The Research of New Natural Spontaneous Fertile Attention: Title Altered Hybrids (Aegilops trivialis Migusch. Et Chak) Using Laser Microscopy and Tandem Mass Spectrometry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates four putative spontaneous fertile Aegilops-Triticum hybrids using a combination of morphological characterization, gliadin protein profiling, laser microscopy, and HPLC-ion trap-MS/MS analysis. The topic has potential value for wheat genetic resources and pre-breeding research.

However, the manuscript is hard to acceptance in current form. The study remains largely descriptive and does not provide sufficient molecular or genetic evidence to support its central claims. The most important weaknesses include insufficient confirmation of hybrid identity, lack of rigorous quantitative analysis, limited mechanistic or molecular insight, overinterpretation of descriptive observations, and formatting problems.

Although the manuscript includes mass spectrometry and microscopy, the scientific contribution remains mainly descriptive. The paper does not sufficiently advance a clear molecular question or provide mechanistic insight. I do not believe this manuscript is suitable for IJMS.

Author Response

Dear Reviewer,

Thank you for your thorough analysis and constructive recommendations. We highly appreciate your contribution to improving our work.

In response to your comments, we would like to note that our article has been significantly revised to incorporate the improvements you suggested. We have added the results of modern statistical analysis methods, including PCA analysis, which has allowed us to provide a more comprehensive understanding of the subject matter.

You expressed a desire to see more of a biotechnological aspect in the study. However, it should be noted that the main goal of this work was to demonstrate the hybrid nature of a unique plant form that was identified in natural conditions. We aimed to show the hereditary nature of this form, which has the potential for further study using modern biotechnological and molecular genetic methods.

Our article focused on the hybrid origin of this plant, which was isolated in natural conditions and successfully propagated in the field for almost ten years. As a result, a hybrid was obtained that produces full-fledged grain, which is confirmed by spectral analysis and mass-spectrometric studies. These data indicate the hybrid origin of the plant and the presence of wheat components in its genome.

We understand that our research is a significant step in the study of hybrid plant forms. The global scientific literature describes first-generation hybrids that were obtained naturally, but they were typically sterile in subsequent cross-breeding, indicating their genetic instability. In contrast, our work demonstrates the possibility of obtaining a stable hybrid form with human participation and long-term selection.

Further research will be aimed at clarifying the embryological nature of this plant, its chromosomal composition and genetic characteristics. We plan to use modern methods such as GISH and FISH technologies, as well as cyto-embryogenetic approaches, to study in more detail the origin and stability of this unique hybrid form.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the study conducted a search and examination of new hybrids of Aegilops and wheat to identify new genetic material for the possible selection of wheat, improving its traits of resistance to abiotic and biotic environmental factors and nutritional value. Important and priority data have been obtained, describing the morphological and biochemical characteristics of the new hybrids of Aegilops and wheat. As a result of reviewing the work, minor questions and comments have arisen:

1. In the Abstract section, only the obtained results should be described; there is no need to list future plans: «These spontaneous Aegilops-wheat hybrids will be used in further work to identify their paternal form. It should be noted that it is advisable to use the studied spontaneous Aegilops-wheat hybrids in future breeding to expand the gene pool of the genus Triticum L. and to obtain new heterogeneous forms». Further prospects for continuing the research can be presented in the discussion section.
2. There are typos in the text: in the Abstract section, the word Triticum in the first paragraph should be written entirely in italics; in section 2.3 – a typo in the word subcomponent 71,
3. It is necessary to translate the captions on the figures and to the figures into English (Figures 1, 3, 4 – Uzbekistan, Figures 2, 3, 4 – caption "scales").
4. In Figures 1-6, some of the provided images are misaligned.
5. Is it possible to provide a more detailed description of the protein spectrum in the results section 2.1? And also the description of the grain in the same section 2.1?

Author Response

Dear Reviewer,

Thank you for your thorough analysis and constructive recommendations. We highly appreciate your contribution to the improvement of our work. We have taken into account all the comments and additions as much as possible.

Our article has been significantly revised and expanded to take into account the improvements you suggested. We have also added the results of modern statistical analysis methods, including PCA analysis, which allowed us to reveal the nature of the object under study in a more profound way. We kindly ask you to re-read and consider the updated manuscript.

In addition, the authors would like to note that the main goal of this work was to demonstrate the hybrid nature of a unique plant form that was identified in natural conditions. We aimed to show the hereditary nature of this form, which has the potential for further study using modern biotechnological and molecular genetic methods.

Our article focused on the hybrid origin of this plant, which was isolated in natural conditions and successfully propagated in the field for almost ten years. As a result, a hybrid was obtained that produces full-fledged grain, which is confirmed by spectral analysis and mass-spectrometric studies. These data indicate the hybrid origin of the plant and the presence of wheat components in its genome.

We understand that our research is a significant step in the study of hybrid plant forms. The global scientific literature describes first-generation hybrids that were obtained naturally, but they were typically sterile in subsequent cross-breeding, indicating their genetic instability. In contrast, our work demonstrates the possibility of obtaining a stable hybrid form with human participation and long-term selection.

Further research will be aimed at clarifying the embryological nature of this plant, its chromosomal composition and genetic characteristics. We plan to use modern methods such as GISH and FISH technologies, as well as cyto-embryogenetic approaches, to study in more detail the origin and stability of this unique hybrid form.

Reviewer 3 Report

Comments and Suggestions for Authors

This study aims to investigate spontaneous hybrids formed by crossing Triticum and Aegilops species. The study was conducted using modern microscopy and mass spectrometry techniques. The relevance of such research is undeniable, as wheat forms the basis of global food programs worldwide. The authors of the study have done excellent and useful work, but the manuscript contains a number of errors, mostly technical in nature.

Abstract.
Spelling of names should be checked: Triticum, Agropyron, etc.

Introduction
Apparently, only one format for citing references should be used. Here we see this format [3] [4] and this format [6,7].
It is also necessary to determine how to cite the authors who first described the species and genus. For example, in the second paragraph, Aegilops is mentioned without the authors, and in the fourth, with the author (L.). Typically, this is done the other way around.

Discussion
In our opinion, the discussion in this section only began with the fourth paragraph. The first three paragraphs contain general information that could have been significantly shortened. As readers of this article, we would have appreciated a more in-depth analysis of the results, a comparison with other studies, and the significance of the new knowledge gained about hybrids.

Graphic Material.

We believe that Figures 1-5 should be revised. The captions should be standardized. Some captions should be rewritten, changing the background to transparent.
In our opinion, including Table 1 in the appendix is ​​pointless, as it contains data already published. Its inclusion can only be justified by the article's readability.

Author Response

Dear Reviewer.

Thank you for your thorough analysis and constructive recommendations. We highly appreciate your contribution to the improvement of our work. We have taken into account all the comments and additions as much as possible.

Our article has been significantly revised and expanded to take into account the improvements you suggested. We have also added the results of modern statistical analysis methods, including PCA analysis, which allowed us to reveal the nature of the object under study in a more profound way. We kindly ask you to re-read and consider the updated manuscript.

In addition, the authors would like to note that the main goal of this work was to demonstrate the hybrid nature of a unique plant form that was identified in natural conditions. We aimed to show the hereditary nature of this form, which has the potential for further study using modern biotechnological and molecular genetic methods.

Our article focused on the hybrid origin of this plant, which was isolated in natural conditions and successfully propagated in the field for almost ten years. As a result, a hybrid was obtained that produces full-fledged grain, which is confirmed by spectral analysis and mass-spectrometric studies. These data indicate the hybrid origin of the plant and the presence of wheat components in its genome.

We understand that our research is a significant step in the study of hybrid plant forms. The global scientific literature describes first-generation hybrids that were obtained naturally, but they were typically sterile in subsequent cross-breeding, indicating their genetic instability. In contrast, our work demonstrates the possibility of obtaining a stable hybrid form with human participation and long-term selection.

Further research will be aimed at clarifying the embryological nature of this plant, its chromosomal composition and genetic characteristics. We plan to use modern methods such as GISH and FISH technologies, as well as cyto-embryogenetic approaches, to study in more detail the origin and stability of this unique hybrid form.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This work holds important theoretical value and practical significance for advancing wheat genetic improvement and safeguarding food security. I believe that after the authors have made efforts to improve their existing data and manuscript, the quality of the paper has been significantly enhanced.

Reviewer 3 Report

Comments and Suggestions for Authors

We have reviewed the revised version of the article. We believe that the revised version is suitable for publication.