Using Marker-Assisted Selection to Develop a Drought-Tolerant Rice Line with Enhanced Resistance to Blast and Brown Planthopper

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper needs to be revised with these major comments.

1.      Line 2: “Drought-Resistant Rice variety” should be changed to be “Drought-tolerant Rice variety”

2.      The introduction needs substantial enhancement. It currently offers only a cursory and inadequate overview of the research topic. Moreover, much of the introduction section has no supporting citations, particularly lines 29-34, lack appropriate citations to support the presented information.

3.      Why the authors only used one single check commercial variety, ‘Hanyou 73’.

4.      Line 68: Further details are required regarding the planting methodology. Please specify, whether direct seeding or transplanting was used, the number of rows per replicate, the distance between hills, the number of plants per hill

5.      Line 68 : What is the rationale for not including replications in yield measurements?

6.      Line 67: What specific agricultural practices are recommended for the successful cultivation of this new variety, including guidelines for fertilizer application and any other essential management techniques?

7.      Line72: Please provide a concise description of key characteristics for both parental genotypes, 'Huhan 1509' and W16.

8.      Line 90: Why was the drought tolerance experiment conducted in only a single season?

9.      Why did the authors not conduct more advanced and verification yield trials for the new cultivar?
Line 91: give more details regarding the drought experiment such as the experimental design, growth conditions, amount of the applied water, etc

10.  Line 108: Please provide more detailed information regarding the evaluation of rice blast, including the methods used for assessment, and the scale of the evaluation.

11.  Line 109: Are the authors conducting an evaluation of rice genotypes against different physiological races of Pyricularia oryzae?

12.  Line 141: please add section for the statistical analysis.

13.  Line 155: The authors should include the maturity date or the duration of the new cultivar.

14.  Line 164: the authors should calculate the water use efficiency of the new cultivar and the check variety and include a new section in the results dedicated to water use efficiency.

15.  Table 4,5,6: please add letters to highlight the differences between the check and the new cultivar.

16.  Line 173 please review.

17.  Line 230: the subtitle should be “Discussion”

18.  The discussion section is somewhat weak and lacks depth and could be strengthened.

19.  The manuscript demonstrates an insufficient number of cited references, only 21 references, indicating a need for more comprehensive literature support.

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

Dear Sir or Madam

Thank you very much for your positive feedback on our revised draft. We greatly appreciate your detailed suggestions throughout the process, which have been instrumental in improving the quality of our work. We will carefully consider the additional comments you have provided and make the necessary changes to further improve the quality of the manuscript. If there are any inappropriate expenditures, we would be grateful for your guidance. Thank you again for your support and guidance.

Reviewer 1:

Comment 1: Line 2: “Drought-Resistant Rice variety” should be changed to be “Drought-tolerant Rice variety”

Response: Thank you for pointing out this issue with the terminology. We have carefully reviewed the entire manuscript and have ensured that all instances of "Drought-Resistant Rice variety" have been appropriately corrected to "Drought-tolerant Rice variety."

Comment 2: The introduction needs substantial enhancement. It currently offers only a cursory and inadequate overview of the research topic. Moreover, much of the introduction section has no supporting citations, particularly lines 29-34, lack appropriate citations to support the presented information.

Response: Thank you for your valuable suggestions on our work. We have added relevant references to the content in lines 29-34 and revised and expanded the introduction to better align with the focus of our research. In particular, we have included additional explanations on the impact of rice blast and brown planthopper on rice. These two pests not only significantly reduce rice yield but also threaten global food security. Therefore, improving resistance to diseases and pests is crucial in rice breeding.

Comment 3: Why the authors only used one single check commercial variety, ‘Hanyou 73’.

Response: Thank you for your insightful question. We chose 'Hanyou 73' as the single check variety because it serves as a representative water-saving and drought-tolerant rice variety and is the designated control in China's drought tolerance regional trials. Additionally, 'Hanyou 73' has been widely cultivated in China, making it a material with significant practical relevance for actual production. Its use as a comparison helps ensure that the results are meaningful and applicable to real-world agricultural conditions.

Comment 4: Line 68: Further details are required regarding the planting methodology. Please specify, whether direct seeding or transplanting was used, the number of rows per replicate, the distance between hills, the number of plants per hill

Response: Thank you for your valuable suggestion. We have added further details regarding the planting methodology in the revised manuscript. Specifically, we used a transplanting method with 7 rows of 4-5 seedlings per row, with a row spacing of 12×30 cm, resulting in a total of 60 rows. The trials were arranged in a completely randomized block design with three replications, and 'Hanyou 73' was used as the control variety. The plot size for the regional trials was 13.34 square meters. For yield tests, the plot area was 333.5 square meters, and yield tests were arranged randomly without replication.

Comment 5: Line 68 : What is the rationale for not including replications in yield measurements?

Response: Thank you for your insightful comment. Regarding the yield measurement, we did not perform multiple small-scale replicated yield measurements. Instead, we harvested the entire planting area to obtain actual yield data, which aligns with our practical yield assessment experience. This approach allows us to reflect more accurately the true production potential of the varieties under field conditions. Additionally, we conducted the yield trials over two consecutive years to further validate yield consistency and account for any potential variations between growing seasons.

Comment 6: Line 67: What specific agricultural practices are recommended for the successful cultivation of this new variety, including guidelines for fertilizer application and any other essential management techniques?

Response: Thank you for your comment. In response, we have added detailed agricultural practices recommended for the successful cultivation of the new variety, 'Huhan 1516'. Specifically, the transplanting method involves medium-density planting, with a recommended planting spacing of 5×8 inches (equivalent to approximately 300 plants per acre) and 4-5 seedlings per hill. The seed sowing rate is 15-20 kg per acre, and seedlings are grown for about 30 days before being transplanted. Fertilization management includes the application of 15 kg of nitrogen per acre, along with basal phosphate and supplemental potassium. The general fertilization principle is to apply 60% of the total fertilizer as a base, with additional nitrogen applied after transplantation at a rate of 5-8 kg/acre to promote tillering. These guidelines are based on extensive field experience to optimize the growth and yield of 'Huhan 1516'.

Comment 7: Line72: Please provide a concise description of key characteristics for both parental genotypes, 'Huhan 1509' and W16.

Response: Thank you for your comment. In response, we have provided a concise description of the key characteristics of both parental genotypes 'Huhan 1509' and W16 in the revised manuscript. Specifically, 'Huhan 1509' carries the Pi2 gene, which confers resistance to rice blast, as well as resistance to bacterial leaf blight and brown planthopper. On the other hand, W16 is derived from the hybridization of indica rice materials, characterized by its typical indica grain shape and good grain quality. These traits were crucial in the development of 'Huhan 1516', contributing to its drought tolerance, disease resistance, and overall rice quality.

Comment 8: Line 90: Why was the drought tolerance experiment conducted in only a single season?

Response: Thank you for your valuable comment. In our study, the drought tolerance experiment was conducted at the Hangzhou of the China National Rice Research Institute, using a pot experiment under controlled greenhouse conditions. The pots were covered with a transparent film to prevent rainfall, allowing us to precisely control soil water content. To ensure accuracy, the experiment was conducted over a single season, during which we applied two different water treatments: (1) a control (CK), maintaining a shallow water layer; and (2) a drought treatment, where the water supply was gradually reduced until the soil water potential reached -50 kPa. Given the precision of the water control system, conducting the experiment in one season was sufficient to achieve reliable and accurate results.

Comment 9: Why did the authors not conduct more advanced and verification yield trials for the new cultivar? Line 91: give more details regarding the drought experiment such as the experimental design, growth conditions, amount of the applied water, etc

Response: Thank you for your insightful comment regarding the need for more advanced and verification yield trials, as well as further details on the drought tolerance experiment. We fully agree that conducting additional yield trials across a broader range of environments would provide a more comprehensive evaluation of the new cultivar ‘Huhan 1516’.

However, due to limited funding and time constraints, we were unable to conduct more extensive trials in this phase of the study. These limitations made it challenging to expand the scope of the yield verification beyond the current locations and growing seasons.

Regarding the drought tolerance experiment, we conducted the trial in 2020 at the Fuyang District base of the China Rice Research Institute, utilizing a pot experiment to assess the drought tolerance of ‘Huhan 1516’ at the flowering stage. The experimental design involved controlling soil moisture and setting up two treatments: a control (CK) group with normal shallow water levels and a drought treatment group where the water layer was removed, and soil moisture was reduced to a level where leaf curling was observed. The main evaluation index was the relative seed-setting rate under drought stress, calculated using the following formula:

Relative seed-setting rate (%) = (Seed-setting rate under drought treatment/Seed-setting rate under control (CK)) × 100%.

Drought tolerance was classified according to the Chinese Agricultural Industry Standard, "Technical Specification for Identification of Drought Tolerance of Water-Saving and Drought-Resistant Rice." The details of the experimental setup and soil conditions, including pot size, soil nutrient levels, and water management, are described in the materials and methods section. The relative seed-setting rate and other physiological indicators were measured to determine the drought tolerance level of the new cultivar.

That said, we recognize the importance of your suggestion and plan to incorporate additional yield trials and more detailed drought tolerance studies in future research as we seek further funding and resources. This will help confirm the robustness of ‘Huhan 1516’ across a wider range of agroecological zones and growing conditions.We sincerely appreciate your valuable feedback and will certainly take this into account in our ongoing and future studies.

Comment 10: Line 108: Please provide more detailed information regarding the evaluation of rice blast, including the methods used for assessment, and the scale of the evaluation.

Response: Thank you for your comments regarding the evaluation of rice blast. We have now revised the manuscript and added Table 2 and Table 3 to provide a detailed explanation of rice blast evaluation. The revised manuscript is as follows:

2.4. Identification method of natural induction of rice blast

In 2019 and 2020, rice blast resistance was evaluated at six identification sites located in six different provinces. Each experimental plot consisted of 10 rows with 7 plants per row, replicated three times using a randomized block design. Fertilization and water management followed standard field practices. The susceptibility of control materials (CK) varied based on the specific planting environment: WH26 was used in Zhejiang, Xiangwanxian 11 in Hunan and Jiangxi, Guangluai 4 in Hubei, Yuanfengzao in Anhui, and Longheinuo 2 in Fujian. Control plants were placed around the ridge as protection rows and in field operation ditches. Rice blast was evaluated according to the National Identification Standard for Rice Fever in China [26]. Specifically, the disease resistance composite index was calculated by evaluating the incidence of spike blight and the rate of spike blight loss in the rice blast natural induced identification nursery. (Table 1 and Table 3). The following formula is used to calculate the comprehensive resistance index of rice blast: Composite Index = Spike plague incidence disease level×50% + Loss Rate of ear blast disease grade × 50%

 

 

Table 2. Standard for classification of incidence of ear blast
level	Incidence of a disease
0	No disease
1	≤5.0%
3	5.1%～10.0%
5	10.1%～25.0%
7	25.1%～50.0%
9	≥50.1%
Table 3. Criteria for grading the rate of loss of spodumene
level	Loss rate of boll weevil
0	No disease
1	≤5.0%
3	5.1%～10.0%
5	10.1%～25.0%
7	25.1%～50.0%
9	≥50.1%

 

Comment 11: Line 109: Are the authors conducting an evaluation of rice genotypes against different physiological races of Pyricularia oryzae?

Response: Thank you very much for your insightful comment. We would like to clarify that the identification of rice blast resistance in our study was based on natural occurrence in six different provinces, aimed at evaluating the variety’s resistance to local strains of rice blast. However, we did not conduct specific tests to evaluate resistance against different physiological races of Pyricularia oryzae in each region. We appreciate your suggestion and will consider improving future evaluations by incorporating both natural pathogen assessments and targeted inoculation tests with a mixture of different physiological races of Pyricularia oryzae using syringe injection methods. Thank you again for your valuable feedback.

Comment 12: Line 141: please add section for the statistical analysis.

Response: Thank you for your comment regarding the need to include a section on statistical analysis. We have now provided a detailed description of the statistical methods used in the study.

Statistical Analysis:Phenotypic data and variance were calculated using Excel 2017 software. Significant differences between treatments were tested using Student’s t-test or Fisher’s least significant difference (LSD) test at both the 5% and 1% probability levels. This approach was used to assess differences in traits such as yield, drought tolerance, and resistance to rice blast and brown planthopper between the new cultivar 'Huhan 1516' and the control variety.

Comment 13: Line 155: The authors should include the maturity date or the duration of the new cultivar.

Response: Thank you very much for your comments on the changes related to rice maturity. We have revised it. The revised manuscript is shown below:

3.1. Main agronomic traits and yield performance

‘Huhan 1516′ is a medium-maturity rice variety that is both water-saving and drought-resistant. In Shanghai, the variety was planted in a single season, sown on May 23, entered the milky ripening stage on August 15, the fruiting period lasted for 28 days. It ripened on September 13, with a total fertility period of 114.5 days, which was 0.5 days earlier than that of the control variety 'Hanyou 73'. The average plant height was 103.2 cm. The average number of effective panicles per hole was 11.6, with an effective panicle length of 25.43 cm. ‘Huhan 1516′ exhibits a moderate plant type, with green bud sheaths, leaf sheaths, and leaves. The stem angle is classified as middle-type, providing the plant with strong elasticity and lodging tolerance. The flag leaf is slightly curled with a light green color, and the color changes noticeably in the later stages of growth. The spike standing characteristic is drooping, and the spike extraction in the secondary branch is good. The color of the spikelet glume is light yellow, and the grains are elongated and spindle-shaped in brown rice, with an average grain length of 6.36 mm (Figure 2 and Table 5).

 

Comment 14: Line 164: the authors should calculate the water use efficiency of the new cultivar and the check variety and include a new section in the results dedicated to water use efficiency.

Response: Thank you for your valuable suggestion regarding the evaluation of water use efficiency. In this study, drought tolerance was assessed based on the seed-setting rate under high-temperature conditions and the drought resistance index, rather than water use efficiency. These indicators were chosen to reflect the cultivar’s ability to maintain yield under drought stress. However, we appreciate your insightful suggestion, and we will certainly consider incorporating water use efficiency as an additional evaluation metric in future experiments to further enhance our understanding of the cultivar's performance under water-limited conditions.

Comment 15: Table 4,5,6: please add letters to highlight the differences between the check and the new cultivar.

Response: Thank you for your suggestion. We have revised Tables 4, 5, 6, and 7 by adding significance letters to highlight the statistical differences between the check variety and the new cultivar 'Huhan 1516'. These letters now clearly indicate the significant differences, making it easier for readers to interpret the results.

Comment 16: Line 173 please review.

Response: Thank you for your comment regarding the content on line 173. We have carefully reviewed and revised the relevant section to improve its clarity and accuracy. The updated description now provides a more precise and comprehensive explanation, aligning with the overall flow of the manuscript.

Comment 17: Line 230: the subtitle should be “Discussion”

Response: Thank you for your suggestion. We have changed the subtitle to "Discussion" as recommended.

Comment 18: The discussion section is somewhat weak and lacks depth and could be strengthened.

Response: Thank you very much for your valuable comments. For this purpose for the discussion part of the article, we have cited literature related to rice flavor and added the current status of water-saving and drought-resistant rice improvement. Please go to the original article to view it.

Comment 19: The manuscript demonstrates an insufficient number of cited references, only 21 references, indicating a need for more comprehensive literature support.

Response: Thank you for your valuable feedback. We agree that the original manuscript lacked sufficient references to support our claims and conclusions. In response, we have thoroughly reviewed the relevant literature and added additional references throughout the manuscript to strengthen the scientific basis of our research and its conclusions. These new citations provide more comprehensive support for the association and relevance of our findings in the context of current studies.

Thank you for your consideration.

Sincerely,

Li Ao, Zhu peiwen

 

Reviewer 2 Report

Comments and Suggestions for Authors

I checked your manuscript and described comments below.

Drought resistance, resistance to blast disease, high yield, and water saving are very important factors for rice. In this paper, we report the development of 'Huhan 1516', a new variety with these properties, through selective breeding. The creation of such a new rice variety is very significant.

I think it would be better to consider the following points.

1.       I think that 'Jiafuzhan' and 'Huanghuazhan' are Oryza sativa subsp. indica. I think it would be better to include the scientific name in the title.

2.       'Jiafuzhan', 'Huanghuazhan', 'Hanyou 73' and 'Huhan 1516' are not well known internationally. I think it would be better if there were photos of these seeds.

3.       There is no description of the reagents or equipment used for PCR. I think it would be better to provide more details.

I don't think this paper has major problems and grammatical problems.

Author Response

Dear Sir or Madam

Thank you very much for your positive feedback on our revised draft. We greatly appreciate your detailed suggestions throughout the process, which have been instrumental in improving the quality of our work. We will carefully consider the additional comments you have provided and make the necessary changes to further improve the quality of the manuscript. If there are any inappropriate expenditures, we would be grateful for your guidance. Thank you again for your support and guidance.

Reviewer 1:

Comment 1: I think that 'Jiafuzhan' and 'Huanghuazhan' are Oryza sativa subsp. indica. I think it would be better to include the scientific name in the title.

Response: Thank you very much for your comments on the title change. We have changed the title as per your comments below:

‘Huhan 1516′, A Water-Saving and Drought-Resistant Rice (Oryza sativa L.) with High Yield, Disease Resistance, and Broad Adaptability

 

Comment 2: 'Jiafuzhan', 'Huanghuazhan', 'Hanyou 73' and 'Huhan 1516' are not well known internationally. I think it would be better if there were photos of these seeds.

Response: Thank you very much for your insightful comment. We apologize for not including photos of the seeds of “Jiafuzhan” and “Huanghuazhan” at the beginning of the experiment. However, we have provided images of the plants in the original manuscript and included detailed descriptions of their agronomic traits to enrich the content of the article. Your suggestion to include seed photos is highly valuable, and we will certainly consider incorporating such details in future studies.

 

Comment 3: There is no description of the reagents or equipment used for PCR. I think it would be better to provide more details.

Response: Thank you very much for your comments. We have added details about the PCR amplifier.

2.6. MAS was used to detect the genes

Functional markers were used to detect the presence of resistance genes related to rice blast and rice quality in BC1F7 population lines. The PCR reaction system (10 μL total volume) consisted of 10.50 ng of DNA template, 1.9 μL of 10× buffer, 0.5 μL of primers, 0.1 μL of Taq, and ddH2O to 10 μL. The PCR protocol included an initial denaturation step at 94 °C for 3 min, followed by 34 cycles of 94 °C for 30 s, annealing (temperature dependent on the primers) for 30 s, and extension at 72 °C for 1 KB/min, with a final extension at 72 °C for 3 min. Restriction enzyme digestion was performed using 1 μg of PCR product, 1 mL of endonuclease, and 1 mL of 10× buffer, incubating at 37 °C for 20 min. Primer sequences for detecting resistance genes are shown in Table 4.

The PCR amplification instrument was a Bio-Rad S1000-T100 Gene Amplifier.

 

Thank you for your consideration.

Sincerely,

Li Ao, Zhu peiwen

 

Reviewer 3 Report

Comments and Suggestions for Authors

The article titled ‘Huhan 1516, A Water-Saving and Drought-Resistant Rice Variety with High Yield, Disease Resistance, and Broad Adaptability’ introduces a new rice variety, "Huhan 1516," developed using molecular marker-assisted selection (MAS) and marker-assisted backcrossing (MABC). The variety addresses several key agricultural challenges, including drought, freshwater scarcity, and diseases such as rice blast and brown planthopper. Huhan 1516 demonstrates strong adaptability to water-limited and drought-prone regions, particularly in the Yangtze and Huai River Basins. Its key attributes include drought tolerance, high yield, and moderate resistance to rice blast and brown planthopper. Field trials revealed a 2.2% yield increase over control varieties, indicating promising potential for wider adoption.

The use of MAS and MABC techniques in breeding Huhan 1516 provides a modern and robust approach to achieving desirable agronomic traits. Furthermore, the multi-site and multi-year trials lend considerable strength to the reliability of the findings. The aims of this study are clear, and the results are noteworthy. I found the manuscript highly engaging and commend the authors on their work. While I do not have major concerns, I have provided a few suggestions below for further improvement.

Although Huhan 1516 exhibits resistance to both rice blast and brown planthopper, the resistance levels are categorised as moderate (MR), which may limit its effectiveness in regions facing high disease pressure. Increasing the resistance levels would likely enhance its value across a broader range of environments. Additional discussion or a future research plan to address this aspect would be helpful.

Despite its many agronomic strengths, Huhan 1516 lacks the aromatic qualities that are becoming increasingly sought after by both consumers and the market. This may reduce its appeal, particularly in markets that place a premium on eating quality and aroma. The study acknowledges areas for potential improvement, such as the introduction of aroma genes and increased disease resistance. Furthermore, the limited focus on biotic stressors beyond rice blast and brown planthopper suggests the possibility of vulnerabilities to other pests and diseases, which should be addressed in future research.

Comments on the Quality of English Language

In general, the manuscript is well-organised, with distinct sections that guide the reader effectively through the introduction, methods, results, and conclusions. However, some paragraphs are rather dense, making it harder for readers to absorb all the details. Breaking up longer paragraphs into more manageable sections would enhance clarity and flow.

The grammar throughout is generally sound, though there are occasional instances of awkward phrasing or less-than-ideal word choices. For example, the phrase "seed-setting rate under control" might be more clearly expressed as "seed-setting rate in the control treatment." I would also recommend reviewing the manuscript for areas where sentence length could be reduced for better clarity. Shorter, more concise sentences will help to maintain the reader’s attention and improve overall comprehension.

Finally, I would suggest a proofreading pass by a native English speaker or a professional editor, which could further refine the tone and readability, particularly in terms of grammatical nuances and idiomatic expressions. This would ensure the manuscript is as polished as possible for its audience.

 

Author Response

Dear Sir or Madam

Thank you very much for your positive feedback on our revised draft. We greatly appreciate your detailed suggestions throughout the process, which have been instrumental in improving the quality of our work. We will carefully consider the additional comments you have provided and make the necessary changes to further improve the quality of the manuscript. If there are any inappropriate expenditures, we would be grateful for your guidance. Thank you again for your support and guidance.

Reviewer 1:

Comment 1: Although Huhan 1516 exhibits resistance to both rice blast and brown planthopper, the resistance levels are categorized as moderate (MR), which may limit its effectiveness in regions facing high disease pressure. Increasing the resistance levels would likely enhance its value across a broader range of environments.

Response: Thank you very much for your insightful comment. Rice blast resistance is a quantitative trait and polymerization of multiple resistance genes can greatly increase the level of resistance. At present, this team, has introduced more broad-spectrum resistance genes such as Pigm, Pi9 and other resistance genes for strengthening the level of resistance, and the improved lines have reached a significant increase in resistance, and in some areas, they have shown a high level of resistance.

Comment 2: Despite its many agronomic strengths, Huhan 1516 lacks the aromatic qualities that are becoming increasingly sought after by both consumers and the market. This may reduce its appeal, particularly in markets that place a premium on eating quality and aroma.

Response: Thank you very much for your long-term goal-oriented suggestions. At present, the improved rice material ‘Huhan 1516’, which contains the flavor gene, has entered into production trials, and there has been a significant improvement in the flavor quality of the rice. Thank you again for your visionary advice. We have added a reference to flavor improvement, specifically in line 354 of the original manuscript.

 

Comment 3: The limited focus on biotic stressors beyond rice blast and brown planthopper suggests the possibility of vulnerabilities to other pests and diseases, which should be addressed in future research.

Response: Thank you very much for your insights. As a matter of fact, we also did experiments on leaf blight and other pests and diseases in the course of our experiment, but they were not reflected in the original manuscript because they are not the mainstream pests and diseases of rice, and currently have less impacts on the growth of rice among the pests and diseases of rice in China. Your comments on other biotic stress issues are very much appreciated.

 

Comment 4: In general, the manuscript is well-organised... However, some paragraphs are rather dense, making it harder for readers to absorb all the details... The grammar throughout is generally sound, though there are occasional instances of awkward phrasing or less-than-ideal word choices... I would also recommend reviewing the manuscript for areas where sentence length could be reduced for better clarity.

Response: Thank you very much for your careful reading and valuable comments on the manuscript. I fully understand your feedback regarding paragraph density and grammatical details, which are essential to enhance the readability and clarity of the article. Regarding paragraph density: I will revisit paragraphs that are too dense with content and try to improve the structure and readability of the article by splitting it up, refining key points, or using subheadings.  Regarding grammar and wording: I will scrutinize the grammar and wording in the essay to ensure that each sentence flows naturally. I will revise any awkward or inappropriate expressions to improve the accuracy of language and clarity of expression. Regarding sentence length: I have taken note of your suggestion regarding sentence length. I will review the sentence structure in the article and shorten excessively long sentences appropriately to enhance the rhythm and readability of the article. Thanks again for your feedback, it's very helpful in improving the manuscript.

 

Thank you for your consideration.

Sincerely,

Li ao, Zhu peiwen

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

The authors have made notable progress in addressing the majority of the suggestions for improving the manuscript. However, a few remaining points still require further refinement
1-The introduction still requires significant improvement.

2-The authors should provide the exact recommended amount of applied water and the number of irrigations required for this variety, in comparison to the check variety Hanyou 73.

3- I recommend the authors revise the title to: “Huhan 1516: A Water-Saving and Drought-Tolerant Rice Line (Oryza sativa L.) with High Yield, Disease Resistance, and Broad Adaptability”

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Sir or Madam

Thank you very much for your positive feedback on our revised draft. We greatly appreciate your detailed suggestions throughout the process, which have been instrumental in improving the quality of our work. We will carefully consider the additional comments you have provided and make the necessary changes to further improve the quality of the manuscript. If there are any inappropriate expenditures, we would be grateful for your guidance. Thank you again for your support and guidance.

Reviewer 1:

Comment 1: The introduction still requires significant improvement.

Response: Thank you very much for your insightful comments. We have revisited the introduction and added a section on molecular marker-assisted breeding. The revised manuscript follows:

 

Compared with traditional breeding methods, MAS technology has significant advantages: it is not only accurate and efficient, but also free from the interference of environmental factors. Through MAS technology, researchers can realize the precise transfer of single resistance genes and even aggregate multiple resistance genes, opening up a “green channel” for rice resistance breeding. In the face of the continuous mutation and evolution of pests and diseases such as rice blast fungus and brown planthopper, varieties with a single resistance gene may face new challenges. Therefore, breeding new pest-resistant varieties by introducing and polymerizing multiple pest-resistant genes has become a more environmentally friendly and effective approach. This study aims to address the growing challenges in rice production posed by water scarcity, drought, and the threats of diseases such as rice blast and brown planthopper. To this end, we developed a new water-saving, Drought-tolerant Rice variety, ‘Huhan 1516’, through molecular marker-assisted selection (MAS) and marker-assisted backcrossing (MABC). Specifically, we evaluated the agronomic performance, drought tolerance, and disease resistance of ‘Huhan 1516’ under a range of environmental conditions. The incorporation of the Pi2 gene for rice blast resistance and BPH genes for brown planthopper resistance into ‘Huhan 1516’ enhances its adaptability to diverse agro-ecological zones, making it a strong candidate for sustainable rice production on a global scale [25].

 

Comment 2: The authors should provide the exact recommended amount of applied water and the number of irrigations required for this variety, in comparison to the check variety Hanyou 73. 

Response: Thank you very much for your advice on water consumption and irrigation. Since the results of the three years of testing, the pattern is basically the same, so the results of the data in 2022 were selected for analysis. Our revised manuscript reads as follows:

2.3. Identification of drought tolerance

In 2020, ‘Huhan 1516’ was assessed for drought tolerance at the flowering stage using a pot experiment at the Fuyang District base of the China Rice Research Institute. Plastic pots with a diameter of 30 cm and a height of 15 cm were used, and each pot was filled with 11 kg of air-dried soil. The soil, collected from the same region, had an organic matter content of 36.1 g/kg, total nitrogen content of 2.70 g/kg, total phosphorus content of 0.62 g/kg, and total potassium content of 20.4 g/kg. Other soil nutrient measurements included available nitrogen (239 mg/kg), available phosphorus (9.8 mg/kg), available potassium (62 mg/kg), and a pH of 6.5. Seedlings were sown on May 12 and transplanted on June 8, with three replications per treatment and two seedlings per pot.

Two water treatments were applied: (1) a control (CK) with shallow water maintained at normal levels, and (2) a drought treatment where water was withheld until the soil moisture level dropped to -50 kPa, which caused the upper leaves of the plants to begin rolling. This drought treatment was sustained for about a week until severe leaf rolling was observed. After rewatering, the plants were allowed to recover and reach full maturity.The main index for identification was the relative seed-setting rate under drought stress, calculated using the following formula:

Relative seed-setting rate (%) = (Seed-setting rate under drought treatment/Seed-setting rate under control (CK)) × 100%.

Drought tolerance was classified according to the Chinese Agricultural Industry Standard, “Technical Specification for Identification of Drought Tolerance of Water-Saving and Drought-Resistant Rice,” with the classification system presented in Table 1. Soil moisture, temperature, and plant growth stages were closely monitored throughout the experiment. The water consumption and irrigation amount of water-saving and drought-resistant rice were measured in ‘Huhan 1516’ and ‘Hanyou 73’, and the test method was referenced to “A cultivation device for water consumption measurement of water-saving and drought-resistant rice and its method of use”[26]. In the years 2020, 2021, and 2022, two rice varieties, ‘Huhan 1516’ and ‘Hanyou 73’, which are both water-saving and drought-resistant, were tested. The two varieties were transplanted into the cultivation equipment and subjected to six irrigation gradients, with the maximum irrigation amount representing precise quantitative irrigation and the remaining gradients decreasing by 10% of the irrigation amount of the previous gradient. The two varieties were irrigated under these conditions. In accordance with the outcomes of the three-year trial, one representative year should be selected for analysis.

 

3.2. Resistance analysis

3.2.1. Drought tolerance performance and water consumption

In a two-year drought resistance evaluation, ‘Huhan 1516’ achieved an average relative seed-setting rate of 63.5% and a drought resistance index of 0.85. In comparison, ‘Hanyou 73,’ another water-saving and drought-resistant variety, recorded an average relative seed-setting rate of 61.85% and a drought resistance index of 1.0. Both varieties were classified as ‘Drought-Resistant’ (DR) according to the evaluation criteria (Table 7). Since the results of the three years of testing, the pattern is basically the same, so the results of the data in 2022 were selected for analysis. As shown in Table 8, with the decrease of irrigation amount, ‘Huhan 1516’ showed a significant difference in the decrease of single plant yield when the water consumption was 4707 m3 ha-1, so its water consumption should be the upper gradient of 4707 m3 ha-1, i.e. 5377.5 m3 ha-1. while the water consumption of ‘Hanyou 73’ should be 7105.5m3 ha-1 (Table 8).

 

Table 8. Water consumption (m³ ha^-1) and yield (g/plant) of two rice varieties.

Treatment gradient	100%	90%	80%	70%	60%	50%
Irrigation	9472.5	8398.1	7201.5	6400.5	5406	4726.5
Permeability	276	166.5	96	36	28.5	19.5
Water consumption	9196.5	8231.6	7105.5	6364.5	5377.5	4707
‘Huhan 1516’	36.1a	36.5a	34.1a	34.2a	34.2a	31.5b
‘Hanyou 73’	37.9a	36.8a	35.4a	34.7a	31.2b	23.7c

Note: Values in the same column with the same letter indicate no significant difference (P > 0.05), while values with different letters indicate a significant difference (P < 0.05).

 

 

Comment 3: I recommend the authors revise the title to: “Huhan 1516: A Water-Saving and Drought-Tolerant Rice Line (Oryza sativa L.) with High Yield, Disease Resistance, and Broad Adaptability”

Response: Thank you for your valuable comments on the article title. We've changed the title of the article to “Huhan 1516: A Water-Saving and Drought-Tolerant Rice Line (Oryza sativa L.) with High Yield, Disease Resistance, and Broad Adaptability”.

 

 

Sincerely,

Li Ao, Zhu peiwen