水稻抗稻瘟病基因Pi47的精细定位和候选基因分析

doi:10.3724/SP.J.1006.2018.00977

作物学报 ›› 2018, Vol. 44 ›› Issue (7): 977-987.doi: 10.3724/SP.J.1006.2018.00977

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

水稻抗稻瘟病基因Pi47的精细定位和候选基因分析

肖湘谊,史学涛,盛浩闻,刘金灵(),肖应辉()

湖南农业大学农学院 / 水稻油菜抗病育种湖南省重点实验室 / 南方粮油作物协同创新中心, 湖南长沙 410128

收稿日期:2017-11-21 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-04-27
通讯作者: 刘金灵,肖应辉
基金资助:
本研究由国家重点研发计划项目(2016YFD0101100), 国家自然科学基金项目(31171834)和湖南省科技重大专项( 2015NK1001-1)资助

Fine Mapping and Candidate Gene Analysis of Rice Blast Resistance Gene Pi47

Xiang-Yi XIAO,Xue-Tao SHI,Hao-Wen SHENG,Jin-Ling LIU(),Ying-Hui XIAO()

Agronomy College of Hunan Agricultural University / Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance / Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China

Received:2017-11-21 Accepted:2018-03-26 Published:2018-07-10 Published online:2018-04-27
Contact: Jin-Ling LIU,Ying-Hui XIAO
Supported by:
This study was supported by the National Key Research and Development Program (2016YFD0101100), the National Natural Science Foundation of China (31171834), and the Science and Technology Major Project of Hunan Province (2015NK1001-2).

摘要/Abstract

摘要：

不断挖掘和克隆抗稻瘟病新基因, 是解析水稻抗病分子遗传机制和培育抗稻瘟病新品种的重要基础。Pi47是笔者从广谱、持久抗稻瘟病湖南地方品种湘资3150中鉴定的稻瘟病抗性基因, 前期研究将其初步定位于第11染色体标记RM224和RM5926间。本研究利用3个Pi47单基因系与感病亲本CO39杂交F₂群体1687个感病单株对Pi47精细定位, 利用6个STS标记对3个单基因系进行背景分析, 采用生物信息学方法进行了候选基因分析。结果表明, Pi47被精细定位于CAPS标记S32与K33间0.24 cM区域的171.2 kb物理区间内, 背景分析将Pi47进一步缩小至SC12和K33间67.8 kb的区间内; 该区间含有8个结构基因, 其中2个编码NBS-LRR抗病类似蛋白, 为Pi47的候选功能基因。稻瘟菌抗谱比较分析发现, Pi47单基因系与其定位区间内4个Pik位点的等位基因Pik、Pikm、Pikh和Pikp的近等基因系抗谱不同。这些结果为进一步克隆Pi47和利用其进行分子标记辅助选择培育抗稻瘟病水稻新品种奠定了基础。

关键词: 水稻, 稻瘟病, 抗病基因, Pi47, 精细定位

Abstract:

Continuous mapping and cloning new blast resistance genes provided an important approach for revealing the molecular mechanism of resistance to rice disease and breeding new varieties resistant to rice blast. Previously, the blast resistance gene Pi47 was mapped between SSR markers RM224 and RM2956 on chromosome 11, from a broad-spectrum and durable resistance native cultivar Xiangzi 3150 in Hunan province. In this study, the fine mapping and candidate gene prediction were performed, showing that Pi47 was mapped in an interval of 0.24 cM between CAPS markers S32 and K33 with 171.2 kb on Nipponbare reference genome, and further narrowed in an interval of 67.8 kb between markers SC12 and K33, through background screening to Pi47 monogenic lines and susceptible parent CO39 with six STS markers. Eight genes were predicted in this region on the reference genome, among them two encoded NBS-LRR resistance-like proteins, which probably were Pi47 functional candidate genes. A blast resistance spectrum evaluation, using Pi47 monogenic lines and four near-isogenic lines with the Pik allelic gene Pik, Pikm, Pikh, and Pikp across Pi47 region, revealed that Pi47 shared different resistance spectra with these four alleles. These results shed light on further molecular cloning of Pi47, and the molecular marker will be useful for molecular maker assisted selection to breed new resistant cultivars.

Key words: rice, rice blast, resistance gene, Pi47, fine mapping

肖湘谊,史学涛,盛浩闻,刘金灵,肖应辉. 水稻抗稻瘟病基因Pi47的精细定位和候选基因分析[J]. 作物学报, 2018, 44(7): 977-987.

Xiang-Yi XIAO,Xue-Tao SHI,Hao-Wen SHENG,Jin-Ling LIU,Ying-Hui XIAO. Fine Mapping and Candidate Gene Analysis of Rice Blast Resistance Gene Pi47[J]. Acta Agronomica Sinica, 2018, 44(7): 977-987.

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株系 Line	接种菌株193-1-1的抗性表型 Phenotype inoculated with isolate 193-1-1	Pi47连锁标记 Markers linked to Pi47			Pi48连锁标记 Markers linked to Pi48
株系 Line	接种菌株193-1-1的抗性表型 Phenotype inoculated with isolate 193-1-1	RM206	RM224	RM5926	RM3102	RM1337	RM7102
CX18	R	+	+	+	-	-	-
CX36	R	+	+	+	-	-	-
CX40	R	+	+	+	-	-	-
CX52	R	+	+	+	-	-	-
CX66	R	+	+	+	-	-	-
CX67	R	+	+	+	-	-	-
CX70	R	+	+	+	-	-	-
CX147	R	+	+	+	-	-	-
CX152	R	+	+	+	-	-	-
CX155	R	+	+	+	-	-	-
CX166	R	+	+	+	-	-	-
CX279	R	+	+	+	-	-	-

F₂群体 F₂ population	抗病单株 Resistant individuals	感病单株 Susceptible individuals	作图群体株数 Individual number of mapping population	卡方值 χ²for 3:1 ratio
CX18/CO39 F₂	1040	329	308	0.68
CX52/CO39 F₂	1116	340	247	2.21
CX155/CO39 F₂	1512	310	0	62.0
CX279/CO39 F₂	3820	1221	1132	1.66
总数 Total	/	/	1687	/

群体 Population	感病单株数目 No. of susceptible individuals	重组子分布 Distribution of recombinants
群体 Population	感病单株数目 No. of susceptible individuals	RM224	S11, S4, S32	S10	K33	K107	K134	RM5926
CX279/CO39F₂	1132	20	4	0	1	1	8	56
CX18/CO39F₂	308	5	2	0	0	0	1	8
CX52/CO39F₂	247	2	1	0	0	0	1	11
合计 Total	1687	27	7	0	1	1	10	75

标记 Marker	引物顺序 Primer sequence (5°-3°)	物理位置 Physical position	扩增大小 Expect size	酶 Enzyme	标记类型 Marker type
RM224	F: TCGATCGATCTTCACGAGG R: TGCTATAAAAGGCATTCGGG	27673251 -27673372	122	/	SSR
S11	F: TCTTTGAACAAGAGGACCAG R: AATTACCGAATTACTTAGCG	27824419 -27825439	1021	Mva I or Hinf I	CAPS
S4	F: CGAATGATGACGAGGACCCG R: ATCAGGCACGGCAGCAGAAG	27827786 -27828843	1058	Hinf I or Rsa I	CAPS
S32	F: GCCCAGTAACCGTAGGAGTC R: TTTGATGAGCGGCAAAGTAG	27835604 -27836295	692	Msp I	CAPS
S10	F: AGAGCAAGAAGGGCACGGTAT R: AGAATGGTCCTCCTGACATCG	27992866 -27993970	1105	Rsa I	CAPS
K33	F: TTTGGTGCTCCTCTTACGGG R: TCGGAGTTCACAGAGCCAAG	28005690 -28006842	1153	Hinf I	CAPS
K107	F: ACATCAATGGCTACAACT R: TGCTAACGGTGCTGGTAT	28012753 -28012940	184/188	/	STS
K134	F: GATGGCGAGATGGTTGTC R: GCCTTTGAGATAGGGATTGC	28163563 -28163742	180/168	/	STS
RM5926	F: TAGGTCCATCCAAATCTCGATCC R: TGGCAGAGGAGATTAGAGTAATACGG	28957564 -28957858	295	/	SSR
S6	F: TCAACCGAACGCAAGAATCAC R: GGCCCTCCAGCTCATCTACCT	27841529 -27842431	903	/	STS
SC1	F: CCCCACAATCCCACGAGAC R: CGGGCCGACAATCCAATAC	27848711 -27849635	925	/	STS
SC3	F: TTTCCCACCGATGACCCAG R: GCTCGCCATCCTTCCCTTT	27879005 -27879906	902	/	STS
SC6	F: TATTGGTAAGTGGTAACGGGTGA R: GCAGATTCGAGGAGGAAGG	27891111 -27892550	1440	/	STS
SC7	F: TGGCGTGGCGGTTTGGTAG R: GCTGGTTGGGCATGGGTTG	27909896 -27910602	707	/	STS
SC12	F: GGGCAACAAGCGAGCCATAA R: GTGGTGAGGCAGCGGAACAG	27939030 -27940473	1444	/	STS

基因名称 Gene name	推测功能 Putative functions
LOC_Os11g46150	Expressed protein
LOC_Os11g46180	Transposon protein
LOC_Os11g46190	Transposon protein
LOC_Os11g46200	Leucine rich repeat family protein, expressed
LOC_Os11g46210	NB-ARC domain containing protein, expressed
LOC_Os11g46220	Hypothetical protein
LOC_Os11g46230	Tetratricopeptide repeat domain containing protein
LOC_Os11g46240	Retrotransposon protein

水稻抗稻瘟病基因Pi47的精细定位和候选基因分析

Fine Mapping and Candidate Gene Analysis of Rice Blast Resistance Gene Pi47

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基因 Gene	Pik1-KA	Pi7-1	Piks-1	Pik-2	Pikp-1	Pikm1-TS	Pi1-5
Pi47-1 DNA	0.997	0.975	0.998	0.997	0.975	0.998	0.997
Pi47-1蛋白 Pi47-1 protein	0.987	0.947	0.993	0.989	0.946	0.994	0.995

基因 Gene	Pik2-KA	Pi7-2	Piks-2	Pik-1	Pikp-2	Pikm2-TS	Pi1-6
Pi47-2 DNA	0.999	0.998	0.999	0.999	0.998	0.999	1.000
Pi47-2蛋白 Pi47-2 protein	0.999	0.996	0.999	0.999	0.996	0.999	1.000

稻瘟菌小种 Isolate	来源 Origin	水稻品种 Rice cultivar
稻瘟菌小种 Isolate	来源 Origin	CO39	CX18 (Pi47)	IRBLk-Ka (Pik)	IRBLkm-Ts (Pikm)	IRBLkh-K3 (Pikh)	IRBLkp-K60 (Pikp)
318-2	中国湖南 Hunan, China	S	R	R	R	R	R
CHL473	中国湖南 Hunan, China	S	R	R	R	R	R
X2007A-3	中国湖南 Hunan, China	S	R	R	R	R	S
CHL440	中国湖南 Hunan, China	S	R	R	R	R	S
CHL438	中国湖南 Hunan, China	S	R	R	R	R	R
X2007A-7	中国湖南 Hunan, China	S	R	R	R	R	R
195-2-2	中国湖南 Hunan, China	S	S	R	R	R	S
87-4	中国湖南 Hunan, China	S	R	R	R	R	R
193-1-1	中国湖南 Hunan, China	S	R	R	R	R	R
110-2	中国湖南 Hunan, China	S	R	R	R	R	R
236-1	中国湖南 Hunan, China	S	R	R	R	R	R
RB4	中国广东 Guangdong, China	S	R	R	R	R	S
RB6	中国广东 Guangdong, China	S	R	R	R	R	S
RB18	中国福建 Fujian, China	S	R	S	R	R	S
RB19	中国福建 Fujian, China	S	R	R	R	R	R
RB12	中国 China	MR	MR	MR	MR	MR	MR
KOH	日本 Japan	S	S	S	S	S	S

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