广谱抗稻瘟病基因PigmR的KASP标记开发及应用

doi:10.16819/j.1001-7216.2025.240104

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (3): 365-372.DOI: 10.16819/j.1001-7216.2025.240104

广谱抗稻瘟病基因PigmR的KASP标记开发及应用

李文奇¹^,²^,³, 许扬¹^,²^,³, 王芳权¹^,²^,³, 朱建平¹^,²^,³, 陶亚军¹^,²^,³, 李霞¹^,²^,³, 范方军¹^,²^,³, 蒋彦婕¹^,²^,³, 陈智慧¹^,²^,³, 杨杰¹^,²^,³^,^*()

¹江苏省农业科学院粮食作物研究所/农业农村部淮河下游种质创制重点实验室（南京）/国家水稻改良中心南京分中心，南京 210014
²生物育种钟山实验室，南京 210014
³扬州大学江苏省粮食作物现代产业技术协同创新中心，江苏扬州 225009

收稿日期:2024-09-08 修回日期:2025-02-25 出版日期:2025-05-10 发布日期:2025-05-21
通讯作者: *email: yangjie168@aliyun.com
基金资助:
江苏省种业振兴揭榜挂帅项目[JBGS（2021）039];江苏省重点研发计划（现代农业）资助项目(BE2021374);国家自然科学基金面上项目(42071023);生物育种钟山实验室项目(BM2022008-03);生物育种钟山实验室项目(1201);生物育种钟山实验室项目(1002);江苏省自然科学基金资助项目(BK20212010)

Development and Application of KASP Marker for Broad-Spectrum Resistance Gene PigmR to Rice Blast

LI Wenqi¹^,²^,³, XU Yang¹^,²^,³, WANG Fangquan¹^,²^,³, ZHU Jianping¹^,²^,³, TAO Yajun¹^,²^,³, LI Xia¹^,²^,³, FAN Fangjun¹^,²^,³, JIANG Yanjie¹^,²^,³, CHEN Zhihui¹^,²^,³, YANG Jie¹^,²^,³^,^*()

¹Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Germplasm Innovation in Downstream of Huaihe River (Nanjing), Ministry of Agricultural and Rural Affairs/ Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, China
²Zhongshan Biological Breeding Laboratory, Nanjing 210014, China
³Jiangsu Co-innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

Received:2024-09-08 Revised:2025-02-25 Online:2025-05-10 Published:2025-05-21
Contact: *email: yangjie168@aliyun.com

1. Liwenqi table S1.doc(129KB)

摘要/Abstract

摘要：

【目的】广谱抗稻瘟病基因PigmR对水稻抗稻瘟病遗传改良具有重要育种价值。根据其编码区变异位点开发竞争性等位基因特异性PCR(Kompetitive allele specific，PCR KASP)标记，有利于提升PigmR育种利用的效率。【方法】通过序列比对明确PigmR基因编码区存在单核苷酸变异，开发KASP分子标记PigmR-K，通过对水稻品种和F₂分离群体进行基因型分析，结合稻瘟病表型鉴定，并开展分子标记辅助选择育种，对标记的精准性和高效性进行验证。【结果】根据PigmR基因编码区序列第2503位存在特异性单核苷酸A>C变异，建立基于KASP的PigmR精准高效基因分型技术体系，通过对159份水稻品种、270份F₂群体单株以及247份BC₁F₃群体单株进行基因型检测，PigmR-K能够高效区分供试水稻材料的纯合抗病、纯合感病和杂合基因型，并与人工接种穗颈瘟症状一致。【结论】利用PigmR基因编码区变异位点开发的KASP标记PigmR-K具有精准高效等优势，有利于稻瘟病抗性遗传改良。

关键词: 水稻, 广谱抗性, 稻瘟病, PigmR基因, KASP, 育种效率

Abstract:

【Objective】The broad-spectrum resistance gene PigmR has important breeding value for the genetic improvement of rice blast resistance. Developing kompetitive allele specific PCR (KASP) markers based on its coding region variation sites is beneficial for improving the accuracy and efficiency of PigmR utilization in breeding.【Method】This study identified single nucleotide variations in the coding region of PigmR through sequence alignment, developed the KASP molecular marker PigmR-K, conducted genotype analysis using rice varieties and F₂ segregating populations, combined these results with rice blast symptom identification, and carried out molecular marker assisted selection breeding to verify the precision and efficiency of this marker.【Result】Based on an A-to-C mutation at position 2503 in the PigmR gene coding region sequence, a precise and efficient PigmR genotyping technology system based on KASP markers was established. The genotyping of 159 rice varieties, 270 F₂ individuals, and 247 BC₁F₃population lines demonstrated that PigmR-K reliably distinguished the homozygous resistant, homozygous susceptible and heterozygous genotypes of PigmR. These results were completely consistent with disease symptoms from artificial panicle blast inoculation.【Conclusion】The KASP marker PigmR-K developed based on the variation site of on the coding region has advantages such as precision and efficiency, which is beneficial for genetic improvement of rice blast resistance.

Key words: rice, broad-spectrum resistance, rice blast, PigmR gene, KASP, breeding efficiency

李文奇, 许扬, 王芳权, 朱建平, 陶亚军, 李霞, 范方军, 蒋彦婕, 陈智慧, 杨杰. 广谱抗稻瘟病基因PigmR的KASP标记开发及应用[J]. 中国水稻科学, 2025, 39(3): 365-372.

LI Wenqi, XU Yang, WANG Fangquan, ZHU Jianping, TAO Yajun, LI Xia, FAN Fangjun, JIANG Yanjie, CHEN Zhihui, YANG Jie. Development and Application of KASP Marker for Broad-Spectrum Resistance Gene PigmR to Rice Blast[J]. Chinese Journal OF Rice Science, 2025, 39(3): 365-372.

图/表 5

图1 谷梅4号中PigmR部分编码区序列变异位点数字为PigmR编码区位置；粗体字为PigmR编码区2503 bp处特异核苷酸变异位点。

Fig. 1. Variation sites of partial PigmR coding region sequence in Gumei 4 The numbers represent the position in the PigmR coding region; Bold letters refer to the specific nucleotide variation site at 2503 bp in the coding region.

表1 PigmR-K标记信息

Table 1. PigmR-K marker information

引物名称 Primer	引物序列(5′→3′) Primer sequence(5′→3′)	引物长度和标记 Primer length and marker	结果 Result
Pigm-KR1	GAAGGTGACCAAGTTCATGCTCCTTTAGTTTACTCCTCCGTAAGTC	46 bp, Fam: Allele-C	C:C为感病基因型 C:C, Susceptible genotype
Pigm-KR2	GAAGGTCGGAGTCAACGGATTTCCTTTAGTTTGCTCCTCCGTAAGTA	47 bp, Hex: Allele-A	A:A为抗性基因型 A:A, Resistant genotype
Pigm-KF	TAACTGGATTGAGCAGCTCACTCACCTG	28 bp	A:C为杂合基因型 A:C, Heterozygous genotype

图2 PigmR-K标记验证 A: PigmR-K标记反应条件优化; B: 水稻品种中PigmR-K分型图。×代表无DNA模板的空白对照, 红色标记点为Hex荧光信号, 表示纯合抗病基因型(A:A); 蓝色标记点为Fam荧光信号, 表示纯合感病基因型(C:C); 绿色标点代表杂合基因型(A:C)。

Fig. 2. PigmR-K marker verification A, Optimization of reaction conditions for the PigmR-K marker; B, Genotyping plot of PigmR-K in rice varieties. × represents blank control without DNA template. The red dots represent the Hex fluorescence signal, indicating homozygous resistant genotypes (A:A); The blue dots represent the Fam fluorescence signal, indicating homozygous susceptible genotypes (C:C); The green dots represent heterozygous genotypes (A:C).

图3 PigmR-K标记检测水稻F2分离群体PigmR基因型 A和B: PigmR-K标记检测淮稻5号/金香玉1号的F2分离群体单株PigmR的基因型; C: PigmR-K标记检测F2 群体的PigmR 三种基因型; D: F2群体部分单株穗颈瘟症状。×代表无DNA模板的空白对照, 红色标记点为Hex荧光信号, 表示纯合抗病基因型(A:A), 蓝色标记点为Fam荧光信号, 表示纯合感病基因型(C:C), 绿色标点代表杂合基因型(A:C)。HS: 高感; S: 感; R: 抗; MR: 高抗。

Fig. 3. Genotyping of PigmR in rice F2 segregating population using PigmR-K marker A & B, Genotyping of PigmR in F2 progeny derived from a cross between Huaidao 5 (susceptible parent) and Jinxiangyu 1 (resistant parent) using the PigmR-K marker. C, Distribution of three PigmR genotypes detected by PigmR-K marker. D, Representative neck blast symptoms of selected F2 plants. × represents blank control without DNA template, the red dots represent the Hex fluorescence signal, indicating homozygous resistant genotypes (A:A), the blue dots represent the Fam fluorescence signal, indicating homozygous susceptible genotypes (C:C), and the green dots represent heterozygous genotypes (A:C).

图4 PigmR-K标记对回交育种材料的PigmR基因型检测 A和B: PigmR-K标记检测南粳53045/金香玉1号的BC1F3群体单株的PigmR基因型。×代表无DNA模板的空白对照，红色标记点为Hex荧光信号表示纯合抗病基因型(A/A), 蓝色标记点为Fam荧光信号, 表示纯合感病基因型(C/C), 绿色标点代表杂合基因型(A/C)。

Fig. 4. Genotyping of PigmR in backcross breeding population using PigmR-K marker A and B, Genotyping of PigmR in the BC1F3 population of Nanjing 53045/Jinxiangyu 1 using PigmR-K markers. × represents blank control without DNA template, the red dots represent the Hex fluorescence signal, indicating homozygous resistant genotypes (A/A), the blue dots represent the Fam fluorescence signal, indicating homozygous susceptible genotypes (C/C), and the green dots represent heterozygous genotypes (A/C).

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广谱抗稻瘟病基因PigmR的KASP标记开发及应用

Development and Application of KASP Marker for Broad-Spectrum Resistance Gene PigmR to Rice Blast

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