Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a GWAS analysis to identify markers associated with grain yield in maize using DArTseq and SNP data. The topic is relevant, and the dataset is substantial. However, there are significant concerns regarding statistical rigor and interpretation, particularly in the GWAS methodology. These issues affect the reliability of the reported associations. Minor revision is required.

Comments and suggestions to the Authors

1. Lack of multiple testing correction
   The study reports 2,478 significant markers using a p-value threshold of 0.05. No multiple-testing correction (e.g., FDR or Bonferroni) is applied, likely resulting in a high number of false positives.
   → Appropriate correction must be applied, and significant associations re-evaluated.
2. Population structure and kinship are not addressed
   The manuscript does not clearly describe how population structure or relatedness were controlled. This is essential in GWAS to avoid spurious associations.
   → Include PCA/STRUCTURE analysis and a kinship matrix, and clarify the statistical model (e.g., MLM with Q+K).
3. Overinterpretation of marker utility
   The identified markers are presented as directly useful for breeding. However, no independent validation or multi-environment testing is provided.
   → Reframe as candidate loci and moderate claims regarding marker-assisted selection.
4. Limited biological interpretation
   Many candidate genes are weakly annotated, and functional links to yield are not sufficiently supported.
   → Strengthen functional annotation and discussion using relevant literature.
5. Missing GWAS quality assessment
   No QQ plot or inflation factor is provided.
   → Include QQ plot to assess model validity.
6. Single-environment limitation
   Phenotypic data are derived from a single environment, yet broader conclusions are made.
   → Acknowledge this limitation and avoid strong generalization.
7. Minor Comments

• The Introduction is overly long and should be more focused.
• Figures (especially dendrograms) require clearer interpretation.
• Large marker tables could be simplified or moved to supplementary materials.
• Minor language editing is needed.

Author Response

Response to Reviewer 1 Comments

 

Reviewer #1

Point 1: This manuscript presents a GWAS analysis to identify markers associated with grain yield in maize using DArTseq and SNP data. The topic is relevant, and the dataset is substantial. However, there are significant concerns regarding statistical rigor and interpretation, particularly in the GWAS methodology. These issues affect the reliability of the reported associations. Minor revision is required.

Response: Thank you very much for all your comments. Below are the responses. We have revised the manuscript based on your suggestions.

 

Comments and suggestions to the Authors

Point 2: 1. Lack of multiple testing correction

 The study reports 2,478 significant markers using a p-value threshold of 0.05. No multiple-testing correction (e.g., FDR or Bonferroni) is applied, likely resulting in a high number of false positives.

 → Appropriate correction must be applied, and significant associations re-evaluated.

Response: We applied correction for multiple testing in our studies. The manuscript includes the paragraph: "The significance of associations between yield and SilicoDArT and SNP markers was evaluated using p-values adjusted for multiple testing with the Benjamini–Hochberg procedure." For further clarification, we have added the following text: "The Benjamini–Hochberg method is a statistical technique that controls the false discovery rate (FDR) during multiple hypothesis testing. It is less stringent than the Bonferroni correction, offering greater power to detect significant differences while reducing the false positive rate."

 

Point 3: 2. Population structure and kinship are not addressed

 The manuscript does not clearly describe how population structure or relatedness were controlled. This is essential in GWAS to avoid spurious associations.

 → Include PCA/STRUCTURE analysis and a kinship matrix, and clarify the statistical model (e.g., MLM with Q+K).

Response: Population structure was estimated by decomposing eigenvalues and then modeled as random effects (MLM). This information was included in the previous version of the manuscript. To emphasize that the eigenvalues were the result of principal component analysis (PCA), this information has been added in the current version of the manuscript.

 

Point 4: 3. Overinterpretation of marker utility

 The identified markers are presented as directly useful for breeding. However, no independent validation or multi-environment testing is provided.

 → Reframe as candidate loci and moderate claims regarding marker-assisted selection.

Response: Multi-environment testing was not conducted because the plant breeder who provided us with the phenotypic data treated it as a trade secret. They were unwilling to share their results with other centers to submit varieties to the Central Variety Registration Center. Therefore, we relied on data from a single location.

 

Point 5: 4. Limited biological interpretation

 Many candidate genes are weakly annotated, and functional links to yield are not sufficiently supported.

 → Strengthen functional annotation and discussion using relevant literature.

Response: I agree with the esteemed reviewer that we should focus on and characterize important genes. We selected chain acyl-coA synthetase (LACS) as the most promising gene because literature reports indicate that it indirectly influences maize yield. We described the role of this gene in the Discussion (Literature reports indicate that this gene may influence maize yield. LACSs, which convert free FAs into fatty acyl-CoA thioesters, play crucial roles in FA catabolism, lipid synthesis and storage, epidermal wax synthesis, and stress tolerance [68-70]. In Arabidopsis, more than three LACS proteins have been shown to be involved in keratin and epidermal wax production. Furthermore, the biochemical homeostasis of epidermal wax in Arabidopsis can enhance plant tolerance to water deficiency, salt stress, drought stress, and ABA treatment [71-73]. These studies strongly suggest that LACS proteins play a vital role in responding to environmental stress via regulating wax biosynthesis. In maize research  levels across diverse tissues demonstrated that the majority of LACS genes exhibited the highest expression in the meiotic tassel. The high demand for LACS activities in floral tissues is linked to the strong expression of other lipid metabolic genes in flowers, indicating their involvement in active FA metabolism during meiotic tassel development. Additionally, all LACS genes showed elevated expression levels during seed development at suggesting their potential role in early FA accumulation in maize seeds [74,75].)

 

Point 6: 5. Missing GWAS quality assessment

 No QQ plot or inflation factor is provided.

 → Include QQ plot to assess model validity.

Response: A Q-Q plot was added to assess model validity.

 

Point 7: 6. Single-environment limitation

 Phenotypic data are derived from a single environment, yet broader conclusions are made.

 → Acknowledge this limitation and avoid strong generalization.

Response: We refined our conclusions by taking into account the limitations of a single environment. This allowed us to avoid overgeneralization.

 

7. Minor Comments

Point 8: The Introduction is overly long and should be more focused.

Response: We have significantly shortened the Introduction.

 

Point 9: Figures (especially dendrograms) require clearer interpretation.

Response: The caption under Figure 3 has been updated to make the message of the different similarities at the phenotypic and genetic levels more visible. The main purpose of presenting the results in the form of dendrograms was to highlight these differences. They make the selection of breeding materials challenging and require simultaneous consideration of both types of differentiation.

 

Point 10: Large marker tables could be simplified or moved to supplementary materials.

Response: The manuscript contains three tables. We believe the information contained within them is so important that it should be included in the main text. We have tried to keep them to a minimum. Therefore, Tables 2 and 3 are separate, although in the first (preliminary) version of the manuscript they were one very large table. If the Editor wishes to move them to the supplementary materials, we will do so.

 

Point 11: Minor language editing is needed.

Response: The manuscript has been revised for English.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

D

 

Dear authors,

This article concerns “Application of NGS Technology, Association Mapping, and Physical Mapping Technologies to Identify Candidate Genes Associated with Maize (Zea mays L.) Hybrid Yield, by Jan Bocianowski, Agnieszka Tomkowiak, Ewelina Wagner and Daniel Lipiński. As potentially interesting data I may recommend it for an international audience in this journal, however several points have to be considered by the authors and a major revision is requested, the present version being not receivable.

 

 

Please notice that in order to bring a broad audience to this article and to this journal, for specialists and non-specialists, the six points of my comments (at the beginning) are very important (mandatory…) for a suitable value of the article. Minor points are also enhanced at the end of this review.

 

Sincerely yours,

 

The six major points, linked together, are:

 

• 1 Since as enhanced by the authors themselves "Notably, genetic grouping did not correspond to phenotypic grouping based on yield performance, which is consistent with findings reported by Kozak et al. [77].", the interest and specificity of new knowledge for maize in this article is unclear.

 

 

• 2 The second point concerns the yield-phenotypic characters: 1/In 4.2, indicate clearly the total number of plants measured (is it 10X3? Statistically speaking, 10 is far too low for each entry, 30 being acceptable sensu lato); 2/ As stated in the abstract I cannot see the "significant differences among hybrids in terms of yield"; 3/As used in other previous articles, yield could be quantified with other characters than just the weight? e.g. number of caryopses, their size, water content or not…

 

 

• 3 Although it is an interesting possibility, the putative relationship between yield and environment is totally un-sustained: 1/As apparently the crossings were grown in the same place (?), i.e. the same environment (?), how can the authors pretend this relationship? 2/Precise the use of figure 5; 3/ In other articles yield can be related with other factors.

 

• 4 This text is not clear at all on the useful application of this study for agronomists: finally, which are the best sequencies for a high yield quantity?

 

 

• 5 The discussion seems not connected to the results, it does not take (at all) in account the figures of the results.

 

• 6 References already taken in account by the authors are of interest, however checking in the web of science and scilit from mdpi, other studies should be updated and used (if relevant) in order to sustain much more and provide a larger view of this article. Among papers are the followings (please check for more):

1. Qi, X.; Zhao, X.; Lyu, M.; Wu, K.; Liu, N.; Li, R.H.; Xu, C.; Shen, R.Y.; Zhou, Z.H.; Wang, C.; et al. Efficient transgenic maize (Zea Mays L.) detection using low-depth next-generation sequencing. Sci Rep-Uk 2025, 15, doi:10.1038/s41598-025-18593-8.

 

2. Tofa, A.I.; Chiezey, U.F.; Babaji, B.A.; Kamara, A.Y.; Adnan, A.A.; Beah, A.; Adam, A.M. Modeling Planting-Date Effects on Intermediate-Maturing Maize in Contrasting Environments in the Nigerian Savanna: An Application of DSSAT Model. Agronomy-Basel 2020, 10, doi:10.3390/agronomy10060871.

 

3. Yang, D.G.; Gao, Z.F.; Liu, Y.Q.; Li, Q.; Yang, J.J.; Wang, Y.B.; Wang, M.Y.; Xie, T.L.; Zhang, M.; Sun, H. Exogenous application of 5-NGS increased osmotic stress resistance by improving leaf photosynthetic physiology and antioxidant capacity in maize. Peerj 2024, 12, doi:10.7717/peerj.17474.

 

 Minor points are:

 

1 Indicate the meaning of the colors in figures 2-3.

 

2 For figure 4, indicate in a caption the meaning of “chr” in full letters.

 

 

3 For tables 1-2, indicate in a caption the meaning of Chr ID LOD in full letters.

 

 

 

 

 

 

 

 

Author Response

Response to Reviewer 2 Comments

 

Reviewer #2

Point 1: This article concerns “Application of NGS Technology, Association Mapping, and Physical Mapping Technologies to Identify Candidate Genes Associated with Maize (Zea mays L.) Hybrid Yield, by Jan Bocianowski, Agnieszka Tomkowiak, Ewelina Wagner and Daniel Lipiński. As potentially interesting data I may recommend it for an international audience in this journal, however several points have to be considered by the authors and a major revision is requested, the present version being not receivable.

Please notice that in order to bring a broad audience to this article and to this journal, for specialists and non-specialists, the six points of my comments (at the beginning) are very important (mandatory…) for a suitable value of the article. Minor points are also enhanced at the end of this review.

Response: We sincerely thank you for your positive review of our manuscript. We appreciate all your comments and suggestions. We have revised the manuscript according to your suggestions. We hope that the new, revised version of the manuscript will be sufficient for publication. Below are our responses to each of your comments.

 

The six major points, linked together, are:

Point 2: 1 Since as enhanced by the authors themselves "Notably, genetic grouping did not correspond to phenotypic grouping based on yield performance, which is consistent with findings reported by Kozak et al. [77].", the interest and specificity of new knowledge for maize in this article is unclear.

Response: The fact that genetic grouping is not the same as phenotypic grouping is crucial information for breeders, including maize breeders. It means that when selecting materials, breeders must consider both types of variability: phenotypic and genetic. Selecting markers to identify candidate genes related to maize yield should facilitate the selection process. It certainly shortens it. We have added this information in the new version of the manuscript.

 

Point 3: 2 The second point concerns the yield-phenotypic characters: 1/In 4.2, indicate clearly the total number of plants measured (is it 10X3? Statistically speaking, 10 is far too low for each entry, 30 being acceptable sensu lato); 2/ As stated in the abstract I cannot see the "significant differences among hybrids in terms of yield"; 3/As used in other previous articles, yield could be quantified with other characters than just the weight? e.g. number of caryopses, their size, water content or not…

Response: Yield assessment was performed on ten plants collected from each plot for each replicate. The total number of plants measured for each hybrid was 30. This information was added in the revised version of the manuscript. Differences between hybrids in yield were statistically significant. To emphasize that these are "statistically" significant differences, the manuscript has been supplemented with this term. We agree that hybrid yield can also be determined based on traits other than yield. As the reviewer mentioned, these can include kernel number, kernel size, and moisture content or lack thereof. However, the most important trait for breeders is seed yield, which is why this trait is given the greatest importance. In our study, we analyzed only hybrid yield because phenotypic data for this trait were obtained from plant breeding stations.

 

Point 4: 3 Although it is an interesting possibility, the putative relationship between yield and environment is totally un-sustained: 1/As apparently the crossings were grown in the same place (?), i.e. the same environment (?), how can the authors pretend this relationship? 2/Precise the use of figure 5; 3/ In other articles yield can be related with other factors.

Response: We agree with the reviewer that the use of the term "genotype-environment interaction" was excessive. This overinterpretation resulted from observing different phenotypic and genetic groupings. The manuscript has been amended to remove the reference to the "genotype-environment interaction." In fact, meteorological conditions were described in the manuscript text. At least for several months of the growing season. We decided to include a figure containing this data because: (1) a graphical presentation of the data is more convincing to most people, and (2) the figure presents data for all months of the growing season. We agree with the opinion that yield may be related to other factors, as mentioned by other authors. In this study, we considered only (or at least as much as) the dependence of yield on molecular markers. Other factors may influence yield. However, factors such as fertilization and similar factors were equal for all hybrids.

 

Point 5: 4 This text is not clear at all on the useful application of this study for agronomists: finally, which are the best sequencies for a high yield quantity?

Response: I agree with the esteemed reviewer that we should include guidelines for breeders regarding which genes can be selected for and which genes are most important. Our research shows that the SilicoDArT marker (Clone ID 24028032), located near (809 bp) the chain acyl-coA synthetase (LACS) gene, can be used to select high-yielding genotypes because literature reports indicate that this gene may influence maize yield. LACSs, which convert free FAs into fatty acyl-CoA thioesters, play crucial roles in FA catabolism, lipid synthesis and storage, epidermal wax synthesis, and stress tolerance.  In Arabidopsis, more than three LACS proteins have been shown to be involved in keratin and epidermal wax production. Furthermore, the biochemical homeostasis of epidermal wax in Arabidopsis can enhance plant tolerance to water deficiency, salt stress, drought stress, and ABA treatment [71-73]. These studies strongly suggest that LACS proteins play a vital role in responding to environmental stress via regulating wax biosynthesis. In maize research  levels across diverse tissues demonstrated that the majority of LACS genes exhibited the highest expression in the meiotic tassel. The high demand for LACS activities in floral tissues is linked to the strong expression of other lipid metabolic genes in flowers, indicating their involvement in active FA metabolism during meiotic tassel development. Additionally, all LACS genes showed elevated expression levels during seed development at suggesting their potential role in early FA accumulation in maize seeds. This information is posted in the Discussion.

 

Point 6: 5 The discussion seems not connected to the results, it does not take (at all) in account the figures of the results.

Response: We have improved the manuscript discussion by referencing relevant figures and tables.

 

Point 7: 6 References already taken in account by the authors are of interest, however checking in the web of science and scilit from mdpi, other studies should be updated and used (if relevant) in order to sustain much more and provide a larger view of this article. Among papers are the followings (please check for more):

Qi, X.; Zhao, X.; Lyu, M.; Wu, K.; Liu, N.; Li, R.H.; Xu, C.; Shen, R.Y.; Zhou, Z.H.; Wang, C.; et al. Efficient transgenic maize (Zea Mays L.) detection using low-depth next-generation sequencing. Sci Rep-Uk 2025, 15, doi:10.1038/s41598-025-18593-8.

Tofa, A.I.; Chiezey, U.F.; Babaji, B.A.; Kamara, A.Y.; Adnan, A.A.; Beah, A.; Adam, A.M. Modeling Planting-Date Effects on Intermediate-Maturing Maize in Contrasting Environments in the Nigerian Savanna: An Application of DSSAT Model. Agronomy-Basel 2020, 10, doi:10.3390/agronomy10060871.

Yang, D.G.; Gao, Z.F.; Liu, Y.Q.; Li, Q.; Yang, J.J.; Wang, Y.B.; Wang, M.Y.; Xie, T.L.; Zhang, M.; Sun, H. Exogenous application of 5-NGS increased osmotic stress resistance by improving leaf photosynthetic physiology and antioxidant capacity in maize. Peerj 2024, 12, doi:10.7717/peerj.17474.

Response: We sincerely appreciate your recommendations for three specific publications. After a thorough analysis, we will certainly use them in our considerations for future research. Tofa et al. (2020) used seasonal analysis to examine the effect of variable sowing windows on grain yield of mid-maturing maize in three agroecologies. The authors studied maize based solely on phenotypic observations. These are very interesting studies, although different from those presented in our manuscript. In their study, Liu et al. (2023) used a similar model to ours. It should be noted, however, that their focus was on the tomato. Although they obtained equally interesting results that may be useful in selection efforts, these species are so different that direct comparisons could only be made at a methodological level. Yang et al. (2024) investigated the effect of 5-NGS on the growth and tolerance of maize seedlings to osmotic stress. This is a very interesting study. Since we are also conducting research on the resistance and telangiectasia of maize hybrids, the results presented in this publication will be inspiring for us in the context of applying additional types of stress.

 

Minor points are:

Point 8: 1 Indicate the meaning of the colors in figures 2-3.

Response: An additional explanation of the colors has been added to the captions under Figures 2 and 3: "Individual colors correspond to six groups of phenotypic similarity of hybrids determined on the basis of yield." and "Individual colors correspond to six groups of hybrid phenotypic similarity determined based on yield (see Figure 2)."

 

Point 9: 2 For figure 4, indicate in a caption the meaning of “chr” in full letters.

Response: Explanation added: chr – chromosome.

 

Point 10: 3 For tables 1-2, indicate in a caption the meaning of Chr ID LOD in full letters.

Response: An explanation of the abbreviations used in the captions of Tables 1-3 has been added.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript combined with re-sequencing, GWAS, and candidate gene prediction to concentrate on the maize yield, and those results are of innovation and significance for maize studies, while some minor modifications must be put forward first.

(1) How could the authors chose the hybrid population ? Did you depend on the standard while we could not only perform the research based on their normal distribution.

(2) Normally, the yield traits are comporsed of number of rows of panicle, number of grains per row, panicle length, panicle diameter, shaft diameter, panicle weight, weight per hundred grains and bulk density, while we never found the specific trait in this study.

(3) The tables should be presented with three-wire format.

(4) We noticed that the authors identify some significant loci, which should be combined with GO and KEGG enrichment analyses for predicting the candidate genes. Why not add some functional prediction figures in this study ?

(5) We suggest to add some verifying experiments, such as the VIGS and qRT-PCR on the candidate genes, or utilize the Markers into the other public population.

(6) The references were lack of issue numbers.

Author Response

Response to Reviewer 3 Comments

 

Reviewer #3

Point 1: This manuscript combined with re-sequencing, GWAS, and candidate gene prediction to concentrate on the maize yield, and those results are of innovation and significance for maize studies, while some minor modifications must be put forward first.

Response: Thank you very much for the positive reception of our manuscript. We also appreciate all your comments. We have revised the manuscript based on your suggestions. Below are our responses to each comment.

 

Point 2: (1) How could the authors chose the hybrid population ? Did you depend on the standard while we could not only perform the research based on their normal distribution.

Response: Hybrids were created by crossing inbred lines of different origins and characterized by extreme yield values in order to obtain diversified offspring. Due to the trade secret of the breeding company, we had no information regarding which specific parental forms were used to obtain the tested hybrids.

 

Point 3: (2) Normally, the yield traits are comporsed of number of rows of panicle, number of grains per row, panicle length, panicle diameter, shaft diameter, panicle weight, weight per hundred grains and bulk density, while we never found the specific trait in this study.

Response: We agree that hybrid yield can also be determined based on traits other than yield. As the reviewer mentioned, these can include number of rows of panicle, number of grains per row, panicle length, panicle diameter, shaft diameter, panicle weight, weight per hundred grains and bulk density. However, the most important trait for breeders is seed yield, which is why this trait is given the greatest importance. In our study, we analyzed only hybrid yield because phenotypic data for this trait were obtained from plant breeding stations.

 

Point 4: (3) The tables should be presented with three-wire format.

Response: Corrected Tables 1 and 2 to three-wire format.

 

Point 5: (4) We noticed that the authors identify some significant loci, which should be combined with GO and KEGG enrichment analyses for predicting the candidate genes. Why not add some functional prediction figures in this study ?

Response: We have supplemented the manuscript by adding the results of the Gene Ontology analysis.

 

Point 6: (5) We suggest to add some verifying experiments, such as the VIGS and qRT-PCR on the candidate genes, or utilize the Markers into the other public population.

Response: We are currently working on a follow-up publication in which we analyzed the expression of all candidate genes using qPCR and analyzed the most important metabolites produced by these genes using mass spectrometry. Due to the clarity of the results and the large number of analyses, we did not want to include all of this in a single manuscript. Of course, we will cite this article in our next publication, emphasizing that this is a continuation of our research.

 

Point 7: (6) The references were lack of issue numbers.

Response: We have supplemented the references with issue numbers.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

In the submitted manuscript, the authors identified molecular markers associated with the yield of 122 maize hybrids.

Major comments.

The yield markers were identified based on data from only one year. This is completely insufficient, as yield of crops is highly dependent on environmental factors and management. Yield may be completely different next year.

 

The Introduction is very extensive and should be shortened. For example, L.94-107, as the manuscript does not compare different NGS technologies; L.189-203, as it does not compare different statistical methods.

L.289-290. "Two SilicoDArT markers (7306 and 2531)…"; L.291. "Marker SilicoDArT 3201…". Are these markers among the 10 markers selected for further study? If so, are they not included in Tables 2 and 3. If not, why are they mentioned among tens of thousands of other markers?

L.332-333. “… sequences of more than 100 plant species have been reported [63,64].” Refs 63 (2008) and 64 (2015) are significantly outdated. The genomes of several thousand plant species have now been sequenced.

Subsection 4.1. It is advisable to shorten the description of hybrid names: instead of listing G01.01, G01.02, …, G01.21, it is sufficient to indicate G01.01-01.21.

Subsection 4.2. What was the duration of the experiment from sowing to harvest? What were the growing conditions: planting density, fertilization, irrigation, etc.?

L.390-392. “The hybrids were developed through crosses between inbred lines of diverse genetic origin, which were comprehensively characterized by Sobiech et al. [78].” Ref. 78 presents data only on resistance to Fusarium. Which of the 186 inbred lines were used to produce the hybrids?

Author Response

Response to Reviewer 4 Comments

 

Reviewer #4

Point 1: The yield markers were identified based on data from only one year. This is completely insufficient, as yield of crops is highly dependent on environmental factors and management. Yield may be completely different next year.

Response: We agree with the reviewer that plant yields are highly dependent on environmental factors and management. Yields may vary significantly from year to year. However, the identified yield-determining markers presented in our manuscript can be considered specific to specific environmental conditions and can be generalized to environments with similar conditions. To select universal markers (independent of environmental conditions), studies should be conducted in various environments (years, locations). The results presented in this manuscript were used to select plant materials for further research by plant breeding. In this regard, these results were sufficient.

 

Point 2: The Introduction is very extensive and should be shortened. For example, L.94-107, as the manuscript does not compare different NGS technologies; L.189-203, as it does not compare different statistical methods.

Response: We have shortened the Introduction by removing the two highlighted sections. We have also removed other sentences that were in the first draft of the manuscript.

 

Point 3: L.289-290. "Two SilicoDArT markers (7306 and 2531)…"; L.291. "Marker SilicoDArT 3201…". Are these markers among the 10 markers selected for further study? If so, are they not included in Tables 2 and 3. If not, why are they mentioned among tens of thousands of other markers?

Response: The first version of the manuscript incorrectly provided the working marker numbers. The revised version provides the correct marker numbers: 4593047, 9693261, and 24028032, respectively.

 

Point 4: L.332-333. “… sequences of more than 100 plant species have been reported [63,64].” Refs 63 (2008) and 64 (2015) are significantly outdated. The genomes of several thousand plant species have now been sequenced.

Response: I agree with the esteemed reviewer that the data on sequenced plant genomes was outdated. The latest information on this topic was cited. “In 2024, the genomes of 500 plant species were published, including 370 sequenced for the first time. Tracking and sharing published plant genomes (currently covering over 1,800 species) is an invaluable service for plant researchers.” Schwacke, R.; Bolger, M.E.; Usadel, B. PubPlant – a continuously updated online resource for sequenced and published plant genomes. Front. Plant Sci. 2025, 16, 1603547. https://doi.org/10.3389/fpls.2025.1603547.

 

Point 5: Subsection 4.1. It is advisable to shorten the description of hybrid names: instead of listing G01.01, G01.02, …, G01.21, it is sufficient to indicate G01.01-01.21.

Response: The description of hybrid names has been shortened as suggested.

 

Point 6: Subsection 4.2. What was the duration of the experiment from sowing to harvest? What were the growing conditions: planting density, fertilization, irrigation, etc.?

Response: The description of the experimental conditions is given in the methodology: Maize is a plant with a long growing season, typically lasting 140 to 180 days in Polish climatic conditions. Maize was sown on April 25th (when the soil temperature at a depth of 5–10 cm reached 10°C). Harvest occurred on October 15th (at full maturity, with grain moisture below 30%). The sowing density was 80,000 plants/ha. The sowing depth was 5 cm. Regarding fertilization, maize has very high nutrient requirements. Nitrogen (150 kg/h) was applied in two doses: pre-sowing and top-dressing (up to the 4–6 leaf stage). Phosphorus was applied at 80 kg/ha, and potassium at 150 kg/ha. In terms of irrigation and water conditions, maize manages water efficiently (low transpiration rate), but due to its enormous biomass, it requires significant amounts of water during critical phases: flowering and intensive growth (July–August). Sprinkler irrigation was used as needed, maintaining soil moisture at 60–80% of field water capacity.

 

Point 7: L.390-392. “The hybrids were developed through crosses between inbred lines of diverse genetic origin, which were comprehensively characterized by Sobiech et al. [78].” Ref. 78 presents data only on resistance to Fusarium. Which of the 186 inbred lines were used to produce the hybrids?

Response: All hybrids that yielded a yield were included in the study. Unfortunately, some hybrids were excluded from further plant breeding studies.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, 

I read this new version, however still important points have to be clarified: as your replies are quite clear, they should appear in the text in appropriate parts, the text being quite obscure in some parts.

 

• For my major point 1, as clearly written by the authors (“The fact that genetic grouping is not the same as phenotypic grouping “), the discussion is still very unclear (at the end of the discussion, the analogy with Arabidopsis genes is somewhat conflictious as it is not the same plant family, be very cautious with these “easy” assimilations) as not enough developed; it is the same for the abstract and conclusions.
•  
• For my major point 2, “However, the most important trait for breeders is seed yield” needs more scientific details as new significant characters are always welcome; moreover my sub-point 2/ (As stated in the abstract I cannot see the "significant differences among hybrids in terms of yield") is still unclear (precise the correspondence between figure 1 (1 groups) and figure 2 (6 groups).
•  
• For my major point 3, environment still appears in several parts where it is very ambiguous as your results do not show this relationship at all (?), so just keep it for former references. Please consider that the objectivity of a paper makes its quality.

Author Response

Response to Reviewer 2 Comments

 

Reviewer #2

Point 1: I read this new version, however still important points have to be clarified: as your replies are quite clear, they should appear in the text in appropriate parts, the text being quite obscure in some parts.

Response: We sincerely thank you for your positive feedback on the revised manuscript. The current version has been supplemented with sections that previously served only as clarifications to individual comments. We hope that the manuscript is now sufficiently clear.

 

Point 2: For my major point 1, as clearly written by the authors (“The fact that genetic grouping is not the same as phenotypic grouping “), the discussion is still very unclear (at the end of the discussion, the analogy with Arabidopsis genes is somewhat conflictious as it is not the same plant family, be very cautious with these “easy” assimilations) as not enough developed; it is the same for the abstract and conclusions.

Response: We thank the Reviewer for this important and constructive comment. We agree that the previous version of the Discussion may have suggested an overly direct functional analogy between Arabidopsis and maize genes. In the revised manuscript, we substantially revised the Discussion, Abstract, and Conclusions to provide a more cautious interpretation of the association mapping results. In particular, we clarified that the identified marker–trait associations represent statistical relationships and do not demonstrate direct causality. We also emphasized that findings reported for Arabidopsis are presented only as supportive background information regarding possible biological functions of LACS genes and should not be interpreted as direct evidence for maize. Additionally, we expanded the discussion of maize-specific studies related to LACS gene expression and highlighted that the identified genomic regions require further functional and multi-environment validation before practical implementation in breeding programs. We also strengthened the discussion concerning the discrepancy between genetic grouping and phenotypic performance, emphasizing the complex polygenic nature of maize yield. These modifications improved the clarity and biological interpretation of the manuscript.

 

Point 3: For my major point 2, “However, the most important trait for breeders is seed yield” needs more scientific details as new significant characters are always welcome; moreover my sub-point 2/ (As stated in the abstract I cannot see the "significant differences among hybrids in terms of yield") is still unclear (precise the correspondence between figure 1 (1 groups) and figure 2 (6 groups).

Response: We agree with the Reviewer that traits other than yield are also interesting and always welcome. These include those related to yield structure and resistance. However, breeders are interested in genotypes characterized by the highest yield. These genotypes have the greatest chance of being registered and, at the same time, achieving commercial success among farmers. There have been cases of varieties being registered that stood out for certain traits (e.g., very low glucosinolate levels in rapeseed), but at the expense of low yields. This ultimately led to a lack of interest among farmers. Therefore, breeding stations primarily focus on high-yielding genotypes. Because we received the data from a breeding company, the result they were interested in was association mapping by yield, and we only received data on yield.

We have revised the abstract to further emphasize that the significant differences in hybrid yield were the result of the analysis of variance. The results of this differentiation are presented in Figure 2. To conduct an analysis of variance, the assumptions of normality and homogeneity of variance must be met. Figure 1 shows the distribution of mean yields for maize hybrids. It can be seen (and was also confirmed by appropriate statistical tests) that the empirical distribution follows a normal distribution. This allowed us to conduct an analysis of variance. As mentioned above, the result of the analysis of variance was a statistically significant (p<0.001) differentiation between hybrids in terms of yield. This result is a necessary condition for conducting association mapping.

 

Point 4: For my major point 3, environment still appears in several parts where it is very ambiguous as your results do not show this relationship at all (?), so just keep it for former references. Please consider that the objectivity of a paper makes its quality.

Response: We have revised the manuscript to remove any information that might suggest an environmental influence on the results. We agree with the Reviewer that the term 'environment' can be very ambiguous when the presented results do not demonstrate any environmental dependence at all.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

All the incorrect portions have been modified in this manuscript, and we believe this revised has met the standard for publication.

Author Response

Response to Reviewer 3 Comments

 

Reviewer #3

Point 1: All the incorrect portions have been modified in this manuscript, and we believe this revised has met the standard for publication.

Response: We would like to thank you very much for your positive assessment of the revised version of the manuscript.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been significantly improved. The origin of 122 maize hybrids (index G) must be indicated. Which inbred lines (indices K and S; Sobieh et al., 2022) were used for hybridization?

Author Response

Response to Reviewer 4 Comments

 

Reviewer #4

Point 1: The manuscript has been significantly improved. The origin of 122 maize hybrids (index G) must be indicated. Which inbred lines (indices K and S; Sobieh et al., 2022) were used for hybridization?

Response: The plant material used in this study was obtained from Plant Breeding Smolice Ltd. (Kobylin, Poland), belonging to the Plant Breeding and Acclimatization Institute – National Research Institute Group. These are hybrids designated with the symbol S in the publication by Sobiech et al. (2022). All genotypes that yielded were included in the study. Unfortunately, it was not possible to analyze all the genotypes presented in the publication by Sobiech et al. (2022), which we deeply regret. Due to internal Plant Breeding Smolice decisions, we had to code the hybrids with new symbols (G).

 

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Good afternoon,

I read this new version, however few point has to be considered:

• In the abstract “however, their usefulness requires further validation” should be more positive including some words like “these first positive results” or something like this.

 

• Put the authors for all species and subspecies plant latin names (Zea mays parviglumis, Cajanus cajan… please check for eventual other ones) as it is requested in Taxonomy (use International Plant Names Index www.ipni.org or equivalent).

Author Response

Response to Reviewer 2 Comments

 

Reviewer #2

Point 1: In the abstract “however, their usefulness requires further validation” should be more positive including some words like “these first positive results” or something like this.

Response: We sincerely thank you for your attention and for shifting the wording to a more positive one in the context of the results obtained. We have corrected the sentence in question, changing it to: "These are some of the first positive results."

 

Point 2: Put the authors for all species and subspecies plant latin names (Zea mays parviglumis, Cajanus cajan… please check for eventual other ones) as it is requested in Taxonomy (use International Plant Names Index www.ipni.org or equivalent).

Response: Authors are provided for the names of all plant species and subspecies mentioned in the manuscript. These are: Zea mays subsp. parviglumis H.H. Iltis & Doebley; Cajanus cajan (L.) Millsp.; Arabidopsis thaliana (L.) Heynh.

 

Author Response File: Author Response.pdf