Review Reports - The High-Amylose Maize Line GEMS-0067 Is Associated with an Intron 8 Insertion in Sbe1a, Altered Native PAGE Zymogram Patterns, and Development of PCR Markers for Breeding Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “The High-Amylose Maize Line GEMS-0067 Is Associated with an Intron 8 Insertion in Sbe1a, Altered Native PAGE Zymogram Patterns, and Development of PCR Markers for Breeding Applications” addresses a useful breeding problem and has a potentially valuable marker, but the association, functional interpretation, and marker validation need to be strengthened before consideration.

Summary of the Manuscript

The manuscript describes a targeted survey of the Sbe1a locus in the high-amylose maize line GEMS-0067. The authors identify an 8-bp polymorphism in intron 8, consisting of a 6-bp TACATA insertion plus a downstream 2-bp GA insertion, which is present in GEMS-0067 and Guatemala 209 but absent from 33 surveyed USDA-GEM releases. They also develop RsaI- and MslI-based CAPS markers, with the MslI assay allowing codominant discrimination of wild-type, mutant, and heterozygous individuals. Native PAGE zymograms show an altered migration pattern in the putative SBE1a region of GEMS-0067. The breeding motivation is clear, and the marker has practical potential for marker-assisted selection.

However, the study remains largely descriptive and associative. The central weakness is that the intron 8 polymorphism is not formally shown to co-segregate with the high-amylose modifier effect in a relevant ae-fixed population, nor is it demonstrated to be causal. The native PAGE evidence is suggestive but does not identify SBE1a or distinguish among splicing, protein structure, and protein-complex effects. Several figures, captions, enzyme names, and gene/protein nomenclatures are inconsistent, and the discrepancy with Gyawali’s earlier report is noted but not resolved. Overall, the work is promising but requires significant revision before its conclusions can be considered robust.

Constructive Feedback for Authors

Strengthen the genotype–phenotype association.
The manuscript repeatedly states that the intron 8 insertion is “associated” with the high-amylose modifier background, but the evidence is mostly presence/absence in a small set of lines. The authors should test the marker in a segregating population where the ae mutation is fixed and amylose content varies due to modifier loci. A chi-square test of marker segregation, correlation/regression between marker genotype and amylose percentage, and/or QTL interval analysis would substantially strengthen the claim. Without this, the marker could simply be linked to the GEMS-0067/Guatemala 209 background rather than the trait itself.
Clarify whether the insertion is causal or merely linked.
The title and discussion imply a functional link between the intron 8 insertion and the high-amylose phenotype. The authors should avoid overclaiming. If causality is not tested, describe the polymorphism as a linked marker. To move toward causality, RT-PCR/cDNA sequencing should be used to test for altered splicing, and the exact intron 8/exon 9 boundary effect should be shown. The 2-bp GA insertion also needs a clearer explanation: is it functionally relevant, or simply co-inherited with the TACATA insertion?
Fix figure, caption, and nomenclature problems.
There are numerous presentation issues: “GEMS-67” vs. “GEMS-0067,” “Sbe1a” vs. “SBE1a” vs. “SBEIa,” “Rsal” vs. “RsaI,” etc...”

Figure numbering and captions are confusing, and some figures appear misplaced or duplicated. Line 98 cites figure 1, while line 105 cite figure 3 directly. The figure 2 was cited by author’s later in line 235 and line 237, please correct cite in sequence.

Figure 2B’s sequence alignment uses dots and asterisks that are not explained in the legends. Supplemental Table 55/56 typos should be corrected. These are not just cosmetic; they affect reproducibility.

Line 276 (Figure 9): Substantiate the native PAGE zymogram results.
The altered migration band is described as “putative SBE1a,” but no immunoblot, mass spectrometry, or specific activity assay confirms its identity. The authors should add SBE1a-specific immunoblotting after native PAGE, or at least use a more specific substrate/inhibitor system. Replication details are also insufficient: report the number of biological replicates, migration distances with means ± SD, and statistical comparisons. The current claim that the band “displayed altered migration” is interesting but remains preliminary.
Resolve the Gyawali discrepancy and germplasm heterogeneity.
The authors note that Gyawali reported the TACATA insertion in NC350 but not in GEMS-0067. This is a significant inconsistency. The authors should sequence multiple GEMS-0067 and NC350 plants from independent seed sources, verify sample identity, and discuss possible explanations such as seed mixing, heterozygosity, sequencing error, or a different GEMS-0067 subline. Since Guatemala 209 is an open-pollinated accession, the authors should also confirm that multiple individuals carry the same insertion.

The Discussion currently lacks depth on modern maize QTL mapping, functional-allele interpretation, and translation to breeding; the authors should cite and discuss: (1) Zhou Yu et al. (2026) Title: “Strategies in Quantitative Trait Locus Mapping for Complex Traits in Maize,”. This paper directly relevant to the unresolved 5S modifier QTL and marker–trait association; (2) Zhu Lin et al. (2026) Title: “Insights into the Regulatory Pattern Classification for Functional Alleles Underlying Trait Variation in Maize and Other Crops”, relevant to interpreting intronic/non-coding variation as functional alleles; and (3) Hu Weijuan et al. (2026) Title: “Artificial intelligence and digital phenotyping shape the future of plant breeding: evolution, challenges, and a roadmap from AI breeding 1.0 to 2.0”, relevant to positioning the CAPS marker within contemporary marker-assisted selection and digital breeding workflows.

Add statistics, replication, and methodological detail.
The amylose colorimetric assay, PCR-CAPS scoring, and zymogram analyses need clearer replication and statistical treatment. For amylose, report technical and biological replicates, standard curve performance, and statistical comparisons among genotypes. For CAPS, report whether scoring was blinded and whether ambiguous bands were sequenced. For native PAGE, include protein loading controls and, if possible, molecular weight markers or reference bands.
Revise conclusions to match the evidence.
The conclusion that the marker “provides a practical tool for marker-assisted breeding” is reasonable, but it should be qualified as requiring further validation. The statement that the results “establish a novel gene-based marker associated with the GEMS-0067 high-amylose modifier background” is acceptable if “associated” is emphasized. The speculative statements about altered pre-mRNA processing, protein structure, or complex assembly should remain clearly labeled as hypotheses for future work.

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, a novel 8 bp insertion polymorphism on intron 8 of the Sbe1a gene in the high amyloid maize germplasm gem-0067 was systematically identified, and a codominant PCR-CAPS labeling system was established for label-assisted selection.The experimental design is reasonable, the methodological approach is clear, and the research results have practical value for the breeding of high-amylose corn.However, there are several aspects that need improvement in terms of mechanical interpretation, data rigor and representation consistency.Specific comments are as follows:

There are SNP markers on exons of the same gene. Currently, there are many methods for detecting SNP markers that are both effective and inexpensive. I wonder if the marker developed in this study has a similar effect to the detection of SNP markers? Is intron 8 insertion completely linkage imbalanced with these exon SNPS? If so, the value of the new tag mainly lies in the convenience of detection, and the "novel" in the title/abstract must be weakened.
Since the authors state that this marker is associated with the high-amylose trait, the description of banding patterns in the Results section should be accompanied by the corresponding amylose content data for each genotype analyzed—or at minimum, an indication of whether each line exhibits high or normal amylose levels.
Gyawali (2016) did not detect the TACATA insertion in his GEMS-0067 sample, whereas the present study detected the insertion in the authors' GEMS-0067 stock. The authors should clarify whether the GEMS-0067 seeds used in this study and those used by Gyawali were obtained from the same source. The difference likely reflects distinct sub-lines or residual
Figure 7 shows a label "H88ae" which looks like a typo; the rest of the manuscript consistently uses "H99ae." Please verify and correct this.
The terms "amlose" and "amylose" appear inconsistently throughout the manuscript. Standardize all instances to "amylose".
Line 447: "4.5 Restriction Fragment Length Polymorphism (CAPS) and Sequencing", the label name is incorrect. Please check.
References need to be uniform. For example, in reference 11, the names of the journals are not abbreviated, but all others are abbreviated. Please check.

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this manuscript entitled, “The High-Amylose Maize Line GEMS-0067 Is Associated with an Intron 8 Insertion in Sbe1a, Altered Native PAGE Zymogram Patterns, and Development of PCR Markers for Breeding Applications.” The study highlights an important subject in maize starch genetics and introduces a potentially valuable PCR-CAPS marker associated with high-amylose germplasms. The combination of sequence analysis, germplasm screening, marker development, and native PAGE analysis is a strength.

However, further clarity is required on marker validation, the association of the Sbe1a polymorphism with high amylose content, experimental replication, and data presentation. The following comments are suggested to further strengthen the manuscript.

The intron 8 insertion in Sbe1a is consistently linked to the tested GEMS-0067 material and its high-amylose derivatives. However, the available data do not support a direct causal link between this insertion and higher amylose content. Therefore, it is recommended that the manuscript include cautious phrasing such as "associated with," "linked to," or "candidate polymorphism," especially in the title, Abstract, and Conclusions sections. This will improve the accuracy of the claims in the manuscript.

The developed MslI PCR-CAPS marker shows potential for detecting the GEMS-0067-related allele. The link between this marker and amylose levels might be strengthened if genotype and amylose-content data from a segregating population were presented. This should provide the number of plants evaluated, their marker genotypes, amylose concentrations, and statistical comparisons between genotype classes.

GEMS-0067 has both the ae/sbe2b mutation and the proposed Sbe1a modifier allele. The authors should clarify whether breeders need to select for both genes to achieve high-amylose maize. They should also explain whether the sbe1a:gm67 marker alone can effectively identify high-amylose plants or if it works in conjunction with the ae mutation.

The altered zymogram band movement found in GEMS-0067 is unique; yet, native PAGE cannot clearly identify this band as SBE1a. As a result, it should be referred to as a "putative SBE1a region" unless validated using another approach. Furthermore, the discussion should proceed with caution when discussing the impacts on splicing, protein structure, or protein-complex formation, as these aspects were not directly examined.

The discussion section should be more based in previous studies by referencing relevant literature to contextualize the findings and show how they align with or differ from existing research. Additionally, the future prospects appear limited; the authors should elaborate on potential applications of their findings in breeding programs or genetic research to strengthen the manuscript's relevance.

The study contains interesting findings; however, the novelty should be more clearly described. To increase their effect in the field, the authors should specifically explain how their findings improve our understanding of the high-amylose phenotype and differentiate their work from earlier research.

The figures and manuscript formatting need enhancement. Specifically, gel images should feature clearer lane labels and molecular-size markers to enhance readability. Additionally, the authors should carefully check for placeholder text, ensure that figure cross-references are accurate, and confirm that the reference list is formatted correctly. These adjustments will improve the overall presentation of the manuscript.