Transposon Polymorphism and Its Potential Impacts on Brown Planthopper (Nilaparvata lugens Stål) Resistance in Rice (Oryza sativa L.)
Round 1
Reviewer 1 Report
The article “Transposon Polymorphism and its Impacts on Brown Planthopper (Nilaparvata lugens Stål) Resistance in Rice (Oryza sativa L.)” deals with the role of transposons towards conferring resistance to BPH. There are major corrections in the materials and method sections, therefore can’t be accepted for publication at this point of time.
1. Abstract should be more concise rather just providing general statements.
2. Line number 20 to 23 “on which chromosomes these six new genes were identified “and without proper validation we can’t say them as new genes. No biotypes were mentioned against which they have taken resistance or susceptible score. No data were provided on BPH rearing.
3. Line number 24 - How much difference in the expression of the Locus “LOC_Os04g02720” was observed between Susceptible and resistant cultivars? It should be mentioned rather writing significant difference was observed.
4. Line number 79- the data should be updated. Now the identified genes are more than 40.
5. The line number 79- 80 does not have any connections with the current study and can be removed.
6. Table 1: classification of resistance level from 0 to 5 (R58) does not make sense as it goes from immune to moderately resistant. It would be good to have a figure demarcating 0 from 1 and others.
7. for quantitative estimation the protocols were not described properly. At what time interval they have taken tissue, what is the number of replications, how many resistant and susceptible lines they have taken for analysis. Therefore, the presented data is imprecise.
average quality
Author Response
Response to Reviewer 1 Comments
The article “Transposon Polymorphism and its Impacts on Brown Planthopper (Nilaparvata lugens Stål) Resistance in Rice (Oryza sativa L.)” deals with the role of transposons towards conferring resistance to BPH. There are major corrections in the materials and method sections, therefore can’t be accepted for publication at this point of time.
Point 1: Line 21-30: Abstract should be more concise rather just providing general statements.
Response 1: Thanks so much for your comments and suggestions. Please see the revised manuscript for details.
Point 2: Line number 25 to 27: “on which chromosomes these six new genes were identified “and without proper validation we can’t say them as new genes. No biotypes were mentioned against which they have taken resistance or susceptible score. No data were provided on BPH rearing.
Response 2: We have revised the sentences for more reasonable. Please see the revised manuscript for details.
Point 3: Line number 29-30: How much difference in the expression of the Locus “LOC_Os04g02720” was observed between Susceptible and resistant cultivars? It should be mentioned rather writing significant difference was observed.
Response 3: We’ve revised these sentences. Quantitative analysis showed significant differences in the expression of this gene between insect-resistant and insect-susceptible varieties (p < 0.05). Please see the revised manuscript for details.
Point 4: Line number 87, the data should be updated. Now the identified genes are more than 40.
Response 4: Corrected. Thanks. More than forty loci have been identified for resistance to BPH.
Point 5: The line number 86-88: does not have any connections with the current study and can be removed.
Response 5: Deleted. Thanks. Please check the revised manuscript for details.
Point 6: Line 121, Table 1: classification of resistance level from 0 to 5 (R58) does not make sense as it goes from immune to moderately resistant. It would be good to have a figure demarcating 0 from 1 and others.
Response 6: Thanks for your suggestion. The resistance classes corresponding to the samples in this paper were obtained from the table of damage classes of control varieties [24]. Details have been added with reference to your suggestions.
Point 7: Line 175-178 and 187-190: for quantitative estimation the protocols were not described properly. At what time interval they have taken tissue, what is the number of replications, how many resistant and susceptible lines they have taken for analysis. Therefore, the presented data is imprecise.
Response 7: Tissue was collected from 15-day-old rice seedling leaves of five insect-susceptible varieties and one insect-resistant variety. Three biological replicates were set. We have added more information in the revised manuscript.
Reviewer 2 Report
Please check the attachment.Comments for author File: 
Comments.pdf
The overall English is ok but it can be improved further
Author Response
Response to Reviewer 2 Comments
Point 1: line 37, plz use full name at first use.
Response 1: Done. Thanks for reminding.
Point 2: line 98, why did you choose these cultivars?
Response 2: Thanks very much for your comments and suggestions. Previously, two candidate genes for BPH resistance were identified by whole-genome resequencing of rice varieties conserved within the subject group and combined with GWAS methods. Based on these data, this paper would like to further explore whether transposon polymorphism has an effect on BPH from the perspective of transposon polymorphism.
Point 3: Line 121, the table must be informative enough without the need to refer to the text. What does R and its following number indicate? what stage the plants were scored? give a ref for the scale.
Response 3: Thanks for your comments.
- The name of each sample ID could be found in the paper of Shi et al. [24].
- We have revised the method as your comments: Seedling trays (50 × 35 × 8 cm) were covered with soil was evenly divided into 11 rows with one rice variety per row, 12 seedlings per variety, and three biological replicates per variety. Each tray contained ten varieties and the susceptible control Taichung Native 1(TN1) in a random block design. Two seedlings of each variety were randomly selected and stored (without roots) at -80°C for subsequent sequencing. The remaining ten seedlings of each variety were artificially inoculated with 8–10 nymphs of 1–2 instar BPHs per seedling. When the mortality of the susceptible TN1 variety reached 70%, the number of dead seedlings of the rest varieties was recorded daily. The mean resistance grade of three biological replicates of each variety was assessed when all TN1 plants were dead. For the varieties with resistance grades of 1 to 5, changes in seedling damage and grading continued to be recorded after the TN1 resistance grade reached 9 (seedling mortality > 70%) to evaluate their durable resistance period (started from the the day 70% of TN1 seedlings were dead, ended to 3 days before 70% of tested variety seedlings were dead).
Point 4: Line 175-178: Did you extract RNA from infected and non-infected plants? when did you take samples?
Response 4: Yes. In this study, the leaf tissues of 5 insect-susceptible and 1 insect-resistant rice varieties were collected at the seedling stage (15 days after sowing). We have added more information in the revised manuscript.
Point 5: Line 188, Why choose UBQ as an internal reference gene?
Response 5: Firstly, the expression stability of UBQ in different tissues and developmental stages has been extensively studied and validated. Secondly, the expression of UBQ in rice is comparable to other commonly used internal reference genes, and its expression level is more stable in this study, so the selection of UBQ in gene expression studies can improve the accuracy and reliability of the data. In addition, the relatively conservative promoter sequence of UBQ makes the design and synthesis of primer sequences relatively simple, which is an important consideration in selecting UBQ as an internal reference gene. In summary, the selection of UBQ as an internal reference gene for rice gene expression studies was based on a combination of factors including its stability, comparable expression, and relatively conserved promoter sequence.
Point 6: Line199 and 207, please use susceptible instead of sensitive
Response 6: Corrected. Thanks for reminding.
Point 7: Line 262-264, why these six?
Response 7: The explanation is given in the discussion section. To make the identified candidate insect resistance genes more accurate, the functions of genes whose gene promoter regions overlapped with TEs were queried in the rice database. LOC_Os10g32600 was associated with an early spike; LOC_Os08g07740 was associated with rice grain yield, plant height, and tassel stage, but apparently, none of these genes were related to insect resistance. In this study, we selected and focused on six candidate genes that might be related to insect resistance: LOC_Os10g17910 (cell stress, signal transduction related), LOC_Os01g11670 and LOC_Os12g22030 (serine metabolism related), LOC_Os03g49040 and LOC_Os07g20360 (transporter protein related), LOC_Os04g02720 (potassium ion channel-related). Please see the revised manuscript for details.
Point 8: Line 275, why did you clone only this?
Response 8: The reason for cloning the LOC_Os04g02720 has been supplemented at the material(2.2.3). The LOC_Os04g02720 is associated with potassium ion channels that enhance cell wall thickness [30]. A large number of studies on rice resistance to BPH have shown that the cell wall plays an important role as a physical barrier against BPH infesting rice[24], blocking BPH from feeding and protecting the plant from pests. Therefore, it is inferred that the LOC_Os04g02720 may be associated with BPH resistance. Please see the revised manuscript for details.
Point 9: Line 300, please discuss your own results
Response 9: Done. Thanks for your advice. We have added more information in the revised manuscript. Please check the revised manuscript for details.
Point 10: Line 403, what are the applications of your study?
Response 10: Done. Please see the revised manuscript for details.
