广谱抗稻瘟病种质75-1-127的褐飞虱抗性基因鉴定及分子标记辅助选择育种

doi:10.16819/j.1001-7216.2019.9005

中国水稻科学 ›› 2019, Vol. 33 ›› Issue (3): 227-234.DOI: 10.16819/j.1001-7216.2019.9005

广谱抗稻瘟病种质75-1-127的褐飞虱抗性基因鉴定及分子标记辅助选择育种

降好宇, 曾盖, 郝明, 黄湘桂, 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, 长沙 410128;

收稿日期:2019-01-08 修回日期:2019-02-18 出版日期:2019-05-10 发布日期:2019-05-10
通讯作者: 肖应辉
作者简介:
^#共同第一作者;
基金资助:
国家重点研发计划资助项目(2016YFD0101100);湖南省科技重大专项(2015NK1001-1);湖南省研究生科研创新项目(CX2015B255)

Identification of Brown Planthopper Resistance Genes in Broad-spectrum Blast Resistant Rice Germplasm 75-1-127 and Its Molecular Marker-Assisted Selection Breeding

Haoyu JIANG, Gai ZENG, Ming HAO, Xianggui HUANG, Yinghui XIAO^*()

Agronomy College, 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, China;

Received:2019-01-08 Revised:2019-02-18 Online:2019-05-10 Published:2019-05-10
Contact: Yinghui XIAO
About author:
^#These authors contributed equally to this work;

摘要/Abstract

摘要：

【目的】水稻品系75-1-127携带广谱抗稻瘟病基因Pi9,已被广泛应用于抗稻瘟病水稻品种改良。笔者育种实践发现75-1-127表现出较强的褐飞虱抗性,因此鉴定该品系中的褐飞虱抗性基因并进行分子辅助选择育种。【方法】根据水稻品系B5中褐飞虱抗性基因Bph14和Bph15的序列,设计引物扩增75-1-127的基因组DNA,并对PCR产物进行测序分析。采用苗期集团法鉴定了75-1-127和B5的褐飞虱抗性表型。利用与Bph14与Bph15连锁的分子标记筛查了75-1-127为稻瘟病抗源回交转育的两系不育系后代,并鉴定了这些后代的稻瘟病抗性、褐飞虱抗性和主要农艺性状。【结果】75-1-127中含有与B5完全一致的Bph14和Bph15序列。75-1-127和B5苗期褐飞虱抗性均为1级。在以75-1-127为抗源改良的两系不育系中,携带Bph14、Bph15的单基因系或双基因系的褐飞虱抗性均得以改良,其中双基因聚合系的死苗率为8.5%,与供体亲本75-1-127以及阳性对照B5差异不显著,进一步证实75-1-127含有褐飞虱抗性基因。【结论】水稻品系75-1-127携带褐飞虱抗性基因Bph14和Bph15,可以作为抗源应用于水稻褐飞虱抗性育种。

关键词: 水稻, 褐飞虱抗性, Bph14, Bph15, 分子标记辅助育种

Abstract:

【Objective】Rice line, 75-1-127, which harbors the broad-spectrum blast resistance gene Pi9, has been widely used in rice variety improvement for blast resistance. Given that 75-1-127 shows strong resistance to brown planthoppers in our previous breeding projects, we identified its resistance genes to brown planthoppers and used it in molecular marker-assisted selection breeding. 【Method】The PCR products, which amplified from the genomic DNA of 75-1-127 with the primers designed according to the sequence of Bph14 and Bph15 genes in rice line B5, were sequenced and analyzed. The brown planthopper resistance of 75-1-127 and B5 were also identified with seedling bulk test method. A series of two-line genic male sterile lines derived from backcross with 75-1-127 as a donor parent, were screened with the molecular markers linked to Bph14 and Bph15 genes. The blast resistance, brown planthopper resistance and main agronomic traits of these progenies were also identified and evaluated. 【Result】The sequences of Bph14 and Bph15 in 75-1-127 were identical with those in B5. The resistance scores at seedling stage of 75-1-127 and B5 were both grade 1. In the progenies with 75-1-127 as resistance resource, the resistances to brown planthoppers of those monogenic lines of Bph14 or Bph15 and those Bph14/Bph15 pyramiding lines were all improved. That the seedling mortality of Bph14/Bph15 pyramiding lines was 8.5%, equivalent to the donor parents 75-1-127 and the positive control B5, further confirming that 75-1-127 contained resistance genes to brown planthoppers. 【Conclusion】Rice line 75-1-127, which carries the genes of Bph14 and Bph15, could be used as resistance resource in breeding.

Key words: rice, brown planthopper resistance, Bph14, Bph15, molecular marker-assisted selection breeding

中图分类号:

降好宇, 曾盖, 郝明, 黄湘桂, 肖应辉. 广谱抗稻瘟病种质75-1-127的褐飞虱抗性基因鉴定及分子标记辅助选择育种[J]. 中国水稻科学, 2019, 33(3): 227-234.

Haoyu JIANG, Gai ZENG, Ming HAO, Xianggui HUANG, Yinghui XIAO. Identification of Brown Planthopper Resistance Genes in Broad-spectrum Blast Resistant Rice Germplasm 75-1-127 and Its Molecular Marker-Assisted Selection Breeding[J]. Chinese Journal OF Rice Science, 2019, 33(3): 227-234.

图/表 9

参考文献 35

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目的基因	序列片段	正向引物序列	反向引物序列	PCR产物
Target gene	Sequence fragment	Forward primer sequence	Reverse primer sequence	PCR product / bp
Bph14-1	Frag. 1	AGCTGCTCAGACTCTTGCCTT	TTGCCGCATATATGATGGCA	1384
	Frag. 2	TACACTGCGTACGAAGACCA	AGCTCTTCTCCTTGTACCTCA	1422
	Frag. 3	TTGCCTGGAAGGACTGGGTA	ATCCAGACGTTGCTGTAGGCT	1438
	Frag. 4	TATCAGACATTGCGGGAGCT	AGCATAAGGATAGTGGCTAGA	1046
Bph14-2	Frag. 1	TCATTCCAGGCAGCAACTCT	TCGATCTCATGATCCTTGGGA	1395
	Frag. 2	ACGCTATATTGAGCAGAAGCA	AGGCAGTTAGCCTAGGACAA	1431
	Frag. 3	TGCAGACTGAAGAAGCTTACCT	TCTCGATGTCATCCAGACGT	1384
	Frag. 4	TGCCCAGGCCTGGTATCCTT	ACAACTCTCATTTGGTCCTG	908
OsLecRK1		GGCCTTGTTGAGTCGTATCCA	ACAATGGTTGCTCTGCTACTC	3729
OsLecRK2		GATCATGTGGTTGTAGAGGAG	AACCGGTTGCCTCGCAGAGA	2861
OsLecRK3		TAGGTAAGCATCAGTGCCCGA	AGCAATGGCTCATCTCCTGT	2543

目的基因	序列片段	正向引物序列	反向引物序列	PCR产物
Target gene	Sequence fragment	Forward primer sequence	Reverse primer sequence	PCR product / bp
Bph14-1	Frag. 1	AGCTGCTCAGACTCTTGCCTT	TTGCCGCATATATGATGGCA	1384
	Frag. 2	TACACTGCGTACGAAGACCA	AGCTCTTCTCCTTGTACCTCA	1422
	Frag. 3	TTGCCTGGAAGGACTGGGTA	ATCCAGACGTTGCTGTAGGCT	1438
	Frag. 4	TATCAGACATTGCGGGAGCT	AGCATAAGGATAGTGGCTAGA	1046
Bph14-2	Frag. 1	TCATTCCAGGCAGCAACTCT	TCGATCTCATGATCCTTGGGA	1395
	Frag. 2	ACGCTATATTGAGCAGAAGCA	AGGCAGTTAGCCTAGGACAA	1431
	Frag. 3	TGCAGACTGAAGAAGCTTACCT	TCTCGATGTCATCCAGACGT	1384
	Frag. 4	TGCCCAGGCCTGGTATCCTT	ACAACTCTCATTTGGTCCTG	908
OsLecRK1		GGCCTTGTTGAGTCGTATCCA	ACAATGGTTGCTCTGCTACTC	3729
OsLecRK2		GATCATGTGGTTGTAGAGGAG	AACCGGTTGCCTCGCAGAGA	2861
OsLecRK3		TAGGTAAGCATCAGTGCCCGA	AGCAATGGCTCATCTCCTGT	2543

引物	目标基因	正向引物序列	反向引物序列
Primer	Target gene	Forward primer sequence	Reverse primer sequence
RM7311	Pi9	AGTGGTCGTTGAACTCGGAG	TCGTGGCGCCTTTAATCTC
RM7178	Pi9	TAACCTTCACAGCGAACGTG	CCGTGAGATGGGCTACCTAC
Feb-76	Bph14	CTGCTGCTGCTCTCGTATTG	CAGGGAAGCTCCAAGAACAG
MS5	Bph15	TTGTGGGTCCTCATCTCCTC	TGACAACTTTGTGCAAGATCAAA

引物	目标基因	正向引物序列	反向引物序列
Primer	Target gene	Forward primer sequence	Reverse primer sequence
RM7311	Pi9	AGTGGTCGTTGAACTCGGAG	TCGTGGCGCCTTTAATCTC
RM7178	Pi9	TAACCTTCACAGCGAACGTG	CCGTGAGATGGGCTACCTAC
Feb-76	Bph14	CTGCTGCTGCTCTCGTATTG	CAGGGAAGCTCCAAGAACAG
MS5	Bph15	TTGTGGGTCCTCATCTCCTC	TGACAACTTTGTGCAAGATCAAA

基因片段	GenBank登录号	序列位置	与B5序列相似度
Gene fragment	GenBank accession No.	Location of sequence/bp	Similarity with B5 sequence/%
Bph14-1	FJ941067.1	3043-7659	100
Bph14-2	FJ941068.1	3753-8387	100
OsLecRK1	KF748957	1-2442	100
OsLecRK2	KF748965	1-2436	100
OsLecRK3	KF748973	1-2436	100

广谱抗稻瘟病种质75-1-127的褐飞虱抗性基因鉴定及分子标记辅助选择育种

Identification of Brown Planthopper Resistance Genes in Broad-spectrum Blast Resistant Rice Germplasm 75-1-127 and Its Molecular Marker-Assisted Selection Breeding

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Metrics

试验材料	株系数	感病级数^#
Experimental material	No. of lines	Disease index^#
CO39	1	8.7±0.6
B191S	1	7.3±0.9
B191S(Pi9)	7	1.6±0.9^**
B191S(Pi9+Bph14)	3	1.3±1.0^**
B191S(Pi9+Bph15)	6	1.6±0.9^**
B191S(Pi9+Bph14+Bph15)	2	1.6±0.9^**

试验材料	Bph基因型	死苗率^#	苗期抗性
Material	Bph genotype	Seedling mortality^#/%	Seedling resistance
S171002	Bph14	41.8±3.4 B b	中抗MR
S171005	Bph14, Bph15	8.5±0.1 C c	高抗HR
S171008	Bph15	11.5±2.6 C c	抗R
B5	Bph14, Bph15	2.8±1.6 C c	高抗HR
75-1-127	Bph14, Bph15	5.0±3.5 C c	高抗HR
B191S		100.0±0.0 A a	感S
TN1		100.0±0.0 A a	感S

试验材料	播始历期	株高	穗长	每穗颖花数	双边柱头外露率	柱头外露
Material	Duration from seeding to heading /d	Plant height	Panicle length /cm	Spikelets per panicle	Percentage of dual exerted stigma/%	Percentage of total exerted stigma/%
		/cm
S171002	90	75.9±3.7^**	24.4±2.9^**	134.5±21.6^**	33.5±1.9^**	70.6±2.5^**
S171005	87	77.1±3.8^**	18.3±0.8^**	122.6±32.7^**	33.2±2.1^**	69.0±2.2^**
S171008	85	72.1±3.3^**	19.0±1.4^**	125.5±14.2^**	34.4±2.3^**	70.2±2.6^**
B191S	81	49.6±3.2	14.4±1.3	74.2±12.6	25.6±1.7	63.3±2.4

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