Review Reports - Comparative Transcriptome Analysis of Sprouting Capabilities of Sweet Potato Storage Roots with Low and High Virus Levels

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s comment:

The manuscript investigates differences in sprouting ability between sweet potato storage roots with low and high virus levels using transcriptome analysis. The topic is relevant for sweet potato seed root production, and the authors combine phenotypic observation, virus detection, and RNA-seq analysis. However, several weaknesses in experimental design, data interpretation, and presentation reduce the reliability of the conclusions.

The following are the section-wise points that need to be considered before acceptance:

Abstract

Long sentence with less clarity: Line 15-18: “With the increasing severity of sweet potato viral diseases, …..potato seedling cultivation.”
Line 26: “…..biological pathways contributing to the differences in sprouting…..” – data only show associations. No functional validation was performed. So can not interpret that these are the cause of the difference in sprouting due to virus load.

Introduction

The term “sprouting capability” is used throughout without a clearly defining through a quantitative criteria. It is not clear why authors assume that virus accumulation is the only cause to reduce sprouting, as other factors like physiological age, storage history, or environmental conditions may also play a role in reducing sprouting.

Materials and Methods

Low-virus and high-virus storage roots were collected from different geographic locations, - so there will be variation in agronomic practice and environmental conditions, that may also contribute for virus accumulation. The logic is not clear. There is no mention about any particular condition that is used during Sprouting Capability Assessment for Each Variety - How it was calculated? Why only five virus? Sweetpotato is infected by many viruses and often as mixed infection. There is no such result. The sprouting assay lacks clarity on the number of individual roots per replicate, as a result biological replication is unclear. Transcriptome analysis does not mention multiple-testing correction for DEG identification.

Results

Sprouting performance is primarily explained through photographs, with limited quantitative data on sprout number, length, or vigour. Samples are primariliy classified as low and high virus titers, and after that, virus detection was done, which confirms the same rather than providing new insights. DEG comparisons between varieties and virus levels are presented together, but varietal genetic effects are not clearly separated from virus-associated effects. KEGG pathway enrichment is interpreted broadly, with large pathways discussed without focusing on specific genes or regulatory points and no mechanistic explanations were given.

Discussion

Transcriptomic changes in starch metabolism and cell wall-related genes are discussed, but in any biotic stress and growth development process, such genes are upregulated. So exclusively, they can not be the drivers of sprouting without supporting enzyme activity or metabolite data. Hormone signalling results, especially for JA and SA pathways, do not clearly distinguish between responses related to virus defense and those directly involved in sprouting. Increased expression of JAZ genes is interpreted as activation of JA signaling but they have known repressor function, so the interpretation should look into this aspect also. There are several interpretations without further validation. So discussion is very weak and need to be rewritten.

Conclusions

The conclusions on “key pathways contributing to sprouting differences,” can not be done as no validation of the pathway was shown. There is no indication about the limitation of such transcriptomics data, as environmental effects, age and many other aspects also influence sprouting.

Comments on the Quality of English Language

Need to improve upon redundancy and language.

Author Response

Reviewer 1：

Reviewer’s comment:

The following are the section-wise points that need to be considered before acceptance:

Response:

Thank you very much for your affirmation of our research and article.

Abstract

Long sentence with less clarity: Line 15-18: “With the increasing severity of sweet potato viral diseases, …..potato seedling cultivation.”

Response:

We thank the reviewer for the suggestion. Based on the comment, we have made modifications for this sentence to clarify its meaning: ‘With the increasing severity of sweet potato viral diseases, the decline of sprouting capability of seed roots leads to severe declines in both the yield and quality of sweet potatoes. It is urgent to uncover the genetic basis and molecular mechanisms underlying the sprouting capability between different virus content storage roots.’ [Lines 15-19].

Line 26: “…..biological pathways contributing to the differences in sprouting…..” – data only show associations. No functional validation was performed. So can not interpret that these are the cause of the difference in sprouting due to virus load.

Response:

We thank the reviewer for this comment. Based on the comment, we have changed “key biological pathways” to “potential biological pathways”: ‘Comparative transcriptome analysis of the differences in sprouting capability between different virus content storage roots revealed that starch metabolism, cellulose metabolism, jasmonic acid (JA) signaling pathway, and salicylic acid (SA) signaling pathways are potential biological pathways contributing to the differences in sprouting capability between different virus content storage roots.’ [Lines 25-29].

Introduction

The term “sprouting capability” is used throughout without a clearly defining through a quantitative criterion. It is not clear why authors assume that virus accumulation is the only cause to reduce sprouting, as other factors like physiological age, storage history, or environmental conditions may also play a role in reducing sprouting.

Response:

We thank the reviewer for pointing this out. We provided a clear defining in Introduction about sprouting capability: ‘Sprouting capability is a key trait for measuring the quality of seed roots, mainly including the time of sprouting, the seedling growth rate, and the number and weight of sprouts produced per kilogram of storage roots.’ [Lines 47-49], ‘This study aims to compare the sprouting capability including the number and weight of seedlings produced per kilogram of storage roots of storage roots with low and high virus levels of two varieties, Jishu 25 and Jishu 26, to observe the differences in sprouting capability between storage roots with low and high virus levels.’ [Lines 72-76].

We agree with the reviewer's point that the sprouting of sweet potatoes is a complex biological pathway, and it can be affected by numerous factors such as physiological age, storage history, and biotic and abiotic stress et al. But viral diseases are the most significant factor affecting the yield and quality of seed roots and impair their sprouting capability. Therefore, in this research, we aimed to explore the molecular mechanism of the impact of viral diseases on sweet potato sprouting capability, so we focused on the factor of viral diseases. However, the impact of other factors on the sprouting of sweet potatoes is equally important and will be an important topic for our future research.

Materials and Methods

Response:

We thank the reviewer for the suggestion. Due to our negligence, we did not clearly explain why the sweet potato storage roots harvested in Yulin were classified as the low virus content group and the sweet potato storage roots harvested in Binzhou were classified as the high virus content group. In fact, we have conducted PCR and qRT-PCR tests for many years on the sweet potato storage roots harvested in the two regions, and carried out statistics on the incidence rate. We found that the virus carrying rate of the Jishu 25 and Jishu 26 storage roots harvested in Binzhou was 17.41% -41.25%, and the virus carrying rate of the storage roots bred in Yulin was 1.43% -8.72%. Therefore, we believe that the virus content of the sweet potato storage roots harvested in Yulin is low, and the virus content of the sweet potato storage roots harvested in Binzhou is high. These results are described in the articles of Li et al (2019). In this manuscript, qRT-PCR is also used for verification, and the results are consistent with the research of Li and others.

As suggested, we added detailed information in Introduction to clarify how "high virus" and "low virus" groups were selected: “In previous study, Li et al [15] found that the virus carrying rate of the Jishu 25 and Jishu 26 storage roots harvested in Binzhou was 17.41% -41.25%, and 1.43% -8.72% in Yulin. Therefore, the virus content of the sweet potato storage roots harvested in Yulin is low, and the virus content of the sweet potato storage roots harvested in Binzhou is high and we sampled the storage roots produced in these two regions for further research.” [Lines 68-72].

The J25 and J26 storage roots used in this research from Yulin and Binzhou were harvested from the same batch of virus-free sweet potato seedlings, which were provided by the Crop Research Institute of Shandong Academy of Agricultural Sciences. We carried out the virus quantification using PCR and qRT-PCR in the leaves of all individual plants which were used to produce storage roots used in this research. Only sweet potato seedlings that have passed our testing will be transported to Yulin and Binzhou for further experiments. In fact, this is currently a mature mode of production of sweet potatoes, as the use of virus-free seedlings can significantly increase the yield and quality of sweet potatoes, and improve economic benefits. Among this mode, the responsibility of our laboratory is to provide virus-free potato seedlings to farmers. Each batch will perform such testing work, and only after passing the testing will it be provided to farmers. Farmers will reproduce asexually in the aphid-proof greenhouses or net houses before planting on a large scale.

Referance:

Li, A. X., Zhang, L. M., Wang, Q. M., Wang, D. L., Xie, B. T., Qin, Z., Hou, F. Y., Dong, S. X. Effect of root seeds of east breeding in west China on occurrence of sweet potato virus disease. J. Shandong Agricu. Sci. 2019, 51, 87-90.

There is no mention about any particular condition that is used during Sprouting Capability Assessment for Each Variety - How it was calculated? Why only five virus? Sweetpotato is infected by many viruses and often as mixed infection. There is no such result. The sprouting assay lacks clarity on the number of individual roots per replicate, as a result biological replication is unclear. Transcriptome analysis does not mention multiple-testing correction for DEG identification.

Response:

We thank the reviewer for pointing this out. In fact, we conducted statistical analysis on the number of sprouts and sprouts weight of the four samples (J25-L, J25-H, J26-L, J26-H) during the experiment. Considering that the photos could intuitively observe their differences in sprouting capability, we did not provide the relevant data. As suggested, we have supplemented the data on the number of sprouts and sprouts weight in Table A2 and described in Results: ‘Figure 1E-H and Table A2 showed that the number and weight of sprouts per kilogram storage roots from J25-L and J26-L was significantly higher than that from J25-H and J26-H, further confirming the superior sprouting capability of storage roots with low virus levels compared to storage roots with high virus levels.’ [Lines 157-160].

In production, the sprouting capability is usually measured by the number and weight of sprouts produced per kilogram of storage roots. In this research, we set up three biological replicates for each sample, and in each biological replicate, we planted 15 kg of uniformly sized storage roots on one square meter of land to evaluate the sprouting capability. Therefore, we did not count the specific number of storage roots. In the Materials and Methods section, we provided a more detailed description of the determination of the number of sprouts and sprouts weight: ‘Following the method described by Hou [6], sprouting was conducted using the sparse arrangement method with a planting density of 15 kg/m². The healthy, medium and similar sized storage roots from two varieties with low and high virus levels (J25-L, J25-H, J26-L, and J26-H) were selected. Three independent biological replicates were taken from each virus levels of each variety, and each biological replicate came from 15 kg storage roots which planted in an area of one square meter. The photography at 15 and 45 DAP (day after planting), and the statistics of number of sprouts and sprouts weight per kilogram of storage roots at 45 DAP were performed for field investigation of sprouting capability.’ [Lines 93-101].

At present, more than 20 types of viruses have been discovered in China that can cause viral diseases in sweet potatoes, mainly including chlorotic stunt virus (CSV), feathery mottled virus (FMV), sweet potato virus G (SPVG), sweet potato latent virus (SPLV) and sweet potato chlorotic fleck virus (SPCFV). Therefore, we selected these five varieties of virus to detection. Our qRT-PCR results showed that J25-H and J26-H have significantly higher levels of these five viruses than J25-L and J26-L. This result can indicate a mixed infection of J25-H and J26-H with viral diseases.

We added the details of multiple-testing correction for DEG identification in Materials and Methods: ‘An FDR < 0.05, as determined by DESeq2 R package, was considered to indicate differential expression genes.’ [Lines 119-120].

Results

Response:

DEG comparisons between varieties and virus levels are presented together, but varietal genetic effects are not clearly separated from virus-associated effects. KEGG pathway enrichment is interpreted broadly, with large pathways discussed without focusing on specific genes or regulatory points and no mechanistic explanations were given.

Response:

We thank the reviewer for this comment. When analyzing the transcriptome, we also considered that there may have differences between J25 and J26 that affect the sprouting capability. In order to eliminate the influence between varieties, we did not compare the J25 and J26. Instead, when conducting differential gene and KEGG enrichment analysis, we took the intersection of J25-L vs J25-H and J26-L vs J26-H to better identify which biological pathways and genes may affect the sprouting capability of storage roots with different virus contents.

The specific potential genes and the primary potential mechanistic explanations were provided in 3.5 section of Results, and 4.1, 4.2, 4.3 and 4.4 sections of Discussion. However, the specific mechanism of the candidate genes in the sprouting capability of sweet potato is not mentioned much in our manuscript, as we still need to clone and validate them through sweet potato genetic transformation. This will be our next focus of work.

Discussion

Response:

We thank the reviewer for pointing this out. In sweet potato, starch is the main nutrient in storage roots and its degradation plays a vital role in providing essential nutrients for sprouting and ensuring the growth of sweet potato seedlings. The cell wall provides shape, volume, structure, rigidity, and strength to plant cells and determines how plants grow. During the growth of above-ground plant parts, plants must loosen and rebuild their cell walls. Therefore, starch and cell wall metabolism play a significant role in the growth and response to stress of sweet potatoes. Sweet potato sprouting is an important growth stage of sweet potato, and it is reasonable that key genes involved in starch and cell wall metabolism play important parts in sweet potato sprouting. Our discussion section also discussed the specific functions of genes involved in starch and cell wall metabolism and their roles in sweet potato sprouting, which is sufficient to demonstrate that key genes for starch and cell wall metabolism are important candidate genes driving sweet potato sprouting.

As suggestion, we will supplement and improve the enzyme activity and metabolite data related to starch metabolism and cell wall metabolism in the subsequent work.

Hormone signalling results, especially for JA and SA pathways, do not clearly distinguish between responses related to virus defense and those directly involved in sprouting. Increased expression of JAZ genes is interpreted as activation of JA signaling but they have known repressor function, so the interpretation should look into this aspect also. There are several interpretations without further validation. So discussion is very weak and need to be rewritten.

Response:

We thank the reviewer for the suggestion. Based on the suggestion, we have added the discussion of the repressor function of JAZ genes in Discussion: ‘When jasmonate levels are low, JAZ proteins inhibit the activation of JA-responsive genes by repressing transcription factors like MYC2.’ [Lines 351-352], and Results: ‘In rice, Lv et al [49] found that OsMYB30 can suppress the expression of β-amylase genes. OsJAZ9 can interact with OsMYB30, inhibiting its transcriptional activation activity. This regulates starch breakdown in grains, affecting rice grain germination.’ [Lines 357-360].

Conclusions

Response:

We thank the reviewer for this comment. Based on the comment, we have changed “key biological pathways” to “The results revealed that starch metabolism, cellulose metabolism, jasmonic acid (JA) signaling pathway, and salicylic acid (SA) signaling pathway are potential biological pathways contributing to the differences in sprouting capability between low and high virus levels seed roots.’ [Lines 398-401].

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper, the authors describe comparative transcriptomic analysis of sweet potato storage roots with low and high virus content. As the result, it was shown that storage roots of two different varieties with low virus levels were characterized by stronger sprouting capability. The key pathways involved in sprouting processes were starch and cellulose metabolism as well as jasmonic and salicylic acid signaling pathways. The results of the study provide the better understanding of the sprouting mechanism in sweet potatoes and look to be of great practical importance. The paper is well-written and could be considered for publication. At the same time, some points can be improved.

In the Introduction, the authors could provide more information on the main viruses infecting sweet potato.
Line 87. The authors mention that 'sample RNA extraction......were conducted using the Agilent 2100 bioanalyzer'. It sounds quite strange. I think the authors should mention the method used for RNA extraction.
How many biological replicates were used for transcriptomic analysis?
In 'Results' the authors describe qRT-PCR detection of five potato viruses, but do not mention anything about these analyses in 'Materials and Methods'. The PCR protocols should be added.
Figure 1, I,J. Obviously, the legend should be 'relative virus content' instead of 'relative expression'.

Author Response

Reviewer 2：

Response:

Thank you very much for your affirmation of our research and article.

In the Introduction, the authors could provide more information on the main viruses infecting sweet potato.

Response:

We thank the reviewer for this comment. Based on the comment, we have added more information on the main viruses infecting sweet potato ‘At present, more than 20 types of viruses have been discovered in China that can cause viral diseases in sweet potatoes, mainly including chlorotic stunt virus (CSV), feathery mottled virus (FMV), sweet potato virus G (SPVG), sweet potato latent virus (SPLV) and sweet potato chlorotic fleck virus (SPCFV).’ [Lines 53-56].

Line 87. The authors mention that 'sample RNA extraction......were conducted using the Agilent 2100 bioanalyzer'. It sounds quite strange. I think the authors should mention the method used for RNA extraction.

Response:

We thank the reviewer for this comment. Based on the comment, we have made careful modifications: ‘Total RNA of 12 samples (two varieties with low and high virus levels performed three biological replicates) were extracted using an RNA isolator, Total RNA Extraction Reagent (Vzayme, Nanjing, China).’ [Lines 106-109].

How many biological replicates were used for transcriptomic analysis?

Response:

We thank the reviewer for pointing this out. We have added the description of biological replicates used for transcriptomic analysis in Materials and Methods: ‘Storage roots from two varieties with low and high virus levels in 2.2 were sampled at 15 DAP. Each virus levels of each variety were performed three biological replicates and each replicate were pooled from five individual storage roots. Total RNA of 12 samples (two varieties with low and high virus levels performed three biological replicates) were extracted using an RNA isolator, Total RNA Extraction Reagent (Vzayme, Nanjing, China).’ [Lines 104-109], and Results: ‘High-throughput transcriptome sequencing was performed on a total of 12 samples from storage roots with low virus levels (J25-L and J26-L with three biological replicates) and storage roots with high virus levels (J25-H and J26-H with three biological replicates) of Jishu 25 and Jishu 26.’ [Lines 181-184].

In 'Results' the authors describe qRT-PCR detection of five potato viruses, but do not mention anything about these analyses in 'Materials and Methods'. The PCR protocols should be added.

Response:

We thank the reviewer for the suggestion. As suggested, we added detailed information to clarify the qRT-PCR used for the detection of five sweet potato viruses: “qRT-PCR was used to the detection of chlorotic stunt virus (CSV), feathery mottled virus (FMV), sweet potato virus G (SPVG), sweet potato latent virus (SPLV) and sweet potato chlorotic fleck virus (SPCFV) in sweet potato storage roots and evaluate the relative expression of DEGs according to Hou et al [6].

Total RNA was extracted from liquid nitrogen-frozen root samples using the Vazyme Total RNA Extraction Kit (Vazyme, Nanjing, China). cDNA was synthesized by reverse transcription using the Takara Reverse Transcription Kit (Takara, Kusatsu, Japan) and used as the template. qRT-PCR was performed using the CFX Connect Real-Time System (Bio-Rad, Hercules, USA) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction program was as follows: initial denaturation at 95 ℃ for 30 seconds, followed by 40 cycles of 95 ℃ for 10 seconds and 60 ℃ for 15 seconds. The IbActin gene in sweet potato was used as an internal reference for calculating relative virus content and the relative expression of DEGs. Table 1 lists the primer sequences for all genes used in this study. Three technical replicates were set up for each experiment, and the relative expression levels of genes were calculated using the comparative C_T method [20].” [Lines 125-140].

Figure 1, I, J. Obviously, the legend should be 'relative virus content' instead of 'relative expression'.

Response:

We thank the reviewer for pointing this out. As suggested, we carefully changed the legend of Figure 1I and J to “Relative Virus Content”

Reviewer 3 Report

Comments and Suggestions for Authors

This study investigated the effect of virus infection on the sprouting of storage roots from two sweet potato ( Ipomoea batatas ) varieties and performed transcriptome (RNA-seq) analysis of leaf samples stratified by virus infection level (i.e., “high” and “low” virus levels).

The results showed that higher virus levels were associated with reduced sprouting capacity. Transcriptome analysis identified differentially expressed genes, including genes involved in starch and cellulose metabolism as well as jasmonic acid and salicylic acid signaling pathways. The authors suggested that differences in gene expression may underlie the observed differences in sprouting between sweet potato tubers with different virus levels.

In general, the study was performed at a high technical level, and the information on differentially expressed genes, sprouting performance, and responses to virus infections would be of interest to the researchers working in this area. However, it is essential to provide additional information on how plant samples were assigned to the "low virus" and "high virus" groups, as the conclusions of this study depend on accurate classifications of the plants to the tratment groups.Major questions:

L.68-71. L 122-123.
Please provide information on how "high virus" and "low virus" groups were selected.
Give details on how the virus levels were determined.
Was virus quantification carried out by RT-PCR?
Was virus quantification carried out in the leaves of all individual plants which were used to produce tubers for further experiments?

L68-70.
Were the J25 and J26 sweet potato plants from different regions genetically identical (i.e. vegetatively propagated clones reproduced by cutting)? IThis point is important because difference in gene expression between varieties sourced in different regions (e.g. J25 from Yulin and Binzhou) could be due to genetic differences between plants rather than virus levels.

L.151-152 / Table 1
How many individual plants were used for each sample?
Do the 12 samples represent individual plants of the pooled of several plants?

L.138 / Figure 1 a-d, 1 e-f.
Provide quantitative analysis of sprouting rates and assess the statistical significance of differences in sprouting, growth rates (number of shoots, timing of sprouting, ?).
Indicate how many tubers were used for the analysis.

Figure 1 i,j / L. 131-137. / L 145-148.
Provide details on how relative levels of virus were calculated (this could be included to the "Materials and methods" section 2.4. " qRT-PCR-Analysis".

Clarify what is meant by "relative levels" (e.g. Delta Delta Ct)?

Figure 1 i,j.
Include data point showing virus levels (e.g.as dots) for each average- virus level bar.
Alternatively, provide a supplementary table showing virus levels for each plants in "low" and "high" virus groups.

L.12-28
To list viruses detected in this study in Abstract

Author Response

Reviewer 3：

This study investigated the effect of virus infection on the sprouting of storage roots from two sweet potato (Ipomoea batatas) varieties and performed transcriptome (RNA-seq) analysis of leaf samples stratified by virus infection level (i.e., “high” and “low” virus levels).

Response:

Thank you very much for your affirmation of our research and article.

68-71. L 122-123.
Please provide information on how "high virus" and "low virus" groups were selected. Give details on how the virus levels were determined. Was virus quantification carried out by RT-PCR? Was virus quantification carried out in the leaves of all individual plants which were used to produce tubers for further experiments?

Response:

In fact, we have conducted PCR and qRT-PCR tests for many years on the sweet potato storage roots harvested in the two regions, and carried out statistics on the incidence rate. We found that the virus carrying rate of the Jishu 25 and Jishu 26 storage roots harvested in Binzhou was 17.41% -41.25%, and the virus carrying rate of the storage roots bred in Yulin was 1.43% -8.72%. Therefore, we believe that the virus content of the sweet potato storage roots harvested in Yulin is low, and the virus content of the sweet potato storage roots harvested in Binzhou is high. These results are described in the articles of Li et al (2019). In this manuscript, qRT-PCR is also used for verification, and the results are consistent with the research of Li and others.

Referance:

L68-70.
Were the J25 and J26 sweet potato plants from different regions genetically identical (i.e. vegetatively propagated clones reproduced by cutting)? IThis point is important because difference in gene expression between varieties sourced in different regions (e.g. J25 from Yulin and Binzhou) could be due to genetic differences between plants rather than virus levels.

Response:

We thank the reviewer for pointing this out. Firstly, the J25 and J26 storage roots used in this research from Yulin and Binzhou were harvested from the same batch of sweet potato seedlings, which were provided by the Crop Research Institute of Shandong Academy of Agricultural Sciences. This is reflected in section 2.1 of Materials and Methods. Although both regions use virus-free seedlings provided by the Crop Research Institute of Shandong Academy of Agricultural Sciences for planting, it was found that sweet potato storage roots in Yulin area have no or very low levels of virus content, while Binzhou area shows high virus content and even virus combination disease. This is because the low temperature in Yulin area is not conducive to the survival of virus transmission vectors such as planthoppers and aphids, so sweet potatoes are almost not infected with viruses.

151-152 / Table 1
How many individual plants were used for each sample? Do the 12 samples represent individual plants of the pooled of several plants?

Response:

138 / Figure 1 a-d, 1 e-f.
Provide quantitative analysis of sprouting rates and assess the statistical significance of differences in sprouting, growth rates (number of shoots, timing of sprouting, ?). Indicate how many tubers were used for the analysis.

Response:

In production, the sprouting capability is usually measured by the number and weight of sprouts produced per kilogram of storage roots. In this research, we set up three biological replicates for each sample, and in each biological replicate, we planted 15 kg of uniformly sized storage roots on one square meter of land to evaluate the sprouting capability. Therefore, we did not count the specific number of storage roots. In the Materials and Methods section, we provided a more detailed description of the determination of the number of sprouts and sprouts weight: ‘Following the method described by Hou [16], sprouting was conducted using the sparse arrangement method with a planting density of 15 kg/m². The healthy, medium and similar sized storage roots from two varieties with low and high virus levels (J25-L, J25-H, J26-L, and J26-H) were selected. Three independent biological replicates were taken from each virus levels of each variety, and each biological replicate came from 15 kg storage roots which planted in an area of one square meter. The photography at 15 and 45 DAP (day after planting), and the statistics of number of sprouts and sprouts weight per kilogram of storage roots at 45 DAP were performed for field investigation of sprouting capability.’ [Lines 93-101].

Figure 1 i,j / L. 131-137. / L 145-148.
Provide details on how relative levels of virus were calculated (this could be included to the "Materials and methods" section 2.4. " qRT-PCR-Analysis". Clarify what is meant by "relative levels" (e.g. Delta Delta Ct)?

Response:

Figure 1 i,j.
Include data point showing virus levels (e.g.as dots) for each average- virus level bar. Alternatively, provide a supplementary table showing virus levels for each plants in "low" and "high" virus groups.

Response:

We thank the reviewer for pointing this out. As suggested, we carefully changed the legend of Figure 1I and J to “Relative Virus Content” and the data points showing relative virus levels display in bar chart.

12-28 To list viruses detected in this study in Abstract.

Response:

We thank the reviewer for the suggestion. As suggested, we listed the virus varieties detected in this study in Abstract: “In this study, storage roots with different virus content of two sweet potato varieties, Jishu 25 (J25) and Jishu 26 (J26), were evaluated for sprouting capability and virus content including chlorotic stunt virus (CSV), feathery mottled virus (FMV), sweet potato virus G (SPVG), sweet potato latent virus (SPLV) and sweet potato chlorotic fleck virus (SPCFV).” [Lines 19-23].

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

MS has been modified substantially and can be accepted.

Author Response

Reviewer 1：

MS has been modified substantially and can be accepted.

Response:

Thank you very much for your affirmation of our research and article.

Author Response File: Author Response.pdf