PONE-D-25-63384 High-density Single Nucleotide Polymorphism markers analysis reveals the genetic diversity and population structure in tropical highland maize (Zea mays L.) inbred lines PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

Reviewer #5: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Lines 127-129 can be elaborated for development of the inbred lines mentioning the hybridization sources and selfed generations.

Lines 131-132 needs elaboration as to what adaptation and disease resistance strategies were adopted for evaluation of inbred lines

Reviewer #2: The work is fundamental and presentation is fine. but authors should give clear high resolution figures. the abstrct shouldbe more informative.If possible the gst and rst value may be added.authors should see if the SNPS can be associated with specific trait. It may be included in discussion.

Reviewer #3: This study presents a comprehensive genetic analysis of 93 tropical highland maize inbred lines using high-density SNP markers. The authors assess genetic diversity, population structure, and linkage disequilibrium, offering valuable insights for maize breeding programs in East African highlands. The manuscript is well-structured and addresses an important topic in crop improvement. However, authors

The authors state that data will be available upon request. However there are major concerns:

1. The LD decay analysis mentions a window size of 50 bp—this seems unusually small for maize. Please confirm and clarify whether this refers to a sliding-window step or to something else.

2. The STRUCTURE analysis used K = 1–10 with 10,000 burn-in and 50,000 MCMC iterations. These are acceptable but relatively low for complex datasets. Consider justifying or noting this as a limitation.

3. The PCA explains only ~8.8% of the total variation (PC1 + PC2). While this is common in highly diverse panels, it may be worth discussing the limitations of PCA in capturing complex structure and the value of complementary methods like STRUCTURE and NJ trees.

4. Table 3 shows chromosome 6 with 0 significant LD pairs—this is unusual and may warrant a check or explanation (e.g., low marker density, filtering artifacts).

5. Please discuss why population 4 might have lower diversity. Could it be due to selection history or genetic drift? What are the practical implications of the LD decay rate for GWAS resolution in this panel? Additionally, consider more explicitly comparing your findings with those from other highland maize studies in Africa or similar environments.

6. Per journal requirement, authors must deposit the SNP data in a public repository (e.g., Figshare, Dryad, or a genomic database) and provide the accession number in the manuscript.

Additionally, some typos must be corrected: Figures are generally clear, but ensure all axes are labeled with units (e.g., Fig 4: Distance in bp). Table 3: The column header "Pr²" is unclear: should it be "Mean r²"? Table S2 (Nm values) is mentioned but not fully described in the text; ensure that all supplementary files are referenced and explained.

In Abstract: The sentence beginning "11,203 single nucleotide polymorphism (SNP) markers genotyped..." is missing a verb (e.g., "were used"). Revise for clarity.

Keywords: Consider adding "GWAS" or "Genomic selection" if relevant to future applications.

Line 185: (additional file1) must be (Additional File S1), and accordingly, the Supporting Information section must be corrected too.

Line 256: "Cluster 1 contained the largest germplasm (54 lines, 58%)" – rephrase to "largest number of lines" or "most lines".

Line 283: "The assessment of genetic diversity..." – this sentence is a general statement; consider moving to the Introduction or shortening.

Technical Corrections

Check for inconsistent spacing (e.g., "p< 0.01" should be "p < 0.01").

Use italic formatting for p-values and FST consistently.

Table 5: The diagonal values (0.001, 0.001, 0.009) are listed under "Sub-populations 1" column, this may confuse readers. Use a standard format with FST above diagonal and genetic distance below.

Reviewer #4: This manuscript presents a comprehensive SNP-based analysis of genetic diversity, population structure, and linkage disequilibrium in tropical highland maize inbred lines. The topic is scientifically relevant and important for maize breeding programs, especially in sub-Saharan Africa. The dataset is substantial, and the analytical approaches are appropriate. However, the manuscript requires major revision to meet PLOS ONE publication standards, particularly in language quality, methodological clarity, data availability, and interpretation of results.

Major Strengths

Scientific relevance:

The study addresses a significant knowledge gap in the genetic characterization of tropical highland maize germplasm, which is important for breeding, conservation, and climate-resilient crop development.

High-quality dataset:

The use of over 11,000 high-quality SNP markers across 93 inbred lines provides robust genomic resolution for population genetic analysis.

Comprehensive analytical framework:

The authors employ multiple complementary analyses, including genetic diversity indices, STRUCTURE clustering, PCA, phylogenetic analysis, AMOVA, and LD decay analysis, which together strengthen the study.

Breeding implications:

The manuscript provides potentially valuable insights for heterotic grouping and parental selection in maize hybrid development.

Major Concerns and Required Revisions

1. Language and Writing Quality (Major Issue)

The manuscript contains numerous grammatical, typographical, and syntactic errors that substantially impair readability and clarity. Professional English language editing is strongly recommended. Several sentences are poorly constructed, repetitive, or ambiguous.

2. Methodological Clarity and Justification

SNP filtering thresholds:

The authors applied several filtering criteria (PIC < 0.1, MAF < 0.05, heterozygosity < 0.02, missing data > 30%), but no justification or citation is provided. These thresholds should be supported with references or methodological rationale.

LD window size:

The manuscript reports an LD window size of 50 bp, which is biologically unrealistic for maize population studies. This likely reflects either a typographical or analytical error. The authors should clarify the actual window size used and revise accordingly.

STRUCTURE parameters:

The burn-in and MCMC iterations appear relatively low for population genetic inference. Justification for these choices should be provided, or additional runs should be performed to ensure robust inference.

3. Interpretation of Linkage Disequilibrium Results

The reported LD decay (~3.33 Mb) is considerably slower than expected for maize and is inconsistent with many previous studies. This may reflect population stratification, recent selection bottlenecks, or marker filtering bias. The authors should compare their findings with published maize LD studies and provide a biological explanation for the unusually slow decay.

4. Population Structure Interpretation

Although three genetic clusters are identified, the biological and breeding significance of these clusters is not adequately discussed. The authors should strengthen interpretation by linking population structure results to pedigree background, geographic origin, adaptation patterns, and implications for heterotic grouping.

5. FST Inconsistencies

The manuscript reports contradictory FST values in different sections. While the text mentions values as high as 0.716, the tables show FST values below 0.01. This discrepancy must be corrected, and population differentiation should be interpreted accordingly.

6. Data Availability (Mandatory Revision)

The current Data Availability Statement does not comply with PLOS ONE policy. All genotype datasets and supporting data must be deposited in a publicly accessible repository, and accession numbers or DOIs must be provided.

Minor Issues:

Several figures require improved resolution and clearer legends.

Some tables contain formatting inconsistencies.

Citations should be updated and formatted consistently.

Reviewer #5: The manuscript tilted “High-density Single Nucleotide Polymorphism markers analysis reveals the genetic diversity and population structure in tropical highland maize (Zea mays L.) Inbred lines” is well written scientifically and analytically. Also, it contributes to the understanding of tropical highland maize germplasm for improvement. The manuscript could benefit more by using some of the comments in the attachment

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Ibrokhim Y. Abdurakhmonov

Reviewer #4: No

Reviewer #5: Yes: Idris Ishola Adejumobi (PhD)

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<p>High-density Single Nucleotide Polymorphism markers analysis reveals the genetic diversity and population structure in tropical highland maize (Zea mays L.) inbred lines

PONE-D-25-63384R1

Dear Dr. Gebre,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Dragan Perovic, Ph.D

Academic Editor

PLOS One

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