Pigm特异性选择标记的开发及其在粳稻穗颈瘟抗性育种中的利用

doi:10.16819/j.1001-7216.2018.7135

摘要/Abstract

摘要：

【目的】Pigm是一个广谱的稻瘟病抗性基因,源自持久抗性品种谷梅4号,与Piz、Piz-t、Pi2、Pi9和Pi40等互为复等位基因但抗谱存在差异。为了更好地在分子辅助选择育种中利用Pigm基因,开发与Pigm特异性标记具有重要意义。【方法】在已有文献报道定位结果的基础上,通过随机测序获得了一段谷梅4号基因组的特异序列,并据此开发了一组用于筛选Pigm基因的分子标记,进一步选取江淮稻区3个不同生态型代表性粳稻品种作为受体,利用分子标记辅助选择结合对抗性基因的背景检测将Pigm基因导入受体品种。【结果】Pigm-4标记位于Pigm基因簇内部,与抗病功能元件PigmR紧密连锁,对不同类型的品种检测发现该标记特异性强,且利用该标记可将Pigm与Piz、Piz-t、Pi2、Pi9以及Pi40区分开来。对受体亲本稻瘟病抗性基因的检测和接种结果分析发现江淮稻区粳稻品种虽然携带了Pib、Pi54、Pita、Pb1中的2~3个基因,但是对强毒力的稻瘟病小种抗性普遍不强,而3种代表性粳稻背景下导入Pigm基因均可显著提高其对穗颈瘟的抗性水平。【结论】Pigm可以作为抗稻瘟病粳稻育种的有利基因资源加以利用,而Pigm-4是分子标记辅助筛选Pigm的优异标记。

关键词: Pigm, 特异分子标记, 粳稻, 穗颈瘟, 抗性

Abstract:

【Objective】 Pigm is a broad-spectrum resistance gene to rice blast, which is isolated from Gumei 4, a variety with durable resistance. Pigm is allelic to Piz, Piz-t, Pi2, Pi9, Pi40 and so on, but shows different resistance spectrum. To breed resistance variety carrying Pigm with molecular marker-assisted selection, discovering Pigm-tightly-linked markers is important.【Method】We identified a unique DNA sequence of Gumei 4 by genomic sequencing, and developed a set of specific markers tagged to Pigm base on the sequence divergence. Then, by means of molecular-assisted selection and background gene screening, Pigm was pyramided to three representative japonica varieties belonging to different ecotypes from Yangtze-Huai River Area. 【Result】 An InDel marker Pigm-4 which is closely linked to the functional unit of Pigm was developed. Pigm-4 showed distinct specificity by genotype screening of various rice varieties, and can distinguish the genotype of Pigm from its alleles Piz, Piz-t, Pi2, Pi9 and Pi40. Although receptors carry with resistant genes such as Pib+Pi54 (Wuyun 2674), Pib+Pita (Wuyunjing 27), Pib+Pi54+Pb1 (Zhendao 18), respectively, but none of them can defense high virulence blast pathogen. Relevant introgression lines were established and inoculations of introgression lines indicate that Pigm can promote resistance level to rice panicle blast significnatly in different japonica backgrounds. 【Conclusion】Pigm can be used as a beneficial gene source in the breeding of blast resistant japonica rice, and Pigm-4 is an excellent marker for the molecular marker-assisted selection of Pigm.

Key words: Pigm, specific-marker, japonica rice, panicle blast, resistance

中图分类号:

曾生元, 李闯, 杜灿灿, 孙立亭, 景德道, 林添资, 余波, 钱华飞, 姚维成, 周义文, 龚红兵. Pigm特异性选择标记的开发及其在粳稻穗颈瘟抗性育种中的利用[J]. 中国水稻科学, 2018, 32(5): 453-461.

Shengyuan ZENG, Chuang LI, Cancan DU, Liting SUN, Dedao JING, Tianzi LIN, Bo YU, Huafei QIAN, Weicheng YAO, Yiwen ZHOU, Hongbing GONG. Development of Specific Markers for Pigm in Marker-Assisted Breeding of Panicle Blast Resistant japonica Rice[J]. Chinese Journal OF Rice Science, 2018, 32(5): 453-461.

图/表 9

表2 本研究用于测序和检测Pigm基因的相关标记

Table 2 Sequencing and selecting markers for Pigm in this study.

标记名称 Name	正向引物(5′-3′) Forward primer (5′-3′)	反向引物(5′-3′) Reverse primer (5′-3′)	染色体位置 Physical location / bp	用途 Purpose
Sepigm-4	TCGCAGCCATCCAAAGTGAGTC	TACACCTGCGAATCAAATCACT	10 435 917―10 436 503	测序Sequencing
Pigm-2	TCTGAATTATTGTGGTCGTG	CCGTTCACATCAGTTTTTCT	10 366 681―10 366 821	检测标记MAS
Pigm-4	ATGCTCGATTCGTTACATTT	CGTCCCACACTTTCTTTTT	10 436 075―10 436 229	检测标记MAS

表3 检测抗稻瘟病基因的引物信息

Table 3 Information of molecular markers for screening rice blast resistance genes.

目标基因 Target gene	标记名称 Marker name	正向引物(5′-3′) Forward primer(5′-3′)	反向引物(5′-3′) Reverse primer(5′-3′)	预计片段大小 Expected fragment / bp	参考文献 Reference
Pib	Pibdom	GAACAATGCCCAAACTTGAGA	GGGTCCACATGTCAGTGAGC	365	[12]
Pi9	PB9-1	TAGACTCCTTCCAAGT TTGACT	TGTGATTTTCAGAATTT TCGT	180	[12]
Pi2	AP22	GTGCATGAGTCCAGCTCAAA	GTGTACTCCCATGGCTGCTC	143	[12]
Pizt	Pizt-PA	ATGTGGATGCTGTGTTAT	TAGTTTGCTGCTCAATAAGTA	176	[20]
Pi5	JJ817	GATATGGTTGAAAAGCTAATCTCA	ATCATTGTCCTTCATATTCAGAGT	1450	[12]
Pi54	Pikh-1	CAATCTCCAAAGTTTTCAGG	GCTTCAATCACTGCTAGACC	216	[12]
Pikm	Pikm1	TGAGCTCAAGGCAAGAGTTGAGGA	TGTTCCAGCAACTCGATGAG	174	[5]
Pikm	Pikm2	CAGTAGCTGTGTCTCAGAACTATG	AAGGTACCTCTTTTCGGCCAG	290
Pita / pita	YL155/YL87	AGCAGGTTATAAGCTAGGCC	CTACCAACAAGTTCATCAAA	1042	[12]
Pita / pita	YL155/YL87	AGCAGGTTATAAGCTAGCTAT	CTACCAACAAGTTCATCAAA	1042
Pb1	Pb1-1	ATCAACGCTACCTTCCC	GTGCCATCACAATTTCTTC	159	[21]

图1 谷梅4号序列与相关亲本的部分序列比对结果

Fig. 1. Sequence alignment of different Piz alleles amplified with Sepigm-4.

表1 代表性品种在Pigm-4座位的带型

Table 1 Typical varieties and their genotype in Pigm-4 locus.

图2 Pigm-4在部分亲本间(A)及分离群体(B)的检测结果 A图1~18泳道分别为亲本谷梅4号、IR65482、C101A51、75-1-127、Toride 1、Fukunishiki、武运2674/谷梅4号BC3F3纯系、9311、镇恢82、镇恢832、成恢177、成恢727、秀水519、武运2674、武运粳27、镇稻88、镇稻18及镇糯19;B图1、2泳道分别为武运2674、谷梅4号,3~18泳道为武运2674/谷梅4号部分BC3F2单株。

Fig. 2. Diversity detection of Pigm-4 marker in relevant parents and segregation population. In Fig. A, Lanes 1 to 18 represent Gumei 4, IR65482, C101A51, 75-1-127, Toride 1, Fukunishiki, BC3F3 homozygous lines of Wuyun 2674/Gumei 4, 9311, Zhenhui 82, Zhenhui 832, Chenghui 177, Chenghui 727, Xiushui 519, Wuyun 2674, Wuyunjing 27, Zhendao 88, Zhendao 18, and Zhennuo 19, repectively.In Fig. B, 1, Wuyun 2674; 2, Gumei 4; Lanes 3 to 18 represent some BC3F2 individuals derived from Wuyun 2674/ Gumei 4.

表4 3个受体亲本所包含的抗稻瘟病基因型及抗性等级

Table 4 Distribution of R genes and resistance level of the three receptors.

受体亲本名称 Variety	亲本生态型 Ecotype	Pi54	Pb1	Pib	Pita	抗性等级 Resistance level
武运2674 Wuyun 2674	迟熟中粳 Late maturing mid-season japonica rice	+	-	+	-	9(HS)
武运粳27 Wuyunjing 27	中熟中粳 Medium maturing mid-season japonica rice	-	-	+	+	7(HS)
镇稻18 Zhendao 18	早熟晚粳 Early maturing late japonica rice	+	+	+	-	5(MS)

表5 Pigm-2及Pigm-4检测不同回交群体的基因型情况

Table 5 Genotype distribution in different backcross population detected by Pigm-2 and Pigm-4.

群体 Population	标记 Marker	各基因型单株数 Individuals with different genotype
群体 Population	标记 Marker	Pigmpigm	pigmpigm
武运2674/谷梅4号 BC₅F₁ Wuyun 2674/Gumei 4 BC₅F₁	Pigm-2	89		62
武运2674/谷梅4号 BC₅F₁ Wuyun 2674/Gumei 4 BC₅F₁	Pigm-4	92		59
武运粳27/谷梅4号 BC₄F₁ Wuyunjing 27/Gumei 4 BC₄F₁	Pigm-2	43		50
武运粳27/谷梅4号 BC₄F₁ Wuyunjing 27/Gumei 4 BC₄F₁	Pigm-4	41		54
镇稻18/谷梅4号 BC₄F₁ Zhendao 18/Gumei 4 BC₄F₁	Pigm-2	35		32
镇稻18/谷梅4号 BC₄F₁ Zhendao 18/Gumei 4 BC₄F₁	Pigm-4	35		32

图3 导入系内不同类型交换单株的抗性

Fig. 3. Resistant level of the recombination individuals between Pigm-2 with Pigm-4 of introgression lines.

图4 不同抗稻瘟病基因组合的抗性等级1–武运2674;2–武运2674(Pib+Pi54+Pigm);3–武运粳27;4–武运粳27(Pi54+Pita+Pigm);5–武运粳27(Pita+Pigm);6–镇稻18;7–镇稻18(Pib+Pi54+Pb1+Pigm);8–镇稻18(Pib+Pi54+Pigm)。

Fig. 4. Resistance level of different combination patterns of R genes. 1, Wuyun 2674; 2, Wuyun 2674(Pib+Pi54+Pigm); 3, Wuyunjing 27; 4, Wuyunjing 27 (Pi54+Pita+Pigm); 5, Wuyunjing 27(Pita+Pigm); 6, Zhendao 18; 7, Zhendao 18(Pib+Pi54+Pb1+Pigm); 8, Zhendao 18(Pib+ Pi54+Pigm).

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