Evaluation of Blast Resistance and Identification of Superior Haplotype of OsDR8 in Rice

doi:10.16819/j.1001-7216.2025.240509

Abstract

Abstract:

【Objective】Rice (Oryza sativa L.) is a crucial staple crop globally. Its production is severely threatened by rice blast disease. Identifying resistance materials and analyzing resistance genes are essential for understanding the mechanisms behind rice blast resistance. This study aims to systematically generate and identify genetic materials of the rice blast resistance gene OsDR8, conduct haplotype and evolutionary analyses to select superior haplotypes, and investigate their subpopulation differentiation. These efforts will lay a theoretical basis for the application of OsDR8 in rice blast resistance breeding programs.【Method】We developed transgenic lines overexpressing the OsDR8 gene and its knockout mutants. We conducted rice blast resistance assays on the overexpression lines, homozygous knockout lines, and corresponding wild-type materials using Magnaporthe oryzae isolates RO1-1 and RB22. Meanwhile, haplotype and evolutionary analyses of the OsDR8 gene were conducted based on the rice super pan-genome variation map.【Result】Disease symptom observation and disease resistance evaluation indicated that knockout mutants had significantly larger lesion sizes and a higher proportion of diseased leaf area compared to the wild-type. In contrast, the overexpression lines showed reduced lesion sizes and a lower proportion of diseased leaf area. Combined with haplotype and evolutionary analyses, the results indicated that materials of the superior haplotype Hap2 show stronger disease resistance, with 95.52% of the indica rice subpopulation being the Hap2 haplotype.【Conclusion】The OsDR8 gene positively regulates resistance to rice blast, and that the superior haplotype Hap2 may be associated with stronger resistance in indica rice.

Key words: rice, rice blast, superior haplotype, breeding application

摘要：

【目的】水稻(Oryza sativa L.)是全球重要的主粮作物，其生产安全受到稻瘟病的严重威胁。抗稻瘟病材料的鉴定及相关抗稻瘟病基因的解析，有助于揭示稻瘟病抗性机制。本研究旨在系统创制和鉴定水稻抗稻瘟病基因OsDR8的遗传材料，并对其进行单倍型和进化分析，以期筛选优异单倍型，探究其亚群分化，为OsDR8基因的抗病育种应用提供理论依据。【方法】创制了OsDR8基因的过表达株系和敲除株系，并用RO1-1和RB22稻瘟菌对过表达株系、纯合敲除株系和对应野生型材料进行稻瘟病抗性鉴定。同时，基于水稻超级泛基因组变异图谱，进行OsDR8基因的单倍型分析和进化分析。【结果】对这些材料进行症状观察和稻瘟病抗性鉴定，发现相比野生型株系，敲除突变体株系病斑显著增大，发病叶面积占比也显著上升；而过表达株系病斑显著减小，发病叶面积占比也显著降低。结合单倍型及进化分析，说明携带优异单倍型Hap2的材料抗病性较强，籼稻亚群中95.52%材料为Hap2单倍型。【结论】OsDR8基因正调控水稻稻瘟病抗性，优异单倍型Hap2可能与籼稻稻瘟病抗性较强有关。

关键词: 水稻, 稻瘟病, 优异单倍型, 育种应用

ZHANG Bintao, LIU Congcong, GUO Mingliang, YANG Shaohua, WU Shiqiang, GUO Longbiao, ZHU Yiwang. Evaluation of Blast Resistance and Identification of Superior Haplotype of OsDR8 in Rice[J]. Chinese Journal OF Rice Science, 2025, 39(3): 343-351.

张彬涛, 刘聪聪, 郭明亮, 杨绍华, 吴世强, 郭龙彪, 朱义旺. 水稻OsDR8基因的稻瘟病抗性评价及优异单倍型鉴定[J]. 中国水稻科学, 2025, 39(3): 343-351.

Figures/Tables 13

Table 1. Primers used in this study

引物名称 Primer name	引物序列 Primer sequence
pEGFP-N3	GTCGCCGTCCAGCTCGACCAG
Ubi-F	TTAGCCCTGCCTTCATACGC
OsDR8-g1-F	CAGCTTGATGGGGCTGAACCTGA
OsDR8-g1-R	AACTCAGGTTCAGCCCCATCAAG
OsDR8-g2-F	CAGCTTGACGATGAGGTCCTCGA
OsDR8-g2-R	AACTCGAGGACCTCATCGTCAAG
OsDR8-F1	CACTCTCCTCATCCCAGAGCA
OsDR8-R1	CGATCATGCCGATGTCCTGCA
OsDR8-F2	TGTTACTTCTGCAGGGATCCATGGCAGCCATGGCCACC
OsDR8-R2	CTCACCATAGGCCTCACGTGGGCGTCCACGATCTCGCC
VKOO5-F	GATGAAGTGGACGGAAGGAAGGAG

Fig. 1. Phylogenetic relationship of OsDR8 proteins in closely related species

Table 2. Cis-acting elements in the promoter region of OsDR8

类型 Type	元件名称 Element	数目 Number	功能注释 Functional annotation
光响应元件 Light-responsive elements	G-Box	5	光响应元件Light-responsive elements
光响应元件 Light-responsive elements	GATA-motif	2	光响应元件Light-responsive elements
激素响应元件 Hormone-responsive elements	ABRE	4	ABA响应元件 ABA-responsive element
	CGTCA-motif	3	MeJA响应的顺式作用元件 MeJA-responsive cis-acting element
	TGACG-motif	3	MeJA响应的顺式作用元件 MeJA-responsive cis-acting element
	as-1	3	水杨酸响应元件 Salicylic acid-responsive element
	TCA	2	水杨酸响应元件 Salicylic acid-responsive element
	MRE	3	雌激素反应元件 Estrogen response element
	ERE	2	雌激素反应元件 Estrogen response element
结合元件 Binding elements	MYB	3	MYB结合元件 MYB binding elements
结合元件 Binding elements	MYC	4	MYC结合元件 MYC binding elements
胁迫相关元件 Stress-responsive elements	LTR	2	低温响应元件 Low-temperature-responsive element
	GC-motif	2	防御与应激反应响应元件 Defense- and stress-responsive element
	TC-rich repeats	2	抗病和胁迫响应元件 Disease- and stress-responsive element
	STRE	5	应激响应元件 Stress-responsive element

Fig. 2. Identification results of OsDR8 overexpression lines Control, The total protein content.

Table 3. Editing efficiency and genotypic distribution of OsDR8 through Agrobacterium-mediated genetic transformation

阳性株系 Transgenic-positive lines	编辑率 Mutation rate(%)	纯合型 Homozygous	杂合型 Heterozygous	野生型 Wild-type
36	77.78	5	23	8

Fig. 3. Identification results of OsDR8 knockout mutants The arrows represent gRNA recognition sequences, and the asterisks denote PAM regions. WT-1 and WT-2, Wild type; KO-1 and KO-2, Knockout mutant lines.

Fig. 4. Disease symptoms (A) and proportion of diseased leaf area (B) in wild-type (WT) and gene-edited materials after infection with Magnaporthe oryzae ** and **** represent significant differences between treatments at the levels of P<0.01 and P<0.0001, respectively. WT, Wild type; KO, OsDR8 knockout mutant lines. RO1-1 and RB22, Magnaporthe isolates.

Fig. 5. Disease symptoms (A) and proportion of diseased leaf area (B) in wild-type (WT) and overexpression rice materials after infection with Magnaporthe oryzae ** represents significant differences between treatments at the level of P<0.01. WT, Wild type; OE, OsDR8 overexpression lines; RO1-1, RB22, Magnaporthe isolates.

Table 4. Haplotype analysis of OsDR8

单倍型Haplotype	物理位置Physical position（bp）			群体数量Number
单倍型Haplotype	20,724,692	20,724,742	20,725,760	群体数量Number
Hap1	CC	AA	AA	58
Hap2	TT	AA	AA	155
Hap3	CC	AA	GG	5
Hap4	CT	AA	AA	8
Hap5	CC	GG	AA	4

Fig. 6. Effect of haplotypes of OsDR8 on rice blast resistance ns, *, **, and **** in the figure represent non-significant difference between haplotypes and significant differences at the levels of P<0.05, P<0.01, and P<0.0001, respectively.

Fig. 7. Haplotype evolutionary analysis of OsDR8 In the figure, Aus, Ind, Jap, Ob, Og, and Or refer to Oryza sativa aus, O. sativa indica, O. sativa japonica, O. barthii, O. glaberrima, and O. rufipogon, respectively.

Fig. 8. Distribution of haplogroups of OsDR8 in population samples

Fig. 9. Differences in resistance levels to rice blast among different subgroups of Asian cultivated rice **** in the figure represents significant differences between subgroups at the level of P<0.0001.

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