稻瘟病广谱抗性基因Pigm特异性分子标记的开发和应用

doi:10.16819/j.1001-7216.2020.9082

中国水稻科学 ›› 2020, Vol. 34 ›› Issue (1): 28-36.DOI: 10.16819/j.1001-7216.2020.9082

稻瘟病广谱抗性基因Pigm特异性分子标记的开发和应用

陈涛^1,², 孙旭超¹, 张善磊¹, 梁文化¹, 周丽慧¹, 赵庆勇¹, 姚姝¹, 赵凌¹, 赵春芳¹, 朱镇¹, 张亚东¹, 王才林^1,^*()

¹江苏省农业科学院粮食作物研究所/江苏省优质水稻工程技术研究中心/国家水稻改良中心南京分中心, 南京 210014
²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:2019-07-12 修回日期:2019-11-20 出版日期:2020-01-10 发布日期:2020-01-10
通讯作者: 王才林
基金资助:
国家重点研发计划重点专项(2017YFD0100305);江苏省重点研发计划资助项目(BE2018357);江苏省农业重大新品种创制项目(PZCZ201703);现代农业产业技术体系建设专项资金资助项目(CARS-01-62);江苏省农业科学院粮食作物研究所科研基金资助项目(LZS17-7)

Development and Verification of Specific Molecular Markers for Pigm Gene Associated with Broad-spectrum Resistance to Rice Blast

Tao CHEN^1,², Xuchao SUN¹, Shanlei ZHANG¹, Wenhua LIANG¹, Lihui ZHOU¹, Qingyong ZHAO¹, Shu YAO¹, Ling ZHAO¹, Chunfang ZHAO¹, Zhen ZHU¹, Yadong ZHANG¹, Cailin WANG^1,^*()

¹Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Jiangsu High Quality Rice R&D Center / Nanjing Branch of China National Center for Rice Improvement, Nanjing 210014, China;
²Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

Received:2019-07-12 Revised:2019-11-20 Online:2020-01-10 Published:2020-01-10
Contact: Cailin WANG

摘要/Abstract

摘要：

【目的】来自籼稻品种谷梅4号的Pigm是一个对稻瘟病菌具有广谱和持久抗性的重要基因。为有效提高Pigm基因的选择效率,有必要开发具有特异性的共显性分子标记进行辅助育种。【方法】本研究根据谷梅4号Pigm位点存在的特异性核苷酸变异,利用Tetra-primer ARMS-PCR和KASP两种不同的基因分型技术开发出分子标记T-Pigm和K-Pigm,对不同品种(品系)以及淮稻9号/谷梅4号的F₂分离群体进行基因型检测,并结合穗颈瘟人工接种鉴定,对标记的准确性进行评价。【结果】序列比对分析表明,谷梅4号Pigm位点中Pigm-Nbs2基因起始密码子上游515 bp处存在一个特殊的单核苷酸变异。利用已开发的两种类型的分子标记能够有效区分3种不同的基因型,且基因型与穗颈瘟人工接种鉴定结果完全一致。【结论】利用分子标记T-Pigm和K-Pigm可以实现对Pigm基因型快速、准确的检测,加快抗稻瘟病水稻新品种的选育进程。

关键词: 稻瘟病, Pigm, 特异性标记, 四引物扩增受阻突变体系PCR, 竞争性等位基因特异性PCR

Abstract:

【Objective】 Pigm from an indica rice variety Gumei 4, is one of the most important genes with broad-spectrum and durable resistance to rice blast. It is very necessary to improve its selection efficiency by developing specific and co-dominant markers in breeding. 【Method】The molecular markers T-Pigm and K-Pigm were developing with Tetra-primer ARMS-PCR and KASP technique, based on the specific nucleotide mutation in Pigm locus. Then, the consistency of the genotype and phenotype was evaluated in different varieties and the F₂ population derived from Huaidao 9/ Gumei 4 by artificial inoculation methods of panicle blast.【Result】The sequence alignment analysis indicated that a specific SNP was at -515 bp of the Pigm-Nbs2 gene initiation codon in Gumei 4. The three different genotypes of Pigm locus could be distinguished by the Tetra-primer ARMS-PCR and KASP markers, which exactly conformed to the results of artificial inoculation.【Conclusion】 T-Pigm and K-Pigm markers could be used to detect the genotype of Pigm gene rapidly and accurately, and improved the selection efficiency of blast resistant rice varieties in breeding.

Key words: rice blast, Pigm, specific marker, tetra-primer ARMS-PCR, competitive allele-specific PCR

中图分类号:

陈涛, 孙旭超, 张善磊, 梁文化, 周丽慧, 赵庆勇, 姚姝, 赵凌, 赵春芳, 朱镇, 张亚东, 王才林. 稻瘟病广谱抗性基因Pigm特异性分子标记的开发和应用[J]. 中国水稻科学, 2020, 34(1): 28-36.

Tao CHEN, Xuchao SUN, Shanlei ZHANG, Wenhua LIANG, Lihui ZHOU, Qingyong ZHAO, Shu YAO, Ling ZHAO, Chunfang ZHAO, Zhen ZHU, Yadong ZHANG, Cailin WANG. Development and Verification of Specific Molecular Markers for Pigm Gene Associated with Broad-spectrum Resistance to Rice Blast[J]. Chinese Journal OF Rice Science, 2020, 34(1): 28-36.

图/表 7

图1 谷梅4号与测序品种及含Pi9、Pi2、Piz、Piz-t、Pi40、Pi50基因品种(系)部分序列的比对数字表示碱基在水稻基因组中的物理位置;斜体碱基表示有差异的核酸变异;灰色碱基表示起始密码子上游515 bp处存在的特异单核苷酸变异。

Fig. 1. Sequence alignment of Gumei 4, sequencing varieties and other varieties(lines) carrying Pi9, Pi2, Piz, Piz-t, Pi40, Pi50. The numbers indicate physical location in rice genomic sequence. The differential bases in sequences are in Italic and the specific single nucleotide variation in -515 bp of Pigm-Nbs2 gene initiation codon is in gray.

表1 检测水稻Pigm基因特异SNP变异的测序引物和相关标记

Table 1 Sequencing primers and markers to detect the specific SNP variation for Pigm gene

标记名称 Marker name	引物序列(5′-3′) Sequence(5′-3′)	物理位置 Physical location / bp
S-Primers	GGAATCGCTCCATTTTCGTA	10 380 398-10 380 417
	GACGGTTTAGGTGCGCTTTA	10 380 779-10 380 798
T-Pigm	T-Pigm-O-F: TAAGAATTGAGGTGGTTAGTTGAACGGAGA	10 380 094-10 380 123
	T-Pigm-O-R: TTGCATGGCTCCACTACCCACTATAAG	10 380 742-10 380 768
	T-Pigm-I-F: TGAAAATAAAAATGGTATGATGGTTACG	10 380 474-10 380 501
	T-Pigm-I-R: TAGGGATGAAACGGCTCGAAAACGATCG	10 380 501-10 380 528
K-Pigm	K-F(G): AAAAATGAAAATAAAAATGGTATGATGGTTTCG FAM-labelled	10 380 469-10 380 501
	K-F(C): AAAAATGAAAATAAAAATGGTATGATGGTTTCC HEX-labelled	10 380 469-10 380 501
	K-R: AGGGATGAAACGGCTCGAAAACGAA	10 380 503-10 380 527

图2 T-Pigm标记对不同水稻品种(系)Pigm基因型的检测 M–DNA分子量标准(DL2000);1–谷梅4号;2–IRBL9-W(Pi9);3–IRBLz5-CA-1(Pi2);4–IRBLz-Fu(Piz);5–IRBLzt-T(Piz-t);6–IR65482-4-13(Pi40);7–二八占(Pi50);8–24–部分籼、粳稻品种(系)依次为93-11、中恢8006、成恢727、红恢589、先恢527、明恢63、广恢998、川香29B、珍汕97B、培矮64S、日本晴、嘉58、淮稻9号、南粳9108、盐丰47、吉粳88、龙粳29。

Fig. 2. Electrophoresis detection of Pigm genotypes by T-Pigm marker in different rice varieties or lines. M, DNA marker (DL2000); 1, Gumei 4; 2, IRBL9-W (Pi9); 3, IRBLz5-CA-1(Pi2); 4, IRBLz-Fu (Piz); 5, IRBLzt-T (Piz-t); 6, IR65482-4-13 (Pi40); 7, Erbazhan; 8-24, Parts of indica and japonica rice varieties or lines, including 93-11, Zhonghui 8006, Chenghui 727, Honghui 589, Xianhui 527, Minghui 63, Guanghui 998, Chuanxiang 29B, Zhenshan 97B, Peiai 64S, Nippobare, Jia 58, Huaidao 9, Nanjing 9108, Yanfeng 47, Jijing 88, Longjing 29.

图3 T-Pigm标记对淮稻9号/谷梅4号F2群体Pigm基因型的检测 M–DNA分子量标准(DL2000);1–谷梅4号;2–淮稻9号;3–淮稻9号/谷梅4号 F1;4–24为部分F2分离单株。

Fig. 3. Electrophoresis detection of Pigm genotypes by T-Pigm marker in F2 population derived from Huaidao 9/Gumei 4. M, DNA marker (DL2000); 1, Gumei 4; 2, Huaidao 9; 3, F1 (Huaidao 9/Gumei 4); 4-24, Parts of plants from F2 population.

图4 K-Pigm标记对不同水稻品种(系)Pigm基因型的检测黑色方框代表无DNA模板的空白对照;蓝色和红色圆点分别代表PigmPigm纯合基因型(碱基G的等位变异)和pigmpigm纯合基因型(碱基C的等位变异)。供试品种(系)分别为谷梅4号、IRBL9-W(Pi9)、IRBLz5-CA-1 (Pi2)、IRBLz-Fu(Piz)、IRBLzt-T(Piz-t)、IR65482-4-13(Pi40)、二八占(Pi50)、镇恢084、93-11、中恢8006、成恢727、宜恢1577、红恢589、先恢527、明恢63、航1号、金恢2号、广恢998、宁香1B、川香29B、荃93-11B、珍汕97B、03S、Bph68S、P88S、培矮64S、日本晴、越光、一品、珍富10号、楚粳39、毕粳44、宝农34、浙粳97、嘉58、南粳505、南粳9108、淮稻9号、豫粳8号、临稻17、圣稻20、宁粳40号、伊粳12号、新稻32号、津粳263、沈农265、辽粳10号、盐丰47、吉粳88和龙粳29。

Fig. 4. Detection of Pigm genotypes by K-Pigm marker in different rice varieties or lines. Black boxes indicate the negative controls with no DNA template; Blue and red dots showed PigmPigm homozygous genotype (allelic variation G) and pigmpigm homozygous genotype (allelic variation C), respectively.The DNA of the tested varieties (lines) are Gumei 4, IRBL9-W(Pi9), IRBLz5-CA-1, IRBLz-Fu (Piz), IRBLzt-T (Piz-t), IR65482-4-13(Pi40), Erbazhan(Pi50), Zhenhui 084, 93-11, Zhonghui 8006, Chenghui 727, Yihui 1577, Honghui 589, Xianhui 527, Minghui 63, Hang 1, Jinghui 2, Guanghui 998, Ningxiang 1B, Chuanxiang 29B, Quan 93-11B, Zhenshan 97B, 03S, Bph68S, P88S, Peiai 64S, Nippobare, Koshihikari, Yipin, Zhenfu 10, Chujing 39, Bijing 44, Baonong 34, Zhejing 97, Jia 58, Nanjing 505, Nanjing 9108, Huaidao 9, Yujing 8, Lindao 17, Shengdao 20, Ningjing 40, Yijing 12, Xindao 32, Jinjing 263, Shennong 265, Liaojing10, Yanfeng 47, Jijing 88 and Longjing 29, respectively.

图5 K-Pigm标记对淮稻9号/谷梅4号F2群体Pigm基因型的检测黑色方框代表无DNA模板的空白对照;蓝色、绿色和红色圆点分别代表PigmPigm纯合基因型(碱基G的等位变异), Pigmpigm杂合基因型(同时具有碱基G/C的等位变异)和pigm pigm纯合基因型(碱基C的等位变异)。

Fig. 5. Detection of Pigm genotypes by K-Pigm marker in F2 population derived from Huaidao 9 and Gumei 4. Black boxes indicate the negative controls with no DNA template; Blue, green and red dots showed PigmPigm homozygous genotype (allelic variation G), Pigmpigm heterozygous (allelic variations G/C) and pigmpigm homozygous genotype (allelic variation C), respectively.

表2 淮稻9号/谷梅4号F2群体中三种Pigm基因型单株对穗颈瘟的抗性表现

Table 2 Resistance to neck blast disease for individual plants of the three different genotypes derived from Huaidao 9 and Gumei 4.

基因型 Genotype	等位变异 Allelic variation	不同基因型单株数 No. of individual plants	不同病情等级的单株数 No. of individual plants with different disease grades
基因型 Genotype	等位变异 Allelic variation	不同基因型单株数 No. of individual plants	高抗HR	抗R	中抗MR	中感MS	感S	高感HS
PigmPigm	GG	62	3	35	24	0	0	0
Pigmpigm	GC	166	4	74	88	0	0	0
pigmpigm	CC	72	0	1	4	31	28	8

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稻瘟病广谱抗性基因Pigm特异性分子标记的开发和应用

Development and Verification of Specific Molecular Markers for Pigm Gene Associated with Broad-spectrum Resistance to Rice Blast

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