, regarding A_N, five significant SNPs were simultaneously detected via combined analyses on chromosomes 2, 3, 6, 8, and 9. Four favorable alleles for increasing A_N were found in the majority of inbreds.

We thank the reviewer for the remark. The tables that once have been supplementary have been included in the manuscript, we have updated the text since it belonged to a previous version of the paper. The only supplementary table has been included as an independent document in the submission process

How long is the range in days to silking ? It was the correlation with photosynthesis parameters significant?. Although the plot correspond with a single line, authors was measured the relationship between plot yield and the parameters?  Is there any indication of effect on performance?

It is mentioned in M&M section a different number of lines evaluated each year (248 in 2017 and 581 and 565 in 2018 and 2019), how the BLUEs were estimated, which number of line was used?

As indicated in the manuscript for each trait, across all environments and by years, we calculated the best linear un-biased estimator (BLUEs) of each inbred across environments (using year as a source of variation) and for each environment, using the mixed model procedure (PROC MIXED) of SAS software version 9.4.

We performed the GWAs across years, considering the three years together and a GWAS for the three years individually.

 Also the number of lines is confused (836 or 731).

The panel is originally composed of 836 lines, however in our study we phenotype and  evaluated 731. This has been clarified in the text. “At the end 731 inbred lines (from the original 836) were investigated in the current study.“

 

Minor comments

Line 33: uppercase number 1 is a footnote? Or is the reference 1. Please fix it.

It has been fixed in the text.

Line 53-59: The English is not fluent in some parts of the text, for ex line 53-59, 177-180,

Following the reviewers’ suggestions, for the whole manuscript the English language and grammar has been review by an English-speaking colleague that is not author of the paper

Line 136, 196: The order of supplementary material is not sequential. Please modify this point.

We have corrected and updated the text with the correct supplementary material, thank you again for the remark.

The text ” The x-axis indicates groups 1, 2, 3, 4, and 5 with inbreds carrying 1, 2, 3, 4, and 5 favorable alleles in a large maize panel, respectively.” in the supplementary Fig 2 legend, could be improved.

The legend has been improved

Line 191: Please remove the uppercase in "The" and change "Among four SNPs, The" by  … Among these four SNPs, the

In the new version of the manuscript, this sentence has been removed

Line 447. The name of the reference has mistaken. Please revise all references to avoid this type of mistakes.

The list of the references has been checked and corrected

 

 

We would like to thank your comments; I hope all your inquiries are properly addressed.

We consider the manuscript has improved substantially with your input.

 

Sincerely,

 

Dr. Ana López Malvar

Author Response File: Author Response.docx

Reviewer 4 Report

I have identified only a few deficiencies.

P4L122: “among five maize inbred lines“ should be “ among six maize inbred lines“.

P6L223: Taiz et al. (2014) is not in References.

P8L322: According to me, the average rainfalls in summer in Spain are not 0.68-2.52 mm.

P8L331: Buckler et al. (2009) is not in References.

P9L349: It is rather inappropriately that a different number of inbred lines were evaluated in each year.

P11L447: “Identi Fi Es“ should be “Identifies“.

P12L500: Citation Romay et al. (2013) is not complete.

Author Response

Dear Reviewer,

Please find enclosed the revised version of the manuscript " Genome-wide association analysis identified newly natural variation of photosynthesis-related traits in a large maize panel " submitted to Agronomy

We have corrected all the deficiencies highlighted by the reviewer

Below you can find a point by point answer

 

Comments and Suggestions for Authors

 

I have identified only a few deficiencies.

P4L122: “among five maize inbred lines“ should be “ among six maize inbred lines“.

Following the reviewer’s correction, we have change it in the manuscript

P6L223: Taiz et al. (2014) is not in References.

This reference corresponded to a previous version of the manuscript, it has been deleted

P8L322: According to me, the average rainfalls in summer in Spain are not 0.68-2.52 mm.

The pluviometry data was recorded by a meteorological station placed at he experimental field of the Misión Biológica de Galicia, in Salcedo, Pontevedra, Galicia which is controlled by AEMET (Agencia Estatal de Meterología) Spain.

P8L331: Buckler et al. (2009) is not in References.

The reference has been included both in the text and in the reference list

P9L349: It is rather inappropriately that a different number of inbred lines were evaluated in each year.

The inbred lines measured each year were determined by their development and climatic circumstances within that year. This laborious work for collecting those more reliable photosynthetic parameters (Pn, Cond, etc.) within limited time compared to the simple and fast indicator Fv/Fm, has to be balanced with the required sample numbers when we used a large maize panel. Indeed, due to the environmental diversity, different inbred lines were available for measuring across years; as indicated in the manuscript. Given non-replicated inbreds among years, 731 inbred lines were investigated in the cur-rent study.

P11L447: “Identi Fi Es“ should be “Identifies“.

It has been corrected in the text.

P12L500: Citation Romay et al. (2013) is not complete.

The reference has been completed.

We would like to thank your comments; I hope all your inquiries are properly addressed.

We consider the manuscript has improved substantially with your input.

 

Sincerely,

 

Dr. Ana López Malvar

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The ms has now been improved and seems suitable for further publication.