水稻Pi9位点6个稻瘟病抗性基因特异分子标记的开发及应用

doi:10.3864/j.issn.0578-1752.2023.21.007

中国农业科学 ›› 2023, Vol. 56 ›› Issue (21): 4219-4233.doi: 10.3864/j.issn.0578-1752.2023.21.007

水稻Pi9位点6个稻瘟病抗性基因特异分子标记的开发及应用

杨好¹(), 黄衍焱¹(), 易春霖¹, 石军², 谭楮湉¹, 任文芮¹, 王文明¹()

¹ 四川农业大学西南作物基因资源发掘与利用国家重点实验室，成都 611130
² 绵阳市农业科学研究院水稻研究所/厅市共建作物特色资源创制及应用四川省重点实验室，四川绵阳 621023

收稿日期:2023-07-23 接受日期:2023-09-06 出版日期:2023-11-01 发布日期:2023-11-06
通信作者:
王文明，E-mail：j316wenmingwang@163.com
联系方式: 杨好，E-mail：2365657091@qq.com。黄衍焱，E-mail：h1985yy@163.com。杨好和黄衍焱为同等贡献作者。
基金资助:
国家自然科学基金(U19A2033); 国家自然科学基金(31901839); 四川省青年科技创新研究团队(2022JDTD0023)

Development and Application of Specific Molecular Markers for Six Homologous Rice Blast Resistance Genes in Pi9 Locus of Rice

YANG Hao¹(), HUANG YanYan¹(), YI ChunLin¹, SHI Jun², TAN ChuTian¹, REN WenRui¹, WANG WenMing¹()

¹ State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu 611130
² Institute of Rice Research, Mianyang Academy of Agricultural Sciences/Crop Characteristic Resources Creation and Utilization Key Laboratory of Sichuan Province, Mianyang 621023, Sichuan

Received:2023-07-23 Accepted:2023-09-06 Published:2023-11-01 Online:2023-11-06

摘要/Abstract

摘要：

【目的】水稻Pi9位点由多个串联的同源NLR基因组成，从中已克隆了超过10个优良的稻瘟病抗性基因。论文旨在鉴别水稻亲本Pi9位点抗性基因的组成，促进该位点基因快速、精准地应用于水稻抗性育种。【方法】对比Pi9位点已克隆抗性基因的序列，从中发掘各基因特异的核苷酸位点；然后将各目标基因分别与数据库（Rice Resource Center）中的155个水稻基因组进行比对分析，进一步从中筛选出特异最强的核苷酸位点，用于Pi2、Piz-t、Pi9、Pi9-type5、PigmR和Pid4 6个抗性基因特异分子标记的开发；以24个抗稻瘟病单基因系、阳性对照和110个四川盆地水稻亲本为鉴定对象，通过优化PCR扩增条件、测序或基因组数据分析检验分子标记鉴定结果的准确性。由于该位点基因的同源性较高、基因间常常拥有一些相同的特异位点，导致许多抗性基因很难用一对分子标记进行精准鉴定，因此采用多对分子标记共同鉴定的方式；另外许多特异位点为单碱基差异，因此需要对差异位点旁的碱基进行特异突变，使引物的3′端具有两个碱基的错配，以提高PCR扩增的特异性。【结果】最终为6个Pi9位点的抗性基因开发了有效的分子标记，发现32.09%的参试水稻材料含有Pi9位点抗性基因。Pi9、Pid4、PigmR、Piz-t、Pi2和Pi9-type5分别存在于1、7、8、14、23和33个水稻材料中，其中Pi9仅存在于单基因系中。这些抗性基因通常两个或多个组合在一起，同时存在于一个水稻材料中。其中Pi9-type5往往与Pi2或Piz-t成对出现，仅在成恢993、HR2168和绵恢365 3个亲本中单独存在。雨恢38含有的Pi9位点抗性基因最多，分别有Pi2、Pi9-type5、PigmR和Pid4。川谷B、川农4B、内香6B和双1B中均同时含有Piz-t、PigmR和Pid4 3个抗性基因。千乡654B中含有Piz-t和Pid4两个抗性基因。【结论】为Pi9位点的6个同源稻瘟病抗性基因开发了特异的分子标记，明确了四川盆地110个水稻亲本Pi9位点的抗性基因组成，揭示了Pi9位点抗性基因丰富的组合形式，为水稻育种的抗原选择提供了明确参考。

关键词: 水稻, 稻瘟病, Pi9位点, 同源基因, 分子标记

Abstract:

【Objective】The Pi9 resistance gene locus, conferring a broad-spectrum resistance against Magnaporthe oryzae, is consist by several tandem homologous genes. Over 10 resistance genes have been cloned from this gene locus. This study aims to clarify the R gene composition at Pi9 locus in rice resource materials and promote the application of those genes in rice resistance breeding.【Method】Comparing the DNA sequence of cloned R genes at Pi9 locus, the specific nucleotide polymorphism sites were screened as the candidate sites. Subsequently, each R gene was blasted with 155 rice genomes in the database of Rice Resource Center. The most specific nucleotide polymorphism sites were picked out from the candidate site in each gene to develop primer pair of molecular markers. The PCR product of primer pairs was used to mark indicated R gene in tested rice materials via parameter optimization. To verify the results, the R genes were cloned from indicated rice variety randomly and examined by Sanger sequencing, or analyzed the R genes from the genome database if the genome sequence of indicated rice variety exists in Rice Resource Center. The R genes in Pi9 locus have high homology, which cause same specific nucleotide polymorphism sites existing in different R genes. Therefore, some R genes are hardly identified by one molecular marker. For this case, several molecular markers were employed to identify the indicated R gene simultaneously. Moreover, some specific nucleotide polymorphism sites are single nucleotide polymorphism (SNP), in where the primers of molecular markers have a mismatched base. In order to improve the specificity of PCR amplification, the adjacent base of SNP was mutated to generate two mismatched bases at 3′ site of primer.【Result】Finally, the valid molecular markers were developed for each R gene and identified 32.09% tested materials containing R genes at Pi9 locus. Pi9, Pid4, PigmR, Piz-t, Pi2 and Pi9-type5 are present in 1, 7, 8, 14, 23 and 33 tested materials, respectively. The Pi9 only presents in monogenic line but not in rice parent lines. The other genes are usually present in two or more gene combinations in rice parent lines. The Pi9-type5 often presents in pair with Pi2 and Piz-t, and presents alone in three rice parents, Chenghui 993, HR2168 and Mianhui 365. Yuhui 38 contains the most R genes at Pi9 locus, including Pi2, Pi9-type5, PigmR and Pid4. Chuangu B, Chuannong 4B, Neixiang 6B and Shuang 1B contain Piz-t, PigmR and Pid4. Qianxiang 654B contains Piz-t and Pid4.【Conclusion】This study successfully developed specific molecular markers for six homologous rice blast resistance genes in Pi9 locus and identified the R gene composition in Pi9 locus for 110 rice parent lines that used in rice breeding in Sichuan basin. It also discovered different types of R genes combination at Pi9 locus and provided a clear reference for choosing the resistance source in rice breeding.

Key words: rice, rice blast, Pi9 locus, homologous gene, molecular marker

杨好, 黄衍焱, 易春霖, 石军, 谭楮湉, 任文芮, 王文明. 水稻Pi9位点6个稻瘟病抗性基因特异分子标记的开发及应用[J]. 中国农业科学, 2023, 56(21): 4219-4233.

YANG Hao, HUANG YanYan, YI ChunLin, SHI Jun, TAN ChuTian, REN WenRui, WANG WenMing. Development and Application of Specific Molecular Markers for Six Homologous Rice Blast Resistance Genes in Pi9 Locus of Rice[J]. Scientia Agricultura Sinica, 2023, 56(21): 4219-4233.

图/表 12

表1

供试水稻品种"

编号 No.	品种 Variety	编号 No.	品种 Variety	编号 No.	品种 Variety	编号 No.	品种 Variety
1	IRBLa-A (Pi-a)	35	绵恢146 Mianhui 146	69	泰恢808 Taihui 808	103	德香074B Dexiang 074B
2	IRBLi-F5 (Pi-i)	36	绵恢725 Mianhui 725	70	五山丝苗Wushansimiao	104	赣香B Ganxiang B
3	IRBLks-F5 (Pi-ks)	37	南恢558 Nanhui 558	71	西科恢768 Xikehui 768	105	广8B Guang 8B
4	IRBLk-Ka (Pi-k)	38	南恢968 Nanhui 968	72	雅禾Yahe	106	泸香618B Luxiang 618B
5	IRBLkp-K60 (Pi-kp)	39	蜀恢257 Shuhui 257	73	雅恢2115 Yahui 2115	107	绵5B Mian 5B
6	IRBLkh-K3 (Pi-kh)	40	蜀恢308 Shuhui 308	74	雅恢2116 Yahui 2116	108	忠香B Zhongxiang B
7	IRBLz-Fu (Pi-z)	41	蜀恢451 Shuhui 451	75	雅恢2117 Yahui 2117	109	内香6B Neixiang 6B
8	IRBLz5-CA (Pi-z5)	42	蜀恢498 Shuhui 498	76	雅恢2118 Yahui 2118	110	千乡654B Qianxiang 654B
9	IRBLzt-T (Pi-zt)	43	蜀恢505 Shuhui 505	77	雅恢2119 Yahui 2119	111	荃9311B Quan 9311B
10	IRBLta-K1 (Pi-ta)	44	蜀恢548 Shuhui 548	78	雅恢2199 Yahui 2199	112	蓉18B Rong 18B
11	IRBLb-B (Pi-b)	45	蜀恢573 Shuhui 573	79	雅恢2275 Yahui 2275	113	蓉7B Rong 7B
12	IRBLt-K59 (Pi-t)	46	蜀恢586 Shuhui 586	80	雅恢2348 Yahui 2348	114	双1B Shuang 1B
13	IRBLsh-B (Pi-sh)	47	蜀恢589 Shuhui 589	81	雅恢2816 Yahui 2816	115	泰丰B Taifeng B
14	IRBL1-CL (Pi-1)	48	猪毛糯Zhumaonuo	82	雅恢2918 Yahui 2918	116	武金4B Wujin 4B
15	IRBL3-CP4 (Pi-3)	49	HR2168	83	雅恢5049 Yahui 5049	117	武香B Wuxiang B
16	IRBL5-M (Pi-5)	50	R900	84	雅恢5217 Yahui 5217	118	雅1B Ya 1B
17	IRBL7-M (Pi-7)	51	R9311	85	雨恢38 Yuhui 38	119	雅3B Ya 3B
18	IRBL9-W (Pi-9)	52	成恢19 Chenghui 19	86	岳恢9113 Yuehui 9113	120	雅7B Ya 7B
19	IRBL12-M (Pi-12)	53	乐恢188 Lehui 188	87	粤农丝苗Yuenongsimiao	121	宜香1B Yixiang 1B
20	IRBL19-M (Pi-19)	54	乐恢584 Lehui 584	88	D297B	122	泰国香稻Thai fragrant rice
21	IRBLkm-Ts (Pi-km)	55	泸恢17 Luhui 17	89	D35B	123	楚粳28 Chugeng 28
22	IRBL20-IR24 (Pi-20)	56	泸恢8258 Luhui 8258	90	D62B	124	野香B Yexiang B
23	IRBLta2-Pi (Pi-ta2)	57	绵恢523 Mianhui 523	91	D79B	125	绵恢6139 Mianhui 6139
24	IRBL11-Zh (Pi-11)	58	绵恢528 Mianhui 528	92	F-32B	126	绵恢313 Mianhui 313
25	E33	59	绵恢662 Mianhui 662	93	F59B	127	绵恢357 Mianhui 357
26	成恢3203 Chenghui 3203	60	明恢63 Minghui 63	94	G46B	128	绵恢365 Mianhui 365
27	成恢727 Chenghui 727	61	明恢86 Minghui 86	95	川106B Chuan 106B	129	绵恢821 Mianhui 821
28	成恢993 Chenghui 993	62	内恢99-14 Neihui 99-14	96	川608B Chuan 608B	130	绵恢823 Mianhui 823
29	德恢6099 Dehui 6099	63	蜀恢137 Shuhui 137	97	川谷B Chuangu B	131	绵恢837 Mianhui 837
30	多恢1号 Duohui 1	64	蜀恢208 Shuhui 208	98	川华B Chuanhua B	132	绵恢857 Mianhui 857
31	航恢1179 Hanghui 1179	65	蜀恢527 Shuhui 527	99	川农1B Chuannong 1B	133	绵恢815 Mianhui 815
32	航恢570 Hanghui 570	66	蜀恢538 Shuhui 538	100	川农4B Chuannong 4B	134	地谷B Digu B
33	华占Huazhan	67	蜀恢569 Shuhui 569	101	川种3B Chuanzhong 3B
34	乐恢433 Lehui 433	68	蜀恢882 Shuhui 882	102	德66B De 66B

表1

表2

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表7

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编号 No.	材料 Material	引物Primer
编号 No.	材料 Material	Pi2/z-t-F Piz-t-R2	Pi2/z-t-F Piz-t-R3	Piz-t-F4 Piz-t-R4
9	IRBLzt-T	√	√	√
42	蜀恢498 Shuhui 498	√	√	√
52	成恢19 Chenghui 19	√	√	√
56	泸恢8258 Luhui 8258	√	√	√
61	明恢86 Minghui 86	√	√	√
63	蜀恢137 Shuhui 137	√	√	√
64	蜀恢208 Shuhui 208	√	√	√
78	雅恢2199 Yahui 2199	√	√	√
97	川谷B Chuangu B	√	√	√
100	川农4B Chuannong 4B	√	√	√
109	内香6B Neixiang 6B	√	√	√
110	千乡654B Qianxiang 654B	√	√	√
113	蓉7B Rong 7B	√	√	√
114	双1B Shuang 1B	√	√	√
阳性对照Positive control	中花11 ZH11	√	√	√

编号 No.	材料 Material	引物Primer
编号 No.	材料 Material	Pi9-type5-F1 Pi9-type5-R1	Pi9-type5-F2 Pi9-type5-R2	Pi9-type5-F3 Pi9-type5-mR
8	IRBLz5-CA	√	√	√
9	IRBLzt-T	√	√	√
25	E33	√	√	√
28	成恢993 Chenghui 993	√	√	√
29	德恢6099 Dehui 6099	√	√	√
31	航恢1179 Hanghui 1179	√	√	√
33	华占Huazhan	√	√	√
40	蜀恢308 Shuhui 308	√	√	√
42	蜀恢498 Shuhui 498	√	√	√
43	蜀恢505 Shuhui 505	√	√	√
44	蜀恢548 Shuhui 548	√	√	√
46	蜀恢586 Shuhui 586	√	√	√
50	R900	√	√	√
52	成恢19 Chenghui 19	√	√	√
56	泸恢8258 Luhui 8258	√	√	√
61	明恢86 Minghui 86	√	√	√
63	蜀恢137 Shuhui 137	√	√	√
64	蜀恢208 Shuhui 208	√	√	√
70	五山丝苗Wushansimiao	√	√	√
72	雅禾Yahe	√	√	√
73	雅恢2115 Yahui 2115	√	√	√
74	雅恢2116 Yahui 2116	√	√	√
77	雅恢2119 Yahui 2119	√	√	√
78	雅恢2199 Yahui 2199	√	√	√
79	雅恢2275 Yahui 2275	√	√	√
82	雅恢2918 Yahui 2918	√	√	√
83	雅恢5049 Yahui 5049	√	√	√
84	雅恢5217 Yahui 5217	√	√	√
85	雨恢38 Yuhui 38	√	√	√
87	粤农丝苗Yuenongsimiao	√	√	√
118	雅1B Ya 1B	√	√	√
128	绵恢365 Mianhui 365	√	√	√
133	绵恢815 Mianhui 815	√	√	√

编号 No.	材料 Material	引物Primer
编号 No.	材料 Material	Pigm-F2+ Pigm-newmR1	Pigm-newmF1+ Pigm-newmR1	Pigm-mF4+ Pigm-mR5	Pigm-mF6+ Pigm-mR7
85	雨恢38 Yuhui 38	√	√	√	√
97	川谷B Chuangu B	√	√	√	√
100	川农4B Chuannong 4B	√	√	√	√
109	内香6B Neixiang 6B	√	√	√	√
112	蓉18B Rong 18 B	√	√	√	√
113	蓉7B Rong 7B	√	√	√	√
114	双1B Shuang 1B	√	√	√	√
134	地谷B Digu B	√	√	√	√
阳性对照Positive control	谷梅4号Gumei 4	√	√	√	√

编号 No.	材料 Material	抗性基因Resistance gene
编号 No.	材料 Material	Pi2	Piz-t	Pi9	Pi9-type5	PigmR	Pid4
18	IRBL9-W			√
8	IRBLz5-CA	√			√
25	E33	√			√
29	德恢6099 Dehui 6099	√			√
31	航恢1179 Hanghui 1179	√			√
33	华占Huazhan	√			√
40	蜀恢308 Shuhui 308	√			√
43	蜀恢505 Shuhui 505	√			√
44	蜀恢548 Shuhui 548	√			√
46	蜀恢586 Shuhui 586	√			√
50	R900	√			√
70	五山丝苗Wushansimiao	√			√
72	雅禾Yahe	√			√
73	雅恢2115 Yahui 2115	√			√
74	雅恢2116 Yahui 2116	√			√
77	雅恢2119 Yahui 2119	√			√
79	雅恢2275 Yahui 2275	√			√
82	雅恢2918 Yahui 2918	√			√
83	雅恢5049 Yahui 5049	√			√
84	雅恢5217 Yahui 5217	√			√
85	雨恢38 Yuhui 38	√			√	√	√
87	粤农丝苗Yuenongsimiao	√			√
118	雅1B Ya 1B	√			√
133	绵恢815 Mianhui 815	√			√
9	IRBLzt-T		√		√
42	蜀恢498 Shuhui 498		√		√
52	成恢19 Chenghui 19		√		√
56	泸恢8258 Luhui 8258		√		√
61	明恢86 Minghui 86		√		√
63	蜀恢137 Shuhui 137		√		√
64	蜀恢208 Shuhui 208		√		√
78	雅恢2199 Yahui 2199		√		√
97	川谷B Chuangu B		√			√	√
100	川农4B Chuannong 4B		√			√	√
109	内香6B Neixiang 6B		√			√	√
113	蓉7B Rong 7B		√			√	√
114	双1B Shuang 1B		√			√	√
110	千乡654B Qianxiang 654B		√				√
28	成恢993 Chenghui 993				√
49	HR2168				√
128	绵恢365 Mianhui 365				√
134	地谷B Digu B					√	√
112	蓉18B Rong 18B					√

水稻Pi9位点6个稻瘟病抗性基因特异分子标记的开发及应用

Development and Application of Specific Molecular Markers for Six Homologous Rice Blast Resistance Genes in Pi9 Locus of Rice

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R基因 R gene	引物名称 Primer name	引物序列 Primer sequence	产物长度 Product length (bp)	退火温度 Annealing temperature (℃)	延伸时间 Extension time (s)
Pi2	Pi2/z-t-F	CTCTTCGTTGTATAGGACAGTTTCA	424	57	30
Pi2	Pi2-R	GTTCCACCATCTGAGATTCCTG	424	57	30
Pi9	Pi9-F	CTCTTCGTTGTAGTAGAAAGTTTGT	788	45	50
Pi9	Pi9-R	TTCCAACCTGCAGCAAGAG	788	45	50
Pi9-type5	Pi9-type5F1	CTAAGAACAATAAGAAGGGCAG	454	62	30
	Pi9-type5R1	CCTACTAGACGATTCCTTTGT	454	62	30
	Pi9-type5F2	CGTTGTATAAGAGAGCTTGAATT	709	60	50
	Pi9-type5R2	GCTGATCCAAATTGAAAATCAC	709	60	50
	Pi9-type5F3	ATCTTTCCTGAGGATTTTGAAATAC	185	55	25
	Pi9-type5mR	CAAGTCTTAATTTTTCCTGCAG	185	55	25
Piz-t	Pi2/z-t-F	CTCTTCGTTGTATAGGACAGTTTCA	548	57	35
	Piz-t-R3	GTAGATCTTCTTCAGGTGCA	548	57	35
	Pi2/z-t-F	CTCTTCGTTGTATAGGACAGTTTCA	502	59	30
	Piz-t-R2	TCCTCAAGAATTCCATCCAACAC	502	59	30
	Piz-t-F4	CAGAGAACGCCACCGAATT	169	59	20
	Piz-t-R4	CACATTGCGAGCTGACTGATTA	169	59	20
Pi9-type9	Pi9-type9F2	AGAATGGGAGAAATTCTATGAAAAG	230	59	25
	Pi9-type9R2	TAGTCATCATCCCAACCTGC	230	59	25
	Pi9-type9F	ACCTGAAGAAGATCTACTTATTGAA	305	59	25
	Pi9-type9R	AATCTCCTTGAGCTTTGGAT	305	59	25
	Pi9-type9mF	ACCTGAAGAAGATCTACTTATTGTA	305	59	25
	Pi9-type9mR	AATCTCCTTGAGCTTTGGTT	305	59	25
PigmR	Pigm-F2	CACCTGAAGAAGTTCTACTTACG	111	59	20
	Pigm-newmR1	ATAAGAATTATGATAAAGAGAAAGGTA	111	59	20
	Pigm-newmF1	TCCTCCTCCCCTACTTAGT	180	59	20
	Pigm-newmR1	ATAAGAATTATGATAAAGAGAAAGGTA	180	59	20
	Pigm-mF4	GGAGAAGCTAGTATTCAAAAGG	297	59	20
	Pigm-mR5	TGATACCTTCGGTCACTGAC	297	59	20
	Pigm-mF6	GTATATGGATAGCAGAAGGGTAG	98	59	20
	Pigm-mR7	CATACTTCGGTTGATTAGCTCAAA	98	59	20
Pid4	Pid4-mF	GAATCTGTTTTTCCTGACTCTACA	797	59	50
Pid4	Pid4-mR	GGATTGTAAAAGAAACTTAATGAGT	797	59	50

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