抗稻瘟病基因Pigm组合标记的开发及应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1228

摘要/Abstract

摘要：

Pigm是水稻抗稻瘟病基因之一,抗性广且持久。针对Pigm基因,已开发多种分子标记,但检测特异性或辨识度尚存不足。通过NCBI对7个Pigm标记进行序列比对分析,包括Pigm-4、T9E4、DG-2、M80410、R060939、GMR-3和T-Pigm,并对40个水稻品种开展检测验证。根据序列分析与检测结果开发了一种组合标记,应用于镇糯19×空育131（Pigm）分子标记辅助选择育种。序列分析结果表明,M80410标记特异性强,但不能区分杂合基因型;Pigm-4、T-Pigm和T9E4等标记能区分杂合基因型,但特异性略有不足。结合水稻品种检测结果,筛选了Pigm-4与M80410作为组合标记,并用于辅助育种,选育了糯稻品系NK29,具有较好稻瘟病抗性。组合标记能快速准确地鉴定Pigm 的纯合显性、纯合隐性和杂合基因型,为Pigm分子育种奠定基础。

关键词: Pigm, 稻瘟病, 糯稻, 组合标记

Abstract:

Pigm is one of the rice blast resistance genes,with broad-spectrum and lasting resistance. A variety of molecular markers have been developed for Pigm gene,but the detection specificity or recognition is still insufficient. Seven Pigm markers were analyzed by NCBI,including Pigm-4,T9E4,DG-2,M80410,R060939,GMR-3 and T-Pigm,and total 40 rice varieties species were detected and verified. According to the sequence analysis and detection results,a combinatorial marker was developed and applied to Zhennuo 19 × Kongyu 131（Pigm）molecular marker-assisted selection. Sequence analysis showed that M80410 marker had strong specificity,but could not distinguish heterozygous genotypes;Pigm-4,T-Pigm and T9E4 etc. markers distinguished heterozygous genotypes,but their specificity was slightly insufficient. Combined with the detection results of rice varieties,Pigm-4 and M80410 were selected as combinatorial marker and used in assisted breeding. The glutinous rice line NK29 was selected,which had good resistance to rice blast. Combinatorial marker can quickly and accurately identify the homozygous dominant,homozygous recessive and heterozygous genotypes of Pigm,which lays a foundation for Pigm molecular breeding.

Key words: Pigm, rice blast, glutinous rice, combinatorial marker

李白, 蔡之军, 王蕾, 陈婕, 曹奎荣, 李军, 种高军. 抗稻瘟病基因Pigm组合标记的开发及应用[J]. 生物技术通报, 2022, 38(7): 153-159.

LI Bai, CAI Zhi-jun, WANG Lei, CHEN Jie, CAO Kui-rong, LI Jun, CHONG Gao-jun. Development and Application of the Combinatorial Marker for the Rice Blast Resistance Gene Pigm[J]. Biotechnology Bulletin, 2022, 38(7): 153-159.

图/表 8

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标记名称 Marker name	正向引物序列 Forward primer sequence（5'-3'）	反向引物序列 Reverse primer sequence（5'-3'）
M80410^[6]	GGATTGTCTTGTCTCTCTCGC	CAGGACTTAGGGTTTCTCTCTTT
Pigm-4^[15]	ATGCTCGATTCGTTACATTT	CGTCCCACACTTTCTTTTT
T9E4^[16]	CAGAGCAGTAACAAACCCTA	TCCGCAAGATCAACATTC
DG-2^[17]	GAGACGCAGGAACAGGGTG	CCGAGTGGCTGAGCAACAA
R060939^[18]	CTCAGCAAACCATGTTCAGT	AAACAGCTCGTGGTTTCGCT
GMR-3^[19]	AGTTCTACTTACGGAGGAGC	AGAATTATGATAAAGAGAAAGGAA
T-Pigm^[20] （ARMS-PCR）	TAAGAATTGAGGTGGTTAGTTGAACGGAGA	TTGCATGGCTCCACTACCCACTATAAG
T-Pigm^[20] （ARMS-PCR）	TGAAAATAAAAATGGTATGATGGTTACG	TAGGGATGAAACGGCTCGAAAACGATCG

标记名称 Marker name	正向引物序列 Forward primer sequence（5'-3'）	反向引物序列 Reverse primer sequence（5'-3'）
M80410^[6]	GGATTGTCTTGTCTCTCTCGC	CAGGACTTAGGGTTTCTCTCTTT
Pigm-4^[15]	ATGCTCGATTCGTTACATTT	CGTCCCACACTTTCTTTTT
T9E4^[16]	CAGAGCAGTAACAAACCCTA	TCCGCAAGATCAACATTC
DG-2^[17]	GAGACGCAGGAACAGGGTG	CCGAGTGGCTGAGCAACAA
R060939^[18]	CTCAGCAAACCATGTTCAGT	AAACAGCTCGTGGTTTCGCT
GMR-3^[19]	AGTTCTACTTACGGAGGAGC	AGAATTATGATAAAGAGAAAGGAA
T-Pigm^[20] （ARMS-PCR）	TAAGAATTGAGGTGGTTAGTTGAACGGAGA	TTGCATGGCTCCACTACCCACTATAAG
T-Pigm^[20] （ARMS-PCR）	TGAAAATAAAAATGGTATGATGGTTACG	TAGGGATGAAACGGCTCGAAAACGATCG

标记名称 Marker name	谷梅4号 Pigm扩增片段 Gumei 4 Pigmamplified fragments			相同或相似扩增片段Identical or similar amplified fragments
标记名称 Marker name	起始 Beginning	终止 Termination	长度 Length/bp	片段名称,NCBI登记号 Fragment name,NCBI accession	起始 Beginning	终止 Termination	长度 Length/bp
Pigm-4	143 136	143 216	81	Pi50,KP985760.1	44 033	44 113	81
				Digu Pid4,MG839283.1	821	901	81
T9E4	115 153	115 945	793	Pi50,KP985760.1	16 082	16 874	793
				Shuhui498,CP018162.1	10 198 813	10 199 605	793
				Zhonghua 11 Pizh,MH807580.1	114 411	115 202	792
				Nipponbare,AP014962.1	10 408 011	10 408 803	793
DG-2	22 033	22 780	748	Pi50,KP985759.1	21 992	22 739	748
				Shuhui498,CP018162.1	10 136 210	10 136 990	781
				Zhonghua 11 Pizh,MH807580.1	73 816	74 596	781
				Pi9,DQ285630.1	66 719	67 468	750
M80410	91 983	92 499	517	Null	-	-	-
R060939	-897 333	-896 515	819	Shuhui498,CP018162.1	9 343 478	9 344 296	819
				Nipponbare,AP014962.1	9 395 525	9 396 343	819
				RP Bio-226,CP012614.1	9 782 855	9 783 673	819
				Zhenshan 97,CP056057.1	9 146 431	9 147 249	819
				Minghui 63,CP054681.1	9 252 055	9 252 873	819
GMR-3	63 184	63 281	98	Pi50,KP985759.1	6 509	6 606	98
				RP Bio-226,CP012614.1	10 611 454	10 611 551	98
				Piz（t）,HE580189.1	2 382	2 479	98
T-Pigm	11 149	11 443	295	Pi50,KP985759.1	11 108	11 402	295

标记名称 Marker name	谷梅4号 Pigm扩增片段 Gumei 4 Pigmamplified fragments			相同或相似扩增片段Identical or similar amplified fragments
标记名称 Marker name	起始 Beginning	终止 Termination	长度 Length/bp	片段名称,NCBI登记号 Fragment name,NCBI accession	起始 Beginning	终止 Termination	长度 Length/bp
Pigm-4	143 136	143 216	81	Pi50,KP985760.1	44 033	44 113	81
				Digu Pid4,MG839283.1	821	901	81
T9E4	115 153	115 945	793	Pi50,KP985760.1	16 082	16 874	793
				Shuhui498,CP018162.1	10 198 813	10 199 605	793
				Zhonghua 11 Pizh,MH807580.1	114 411	115 202	792
				Nipponbare,AP014962.1	10 408 011	10 408 803	793
DG-2	22 033	22 780	748	Pi50,KP985759.1	21 992	22 739	748
				Shuhui498,CP018162.1	10 136 210	10 136 990	781
				Zhonghua 11 Pizh,MH807580.1	73 816	74 596	781
				Pi9,DQ285630.1	66 719	67 468	750
M80410	91 983	92 499	517	Null	-	-	-
R060939	-897 333	-896 515	819	Shuhui498,CP018162.1	9 343 478	9 344 296	819
				Nipponbare,AP014962.1	9 395 525	9 396 343	819
				RP Bio-226,CP012614.1	9 782 855	9 783 673	819
				Zhenshan 97,CP056057.1	9 146 431	9 147 249	819
				Minghui 63,CP054681.1	9 252 055	9 252 873	819
GMR-3	63 184	63 281	98	Pi50,KP985759.1	6 509	6 606	98
				RP Bio-226,CP012614.1	10 611 454	10 611 551	98
				Piz（t）,HE580189.1	2 382	2 479	98
T-Pigm	11 149	11 443	295	Pi50,KP985759.1	11 108	11 402	295

品种 Variety	T9E4	DG-2	Pigm-4	M80410	R060939	GMR-3	T-Pigm
9311	-	-	-	-	+	-	-
IR64	-	-	-	-	-	+	-
常农粳8号 Changnongjing 8	-	-	-	-	+	-	-
谷梅4号 Gumei 4	+	+	+	+	+	+	+
淮稻5号 Huaidao 5	-	-	-	-	+	-	-
黄华占 Huanghuazhan	-	-	-	-	+	-	-
嘉58 Jia 58	+	+	-	-	+	-	-
嘉丰优2号 Jiafengyou 2	-	-	-	-	±	-	-
嘉禾218 Jiahe 218	-	-	-	-	+	-	-
嘉禾优001 Jiaheyou 001	-	-	-	-	±	-	-
嘉花1号 Jiahua 1	+	+	-	-	+	-	-
嘉科优11 Jiakeyou 11	+	+	-	-	±	-	-
嘉优中科1号 Jiayouzhongke 1	-	-	-	-	±	+	-
嘉育938 Jiayu 938	+	+	-	-	+	-	-
空育131（Pigm）Kongyu131（Pigm）	+	+	+	+	+	+	+
连粳11号 Lianjing 11	-	-	-	-	+	-	-
南粳46 Nanjing 46	-	-	-	-	+	-	-
南粳9108 Nanjing 9108	-	-	-	-	+	-	-
宁84 Ning 84	+	+	-	-	+	-	-
日本晴 Nipponbare	+	-	-	-	+	-	-
苏香粳100 Suxiangjing 100	-	-	-	-	+	-	-
苏秀867 Suxiu 867	+	+	-	-	+	-	-
太湖糯 Taihunuo	-	-	-	-	+	-	-
特青 Teqing	-	-	-	-	+	-	-
天优华占 Tianyouhuazhan	+	+	-	-	±	-	-
皖稻68 Wandao 68	-	-	-	-	+	-	-
武运粳23 Wuyunjing 23	-	-	-	-	+	-	-
湘晚籼17 Xiangwanxian 17	-	-	-	-	+	-	-
祥湖84 Xianhu 84	-	-	-	-	+	-	-
秀水134 Xiushui 134	+	+	-	-	+	-	-
秀优7113 Xiuyou 7113	+	+	-	-	±	+	-
徐稻3号 Huaidao 3	-	-	-	-	+	-	-
甬优9 Yongyou 9	-	-	-	-	±	+	-
玉针香 Yuzhenxiang	-	-	-	-	+	-	-
浙粳88 Zhejing 88	+	+	-	-	+	-	-
镇稻11 Zhendao 11	-	-	-	-	+	-	-
镇糯19 Zhennuo 19	-	-	-	-	+	-	-
中禾优1号 Zhongheyou 1	-	-	-	-	±	-	-
中花11 Zhonghua 11	+	+	-	-	+	-	-
中嘉8号 Zhongjia 8	-	-	-	-	+	-	-