Review Reports - Dwarf Tomato Plants Allow for Managing Agronomic Yield Gains with Fruit Quality and Pest Resistance through Backcrossing

Round 1

Reviewer 1 Report

Reviewers Comments:

In the present manuscript, the authors aimed to improve fruit quality and pest resistance through successively backcrossing of dwarf Santa Cruz tomato populations with agronomic potential. The authors claimed that they evaluated six dwarf tomato populations obtained from the first backcrossing, 13 dwarf populations obtained from the second backcrossing, the donor parent, and the commercial cultivar ‘Santa Clara’ as check, totaling 21 treatments. Moreover, Univariate analysis and computational intelligence are also used to evaluate the best genotypes. Authors also claim that all agronomic variables showed significant and progressive increases after the first and second backcrossing. The highlighted BC2 populations were Sci#16.1-2; Sci#25.1,1-2; Sci#25.1,2-2; Sci#3.1,1-2; Sci#3.1,2-2; Sci#8.3,1-27 2; and Sci#8.3,2-2, with significant increases in mean fruit weight, pulp thickness, fruit length and diameter, and acyl sugar content. They also concluded that the selected BC2 populations can be used as male parents to obtain normal hybrids with the aim of achieving greater productivity, nutritional quality, and broader spectrum of pest resistance due to the presence of acyl sugars in leaflets.

Question # 1: It is known that ASs are allelochemicals having deleterious effects on the life cycle of pest arthropods but the results, conclusions and interpretations are not supported by the experimental data shown.

Question # 2: An English editing of the text must be done as some sentence must be rewritten (in instance, lines 22, 158, 189, 260, 263) Therefore, the article cannot be accepted in its present form.

Question # 3: The materials and methods section need to be rewritten and should be properly divided into sub headings especially from line 118-144.

Question # 4: How many biological and technical replicates were used should be clearly written in the materials and methods section.

Question # 5: Fruit diameter, fruit length, fruit shape, pulp thickness and internode length etc. are all measured by authors, but none of them is shown in the figure, only the figure 1 is not enough for phenotypic comparison, Adding more figures of phenotypic comparison will further strengthen the reported results.

Question # 6: It is stated by the authors that fruits at full maturity stage were used for β-carotene (CC) and lycopene (LC) extraction. My question is why fruits at “few days after breaker stage” were not utilized for β-carotene and lycopene where the fruits are properly ripped? Authors should refer to the article doi.org/10.1016/j.jplph.2019.152997 for further details. And also cite this article to support you results.

Question # 7: The authors should also cite the article doi.org/10.1007/s00427-019-00643-7 which is purely based on the phenotypic comparison between wild type and dwarf transgenic tomato plants.

Question # 8: Line 96-116, methodology should be briefly explained so that the readers may easily get the idea of methodology followed.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Authors thank the reviewer for the time dedicated to the current manuscript and the valuable comments made.

Response 1: We understand Reviewer 1's concern. We were able to provide a rationale for the issue. The focus of the research was to perform the introgression of dwarfism genes and increase agronomic traits with backcross. When crossing a normal plant with a dwarf plant, there is segregation of the size of the plants in the segregating generation (F₂) regarding the normal and dwarf size. To advance each generation and obtain BC₁ and BC₂, it was necessary to discard normal plants and select dwarf plants. The study of dwarf size inheritance was elucidated by Maciel et al. (2015) as recessive single-gene inheritance for dwarf size. Therefore, the main focus in plant selection within each backcross was for “plant size” (selecting dwarf plants and discarding normal plants). This selection is carried out in the seedling phase, with only dwarf plants being transplanted to set up the experiment (in F₂BC₁ and F₂BC₂). After guaranteeing 100% of dwarf plants for each backcross (BC₁ and BC₂), the experiment is carried out together with commercial, parental, donor and recurrent controls. The second focus from the 100% guarantee of dwarf plants was the agronomic characteristics. As is known, the agronomic traits evaluated are of polygenic inheritance. This means that an increase is expected for each backcross, as we use a recurrent parent with agronomic potential. It is worth mentioning that depending on the progress of each backcross, that is, BC₁ and BC₂, it is necessary to resort to the cross between a dwarf plant and another normal plant. The consequence will be the emergence of segregation (normal and dwarf plants) in F₂BC₁ and F₂BC₂. Resorting to the recurrent parent implies achieving greater increments in agronomic traits after establishing the dwarf size. It is worth remembering here that at the beginning of the research a dwarf plant (parental donor) with small fruits was used. From the results, the increase in agronomic requirements in relation to the parental donor was evident. Regarding acylsugars, the idea is not the same, as the inheritance is not polygenic. Depending on the type of inheritance reported, Gonçalves et al. (2007) cites that “Through genetic models by the likelihood function, the hypothesis of single-gene inheritance was confirmed. Tomato plants with good resistance to arthropod pests, mediated by the allelochemical acylsugar, can be efficiently obtained in backcross populations from the interspecific cross L. esculentum x L. pennellii”. Therefore, our work showed that it was possible to fix the dwarf size at each backcross and improve the agronomic requirements (polygenic inheritance) after two backcrosses. We emphasize that there is no Santa Cruz dwarf plant to obtain hybrids according to Finzi et al. (2019) performed for minitomatoes with several additional advantages. Therefore, it was not the aim of the research to demonstrate increases in acylsugars from backcrosses and, therefore, the article does not support this idea. The focus of the research was to fix the dwarf size in the plants and verify in which magnitude there is an increase in agronomic traits after backcrossing. From the results obtained, it is evident that it is possible to fix the dwarf size and increase the fruit. In addition, it was possible to verify high levels of acylsugars in some populations, allowing them to be used as a male parent (dwarf plants) to obtain hybrids from dwarf plants with a broad spectrum of resistance to pests, as reported by Maluf et al. (2010), even using normal plants with low content (since hybrids with intermediate levels of acylsugars promote a broad spectrum of resistance to pests). The analysis of acylsugars was only performed in this research due to the results obtained by Oliveira et al. (2021) who reported that the dwarf plant used in this research is rich in acylsugars.

Question # 2: An English editing of the text must be done as some sentence must be rewritten (in instance, lines 22, 158, 189, 260, 263) Therefore, the article cannot be accepted in its present form.

Response 2: The article was subjected a new revision of the text in English by a specialized company, including all phrases cited by the reviewer.

Question # 3: The materials and methods section need to be rewritten and should be properly divided into sub headings especially from line 118-144.

Response 3: The materials and methods section was divided into sub headings.

Question # 4: How many biological and technical replicates were used should be clearly written in the materials and methods section.

Response 4: Information has been added to the text. Lines 121 and 126.

Response 5: We understand the reviewer's concern. However, one figure for each response variable could take up too much space in the text and make it tedious. We understand that Figure 1 fully illustrates the plant (evaluated germplasm) and that greater details are observed quantitatively in the tables.

Response 6: We consulted the indicated articles and they are indeed excellent. However, in Brazil there is a preference for the consumption of ripe fruits exactly at the point evaluated. In fact, on other occasions, several stages of maturation could be evaluated and the levels of beta-carotene and lycopene could be monitored. For the present manuscript, we would like to keep the evaluation because it represents in an important way the level of biofortification in each evaluated genotype.

Question # 7: The authors should also cite the article doi.org/10.1007/s00427-019-00643-7 which is purely based on the phenotypic comparison between wild type and dwarf transgenic tomato plants.

Response 7: We thank the reviewer for indicating the article, as it is indeed very important and its citation in the text will enrich the paper.

Included in line 268 (in blue): “The morphology of the leaves and leaflets (Figure 1) were very different when compared to the findings of recent research carried out with dwarf plants [21].

Naeem, M.; Waseem, M.; Zhu, Z.; Zhang, L. Downregulation of SlGRAS15 manipulates plant architecture in tomato (Solanum lycopersicum). Development Genes and Evolution 2020, 230, 1-12. https://doi.org/10.1007/s00427-019-00643-7

Question # 8: Line 96-116, methodology should be briefly explained so that the readers may easily get the idea of methodology followed.

Response 8: We understand the reviewer's concern and have made some improvements to the text. The methods cited in lines 102-123 were developed as detailed. Because it deals with traditional methodologies and has been published in several articles, we prefer to keep it this way, with minor adjustments, as requested by the reviewer.

Author Response File: Author Response.docx

Reviewer 2 Report

General comments:

The Materials and Methods section requires secondary headings, such as study sites and material profiles , experiment design, sample collection and determination, statistical analyses.

For discussion,

Too many paragraphs during this section, suggested paragraph reductions. Maybe, we could divide the discussion into several parts, such as the feasibility of the method, the effect of backcrossing on agronomic traits, resistance and nutritional quality of tomato.

Furthermore, the discussion in the manuscript should add the reasons for the improved resistance and nutritional quality after backcrossing, rather than just discussing the advantages of the backcrossing method.

Special comments:

Abstract:

Line 22, change “Were” to “We”

In general, the need for references cannot be used directly to refer to specific studies, please correct. Such as Line 260, 268...

Author Response

Response to Reviewer 2 Comments

Authors thank the reviewer for the time dedicated to reviewing the current manuscript and the valuable comments made:

Comment # 1:The Materials and Methods section requires secondary headings, such as study sites and material profiles, experiment design, sample collection and determination, statistical analyses.

Response 1: We appreciate the reviewer's comment and have inserted secondary headings in the Materials and Methods section.

Comment # 2: For discussion, too many paragraphs during this section, suggested paragraph reductions. Maybe, we could divide the discussion into several parts, such as the feasibility of the method, the effect of backcrossing on agronomic traits, resistance and nutritional quality of tomato.

Response 2: As suggested, we have also inserted secondary headings in the Discussion section.

Comment # 3: Furthermore, the discussion in the manuscript should add the reasons for the improved resistance and nutritional quality after backcrossing, rather than just discussing the advantages of the backcrossing method.

Response 3: We understand Reviewer 2's concern. We were able to provide a rationale for the issue. The focus of the research was to perform the introgression of dwarfism genes and increase agronomic traits with backcross. When crossing a normal plant with a dwarf plant, there is segregation of the size of the plants in the segregating generation (F₂) regarding the normal and dwarf size. To advance each generation and obtain BC₁ and BC₂, it was necessary to discard normal plants and select dwarf plants. The study of dwarf size inheritance was elucidated by Maciel et al. (2015) as recessive single-gene inheritance for dwarf size. Therefore, the main focus in plant selection within each backcross was for “plant size” (selecting dwarf plants and discarding normal plants). This selection is carried out in the seedling phase, with only dwarf plants being transplanted to set up the experiment (in F₂BC₁ and F₂BC₂). After guaranteeing 100% of dwarf plants for each backcross (BC₁ and BC₂), the experiment is carried out together with commercial, parental, donor and recurrent controls. The second focus from the 100% guarantee of dwarf plants was the agronomic characteristics. As is known, the agronomic traits evaluated are of polygenic inheritance. This means that an increase is expected for each backcross, as we use a recurrent parent with agronomic potential. It is worth mentioning that depending on the progress of each backcross, that is, BC₁ and BC₂, it is necessary to resort to the cross between a dwarf plant and another normal plant. The consequence will be the emergence of segregation (normal and dwarf plants) in F₂BC₁ and F₂BC₂. Resorting to the recurrent parent implies achieving greater increments in agronomic traits after establishing the dwarf size. It is worth remembering here that at the beginning of the research a dwarf plant (parental donor) with small fruits was used. From the results, the increase in agronomic requirements in relation to the parental donor was evident. Regarding acylsugars, the idea is not the same, as the inheritance is not polygenic. Depending on the type of inheritance reported, Gonçalves et al. (2007) cites that “Through genetic models by the likelihood function, the hypothesis of single-gene inheritance was confirmed. Tomato plants with good resistance to arthropod pests, mediated by the allelochemical acylsugar, can be efficiently obtained in backcross populations from the interspecific cross L. esculentum x L. pennellii”. Therefore, our work showed that it was possible to fix the dwarf size at each backcross and improve the agronomic requirements (resistance and nutritional quality - polygenic inheritance) after two backcrosses. We emphasize that there is no Santa Cruz dwarf plant to obtain hybrids according to Finzi et al. (2019) performed for minitomatoes with several additional advantages. Therefore, it was not the aim of the research to demonstrate increases in acylsugars from backcrosses and, therefore, the article does not support this idea. The focus of the research was to fix the dwarf size in the plants and verify in which magnitude there is an increase in agronomic traits after backcrossing. From the results obtained, it is evident that it is possible to fix the dwarf size and increase the fruit. In addition, it was possible to verify high levels of acylsugars in some populations, allowing them to be used as a male parent (dwarf plants) to obtain hybrids from dwarf plants with a broad spectrum of resistance to pests, as reported by Maluf et al. (2010), even using normal plants with low content (since hybrids with intermediate levels of acylsugars promote a broad spectrum of resistance to pests). The analysis of acylsugars was only performed in this research due to the results obtained by Oliveira et al. (2021) who reported that the dwarf plant used in this research is rich in acylsugars.

Special comments:

Abstract:

Question # 1: Line 22, change “Were” to “We”

Response 1: The article was subjected a new revision of the text in English by a specialized company. The sentence has been rewritten.

Question # 2: In general, the need for references cannot be used directly to refer to specific studies, please correct. Such as Line 260, 268...

Response 2: The article was subjected a new revision of the text in English by a specialized company. The pointed highlights have been corrected.

Reviewer 3 Report

Decent article that deals with research on improving fruit quality and pest resistance by backcrossing Santa Cruz dwarf tomato populations. I have no comments. Only the Discussion could be more extensive Słuchaj

Author Response

Response to Reviewer 3 Comments

Authors thank the reviewer for the time dedicated to the current manuscript. We understand the concern. Other reviewers pointed out changes in the text and requested reduction of paragraphs.

Reviewer 4 Report

In this manuscript, the authors obtained the dwarf germplasm materials from the first and second backcrossing. They founded that the all agronomic variables showed significant and progressive increases after the first and second backcrossing. The results are something interesting. However, the relevant experimental verification is lacking. For example, although the authors evaluated genotypes using the Kohonen SOM approach, the results were insufficient to support the results described by the authors. Some obvious issues, but not limited, are listed below.

Major comments in addition to above:

1．The authors need to supplement genotyping methods in detail with results. In addition, more experimental methods are needed to analyze the genotypes of genetic materials.

2. The authors need to supplement relevant insect resistance experiments.

Minor comments:

1. Line22 Were evaluated six？

2. Line23-24,74 “six dwarf tomato populations and13 dwarf populations obtained”

How does the populations here understand that? How big are your two opulations? How many lineages does each population contain? Should it be replaced with pedigree? The author needs to explain in detail.？

3. Line27-28 Sci#16.1-2; Sci#25.1,1-2; Sci#25.1,2-2; Sci#3.1,1-2; Sci#3.1,2-2; Sci#8.3,1- 27-2; and Sci#8.3,2-2, How do you understand the names of the families? Does it need to be rewritten? The author needs to explain in detail.

4. Table 1 has a problem. It should be in three-line grid format.

7. Figure 2 requires a vertical coordinate to be normalized. Is it statistically significant that the error line is larger?

8. Table 2 has a problem. It should be in three-line grid format.

9. Figure 3 has a low resolution and is not clear. In addition, the author needs to explain the results of Figure 3 in detail. Tell the reader how to understand the diagram.

10. The reference format is inconsistent.

Author Response

Response to Reviewer 4 Comments

Authors thank the reviewer for the time dedicated to the current manuscript and the valuable comments made:

Major comments in addition to above:

Question # 1: The authors need to supplement genotyping methods in detail with results. In addition, more experimental methods are needed to analyze the genotypes of genetic materials.

Response 1: We understand the reviewer's notes. In fact, there are many ways to evaluate the germplasm bank in plants. The methodologies used in this manuscript are also techniques frequently applied and published in several journals. It is important to emphasize that there is a scarcity of research in relation to characterizing dwarf tomato germplasm. It should be noted that the result of this article may encourage future research in the area of genetic improvement in order to obtain hybrids. As an example, we reinforce that the present manuscript is demonstrating the potential of a germplasm of Santa Cruz dwarf plants for use as male parents, which can be crossed with normal plants and obtain hybrids with several additional advantages as reported by Finzi et al. (2019). Therefore, among several techniques, we used those of the present manuscript from which we observed excellent responses. Additionally, we inform that there is a greater complexity when we characterize dwarf tomato germplasm, as they are plants that do not have commercial potential because they are dwarfs, but excellent parents to produce normal hybrids.

Question # 2: The authors need to supplement relevant insect resistance experiments.

Response 2: We understand the reviewer's concern. However, to carry out larger experiments, a larger amount of seeds is needed. In the case of the present research, when performing hybridizations, the number of seeds is smaller. Especially due to the low seed production of the dwarf plant. If we consider the research carried out by Maluf et al. (2010), which reports that intermediate levels of acylsugars are sufficient to provide a broad spectrum of resistance to pests, it is possible to assume that in the present research there are genotypes with high potential.

Minor comments:

Question # 3: Line22 Were evaluated six？

Response 3: Yes. We check and verify that our text is correct. Six BC₁populations were evaluated:

“The genetic material evaluated included six dwarf tomato populations obtained from the first backcross (BC₁), 13 dwarf tomato populations obtained from the second backcross (BC₂), the donor parent (DP), and the commercial cultivar ‘Santa Clara’ as the check, totaling 21 treatments.”

The sentence has been rewritten (line 22): “Six and 13 dwarf tomato populations obtained from the first and second backcrossing, respectively, the donor parent, and the commercial cultivar ‘Santa Clara’ as the check, totaling 21 treatments, were evaluated”.

Question # 4: Line23-24,74 “six dwarf tomato populations and13 dwarf populations obtained”

Response 4: In the Materials and Methods section, it is specified that 6 populations were obtained for BC₁ and 13 populations for BC₂, totaling 19 populations that, together with the controls, are 21 treatments, as follows:

Lines 76-81: “The genetic material evaluated included six dwarf tomato populations obtained from the first backcross (BC₁), 13 dwarf tomato populations obtained from the second backcross (BC₂), the donor parent (DP), and the commercial cultivar ‘Santa Clara’ as the check, totaling 21 treatments. BC₁ and BC₂populations were obtained after hybridization between a homozygous pre-commercial strain of Santa Cruz fruits (UFU-TOM-Mother-2) and the dwarf strain UFU MC TOM1 [9].”

Additionally, in the Materials and Methods section, the information requested by the reviewer is presented and we believe that it would solve the doubts listed.

Lines 93-96: “…The experiment followed a randomized block design with 21 treatments and 4 replicates. The experimental plots comprised six plants distributed in double rows with 0.3 × 0.3 m spacing. The spacing between the double rows was 0.8 m, totaling 504 plants.”

Question # 5: Line27-28 Sci#16.1-2; Sci#25.1,1-2; Sci#25.1,2-2; Sci#3.1,1-2; Sci#3.1,2-2; Sci#8.3,1- 27-2; and Sci#8.3,2-2, How do you understand the names of the families? Does it need to be rewritten? The author needs to explain in detail.

Response 5: The codes applied in the experiment were obtained from the advancement of each generation. These codes are automatically generated by the BG Alfa Biofort software registered at INPI (BR512019002403-6) developed by the Federal University of Uberlândia and used to archive the accessions of the vegetable germplasm bank of this institution.

Question # 6: Table 1 has a problem. It should be in three-line grid format.

Response 6: Table 1 was not constructed in three-line grid format. We followed the guidelines listed in the “Instructions for Authors” and used the table template available in the Microsoft Word template (https://www.mdpi.com/journal/agronomy/instructions):

Table 1. This is a table. Tables should be placed in the main text near to the first time they are cited.

Title 1	Title 2	Title 3
entry 1	data	data
entry 2	data	data ¹

¹ Tables may have a footer.

Question # 7: Figure 2 requires a vertical coordinate to be normalized. Is it statistically significant that the error line is larger?

Response 7: The reviewer's observation is very important. We authors would like to present a justification for keeping figure 2. This figure, which deals with the magnitudes between the donor parent, BC1 and BC₂, was made from the calculation of the relative superiority of the backcrosses in relation to the donor parent. It expresses exactly in magnitude (%) the superiority, the increment at each backcross. The relative superiority calculation [(BC_/DG)-1]x(100) is used in the scientific dissemination of several researches/articles. Basically, Figure 2 demonstrates the bar graphs in the box plot format. The bars are the averages of DG, BC₁ and BC₂ for each response variable. The advantages of the box plot are also well known, as they provide not only the mean but also the variance and presence of out liners. We emphasize that the evaluation of germplasm of dwarf plants is very complex, because the plant does not allow ample development and productivity, reducing the magnitude of the effects. Therefore, we would like to maintain the traditional way of presenting relative superiority.

Question # 8: Table 2 has a problem. It should be in three-line grid format.

Response 8: Table 2 was not constructed in three-line grid format. We followed the guidelines listed in the “Instructions for Authors” and used the table template available in the Microsoft Word template (https://www.mdpi.com/journal/agronomy/instructions):

Table 1. This is a table. Tables should be placed in the main text near to the first time they are cited.

Title 1	Title 2	Title 3
entry 1	data	data
entry 2	data	data ¹

¹ Tables may have a footer.

Question # 9: Figure 3 has a low resolution and is not clear. In addition, the author needs to explain the results of Figure 3 in detail. Tell the reader how to understand the diagram.

Response 9: We changed Figure 3 and improved its resolution. We have inserted in the text the explanation of the results in detail for a better understanding of the diagram.

Text has been inserted in lines 246-254 (in blue): “In a Kohonen SOM, genotypes that are most similar are grouped within the same neuron. In contrast, genotypes clustered in different neurons show genetic dissimilarity. Neurons consist of individuals that have some similarity with the neighboring class, and the most divergent and intermediate classes constitute the extreme and central regions of the map, respectively. An output layer comprising 4 rows × 4 columns was obtained from the Kohonen SOM. The genotypes evaluated in this study were grouped into 11 distinct neurons (Figure 3). Each hexagon represents a neuron and the amount of area filled within a hexagon indicates the concentration of grouped genotypes in that neuron. Thus, the greater the number of genotypes grouped in a neuron, the greater is the filled area.”

Question # 10: The reference format is inconsistent.

Response 10: We inform you that we have made changes by inserting the Abbreviated Journal Name. We followed the Microsoft Word template to prepare their manuscript (https://www.mdpi.com/journal/agronomy/instructions):

“References must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript. We recommend preparing the references with a bibliography software package, such as EndNote, Reference Manager or Zotero to avoid typing mistakes and duplicated references. Include the digital object identifier (DOI) for all references where available.”

“In the text, reference numbers should be placed in square brackets [ ] and placed before the punctuation; for example [1], [1–3] or [1,3]. For embedded citations in the text with pagination, use both parentheses and brackets to indicate the reference number and page numbers; for example [5] (p. 10), or [6] (pp. 101–105).”

Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
Author 1, A.; Author 2, B. Title of the chapter. In Book Title, 2nd ed.; Editor 1, A., Editor 2, B., Eds.; Publisher: Publisher Location, Country, 2007; Volume 3, pp. 154–196.
Author 1, A.; Author 2, B. Book Title, 3rd ed.; Publisher: Publisher Location, Country, 2008; pp. 154–196.
Author 1, A.B.; Author 2, C. Title of Unpublished Work. Abbreviated Journal Name year, phrase indicating stage of publication (submitted; accepted; in press).
Author 1, A.B. (University, City, State, Country); Author 2, C. (Institute, City, State, Country). Personal communication, 2012.
Author 1, A.B.; Author 2, C.D.; Author 3, E.F. Title of Presentation. In Proceedings of the Name of the Conference, Location of Conference, Country, Date of Conference (Day Month Year).
Author 1, A.B. Title of Thesis. Level of Thesis, Degree-Granting University, Location of University, Date of Completion.
Title of Site. Available online: URL (accessed on Day Month Year).

Round 2

Reviewer 4 Report

My suggestions were dealt with seriously in the revised manuscript.