SNP- and Haplotype-Based GWAS of Flowering-Related Traits in Maize with Network-Assisted Gene Prioritization

Round 1

Reviewer 1 Report

Page 1; Line 36: You started the sentence by mentioning “maize” and then at the end mentions “corn”. Are they being used interchangeably? If so, consider using just maize, it reads better.

Page 2; Lines 49-51: It is stated that there are certain limitations present in forward and reverse genetics. What are some of these limitations? How does the new technology provide insight?

Page 3 Line 95: What version of TASSEL was used? There are a number of versions of TASSEL and they differ from each other. If some one was to repeat this experiment it would be helpful to know the version used in this study.

Table 1: In the sentence “N°SNP indicates the number of SNPs detected in each chromosome” consider changing “in each chromosome” to “on each chromosome”.  The expression is found elsewhere in the text and should be corrected.

Table 1: “Max_HB correspond to the maximum number of SNPs forming the haplotype blocks”. The acronym Max_HB is not in Table 1. This should be corrected.

Page 4 Line 145-148: Approximately 291 000 SNPs were used to construct haplotype blocks. However, it was reported that the two sites had different numbers of detected haplotypes. I think it should be better explained or expanded upon on how the number of haplotypes might differ between sites. Were the same lines and number of lines evaluated at both sites?

Table S1: “BIN” is not described in the table description.

Page 4 Lines 160-162: Were the genomic regions identified for FF the same for both sites? Were the SNPs and HB on Chr9 the same at both sites or were they for one site?

Page 5 Lines 192-193: “Importantly, three HB had PV% ranging from 11% to 17%”. This sentence makes it seem as though the three HBs had multiple values between 11% and 17%. Either state the three values or state that the three HBs had values higher than 10%.

Page 9 Line 337-339: Citation is incorrect.

Author Response

Answers to the general comments:

Thank you so much for your comments and suggestions on the manuscript “SNP- and Haplotype-Based GWAS of Flowering-Related Traits in Maize with Network‐Assisted Gene Prioritization”. We have given careful attention to the input, and we have included a point-by-point response to each of the reviewer's comments. All modifications are highlighted by Track Changes function in the text.

Reviewer: Page 1; Line 36: You started the sentence by mentioning “maize” and then at the end mentions “corn”. Are they being used interchangeably? If so, consider using just maize, it reads better.

Authors’ answer: This part was amended according to the reviewer’s comment.

Reviewer: Page 2; Lines 49-51: It is stated that there are certain limitations present in forward and reverse genetics. What are some of these limitations? How does the new technology provide insight?

Authors’ answer: Some of these limitations were included in the text, which have been discussed by Lee et al., among other. Network‐assisted gene prioritization system facilitates the genetic analysis through supporting candidate genes based on network neighbors with known traits or functions, and aid in identifying potential candidate genes that are highly likely to be causal to the phenotype of interest. These aspects were included in the new version.

Reviewer: Page 3 Line 95: What version of TASSEL was used? There are a number of versions of TASSEL and they differ from each other. If some one was to repeat this experiment it would be helpful to know the version used in this study.

Authors’ answer: The HB- and SNP-based association analyses were performed using a mixed linear model (MLM) in TASSEL 3.0 and TASSEL 5.2, respectively. This information was included in the text according to the reviewer’s comment.

Reviewer: Table 1: In the sentence “N°SNP indicates the number of SNPs detected in each chromosome” consider changing “in each chromosome” to “on each chromosome”.  The expression is found elsewhere in the text and should be corrected.

Authors’ answer: The expression was amended.

Reviewer: Table 1: “MaxHB correspond to the maximum number of SNPs forming the haplotype blocks”. The acronym MaxHB is not in Table 1. This should be corrected.

Authors’ answer: The acronym was corrected in the Table 1 according to the reviewer’s observation.

Reviewer: Page 4 Line 145-148: Approximately 291 000 SNPs were used to construct haplotype blocks. However, it was reported that the two sites had different numbers of detected haplotypes. I think it should be better explained or expanded upon on how the number of haplotypes might differ between sites. Were the same lines and number of lines evaluated at both sites?

Authors’ answer: We agree with the reviewer´s comment. Haplotype blocks were constructed considering 319 and 293 inbred lines in the sites Cambira and Sabaudia (respectively), and were constructed according this number of lines in each site. Thus, allelic variants and the number of haplotype blocks were dependent on each site. This information was included in the text for better explanation and thus avoids confusion.

Reviewer: Table S1: “BIN” is not described in the table description.

Authors’ answer: This description was included in the Table S1.

Reviewer: Page 4 Lines 160-162: Were the genomic regions identified for FF the same for both sites? Were the SNPs and HB on Chr9 the same at both sites or were they for one site?

Authors’ answer: The genomic regions identified for FF were not the same for both sites. This explanation has been highlighted as follows: "Interestingly, all associations were environment specific, confirming the existence of a significant and complex genetic-by-environment interaction". The sentence about chromosomal positions was also modified for better understanding.

Reviewer: Page 5 Lines 192-193: “Importantly, three HB had PV% ranging from 11% to 17%”. This sentence makes it seem as though the three HBs had multiple values between 11% and 17%. Either state the three values or state that the three HBs had values higher than 10%.

Authors’ answer: This sentence was corrected in the text according to the reviewer’s suggestion.

Reviewer: Page 9 Line 337-339: Citation is incorrect.

Authors’ answer: The citation was corrected.

Author Response File: Author Response.docx

Reviewer 2 Report

The paper titled "SNP- and Haplotype-based GWAS of Flowering-Related Traits in Maize with Network-Assisted Gene Prioritization" analyses the relationship of genotypic variations and flowering-related traits in maize. They use a good methodology (GWAS and network-assisted gene prioritization) to understand the genetic basis of flowering-related traits, which is really useful in a maize breeding program. Regarding the identification of four genes related to female flowering, they are described from a bioinformatic point of view. However, they should be validated by other methodologies to confirm their relationship with the female flowering.

Lines 70-71: Information about the material used in the extraction of DNA is not included in the manuscript.

Lines 141-143: Clarify this sentence.

The number of SNPs, included in the Table 1, are the mean of both species? How many SNPs were identified in each location? The position of the haplotype blocks or SNPs in each chromosome should be included in the manuscript. 

Line 150: What do the authors mean when they say “in both sites”? both locations?

Lines 151-153: The authors should include more information about this results and discussion.

Author Response

Answers to the general comments:

Thank you so much for your comments and suggestions on the manuscript “SNP- and Haplotype-Based GWAS of Flowering-Related Traits in Maize with Network‐Assisted Gene Prioritization”. We have given careful attention to the input, and we have included a point-by-point response to each of the reviewer's comments. All modifications are highlighted by Track Changes function in the text.

Reviewer: The paper titled "SNP- and Haplotype-based GWAS of Flowering-Related Traits in Maize with Network-Assisted Gene Prioritization" analyses the relationship of genotypic variations and flowering-related traits in maize. They use a good methodology (GWAS and network-assisted gene prioritization) to understand the genetic basis of flowering-related traits, which is really useful in a maize breeding program. Regarding the identification of four genes related to female flowering, they are described from a bioinformatic point of view. However, they should be validated by other methodologies to confirm their relationship with the female flowering.

Authors’ answer: We agree with the reviewer’s suggestion; the methodology implemented in this study could be validated with other methodologies. However, to our knowledge, this study is one of the first that examines the inclusion of haplotype blocks in GWAS of flowering-related traits in maize, in combination with Network‐Assisted Gene Prioritization (NAGP). Traditional GWAS have usually implemented only the first approach. As we present specific information on the first GWAS/NAGP (focusing on important traits) we present our results in the form of a Short Communication. Additionally, we used two GWAS approaches to give more statically power, SNP- and Haplotype-based GWAS. Please consider also that GWAS is per se a widely used and validated genetic technique, which has become a practical method for identifying candidate maize genes that are associated with complex traits of interest. Moreover, the co-functional networks of MaizeNet were based on the integration of multiple types of interaction data, such as 26,624 coding genes, gene expression omnibus, co-expression, co-citation, among others.

Reviewer: Lines 70-71: Information about the material used in the extraction of DNA is not included in the manuscript.

Authors’ answer: This information was included in the text according to the reviewer’s comment.

Reviewer: Lines 141-143: Clarify this sentence.Authors’ answer: This sentence was modified for a better understanding.

Reviewer: The number of SNPs, included in the Table 1, are the mean of both species? How many SNPs were identified in each location? The position of the haplotype blocks or SNPs in each chromosome should be included in the manuscript.

Authors’ answer: We agree with the reviewer´s comment. Haplotype blocks were constructed considering 319 and 293 inbred lines in the sites Cambira and Sabaudia (respectively), and were constructed according this number of lines in each site. Thus, allelic variants and the number of haplotype blocks were dependent on each site. This information was included in the text for better explanation and thus avoids confusion.

Additionally, the information about the position of SNPs in each chromosome was included in the Table for a better understanding.

Reviewer: Line 150: What do the authors mean when they say “in both sites”? both locations?

Authors’ answer: This part was amended in all text. Site was changed by location.

Reviewer: Lines 151-153: The authors should include more information about this results and discussion.

Authors’ answer: More information was included in this part according to the reviewer’s suggestion.

Author Response File: Author Response.docx

Reviewer 3 Report

Thanks to the authors for conducting such an interesting research. I would suggest to check the language in a few sentences (e.g. Line 76-77) and improve the  "Network" section of the methodology. I strongly believe that this article will create interest to the readers.

Author Response

Reviewer: Thanks to the authors for conducting such an interesting research. I would suggest to check the language in a few sentences (e.g. Line 76-77) and improve the "Network" section of the methodology. I strongly believe that this article will create interest to the readers.

Authors’ answer: The English of the text was checked, and in the "Network" section was modified for a better understanding. Thanks for your favorable revision.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I agree with all the information included by the authors.