Assessment of Rice Amylose Content and Grain Quality Through Marker-Assisted Selection

Dear Reviewer,

Thank you very much for your time and effort in reviewing our manuscript. We sincerely appreciate your critical and detailed feedback. Although some of your comments were particularly challenging, they have greatly contributed to improving the clarity, rigor, and overall quality of our work.

We have carefully considered each of your suggestions and have revised the manuscript accordingly. Below, we provide a detailed, point-by-point response to all of your comments. We hope that the changes we have made adequately address your concerns.

General Evaluation Questions

1)      Is the research design appropriate and are the methods adequately described?

Ø  The description of the material does not specify whether the material studied originated from a single field experiment or whether there were repetitions. Furthermore, the study does not elucidate whether the conditions under which the plants were cultivated were comparable.

The materials were harvested by the INIAP’s crew and then we proceeded to evaluate the morphological, milling and culinary traits.

Ø  The text is deficient in terms of the provision of information regarding the sequences of the markers utilised.

The markers employed are specified in lines 140-141. Thanks for your comment.

Ø  It is imperative to ascertain which isolation kit was utilised in this procedure.

The DNA kit is specified in lines 131-132. Thanks for your comment.

Ø  It is evident that the methodology described in the experiment does not provide sufficient evidence to demonstrate that the experiment was planned and executed in accordance with the established scientific standards.

Thank you for your valuable observation. We fully recognize the importance of presenting a clear and comprehensive methodological framework to ensure scientific rigor and reproducibility. In response, we have expanded and clarified several aspects of the methodology, directly addressing the points you raised. Your feedback has been instrumental in enhancing the overall clarity, precision, and robustness of our research, and we sincerely appreciate your contribution.

2)      Are the results presented clearly and in sufficient detail, are the conclusions supported by the results and are they put into context within the existing literature?

Ø  The electrophoretic images display substandard quality, which is not attributable to the quality of the photographic images, but rather to the quality of the separation process. The ability of the authors to perform molecular analysis on the basis of such electropherograms is therefore called into question. Consequently, the reliability of the molecular analysis is questionable.

We appreciate your valuable observation regarding the electrophoretic images. We recognize that the resolution of the bands could be improved to enhance the visual clarity of the separation process. This limitation appears to be related to specific conditions during electrophoresis rather than issues with image capture it could be attributed to a little DNA excess. However, the alleles were accurately scored using the appropriate visualization software provided by the gel analyzer system, which allowed for precise band interpretation despite the suboptimal separation. The scoring was further verified because we used three repetitions for each run and gel. For future work, we would optimize parameters such as voltage, and DNA concentration to improve band resolution. Your feedback is important and helps us strengthen the methodological rigor of our molecular analysis.

3)      Does this article provide a relevant contribution to the scientific discussion of this topic?

Ø  The discussion did not adequately address the influence of environmental conditions on the variability of results in the context of molecular markers. The authors should have provided more detailed descriptions of the impact of these factors on the stability of markers and the interpretation of the results obtained. Furthermore, greater emphasis should be placed on the practical ramifications of the findings, particularly about the cultivation of varieties tailored to specific market preferences. The authors should identify particular scenarios and the associated challenges that may arise during the practical implementation of these results. It is crucial to delve into a more comprehensive discussion on the potential limitations associated with the practical implementation of molecular markers in routine breeding programs. Factors such as cost, technological availability, and required expertise should be thoroughly considered, as they can significantly impact the feasibility of the research. It is crucial that the authors provide a more detailed analysis of potential sources of error in the molecular techniques used. This will help to provide a more thorough understanding of the research methods and results, and enhance the credibility of their findings. It is important to place greater emphasis on comparing phenotypic with molecular results, particularly in the context of varieties that have demonstrated inconsistencies in classification. A detailed analysis of such cases could provide valuable insights for future research and enhance the robustness of the authors' findings.

Thank you. We appreciate your thoughtful observation on the importance of addressing environmental influences on molecular marker variability. In response, we have incorporated this into our discussion in lines 426–428, 433–435, and 517–518, where we highlight how phenotypic expression may be affected by environmental conditions, potentially influencing the amylose content and its association with molecular markers. We also emphasized that while environmental factors can alter phenotypic traits, the stability of DNA allows molecular markers to serve as reliable tools in selection. Nonetheless, we acknowledge the value of expanding on this point and in our revised version further clarify the interplay between genotype, environment, and marker performance. Your comment has helped to reinforce the scientific rigor of our discussion.

We fully agree that emphasizing the practical implications of our findings is crucial, particularly in the development and selection of rice varieties tailored to specific market preferences. In this regard, our results are intended to support Ecuadorian rice breeding programs focused on the development of high- or intermediate-amylose varieties that meet both regional and international market demands. Moreover, we recognize that the practical implementation of these findings may be influenced by factors such as genotype–environment interactions, variability in consumer preferences, and post-harvest processing conditions.

We believe that our findings could be of interest to seed companies and agricultural stakeholders as a tool to address these challenges and guide decision-making processes in variety development, thereby strengthening the applied relevance of our research in breeding and commercialization strategies. While the use of SSR markers and enzyme-based assays offers high precision and reproducibility, it is crucial to promote the technification of breeding processes in developing countries and to overcome existing barriers such as implementation cost, limited laboratory infrastructure, and lack of trained personnel.

We agree that identifying potential sources of error in molecular techniques is essential to ensure methodological transparency and scientific rigor. In response, we have expanded the discussion to include specific factors that may have influenced the accuracy of the molecular results, such as electrical voltage and DNA concentration. Additionally, we have emphasized the importance of comparing phenotypic and molecular data, particularly in cases like Impacto, where inconsistencies in amylose content classification were observed. These discrepancies have been carefully analyzed and contextualized as possible results of environmental influences on phenotypic expression or limitations in marker resolution. We believe that this detailed comparison provides valuable insights for improving the integration of molecular and phenotypic data in rice breeding programs and enhances the overall robustness of our conclusions.

4)      Is the quality and presentation of the figures satisfactory?

The quality of the attached drawings and photographs is substandard. The photographs contained within Figure 1, in their present state, contribute nothing of value to the work as a whole. The disparities between these images are not readily apparent.

Thanks for your evaluation on our MS, because it made a better paper after we intended to respond to your insightful comments, correcting the weaknesses as much as we could. 

Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

General Evaluation Questions

a)       Is the research design appropriate and are the methods adequately described?

Ø  In “Statistical analysis” the amylose content (AC) evaluation has a different classification from the one detailed in “Introduction”. If the classification described by Juliano (1985) is better for the statistical analysis, it has to be explained in the text. This is the main trait evaluated and it has to be clear how the authors evaluated it.

We thank the reviewer for this important observation. The concern regarding the classification of amylose content (AC) in the “Statistical Analysis” section has been addressed in the detailed comments. We have clarified the use of Juliano’s (1985) classification and explained its relevance and consistency throughout the manuscript. Additionally, we have ensured that the methodology for AC evaluation is clearly described and aligned with the classification used.

b)       Are the results presented clearly and in sufficient detail, are the conclusions supported by the results and are they put into context within the existing literature?

Ø  Line 318. “Table 1. Descriptive statistics for “ten” quantitative traits characteristics…” Line 324. “Table 2. Matrix of Pearson’s correlation coefficient (r) between “ten” …” Table 4. AC% for Impacto variety should be classified as high (26.5 %) considering any of the classification described in “Introduction” or “Material and Methods”. This genotype would be the only one with high AC, and this would change the discussion and conclusion as well. Milling quality is discussed in term of weight loss percentage (lines 362 and 363), a trait you cannot see in any Table describing the results.

We appreciate the reviewer’s careful reading and constructive comments. The points raised regarding Tables 1, 2, and 4, as well as the discussion of milling quality, are all addressed in detail in the "Detailed Response to Reviewer Comments" section. We have provided clarifications and made the necessary adjustments in the manuscript to ensure consistency and accuracy throughout the tables and corresponding discussions.

c)       Is the quality and presentation of the figures satisfactory?

Ø  Figure 1 and 2 quality are not good to observe the differences among rice cultivars or to read the information about the PC analysis.

We thank the reviewer for the observation regarding the quality of Figures 1 and 2. This concern has been addressed in the "Detailed Response to Reviewer Comments" section. The figures have been improved to enhance clarity, resolution, and readability of the information related to the principal component analysis and the differentiation among rice cultivars.