粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

doi:10.11983/CBB16126

植物学报 ›› 2017, Vol. 52 ›› Issue (1): 61-69.DOI: 10.11983/CBB16126

所属专题：水稻生物学专辑 (2017年52卷1期)

粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

胡朝芹, 刘剑宇, 王韵茜, 杨睿, 汪秉琨, 何月秋, 曾千春, 罗琼^*()

云南农业大学农业生物多样性与病虫害控制教育部重点实验室
云南生物资源保护与利用国家重点实验室, 昆明 650201

收稿日期:2016-06-07 接受日期:2016-07-29 出版日期:2017-01-01 发布日期:2017-01-23
通讯作者: 罗琼
作者简介:
# 共同第一作者
基金资助:
国家重点研发计划(No.2016YFD0100601)和国家自然科学基金(No;U1302261, No.31160223)

Mapping of Pizy6(t), a Gene Conferring Resistance to the Rice Blast Strain LP11, in Oryza sativa subsp. japonica Cultivar Ziyu44

Hu Chaoqin, , Liu Jianyu, , Wang Yunqian, Yang Rui, Wang Bingkun, He Yueqiu, Zeng Qianchun, Luo Qiong^*()

State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan
Ministry of Education Key Laboratory of Agriculture Biodiversity for Plant Disease Management, Yunnan Agricultural University, Kunming 650201, China

Received:2016-06-07 Accepted:2016-07-29 Online:2017-01-01 Published:2017-01-23
Contact: Luo Qiong
About author:
# Co-first authors

摘要/Abstract

摘要： 稻瘟病是世界范围内严重威胁水稻(Oryza sativa)生产可持续发展的主要病害之一, 每年造成10%-30%的水稻产量损失。抗瘟水稻品种的培育和育种利用是解决稻瘟病危害最经济有效的方法。对新的致病性菌株进行分离和筛选是定位与克隆抗病新基因及抗病育种的基础。选择分离自不同稻瘟病发生重灾区的单孢菌株, 对广谱抗瘟水稻子预44和感病水稻江南香糯进行致病性鉴定, 筛选出两材料间致病性差异明显的5个菌株; 进一步利用子预44、湘资3150、9311、日本晴、丽江新团黑谷、中花11、TP309和江南香糯8个抗瘟性不同的水稻材料, 对筛选的菌株进行致病性鉴定。结果显示, LP11能使广谱抗瘟籼稻湘资3150严重发病, 推测其很可能是新进化出来的强致病菌株。利用子预44和江南香糯杂交构建的F₂群体进行抗性遗传分析, 结果表明子预44对LP11菌株的抗性是由单显性基因控制。利用SSR分子标记和图位克隆方法在子预44中定位了1个抗稻瘟病基因Pizy6(t)。研究结果不仅为抗病相关研究提供了有价值的新菌株, 而且为子预44中抗稻瘟病基因Pizy6(t)的克隆奠定了基础。

Abstract: Rice blast, caused by Magnaporthe oryzae infection, is one of the major diseases threatening the sustainable development of rice (Oryza sativa) production worldwide. Between 10% and 30% of the annual rice harvest is lost due to infection by the fungus. Development and breeding utilization of rice-resistant resources are considered the most effective and economic means to control rice blast. Isolating new virulent blast strains from the pathotype Oryza is the basis for cloning novel optimal genes resistant to rice blast and rice breeding for disease resistance. In the present study, we isolated and screened blast strains and identified new genes resistant to rice blast. Overall, 5 strains with significantly different pathogenicity between Ziyu44 and Jiangnanxiangnuo (JNXN) were identified. Eight rice varieties, including Ziyu44, Xiangzi3150, 9311, Nipponbare, Lijiangxintuanheigu (LTH), Zhonghua11, JNXN and TP309, were challenged with the 5 blast strains by spray-inoculation at seedling stage. Xiangzi3150, a variety with broad-spectrum blast resistance, showed obvious attacks after inoculation with LP11, so LP11 is probably a newly emerged strong virulent strain. The F₂ progenies derived from the cross between Ziyu44 and JNXN were inoculated with the LP11; genetic analysis and gene mapping indicated that the resistance phenotype of Ziyu44 to LP11 was controlled by a single dominant gene, and the resistance gene Pizy6(t) was located on rice chromosome 6. These results provided valuable blast strains for genetic studies of disease resistance and laid the foundation for Pizy6(t) gene cloning in Ziyu44.

胡朝芹, 刘剑宇, 王韵茜, 杨睿, 汪秉琨, 何月秋, 曾千春, 罗琼. 粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位. 植物学报, 2017, 52(1): 61-69.

Hu Chaoqin, , Liu Jianyu, , Wang Yunqian, Yang Rui, Wang Bingkun, He Yueqiu, Zeng Qianchun, Luo Qiong. Mapping of Pizy6(t), a Gene Conferring Resistance to the Rice Blast Strain LP11, in Oryza sativa subsp. japonica Cultivar Ziyu44. Chinese Bulletin of Botany, 2017, 52(1): 61-69.

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https://www.chinbullbotany.com/CN/Y2017/V52/I1/61

图/表 11

表1 无毒基因及其PCR引物

Table 1 Avr genes and primers used in this study

Avr genes	Primers	Sequence (5'-3')
AVR_Pita	F	GACCCGTTTCCGCCTTTATT
AVR_Pita	R	GATTCCCTCCATTCCAACAC
AVR_PWL2	F	CTCCGCCACTTTTCTCATTC
AVR_PWL2	R	GCCCTCTTCTCGCTGTTCAC
AVR_Pik	F	GTCAACCAAGCGTAAACCTC
AVR_Pik	R	CGATTCAGAAGTTAGGCATT
AVR_pii	F	GAGGCCGATATGTTACGATT
AVR_pii	R	CTCTGCTCTCACGCTTTACC
AVR_Pia	F	GCCGCTAGCTGTATAGACAA
AVR_Pia	R	TCATCGTCGAGTGGTGTAGG
AVR_ACE1	F	GAGGTGCCAGATATGTCGTC
AVR_ACE1	R	GGATGAGCAGATGAGCAACA

图1 子预44和江南香糯水稻接种不同稻瘟病菌株^Z: 子预44; J: 江南香糯; H53: 哈尔滨53号菌株; YC119: 盐城119号菌株; LP11: 罗平11号菌株; LP33: 罗平33号菌株; LP174: 罗平174号菌株

Figure 1 Ziyu44 and JNXN were inoculated with different rice blast strains^Z: Ziyu44; J: JNXN; H53: No.53 strain from Harbin; YC119: No.119 strain from Yancheng; LP11: No.11 strain from Luo- ping; LP33: No.33 strain from Luoping; LP174: No.174 strain from Luoping

图2 8个水稻材料接种稻瘟病菌株LP11^Z: 子预44; J: 江南香糯; 9: 9311; H: 中花11; N: 日本晴; X: 湘资3150; L: LTH; T: TP309

Figure 2 Eight rice varieties were inoculated with Magnaporthe oryzae LP11^Z: Ziyu44; J: JNXN; 9: 9311; H: Zhonghua11; N: Nipponbare; X: Xiangzi3150; L: LTH; T: TP309

图3 子预44、江南香糯及其F2群体单株喷雾接种稻瘟病菌LP11^Z: 子预44; J: 江南香糯; N: 日本晴; 9: 9311; R: F2群体中的抗病单株; S: F2群体中的感病单株

Figure 3 Ziyu44, JNXN and the F2 progenies were inoculated with Magnaporthe oryzae LP11 by spraying^Z: Ziyu44; J: JNXN; N: Nipponbare; 9: 9311; R: The resistant individual plants of F2 population; S: The susceptible individu- al plants of F2 population

表2 子预44/江南香糯F2群体对LP11的抗性分离

Table 2 Segregation ratios of resistance to blast isolate LP11 in the F2 population of Ziyu44/JNXN

Parents and F₂ population	Number of total plants	Number of R plants	Number of S plants	Expratio (R:S)	Χ²	X²_0.05(1)
JNXN	25	0	25
9311	10	10	0
Nipponbare	14	0	14
Ziyu44/JNXN F₂	489	370	119	3:1	0.012	3.84

图4 RM276在接种LP11的水稻F2群体中的分离^Z: 子预44; J: 江南香糯; S1-S20: F2群体中的感病单株; * : 重组单株

Figure 4 Segregation of RM276 in F2 population of rice treated by LP11^Z: Ziyu44; J: JNXN; S1-S20: Susceptible plants of F2 population; * : Recombinant plants

图5 RM3628在接种LP11的水稻F2群体中的分离^Z: 子预44; J: 江南香糯; R1-R15: F2群体中的抗病单株; S1-S20: F2群体中的感病单株; * : 重组单株

Figure 5 Segregation of RM3628 in F2 population of rice treated by LP11^Z: Ziyu44; J: JNXN; R1-R15: Resistant plants of F2 population; S1-S20: Susceptible plants of F2 population; * : Recombinant plants

图6 Indel06081、RM20023和RM3827在接种LP11的水稻F2群体中的分离^Z: 子预44; J: 江南香糯; S1-S20: F2群体中的感病单株; *: 重组单株

Figure 6 Segregation of Indel06081, RM20023 and RM3827 in F2 population of rice treated by LP11^Z: Ziyu44; J: JNXN; S1-S20: Susceptible plants of F2 population; * : Recombinant plants

表3 LP11和LP33菌株的36个抗病单基因系鉴定

Table 3 Pathogenic identification of LP11 and LP33 with 36 single gene lines of blast resistance

Variety/R-gene	LP11	LP33	Variety/R-gene	LP11	LP33
LTH/-	4	3	IRBL-7/Pi-k^p	4	1
F80-1/Pi-k	4	2	IRBL-8/Pi-k^h	3	1
F98-7/Pi-k^m	4	2	IRBL-9/Pi-z	3	4
F124-1/Pi-ta	4	1	IRBL-10/Pi-z⁵	4	3
F128-1/Pi-ta²	2	0	IRBL-11/Pi-z^t	4	2
F129/Pi-kp	4	3	IRBL-12/Pi-ta	4	4
F145-2/Pi-b	4	2	IRBL-13/Pi-ta	4	4
C039/Pi-CO39	4	0	IRBL-14/Pi-b	3	1
C101A51/Pi-2	3	1	IRBL-15/Pi-t	4	2
C101LAC/Pi-1	3	1	IRBL-16/Pi-sh	3	1
C101PKT/Pi-4a	2	1	IRBL-17/Pi-sh	4	2
C104PKT/Pi-3	4	2	IRBL-18/Pi-1	3	1
IRBL-1/Pi-a(1)	4	1	IRBL-19/Pi-3	3	2
IRBL-2/Pi-a(2)	4	2	IRBL-20/Pi-5(t)	3	1
IRBL-3/Pi-i	3	1	IRBL-21/Pi-7(t)	4	1
IRBL-4/Pi-k^s	3	0	IRBL-22/Pi-9(t)	2	1
IRBL-5/Pi-k^s	4	1	IRBL-23/Pi-12(t)	2	0
IRBL-6/Pi-k	4	2	IRBL-24/Pi-19(t)	3	1

图7 不同无毒基因位点在稻瘟病菌株LP33和LP11中的PCR扩增

Figure 7 PCR amplification of Avr genes in LP33 and LP11 strains

图8 LP11和LP33对8个水稻品种的致病性^Z、9、X、H、N、T、L和J同图2。

Figure 8 The pathogenicity of LP11 and LP33 to 8 rice varieties^Z, 9, X, H, N, T, L and J see Figure 2.

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粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

Mapping of Pizy6(t), a Gene Conferring Resistance to the Rice Blast Strain LP11, in Oryza sativa subsp. japonica Cultivar Ziyu44

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