Genome-Wide InDel Marker Development and Genetic Diversity Analysis of 52 Tomato Germplasm Accessions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, 285,796 InDel loci were initially identified based on genome sequences of five tomato varieties. Subsequently, 63 InDel loci were selected based on the number of InDel bases, polymorphism, and amplification electrophoresis performance. These 63 loci were evaluated and analyzed using 52 tomato accessions, aiming to provide marker support for research on the genetic diversity of tomato germplasm. The manuscript requires substantial revisions, such as providing more detailed descriptions of the loci development and evaluation process, offering more comprehensive data analysis, explaining how morphological data were utilized, and clarifying the application of the 63 loci and the specific problems they can address. The details are as follows.

1. Abstract:The results section contains limited information, only presenting PIC values and the four groups. It is recommended to include more result data.
2. Introduction:It is suggested to further clarify the main application areas and the problems that can be solved by the InDel markers developed in this study. According to the information provided in the manuscript, the 63 InDel loci have little correlation with molecular marker assisted selection breeding and fine trait mapping for excellent agronomic traits.
3. Materials and Methods:The InDel loci mining and evaluation process is too simple. It is recommended to add detailed steps of the screening scheme. The volume of the PCR reaction system is 20 μL, but the total amount of each component exceeds 20 μL. The data analysis section should clearly state which materials and which loci were analyzed, and what specific analyses were performed.
4. Results:

(1) The distribution of the 63 InDels across chromosomes is significantly uneven. According to Figrue3, there are 11 loci on chromosome 9 and only 2 loci chromosome 12.

(2) Some loci have low PIC values, such as three pairs of primers with PIC values below 0.1, which are 0.038, 0.074, and 0.090, respectively.

(3) In section 2.4, it is difficult to explain the narrow genetic background of G1 group and the rich genetic diversity of G4 group based on the results presented. It is recommended to analyze the genetic diversity and other parameters of each group.

(4) It is suggested that the colors used for each group in Figures 4 and 5 should be consistent.

(5) The supplementary table lists the morphological data of 52 materials. It is recommended to use morphological data for statistical analysis or chart display to analyze whether the genotype results are consistent with the phenotype. It is necessary to consider how the morphological data can be integrated and utilized. At present, the morphological data has not been integrated into the article.

(6) In section 2.5, the description of morphological traits appears weakly correlated with the title and main research objectives of this article. If it is necessary to describe the morphological characteristics, the representativeness of the selected 52 materials can be reflected in the material section by describing the morphological characteristics.

(7) Only 24 pairs of primers were used to construct the fingerprint. Is it recommended to use these 24 pairs of primers for variety identification? Is the variety discrimination ability of these 24 pairs of primers consistent with that of 63 pairs of primers?

 

Author Response

Comments 1: Abstract:The results section contains limited information, only presenting PIC values and the four groups. It is recommended to include more result data.

Response 1:Thank you for pointing out the issue. We agree with the comments. We have revised this section to provide additional core results, including: developing 255 molecular markers from 285,796 Indel sites based on genome alignment results; screening 63 pairs of core Indel markers based on Indel length, polymorphism, and electrophoretic performance; collecting 52 tomato germplasm resources and conducting trait investigations on them; performing cluster analysis on the 52 tomato germplasm resources using 63 pairs of core Indel markers and conducting genetic diversity analysis in combination with the surveyed traits; selecting a set of core Indel primer combinations to construct a DNA fingerprint map.

Comments 2:Introduction:It is suggested to further clarify the main application areas and the problems that can be solved by the InDel markers developed in this study. According to the information provided in the manuscript, the 63 InDel loci have little correlation with molecular marker assisted selection breeding and fine trait mapping for excellent agronomic traits.

Response 2:Thank you for pointing out the issue. We agree with the reviewer's comments. We have revised this section to specifically describe the advantages of InDel markers, as well as the areas of application and problems they can address. We also noted that, compared with other molecular markers, InDel markers are widely distributed in the genome, easy to detect, and highly reproducible, thus possessing unique advantages.

Comments 3:Materials and Methods:The InDel loci mining and evaluation process is too simple. It is recommended to add detailed steps of the screening scheme. The volume of the PCR reaction system is 20 μL, but the total amount of each component exceeds 20 μL. The data analysis section should clearly state which materials and which loci were analyzed, and what specific analyses were performed.

Response 3:Thank you for pointing out the issue. We agree with the comment. We have made revisions in the relevant sections and supplemented the results. Firstly, we have changed section 4.3 from 'Primers of InDel markers design' to 'Indel Molecular Marker Screening and Primer Design,' and we have added an explanation of how 285,796 InDel loci were filtered through a series of stepwise selection processes to obtain candidate loci suitable for subsequent marker development. This includes steps such as basic filtering, genome distribution screening, primer design and amplification capability assessment, population polymorphism and experimental verification, ultimately selecting the target loci.

Secondly, the PCR reaction system was a typographical error; thank you for pointing it out, and it has been corrected to the correct system.

Finally, regarding data analysis, in sections 4.1 and 4.2, we have provided detailed explanations of the tomato materials used for marker development and the 52 tomato germplasm resources used for InDel marker identification.

Comments 4：Results:

(1)The distribution of the 63 InDels across chromosomes is significantly uneven. According to Figrue3, there are 11 loci on chromosome 9 and only 2 loci chromosome 12.

Response 4.1: Thank you for your valuable suggestion. In the early stages of marker development, we tried to keep the distribution of markers on each chromosome as uniform as possible. Therefore, the 255 molecular markers we developed are distributed as evenly as possible, while the 63 pairs of markers are selected for high polymorphism among the 52 tomato varieties mentioned in the text. When identifying other tomato varieties, suitable markers can be selected from the 255 pairs based on the germplasm resources collected.

(2) Some loci have low PIC values, such as three pairs of primers with PIC values below 0.1, which are 0.038, 0.074, and 0.090, respectively.

Response 4.2: Thank you for your valuable suggestion. We have also noticed the three pairs of primers with relatively low PIC values that you mentioned. They cannot completely distinguish among these 52 tomato varieties, but when screening certain tomatoes, due to their obvious band differences and ease of amplification, we believe they have the ability to differentiate the genetic polymorphism of these tomatoes.

(3) In section 2.4, it is difficult to explain the narrow genetic background of G1 group and the rich genetic diversity of G4 group based on the results presented. It is recommended to analyze the genetic diversity and other parameters of each group.

Response 4.3: Thank you for your valuable suggestions. We agree that including genetic diversity parameters can provide a more comprehensive data analysis. However, the main objective of this study is to develop a set of InDel markers that can be used for subsequent genetic research, rather than conducting a systematic population genetic analysis. The parameters we chose to present are all directly related to the evaluation and validation of marker quality. To avoid deviating from the main focus of the study and to control the length, we did not further expand the diversity parameter analysis.

(4) It is suggested that the colors used for each group in Figures 4 and 5 should be consistent.

Response 4.4: Thank you for pointing out the issue. We agree with the comment. We have made revisions in this section.

(5) The supplementary table lists the morphological data of 52 materials. It is recommended to use morphological data for statistical analysis or chart display to analyze whether the genotype results are consistent with the phenotype. It is necessary to consider how the morphological data can be integrated and utilized. At present, the morphological data has not been integrated into the article.

Response 4.5: Thank you for your valuable suggestions. In section 2.4 Application of InDel markers in tomato germplasm analysis, we mentioned that the grouping results based on genotype analysis are relatively consistent with the surveyed morphological data. Therefore, the morphological data can demonstrate that the results of our molecular markers in identifying the genetic relationships of tomatoes are relatively reliable.

(6) In section 2.5, the description of morphological traits appears weakly correlated with the title and main research objectives of this article. If it is necessary to describe the morphological characteristics, the representativeness of the selected 52 materials can be reflected in the material section by describing the morphological characteristics.

Response 4.6: Thank you for pointing out the issue. We agree with the comment. We have made revisions in the relevant section and supplemented section 4.1 Test Materials, introducing 52 types of tomatoes, including various wild small-fruited tomatoes, cherry tomatoes, and high-generation homozygous inbred lines, which are common varieties in tomato research.

(7) Only 24 pairs of primers were used to construct the fingerprint. Is it recommended to use these 24 pairs of primers for variety identification? Is the variety discrimination ability of these 24 pairs of primers consistent with that of 63 pairs of primers?

Response 4.7: Thank you for your valuable suggestion. We selected 24 pairs of primers to construct the fingerprint because these 24 markers have a significant identification effect for the 52 tomato varieties we collected, but this does not negate the importance of the other primers. The molecular marker system we established is also applicable to the identification of tomato varieties beyond these 52, and all 63 pairs of primers are important for the identification of different tomato varieties.

Reviewer 2 Report

Comments and Suggestions for Authors

Tomato is an important economic crop. Development of molecular markers is valuable for genetic diversity analysis, germplasm identification, and assisted breeding. In this manuscript, InDel markers were developed from 5 tomato variety genomes to analyze the genetic diversity and fingerprints of 52 tomato germplasms. The results provide useful molecular tools for genetic research and breeding of tomatoes. 

1. The introduction part is too general. It should focus on the practical applications of molecular marker technologies such as RFLP, SSR, AFLP, CAPs, and SNP in tomatoes, and highlight the deficiencies or current requirements to support the necessity of developing InDels markers for this manuscript.

2. It is recommended to add a description of the distribution pattern of InDels in the part 2.1.

3. Figure 1 should adjust the color scheme and improve its clarity.

4. In table 1, what is th meaning of missing Deletion?

5. The developed InDel set should be listed as a supplementary table.

6. How the 255 candidate InDel loci were selected from the InDel set? Rondomly? The authors should describe in the Methods part.

7. Table 2 can be considered as a supplementary table.

8. More genetic diversity parameters should be analyzed. 

9. In part 4.2，the authors only provided the sources of the reference genomes, but did not provide the method for InDel mining.

Author Response

Comments 1: The introduction part is too general. It should focus on the practical applications of molecular marker technologies such as RFLP, SSR, AFLP, CAPs, and SNP in tomatoes, and highlight the deficiencies or current requirements to support the necessity of developing InDels markers for this manuscript.

Response 1: Thank you for pointing out the issue. We agree with the comment. We have made revisions in that section and added an Indel marker in the second paragraph of the introduction to illustrate cases in modern tomato breeding, further clarifying the necessity of the developed InDel marker.

Comments 2: It is recommended to add a description of the distribution pattern of InDels in the part 2.1.

Response 2: Thank you for pointing out the issue. We agree with this comment. We have made revisions in this section to describe the Indel distribution pattern.

Comments 3:Figure 1 should adjust the color scheme and improve its clarity.

Response 3: Thank you for pointing out the issue. We agree with the comment. We have made revisions in this section.

Comments 4: In table 1, what is th meaning of missing Deletion?

Response 4: Thank you for pointing out the issue. We agree with the comment. We have made changes in this section; this was a typographical error and has been corrected to 'Deletion'.

Comments 5:The developed InDel set should be listed as a supplementary table.

Response 5: Thank you for pointing out the issue. We agree with this comment. We have supplemented this part in the attachment (Table S1).

Comments 6:How the 255 candidate InDel loci were selected from the InDel set? Rondomly? The authors should describe in the Methods part.

Response 6: Thank you for pointing out the issue. We agree with the comments. We have made revisions in this section to supplement the results. First, we changed '4.3 Primers of InDel markers design' to 'InDel Molecular Marker Screening and Primer Design' and added an explanation of how 285,796 InDel loci were processed through a series of stepwise screening procedures to obtain candidate sites suitable for subsequent marker development. This includes basic filtering, genome distribution screening, primer design and amplification capacity assessment, population polymorphism, and experimental validation, ultimately selecting the target sites.

Comments 7：Table 2 can be considered as a supplementary table.

Response 7: Thank you for pointing out the issue. We agree with the comment. We have revised this part and moved it to the supplementary material (Table S2).

Comments 8：More genetic diversity parameters should be analyzed. 

Response 8: Thank you for your valuable suggestions. We agree that including genetic diversity parameters can provide a more comprehensive data analysis. However, the main objective of this study is to develop a set of InDel markers that can be used for subsequent genetic research, rather than conducting a systematic population genetic analysis. The parameters we chose to present are all directly related to the evaluation and validation of marker quality. To avoid deviating from the main focus of the study and to control the length, we did not further expand the diversity parameter analysis.

Comments 9：In part 4.2，the authors only provided the sources of the reference genomes, but did not provide the method for InDel mining.

Response 9: Thank you for your valuable suggestions. We agree that including genetic diversity parameters can provide a more comprehensive data analysis. However, the main objective of this study is to develop a set of InDel markers that can be used for subsequent genetic research, rather than conducting a systematic population genetic analysis. The parameters we chose to present are all directly related to the evaluation and validation of marker quality. To avoid deviating from the main focus of the study and to control the length, we did not further expand the diversity parameter analysis.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript deals with an InDel marker development in tomatoes with a perspective for use in diversity studies. As such it represents a rather conventional approach, which nonetheless remains relevant for practical breeding purposes.

Overall quality is good with a few minor corrections needed:

1. Cleaved Amplified Polymorphic Sequences are conventionally abbreviated as CAPS, not CAPs as is in Line 64.
2. Table 1 does not contain any statistical analysis and presents rather a simple tabulation, so it should be renamed appropriately.
3. Table S1 does not contain any place of origin data, which makes difficult to evaluate the PCA and clustering analyses outcomes.

Overall, the system proposed for use is quite laborous with 24 pairs of InDel primers to be used each time for fingerprinting.

Author Response

Comments 1：Cleaved Amplified Polymorphic Sequences are conventionally abbreviated as CAPS, not CAPs as is in Line 64.

Response 1: Thank you for pointing out the issue, we have made corrections.

Comments 2：Table 1 does not contain any statistical analysis and presents rather a simple tabulation, so it should be renamed appropriately.

Response 2: Thank you for pointing out the issue; it has been renamed.

Comments 3：Table S1 does not contain any place of origin data, which makes difficult to evaluate the PCA and clustering analyses outcomes.

Response 3: Thank you for your valuable comments. As shown in Table S1, the agronomic traits of 52 tomato varieties in terms of similarity and difference can, to some extent, reflect the genetic relationships between varieties, thereby supporting the consistency between the clustering analysis results and trait performance.