抗稻瘟病Pi2/9/z-t基因特异性分子标记的开发

doi:10．3969/j．issn．1001G7216．2015．03．010

摘要/Abstract

摘要：

通过对已克隆的抗稻瘟病基因Pi2、Pi9以及Piz-t 进行序列比对,寻找各自特异的核苷酸差异,成功开发了基于PCR技术以及电泳检测技术的 Pi2/Pi9/Piz-t 以及Piz-t 的基因特异性分子标记,能有效地将 Pi2/Pi9/Piz-t 与该位点上的其他抗性等位基因及感病等位基因区分开,这为分子标记辅助育种及抗病基因聚合提供有效的分子标记。用 Pi2/Pi9/Piz-t 基因特异性分子标记对来自全国各稻区的共101份水稻品种和育种亲本进行分子检测,结果发现,除2个品种检测到Piz-t带型之外,大部分水稻品种不携带这3个抗性基因,这为有目的地开展品种的抗性改良提供了重要的参考信息。

关键词: 水稻, 稻瘟病, 抗性基因, 分子标记, 分子标记辅助选择育种, Pi2/9 位点

Abstract:

Specific molecular markers for detecting the alleles of Pi2/Pi9/Piz-t, designated as Pi2SNP, Pi9SNP and Pizt-PA, respectively, have been successfully developed through sequence comparison method. These three specific markers can distinctly distinguish target genes from others mapped on Pi2/9 locus, which facilitate the molecular marker-assistant selection and the development of pyramiding cultivars combined with other allele R genes. Additionally, 101 main cultivars and breeding parents collected from different rice-planting areas have been diagnosed with these three specific markers, and the results showed that only 2 out of 101 cultivars carried Piz-t, but neither Pi2 nor Pi9 band had been detected. So far, it indicated that most of the main cultivars and breeding parents in China do not carry Pi2/Pi9/Piz-t. These results had provided important information for the application of Pi2/Pi9/Piz-t in the blast resistance breeding programs.

Key words: rice, blast, resistance gene, molecular marker, MAS, Pi2/9 locus

中图分类号:

华丽霞, 汪文娟, 陈深, 汪聪颖, 曾烈先, 杨健源, 朱小源, 苏菁. 抗稻瘟病Pi2/9/z-t基因特异性分子标记的开发[J]. 中国水稻科学, 2015, 29(4): 305-310.

Li-xia HUA, Wen-juan WANG, Shen CHEN, Cong-ying WANG, Lie-xian ZENG, Jian-yuan YANG, Xiao-yuan ZHU, Jing SU. Development of Specific DNA Markers for Detecting the Rice Blast Resistance Gene Alleles Pi2/9/z-t[J]. Chinese Journal OF Rice Science, 2015, 29(4): 305-310.

图/表 7

表1 Pi2/Pi9/Piz-t 基因特异性分子标记相关信息

Table 1 Information of specific markers for Pi2/Pi9/Piz-t.

引物名称 Primer name	引物序列(5'-3') Sequences (5'-3')	退火温度 Annealing temperature /℃	预期片段大小 Expected size /bp	标记类型 Marker type	内切酶 Cutting enzyme
Pi9SNP	F:CGCCGGTTGATAAGTAAAAGCT	60	126	dCAPS	Hind Ⅲ
	R:CAAGAACTAATATCTACCCATGG
Pi2SNP	F:TACTCTTCGTTGTATAGGAC	58	462	CAPS	Hinf Ⅰ
	R:GGAGGAGGAGATGAAATAGAATC
Pizt PA	F:ATGTGGATGCTGTGTTAT	62	176	Presence/	—
	R:TAGTTTGCTGCTCAATAAGTA			Absence

图1 Pi9 部分序列比对结果(小写字母“t”代表Pi9特异SNP,斜体字母“G”代表在正向引物3'端引入的错配碱基,下划线表示的位点是限制性内切酶 Hind Ⅲ识别位点。)

Fig. 1. Sequence alignment of Pi9 and its orthlogs. (The Pi9-specific SNP is indicated in lowercase. A forward primer with a mismatch base (in italic and bold) is designed adjacent to Pi9-specific SNP, creating a Hind Ⅲ recognition site that is dependent on this specific SNP.)

图2 Pi9特异性分子标记Pi9SNP的多态性检测(M-D600;泳道1-IRBL9-W (Pi9+);泳道2-C101A51 (Pi2);泳道3-IRBLz-Fu (Piz );泳道4-IRBLzt-T (Piz-t);泳道5-谷梅2号(Pi26);泳道6-谷梅4号 (Pigm);泳道7-EBZ (Pi50);泳道8-日本晴;泳道9-LTH。)

Fig. 2. Polymorphism validation of Pi9-specific marker. (M, D600; Line 1, IRBL9-W (Pi9+); Line 2, C101A51 (Pi2); Line 3, IRBLz-Fu (Piz ); Line 4, IRBLzt-T (Piz-t); Line 5, Gumei 2 (Pi26); Line 6, Gumei 4 (Pigm); Line 7, EBZ (Pi50); Line 8, Nipponbare; Line 9, LTH.)

图3 Pi2与等位基因之间的序列比对(小写字母代表Pi2特异SNP,下划线所表示的位点是限制性内切酶 Hinf I识别位点。)

Fig. 3. Sequence alignment of Pi2 and its orthlogs. (The Pi2-specific SNP is indicated in lowercase. Underlined bases refer to a Hinf I recognition site that is dependent on the Pi2-specific SNP.)

图4 Pi2特异性分子标记Pi2SNP的多态性检测(M-D600; 泳道1-C101A51 (Pi2+); 泳道2-IRBL9-W (Pi9); 泳道3- IRBLz-Fu ( Piz ); 泳道4-IRBLzt-T (Piz-t ); 泳道5-Gumei 2 (Pi26 );泳道6-Gumei 4 (Pigm); 泳道7-EBZ (Pi50); 泳道8- Nipponbare; 泳道9- LTH。)

Fig. 4. Polymorphism validation of Pi2-specific marker. (D600; Line 1, C101A51 (Pi2+); Line 2, IRBL9-W (Pi9); Line 3, IRBLz-Fu ( Piz ); Line 4, IRBLzt-T (Piz-t); Line 5, Gumei 2 (Pi26 ); Line 6, Gumei 4 (Pigm); Line 7, EBZ (Pi50); Line 8, Nipponbare; Line 9, LTH.)

图5 Piz-t与等位基因之间的序列比对(Piz-t 特异SNP用小写字母表示,下划线表示Piz-t 特异引物对序列(左侧为正向引物,右侧为反向引物),Piz-t 的特异性SNP均位于引物的3'末端。虚线代表引物之间的序列。)

Fig. 5. Sequence alignment for Piz-t and its orthlogs. (The Pizt-specific SNPs are noted in lowercase. The designed Pizt-specific primers are underlined (left, forward primer; right, reverse), harbouring the Pizt-specific SNPs at the end of the 3'primer. Dash lines indicate the elliptical base pairs between the primer pair.)

图6 Piz-t 特异性分子标记Pizt-PA的多态性检测(M, DL2000; 泳道1-IRBLzt-T (Piz-t+); 泳道2-IRBL9-W (Pi9); 泳道3-IRBLz-Fu ( Piz ); 泳道4-C101A51 (Pi2); 泳道5-Gumei 2 ( Pi26 ); 泳道6-Gumei 4 ( Pigm ); 泳道7-EBZ (Pi50); 泳道8-Nipponbare; 泳道9-LTH。)

Fig. 6. Specific SNP diagnosis for Piz-t and its polymorphism validation. (M, DL2000; Line 1, IRBLzt-T (Piz-t+); Line 2, IRBL9-W (Pi9); Line 3, IRBLz-Fu ( Piz ); Line 4, C101A51 (Pi2); Line 5, Gumei 2 ( Pi26) ; Line 6, Gumei 4 ( Pigm ); Line 7, EBZ (Pi50); Line 8, Nipponbare; Line 9, LTH.)

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