Review Reports - Genome-Wide Development of InDel-SSRs and Association Analysis of Important Agronomic Traits of Taro (<i>Colocasia esculenta</i>) in China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The gene and species names should be in italic form. Revise this in title and in the whole text of the MS.

The species name when once used in full, used the abbreviation followed by a period for the name of genus.

What are the limitations in current works on Taro and why it is needed to develop SSR molecular markers? Authors are directed to provide the results and limitations of the previous study in Introduction section and update it.

Table 1. Why the accessions are random? Also provide the details of the abbreviation used in the legends.

Table 2. Update the legends for the missing values in the last two columns.

Figure 1 E is not clear. Also update the figure legends for Figure 1 E.

Table 3. Update the legends. Do as directed for Table 1.

Line 140…………….Divided into 3 groups based on…???? Provide justification for your groups.

Figure 2A, B, C. Update the legends in details. Explain what the different colors represents. Provide the data of PC analysis in excel as supplementary information.

Table 4 and 5 . Auhtors need to use specific valid test to compare the means through LSD or Duncan tukey test and update the table with alphabetic letters to show the statistical differences.

The manuscript should be checked for English grammar and editing.

Author Response

Dear Reviewer,

Thank you for your thorough review and constructive comments on our manuscript. We appreciate your valuable feedback and have addressed each of your comments in detail below. A point-by-point response, along with the revised manuscript, is included in the attached document.

Please see the attachment.

Comments 1: The gene and species names should be in italic form. Revise this in title and in the whole text of the MS.

Response 1: Thank you for your observation. We have revised the entire manuscript to ensure that all gene and species names are in italic form, including the title. These updates can be found in manuscript (in title and in lines32, 385-410).

Comments 2: The species name when once used in full, used the abbreviation followed by a period for the name of genus.

Response 2: We appreciate this clarification. We have updated the manuscript to adhere to the specified format, ensuring that the genus name is abbreviated appropriately after its first full mention (in lines 312, 348).

Comments 3: What are the limitations in current works on Taro and why it is needed to develop SSR molecular markers? Authors are directed to provide the results and limitations of the previous study in Introduction section and update it.

Response 3: Thank you for your suggestion. We have updated the Introduction section to demostrate the limitations in current works on Taro and the need to develop SSR molecular markers. Firstly, we highlighted that previous genetic studies on taro had a limited number of molecular markers and lacked comprehensive genomic information, hindering the effective exploration and utilization of taro germplasm resources (in lines 62-65, 83-84). With the recent publication of the taro genome sequence, which includes extensive InDel loci and a high repeat sequence content of 88.43%, the development of SSR and InDel markers has become feasible and advantageous (in lines 69-76). This comprehensive marker development is crucial for providing in-depth genetic information and improving efficiency in marker development, especially for yield-related traits that have seen limited association analysis in past studies.

Comments 4: Table 1. Why the accessions are random? Also provide the details of the abbreviation used in the legends.

Response 4: Thank you for your observation. We have clarified in Section 2.2 "DNA Extraction and Genome Resequencing" that the accessions were selected based on significant phenotypic variation rather than at random, focusing on a subset of 10 materials with distinct phenotypic characteristics (in lines 109-112). Additionally, we have updated the legend in Table 1 to include a note specifying the names of these 10 materials (in lines 207-209).

Comments 5: Table 2. Update the legends for the missing values in the last two columns.

Response 5: Thank you for your feedback. We have updated the legend for Table 2 to clarify that the "Sum" represents the overall calculation across all chromosomes, while the "Average" is derived by dividing the total by the 14 chromosomes in table2. Consequently, the values for InDel-SSR density/Mb and InDel-SSR/INDEL reflect the same overall ratio as the average.

Comments 6: Figure 1 E is not clear. Also update the figure legends for Figure 1 E.

Response 6: Thank you for your observation. To enhance clarity, we have split the original Figure 1 into four separate figures, now labeled as Figures 1-3 and Supplementary Figure S1. The original Figure 1E has been moved to Supplementary Figure S1. Additionally, we have updated Supplementary Table 2 to include the corresponding names for each material identifier. While the legends in the original Figure 1E were too much in the new figures, they can now be found in Supplementary Table 2 for reference.

Comments 7: Table 3. Update the legends. Do as directed for Table 1.

Response 7: Thank you for your suggestion. We have updated the legend for Table 3 to include the full names corresponding to the abbreviations Na, Ne, Ho, He, and PIC, similar to the updates made for Table 1. This ensures consistency and clarity for readers (in lines 244-246).

Comments 8: Line 140…………….Divided into 3 groups based on…???? Provide justification for your groups.

Response 8: Thank you for your comment. In the revised manuscript, we have added Supplementary Figures S2 and S3 to the "Population Genetic Structure" analysis. We now provide a detailed explanation in the text (in lines 248-254) regarding the basis for dividing the samples into three groups, including what each color represents. Additionally, we have clarified that the analysis of population genetic structure and cluster analysis indicates that the optimal division into three groups reflects differences in tillering ability, with distinct groups clustering together based on this trait (in lines 255-256).

Comments 9: Figure 2A, B, C. Update the legends in details. Explain what the different colors represents. Provide the data of PC analysis in excel as supplementary information.

Response 9: Thank you for your feedback. We have added Supplementary Figure S3 to provide additional clarification for Figure 2A, including corresponding identifiers. The text has also been updated (in lines 248-254) to explain the significance of each color in the figures. Furthermore, we have created Supplementary Table 3, which contains the PC analysis data in Excel format for easy reference. This ensures that all relevant information is accessible and clearly presented.

Comments 10: Table 4 and 5 . Auhtors need to use specific valid test to compare the means through LSD or Duncan tukey test and update the table with alphabetic letters to show the statistical differences.

Response 10: Thank you for your suggestion. In Table 4, we have included the results of a one-way ANOVA analysis. The text has been updated to indicate that there were no significant differences in traits between the two years (in lines 283-286). Due to the lack of significant differences between the two groups, we were unable to perform post-hoc tests such as LSD or Duncan tukey range test. However, we would like to clarify that Table 5 presents data on significantly associated loci and is not based on calculated means from multiple data points. As such, post-hoc tests like LSD or Duncan Tukey cannot be applied to this table.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study aimed to develop a set of "high-density InDel-SSR molecular markers covering the taro whole genome to analyze the genetic diversity of 121 taro genetic resources and mining candidate genes regulating the agronomic traits of taro leaves and corms." Agronomic traits were also studied. the authors tell the reader that "due to the lack of enough molecular markers, the genetic diversity, germplasm identification, and molecular breeding of taro were greatly limited." However, as written and because several prior studies have already developed markers for taro, it isn't easy to see this research's novelty or unique contribution. Overall, the authors have used a reference genome to pick new primers that were used to characterize a germplasm collection. The reason for this or why these primers are better than the others is not stated.

Additionally, methods have been poorly described, making it difficult to understand what was done, how the results were obtained, or even how to replicate the study. For instance, several softwares are mentioned but not the specific parameters used in each one. Authors have also stated that several agronomic traits were measured; however, how, and in how many individuals is simply missing in the text. The statistical approach followed is also not described. Thus, within this general context, the reader is left to guess where and how results were obtained.

Comments on the Quality of English Language

Several sentences have typos and grammatical mistakes.

Author Response

Dear Reviewer,

Please see the attachment.

Comments 1: This study aimed to develop a set of "high-density InDel-SSR molecular markers covering the taro whole genome to analyze the genetic diversity of 121 taro genetic resources and mining candidate genes regulating the agronomic traits of taro leaves and corms. " Agronomic traits were also studied. The authors tell the reader that "due to the lack of enough molecular markers, the genetic diversity, germplasm identification, and molecular breeding of taro were greatly limited. " However, as written and because several prior studies have already developed markers for taro, it isn't easy to see this research's novelty or unique contribution. Overall, the authors have used a reference genome to pick new primers that were used to characterize a germplasm collection. The reason for this or why these primers are better than the others is not stated.

Response 1: Thank you for your valuable feedback on our research. We deeply respect your interest in the background of molecular markers for taro and will try our best to clarify the uniqueness of our study. While previous research has indeed developed molecular markers for taro, those markers were not genome-based, resulting in a limited number and sparse distribution (in lines 62-65). In contrast, our study focuses on developing high-density InDel-SSR molecular markers based on a reference genome, which provides a more comprehensive and densely packed genetic resource (in lines 69-76). Additionally, our research offers insights into the evolutionary direction of taro at the molecular level, yielding a more thorough understanding of genetic diversity, which has not been extensively explored in existing literature (in lines 366-367). Additionally, the primers we developed combine both InDel and SSR markers, which effectively reduces development costs and results in a higher polymorphism rate compared to other SSR markers derived from whole-genome studies (in lines 347-349). All 121 primer pairs successfully amplified, demonstrating good stability and polymorphism. Furthermore, there has been no prior association analysis linking molecular markers to agronomic traits related to taro yield (in lines 83-84). Our study fills this gap by performing such analyses with the developed InDel-SSR molecular markers, providing a theoretical basis for uncovering candidate genes regulating important agronomic traits in taro (in lines 410-412). We appreciate your attention to our work and look forward to your further insights.

Comments 2: Additionally, methods have been poorly described, making it difficult to understand what was done, how the results were obtained, or even how to replicate the study. For instance, several softwares are mentioned but not the specific parameters used in each one. Authors have also stated that several agronomic traits were measured; however, how, and in how many individuals is simply missing in the text. The statistical approach followed is also not described. Thus, within this general context, the reader is left to guess where and how results were obtained.

Response 2: Thank you for your constructive feedback regarding the methods section. We have made several updates to enhance clarity and reproducibility. We have included references for the software tools used, specifically citing GenoDive, Polygene(in line 146), and CLUMPP(in line 157), and have noted the version of Tassel (version5.2.81) (in line 176). Additionally, we provided specific parameters for BWA-MEM, SAMtools, BCFtools, and Structure to clarify our analysis procedures(in lines 116-119,152-155). The methodology for measuring these traits has been detailed in Section 2.4 "Genetic Diversity Analysis of Taro," where we explain how the materials were selected and the tools used for measurement(in lines 169-172). Regarding the agronomic traits measured, we specified that leaf area was assessed in 2021, while the other nine traits were evaluated over two years. More than 90% of the individuals in our study have associated trait data (in lines 276-278). These revisions aim to ensure that readers can better understand our methods and replicate the study. Thank you for your valuable input!

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study is aimed to develop a high-density InDel-SSR molecular markers covering the whole genome in taro. The reason of the work is well placed, in fact, there are few studies about this topic.

The methodological approach is innovative and consistent with the aims and results of the research.

Results are significant, in fact, elucidate the genetic diversity and population structure of taro, and reveal the changes of leaf and cormel morphology during the domestication process.

The manuscript is well organized and the conclusions are coherent with results.

Tables and figures are comprehensive.

However, if authors will take into account the following suggestions, the quality of the work could be improved:

• In the “Introduction” section,

o Authors could cite some other research studies in the field, such as those available at https://sabraojournal.org/wp-content/uploads/2021/09/SABRAO-J-Breed-Genet-533-447-458-OKTAVIANINGSIH.pdf , about a taro genetic diversity investigation based on RAPD markers.

o Similarly, for https://www.cabidigitallibrary.org/doi/pdf/10.5555/20209905034

• In the ‘Results’ section,

o Some figures are few readable. I suggest to split them in sub-figures.

• In the ‘Discussion’ section,

o It could be suitable to underline even better the added value of the work compared to the existing studies.

Author Response

Dear Reviewer,

Please see the attachment.

Comments 1: In the “Introduction” section,Authors could cite some other research studies in the field, such as those available at https://sabraojournal.org/wp-content/uploads/2021/09/SABRAO-J-Breed-Genet-533-447-458-OKTAVIANINGSIH.pdf , about a taro genetic diversity investigation based on RAPD markers.

Similarly, for https://www.cabidigitallibrary.org/doi/pdf/10.5555/20209905034.

Response 1: Thank you for suggesting relevant studies to enhance the Introduction section. We have added the first suggested reference from SABRAO Journal of Breeding and Genetics (Oktavianingsih et al., 2021) to our manuscript as Reference 18 (in line 60). This citation contributes valuable context on genetic diversity studies in taro based on RAPD markers. However, we were unable to locate the second reference based on this website (https://www.cabidigitallibrary.org/doi/pdf/10.5555/20209905034). If possible, could you provide the details of this article information to help us locate it? Thank you again for your helpful recommendations.

Comments 2: In the ‘Results’ section, Some figures are few readable. I suggest to split them in sub-figures.

Response 2: Thank you for your observation. To enhance clarity, we have split the original Figure 1 into four separate figures, now labeled as Figures 1-3 and Supplementary Figure S1. The original Figure 1E has been moved to Supplementary Figure S1. Additionally, we have updated Supplementary Table 2 to include the corresponding names for each material identifier. While the legends in the original Figure 1E were too much in the new figures, they can now be found in Supplementary Table 2 for reference.

Comments 3: In the ‘Discussion’ section,it could be suitable to underline even better the added value of the work compared to the existing studies.

Response 3: Thank you for your valuable suggestion. In the Discussion section, we have emphasized the added value of our work compared to existing studies. We state that our research provides a more comprehensive classification of genetic diversity in taro (in lines 346-348, 367-368) and reveals changes in leaf and cormel morphology during the domestication process (in lines 377-379). This contributes to the classification, preservation, and utilization of taro germplasm resources. Additionally, we highlight the significance of exploring the regulatory genes associated with important agronomic traits of cormels and leaves. Our GWAS, based on the developed InDel-SSR markers, comprehensively analyzes these traits, offering a theoretical basis for identifying candidate genes that regulate key agronomic characteristics in taro (in lines 411-413). We believe these clarifications strengthen the contribution of our study and align it more closely with the broader context of taro research. Thank you for your feedback!

Author Response File: Author Response.pdf