Development and Application of Gene-Specific Markers for Tomato Yellow Leaf Curl Virus Resistance in Both Field and Artificial Infections

Round 1

Reviewer 1 Report

The manuscript describes development of TYLCV resistance gene markers and used these markers on a set of breeding lines and commercial cultivars. The work is simple and may be of value for tomato breeding programs. I do feel that it is not very novel because in the literature, markers for TYLCV genes have been described. I would have loved to see if authors have compared the markers or SNPs reported in literature with their newly developed markers. However, it is a good effort to design new markers as described in this manuscript. Few additional things include:

1. At many places authors have mentioned “data not shown”. I will be interested to see the sequence data (used for primer design) as supplementary data.
2. Authors did not see amplification of CI gene by PCR for TYLCV resistance genes. The fact that ty-5 is a weak gene and Ty-3 is also not completely effective in controlling the virus, I would have seen some weak amplification of virus CI gene.
3. Similarly, ty-5 containing plants did not show symptoms under natural infections. Are there escapes in natural infections. Authors have not discussed about escape plants in the manuscript.

Minor comments:

Page 1, line 38: “of” instead of “about”

Page 2, line 68: “TYLCV” instead of “TVLCV”

Page 4: Figure 2 legend does not indicate solid line. Please make it more clear.

Page 4 line 128: sentence “the SNP polymorphism ………..to threonine” should be written correctly. For example, SNP change results in a.a. change not a.a. change results in SNP.

Page 5, line 143: For ty-5, authors compared only one susceptible line. Based on one susceptible line data, author can not claim that their marker will differentiate R and S lines with accuracy.

Page \6, line 181-183: I don’t believe that ty-5 is strong enough to control virus completely but the PCR data indicate that there was no amplification from ty-5 containing lines.

Page 9, line 236: Does the marker used for TY-1/3 distinguish between Ty1 and Ty3 genes.

Page 9, line 241:  Another reason for infrequent use of Ty-2 gene is its narrow range against TYLCV strains.

Page 10, line 281: Why there were no symptoms with plants containing only ty-5?

Author Response

The manuscript describes development of TYLCV resistance gene markers and used these markers on a set of breeding lines and commercial cultivars. The work is simple and may be of value for tomato breeding programs. I do feel that it is not very novel because in the literature, markers for TYLCV genes have been described. I would have loved to see if authors have compared the markers or SNPs reported in literature with their newly developed markers. However, it is a good effort to design new markers as described in this manuscript. Few additional things include: 

• Thank you. We have added Supplemental Figure 2 to show the efficacy of these markers using genotypes well-known for TYLCV resistances. Full sequence information used for each marker development is displayed in current figures (Figures 2, 3, and 4) and explained in the text (page 3-5). 

At many places authors have mentioned “data not shown”. I will be interested to see the sequence data (used for primer design) as supplementary data. 

• The full sequence information used for primer design for each marker is displayed in current figures (Figures 2, 3, and 4). Sequence data for Ty4 marker development has been provided as Supplementary Figure 1. 

Authors did not see amplification of CI gene by PCR for TYLCV resistance genes. The fact that ty-5 is a weak gene and Ty-3 is also not completely effective in controlling the virus, I would have seen some weak amplification of virus CI gene. Similarly, ty-5 containing plants did not show symptoms under natural infections. Are there escapes in natural infections. Authors have not discussed about escape plants in the manuscript. 

• We have replaced the semi-quantitative PCR data with quantitative real-time PCR in naturally infected plants, which better reflects viral DNA accumulation. The weak implication of viral DNA in TY172 with ty5 resistance is detected in both natural infection and agro-infection conditions. Statistically more amplification of viral DNA is detected for the ty5 genotype in natural infection (Fig6C), although a significant difference was not detected when compared with other resistance genotypes in agro-infection conditions (Fig 7C).  

 

Minor comments: 

 

Page 1, line 38: “of” instead of “about”  

• corrected

Page 2, line 68: “TYLCV” instead of “TVLCV” 

• corrected

Page 4: Figure 2 legend does not indicate solid line. Please make it more clear. 

• revised

Page 4 line 128: sentence “the SNP polymorphism ………..to threonine” should be written correctly. For example, SNP change results in a.a. change not a.a. change results in SNP. 

• corrected

Page 5, line 143: For ty-5, authors compared only one susceptible line. Based on one susceptible line data, author cannot claim that their marker will differentiate R and S lines with accuracy. 

• In this study, TY172 was used for the ty5 resistant line, which was derived from four different wild-type tomatoes and used for characterizing ty5 resistance (Lapidot et al., 2015). A marker for ty5 was designed based on a single SNP detected from sequence comparison between TY172 with ty5 resistance and other genotypes. We have added Supplemental Figure 2 to show the efficacy of markers developed in this study using known resistance sources. The figure clearly shows that the ty5 marker can differentiate TY172 from all other genotypes as intended.

 

Page 6, line 181-183: I don’t believe that ty-5 is strong enough to control virus completely but the PCR data indicate that there was no amplification from ty-5 containing lines. 

• We have replaced the semi-quantitative PCR data with quantitative real-time PCR in naturally infected plants, which better reflects viral DNA accumulation. Weak application of viral DNA in TY172 with ty5 resistance is detected in both natural infection and agro-infection conditions. Statistically more amplification of viral DNA is detected for ty5 genotype in natural infection (Fig 6C), although a significant difference was not detected when compared with other resistance genotypes in agro-infection conditions (Fig 7C). 

Page 9, line 236: Does the marker used for TY-1/3 distinguish between Ty1 and Ty3 genes. 

• The marker we used for TY-1/3 cannot distinguish Ty3 allele from Ty1 allele. 

Page 9, line 241:  Another reason for infrequent use of Ty-2 gene is its narrow range against TYLCV strains. 

• Thank you for this comment. The resistance range of Ty2 gene is now incorporated with proper citation(Mejia et al., 2005) 

Page 10, line 281: Why there were no symptoms with plants containing only ty-5? 

• Although viral DNA accumulation was detected in the ty5 genotype, the amount of viral gene was significantly lower than the susceptible genotypes. It can be assumed that the amount of virus that existed in the ty5 genotype was not enough to cause symptom development under the given environmental condition. 

Reviewer 2 Report

The main goal and consequently, the final result is not clear. The result section contains material and methods used in the work, rather than real outcomes. It seems that authors intended to include DNA marker development as one of the outcomes in the study; however, they didn’t make that clear on the title or in the summary. On the other hand, Most of the TYLCV resistant genes (except Ty-4) have been identified and their effectiveness against TYLCV has been characterized. There are published and well documented DNA markers for all the TYLCV resistant genes used in the study and developing a new set of DNA markers might not be a very significant accomplishment. To characterize the effectiveness of gene pyramiding against TYLCV, the study needs to be done by using isogenic populations varying only for the genes under study.

Author Response

The main goal and consequently, the final result is not clear. The result section contains material and methods used in the work, rather than real outcomes. It seems that authors intended to include DNA marker development as one of the outcomes in the study; however, they didn’t make that clear on the title or in the summary. On the other hand, Most of the TYLCV resistant genes (except Ty-4) have been identified and their effectiveness against TYLCV has been characterized. There are published and well documented DNA markers for all the TYLCV resistant genes used in the study and developing a new set of DNA markers might not be a very significant accomplishment. To characterize the effectiveness of gene pyramiding against TYLCV, the study needs to be done by using isogenic populations varying only for the genes under study. 

• We appreciate all the comments by the reviewer 2. The title has been changed to “Development and application of gene-specific markers for tomato yellow leaf curl virus resistance in both field and artificial infections”. We have revised our data and included new data as suggested by other reviewers, which include quantitative real-time PCR to better evaluate the relative amount of viral DNA in both field and artificial infections. Full sequence information used for all the markers developed in this study is provided. To show the efficacy of the markers developed in this study, previously known resistance sources are included and tested with these markers (Supplementary Figure 2). Those are the main improvements in this revised version. 
• Although the coding genes for Ty2 and ty5 resistance have been previously identified and published, efficient marker system to select each TYLCV resistance genotypes is still needed. In addition, the effectiveness of these resistance genes against TYLCV in field condition still required further verifications. We do agree that isogenic populations would be ideal to precisely evaluate gene pyramiding effects so that we are in the process of combining more resistance genes in the minimized genetic background diversity. We used plant materials currently available for us in this study. 

Reviewer 3 Report

The paper considered to be published in the Plants show a development of markers for TY genes that mediated resistance against TYLCV and used them to genotype tomato accession and commercial cultivar regards to presence or absence of those genes. They also performed a resistance screening under controlled and natural conditions against TYLCV of those genotypes. TYLCV   is one of the most important tomato pathogens nowadays and has been spread across all world affecting the tomato production. The development and characterization of genotypes resistant to TYLCV is needed, because is the most effective and sustainable way to control this pathogen. Although the authors show a relevant work, some issues have to be addressed before the acceptance of Plants to publish this paper. In this way follow some comments and suggestion to be addressed for a better understanding of the paper.

 

 

Line 1 – The title passes a wrong idea about what the author had performed, as no breeding system has been developed. In this way, a better title would be only addressing the development of markers and characterization of tomato genotypes resistance against TYLCV.

Line 37 – the author should use “TYLCV is transmitted by different whiteflies specie within Bemisia tabaci complex” in place of “the vector for TYLCV infection in tomato is a whitefly (Bemisia tabaci)”

Line 39 -  Whitefly is a common name for a group of insects involving more than 1000 species. The author should be more specific and mention B. tabaci complex. Furthermore, only B. tabaci complex transmits more than 400 viruses species across six different genera of plant viruses.

Line 40 – The author should use the right and most updated nomenclature that is B. tabaci Middle East Asian Minor 1 (MEAM1) and B. tabaci MEDITERRANEAN (MED) for biotype B and Q, respectively. The Q and B biotypes are outdated as this two groups are known to constitute two distinct species.

Line 41 and 42 – The author should clarify what do they mean with “due to the invasiveness of the infection and the persistent circulation of the virus” who is invasive is the virus not the infection that is a process. It should be better if they use  “due to the invasiveness and the frequent circulation of the virus”.

Line 51 – the author should add a citation at the end of this paragraph.

Line 57 – use “The gene Ty-2 is originated in S. habrochaites “B6013” located on the long arm of chromosome 11”, instead “The gene 57 Ty-2 originated in S. habrochaites “B6013”, and it is located on the long arm of chromosome 11”.

Line 75 – This paragraph should be removed from introduction, and perhaps added to the discussion. In addition, the sentence “TYLCV viruliferous whitefly-mediated inoculation is considered to be analogous to the natural infection process”. What the author wanted to say with this sentence?

Line 115 – The figure legend mention “White boxes indicate coding sequences (exons), solid line indicates non-coding 119 sequences (introns)”, there is no intron in this gene. This should be removed.

The paper has a many figures, 7 at the total, I suggest that Figures 2 , 3 and 4  to be combined in only one that would be Figure 2, with multiple panels (A, B, C, …), as they are showing very similar information and could share the same legend.

Line 133 – Figure 3 – the reverse primer  in not represented.

While the section 2.1 about the development of gene specific markers has been well developed by the authors, before the application of those markers testing any accessions or cultivar, they should have been validated by using plant genotypes that is well known to carry a given gene. In this way the assay would be validated, and so could be used for further genotyping. I suggest the authors to include this validation assay, including well known genotypes that carrying all genes tested  and also negative controls. While the accession LA4440 is known to carry TY-4, what could be used for validation, for other genes, TY-2 and TY-5, this was not the case.  This should be presented in the section 2.1.

Line 158 – The number of accessions and cultivar does not match with those showed at the Table 1 and in Methods section. This information must be verified.

Line 172 - What was the criteria used to select plants to perform the experiment in natural conditions? Why not all of them? This information should be on the section 2.3.

Also, the inoculum pressure and whiteflies abundance may vary from season to season. In this study the author performed a field trial using a limited number of plants (3 plants for each cultivar/accession tested) and only one repetition was conducted during 2018. Based on that, why use only three plants, was it based in what? The authors should mention how many plants of the positive control was infected, so would give the readers an idea about how the inoculum source in those natural condition.

Lines 175 and 176 – Why use semi quantitative PCR? It is already well known that semi-quantitative PCR  is not accurate, and the authors has the possibility to perform real time quantitative PCR, as was performed for experiment using infectious clone. This experiment should be changed by a qPCR.

Line 182 - Were all plants tested for infection based on PCR or the status of infection was verified only based on symptoms? This information is missing. The semi-quantitative PCR was performed for all three plants or not?

Lines 190 and 191 – “Semi-quantitative PCR analysis of the mRNA levels of TYLCV replication-associated protein gene (C1) in leaves. DNA was extracted …”. This sentence is very confused, the author first said they used mRNA to perform semi-quantitative PCR and after they mention DNA extraction? First, what was really done? If mRNA was used, they should explain what the reason for doing that, as TYLCV is a DNA virus.

Line 193 and 194 – Is it the mean for all three plants?

Line 198 – What did the author mean with “Natural infection of TYLCV is caused by multiple whiteflies and the artificial infection is performed with the single agro-injection”. Usually, when performing artificial inoculation either by agro-injection or biolistic, the amount of viruses is much higher compared to natural infection. If the difference in symptoms  is due to the difference in viral titer due difference in inoculation process, they should perform a qPCR between plants agro-injected and and natural inoculated and show that is the case. In the other hand,if they were referring  the variability of the virus population circulating in natural fields is higher and may explain that phenotype, they should address this hypothesis.

Line 214 – Why did they perform a relative PCR using a 2ddCT? What would have happened if you change your control? The author should have used an absolute quantification by performing a standard curve  and give the absolute number of copies of viral genomes. Thus, this would be easily comparable across different studies independent of control used. If the author would like to normalize the amount of virus per plant DNA, they should have done a standard curve for plant DNA and give the number of virus copy per plant genome copy, what would still be comparable.

Line 216 – 218 – If the authors applied a statical test and it was not significant they should not mention that there was difference in DNA levels but was not significant.

Line 259 – “Natural infection with TYLCV in the field performed over multiple years”, the authors only show results for only one year, that was 2018, is it correct?

Line 273 – What would be these numerous factors?

Line 279 and 280 – If the author applied a statical test, they should not keep saying that there was difference, but it was not significantly difference.

Line 331 – 332 – Again, the same as before (Line 279 and 280).

Line 346 – CTAB instead “C-TAB”

Line 356 – Is Money Make an accession or cultivar?

Line 360 – The author gave a reference for all those primers used in this work. What would that references mean, if the authors every moment say “development of a marker”? This should be clarified, was those markers developed in this paper or not?

Line 363 – “For natural infection by whitefly, 18 cultivars with three different plants of each” – in the Figure 6 panel C only 13 images are shown.

Line 379 –“at least three independent inoculations were performed for each plant genotype” this information does not match with that provided in the Supplementary Table 2, for instance No 10 and 16, at the first line.

Line 399 – This section should be better described with more details.

 

Author Response

The paper considered to be published in the Plants show a development of markers for TY genes that mediated resistance against TYLCV and used them to genotype tomato accession and commercial cultivar regards to presence or absence of those genes. They also performed a resistance screening under controlled and natural conditions against TYLCV of those genotypes. TYLCV   is one of the most important tomato pathogens nowadays and has been spread across all world affecting the tomato production. The development and characterization of genotypes resistant to TYLCV is needed, because is the most effective and sustainable way to control this pathogen. Although the authors show a relevant work, some issues have to be addressed before the acceptance of Plants to publish this paper. In this way follow some comments and suggestion to be addressed for a better understanding of the paper. 

•  We appreciate all the comments by the reviewer 2. 

Line 1 – The title passes a wrong idea about what the author had performed, as no breeding system has been developed. In this way, a better title would be only addressing the development of markers and characterization of tomato genotypes resistance against TYLCV. 

• The title has been revised as suggested. The current title is “Development and application of gene-specific markers for tomato yellow leaf curl virus resistance in both field and artificial infections”. 

Line 37 – the author should use “TYLCV is transmitted by different whiteflies specie within Bemisia tabaci complex” in place of “the vector for TYLCV infection in tomato is a whitefly (Bemisia tabaci)” 

• Corrected.

Line 39 -  Whitefly is a common name for a group of insects involving more than 1000 species. The author should be more specific and mention B. tabaci complex. Furthermore, only B. tabaci complex transmits more than 400 viruses species across six different genera of plant viruses. 

• Corrected and proper citations have been added accordingly.

Line 40 – The author should use the right and most updated nomenclature that is B. tabaci Middle East Asian Minor 1 (MEAM1) and B. tabaci MEDITERRANEAN (MED) for biotype B and Q, respectively. The Q and B biotypes are outdated as this two groups are known to constitute two distinct species. 

• Corrected and proper citations have been added accordingly.

Line 41 and 42 – The author should clarify what do they mean with “due to the invasiveness of the infection and the persistent circulation of the virus” who is invasive is the virus not the infection that is a process. It should be better if they use  “due to the invasiveness and the frequent circulation of the virus”. 

• Corrected.

Line 51 – the author should add a citation at the end of this paragraph. 

• The proper citation has been added.

Line 57 – use “The gene Ty-2 is originated in S. habrochaites “B6013” located on the long arm of chromosome 11”, instead “The gene 57 Ty-2 originated in S. habrochaites “B6013”, and it is located on the long arm of chromosome 11”. 

• Corrected.

Line 75 – This paragraph should be removed from introduction, and perhaps added to the discussion. In addition, the sentence “TYLCV viruliferous whitefly-mediated inoculation is considered to be analogous to the natural infection process”. What the author wanted to say with this sentence? 

• The paragraph has been removed from the introduction and partially integrated into the discussion.

Line 115 – The figure legend mention “White boxes indicate coding sequences (exons), solid line indicates non-coding 119 sequences (introns)”, there is no intron in this gene. This should be removed. 

• Corrected.

The paper has a many figures, 7 at the total, I suggest that Figures 2 , 3 and 4  to be combined in only one that would be Figure 2, with multiple panels (A, B, C, …), as they are showing very similar information and could share the same legend. 

• We have agreed on this comment and merged these three figures into one single figure as suggested. However, we found that it is more explicit when they are in separate figures. 

Line 133 – Figure 3 – the reverse primer  in not represented. 

• Figure 3 has been revised accordingly.

 

While the section 2.1 about the development of gene specific markers has been well developed by the authors, before the application of those markers testing any accessions or cultivar, they should have been validated by using plant genotypes that is well known to carry a given gene. In this way the assay would be validated, and so could be used for further genotyping. I suggest the authors to include this validation assay, including well known genotypes that carrying all genes tested and also negative controls. While the accession LA4440 is known to carry TY-4, what could be used for validation, for other genes, TY-2 and TY-5, this was not the case.  This should be presented in the section 2.1. 

• Supplemental Figure 1 is added. Newly developed markers are tested with known sources for TYLCV resistances.  

Line 158 – The number of accessions and cultivar does not match with those showed at the Table 1 and in Methods section. This information must be verified. 

• We have double-checked. There were some minor typos found, but the list in the Table matches with the cultivars used in Figure 5. The order of the list in the table is not maintained in the Figures.

Line 172 - What was the criteria used to select plants to perform the experiment in natural conditions? Why not all of them? This information should be on the section 2.3. Also, the inoculum pressure and whiteflies abundance may vary from season to season. In this study the author performed a field trial using a limited number of plants (3 plants for each cultivar/accession tested) and only one repetition was conducted during 2018. Based on that, why use only three plants, was it based in what? The authors should mention how many plants of the positive control was infected, so would give the readers an idea about how the inoculum source in those natural condition. 

• Limited space in the field was allowed for testing this natural  Threedifferent susceptible genotypes were included as the positive control and three plants per each genotype were used. As shown in quantitative real-time PCR result, a very small amount of SE was detected indicating that all the susceptible controls were fully susceptible when no or statistically meaningless amount of viral DNA amplification was detected in resistant genotypes except TY172 with ty5 resistance. 

Lines 175 and 176 – Why use semi quantitative PCR? It is already well known that semi-quantitative PCR  is not accurate, and the authors has the possibility to perform real time quantitative PCR, as was performed for experiment using infectious clone. This experiment should be changed by a qPCR. 

• We have replaced the semi-quantitative PCR data with quantitative real-time PCR in naturally infected plants as suggested. 

Line 182 - Were all plants tested for infection based on PCR or the status of infection was verified only based on symptoms? This information is missing. The semi-quantitative PCR was performed for all three plants or not? 

• DNA extracted for all three plants were used in this quantitative real-time PCR assay. 

Lines 190 and 191 – “Semi-quantitative PCR analysis of the mRNA levels of TYLCV replication-associated protein gene (C1) in leaves. DNA was extracted …”. This sentence is very confused, the author first said they used mRNA to perform semi-quantitative PCR and after they mention DNA extraction? First, what was really done? If mRNA was used, they should explain what the reason for doing that, as TYLCV is a DNA virus. 

• Corrected.

Line 193 and 194 – Is it the mean for all three plants? 

• Revised.

Line 198 – What did the author mean with “Natural infection of TYLCV is caused by multiple whiteflies and the artificial infection is performed with the single agro-injection”. Usually, when performing artificial inoculation either by agro-injection or biolistic, the amount of viruses is much higher compared to natural infection. If the difference in symptoms  is due to the difference in viral titer due difference in inoculation process, they should perform a qPCR between plants agro-injected and and natural inoculated and show that is the case. In the other hand, if they were referring  the variability of the virus population circulating in natural fields is higher and may explain that phenotype, they should address this hypothesis. 

• The sentence has been revised. Quantitative real-time PCR results in both naturally infected plants and agro-injected plants are provided.

 

Line 214 – Why did they perform a relative PCR using a 2ddCT? What would have happened if you change your control? The author should have used an absolute quantification by performing a standard curve  and give the absolute number of copies of viral genomes. Thus, this would be easily comparable across different studies independent of control used. If the author would like to normalize the amount of virus per plant DNA, they should have done a standard curve for plant DNA and give the number of virus copy per plant genome copy, what would still be comparable. 

• We totally agree with your suggestion. We’ve performed a standard curve. However, we prefer to present relative difference of the viral DNAs. Indeed, we conducted this relative analysis as a key reference (Yan et al, 2018).

Line 216 – 218 – If the authors applied a statical test and it was not significant they should not mention that there was difference in DNA levels but was not significant. 

• We’d like to keep this comment as it is.

Line 259 – “Natural infection with TYLCV in the field performed over multiple years”, the authors only show results for only one year, that was 2018, is it correct? 

• Yes, we are showing natural infection results from 2018.

Line 273 – What would be these numerous factors? 

• Numerous factors affecting viral transmission in nature which can be neglected by Agro-mediated inoculation of TYLCV could be 1) roles of various plant-volatiles attracting or repelling insects, 2) surface structure of plants such as trichomes affecting plant-insect interactions, 3) color changes (chlorosis/yellowing/mosaic) caused by viral infection affecting insect behavior, 4) sugar changes caused by viral infection affecting insect feeding behavior, and so on. 

Line 279 and 280 – If the author applied a statical test, they should not keep saying that there was difference, but it was not significantly difference. 

• We’d like to keep this comment in the discussion.

Line 331 – 332 – Again, the same as before (Line 279 and 280). 

• We’d like to keep this comment in the discussion.

Line 346 – CTAB instead “C-TAB”                                          

• Corrected.

Line 356 – Is Money Make an accession or cultivar? 

• Money Maker can be considered as an accession.

Line 360 – The author gave a reference for all those primers used in this work. What would that references mean, if the authors every moment say “development of a marker”? This should be clarified, was those markers developed in this paper or not? 

• All the markers used in this assay were developed in this study except the Ty1/3 marker. Properly revised. Thank you for this comment.

Line 363 – “For natural infection by whitefly, 18 cultivars with three different plants of each” – in the Figure 6 panel C only 13 images are shown. 

• 18 cultivars with three different plants were used for natural infection as shown in Fig 6C, however, phenotypic images from 13 cultivars are displayed in Fig 6B as pointed out.

Line 379 –“at least three independent inoculations were performed for each plant genotype” this information does not match with that provided in the Supplementary Table 2, for instance No 10 and 16, at the first line. 

• Supplementary Table 2 has been removed. 

Line 399 – This section should be better described with more details. 

• Revised

Round 2

Reviewer 2 Report

TYLCV can be found in tropical and subtropical regions causing severe economic losses. The authors have done a significant amount of research efforts contributing to disease mitigation strategies. There are published DNA markers associated with most of the Ty genes and some of the newly designed DNA markers in this study (Ty-2 and ty-5) might be useful in marker-assisted tomato breeding as well.

Designing a DNA marker, directly based on a genetic variation causing the resistance will discriminate susceptible from the resistant allele; however, for an unknown resistance locus before, a strong association between a DNA marker and resistant phenotype (in a segregating population) needs to be established and confirmed before introducing the new DNA marker to the public. The DNA marker developed for Ty-4 may identify a genetic polymorphism in LA4440 but it cannot be used in marker-assisted selection with much confidence.  The Authors did not confirm the usefulness of the Th-4 marker in such a manner; furthermore, they were not able to evaluate the resistance associated with Ty-4 locus in LA4440. Consequently, the authors need to consider potential recombination events between the Ty-4 marker and the resistance associated with the Ty-4 locus and then address the issue in the study.

If the marker/resistance association is not confirmed, utilizing the DNA marker published in this article may result in the waste of time and resources when plant breeders rely on them to incorporate Ty-4 into their germplasm by marker-assisted selection.

Author Response

• We appreciate this comment about suggesting an association study between the resistance phenotype and the Ty-4 markers developed in this study. In the case of Ty-4 resistance, the resistance gene exists within the 550 kb region between C2_At4g17300 and C2_At5g60160 on chromosome 3 (Ji et al., 2009). Ty4 markers developed in this study are designed based on sequence variations of the candidate genes located within the 550 kb region, therefore, we do believe that the Ty-4 markers can be used in marker-assisted selection with much confidence. In addition to LA4440, the Ty-4 markers are polymorphic in LA1932 and LA2779 (Supplementary Fig. 2). We agree that the validation of these markers in a segregation population would improve the selection confidence. To improve the selection accuracy by generating a functional marker and to characterize the Ty-4 gene itself, we also think that an association study would be necessary and are in the process of generating a population suitable for the purpose. Unfortunately, we are not able to validate this within the permitted revision timeline. In addition, Ty-4 confers a lesser effect on TYLCV resistance when combined with Ty-3 resistance (Ji et al., 2009), which suggests validation of the Ty-4 markers in combination with other TYLCV resistance genes in terms of gain of resistance.

Round 3

Reviewer 2 Report

Ty-4 is a minor TYLCV resistant gene yet to be identified. Tomato breeders are already using flanking markers to introgress Ty-4 locus into new breeding material. Ty-4 was mapped to the 2.3-cM marker interval between C2_At4g17300 and C2_At5g60160 in the introgression (Ji et al., 2009). Using 2 flanking markers gives breeders more confidence that the entire introgression carrying Ty-4 is transferred into new breeding material. instead of building upon the previous study, the single marker introduced in this study brings more disadvantages for marker-assisted selection to incorporate Ty-4 into new breeding material. 

From my point of view, one of the following suggestions may fix the problem :

1. Clearly mention in the text (Summery, Result, and Conclusion) that the newly designed Ty-4 marker is not tested and may not tightly linked to the Ty-4 locus.
2. Design 2 flanking markers covering upper and lower limits of the Ty-4 introgression.
3. Remove entire Ty-4 marker aspect of the article and resubmit it again.

Author Response

Thank you for your kind comments. Two Ty4 markers developed in this study are located between C2_At4g17300 and C2_At5g60160 on chromosome 3 (Ji et al., 2009), however, the Ty4 markers have not been tested with the segregating population for Ty4 resistance as the reviewer pointed out. It is now clearly mentioned in the text (line 155-157 and 247-250) as the reviewer suggested.