云南地方稻种抗稻瘟病基因Pi-d3序列变异分析

doi:10.16819/j.1001-7216.2016.5092

摘要/Abstract

摘要：

对来自云南的80份地方稻种抗稻瘟病基因Pi-d3编码区序列进行了PCR扩增并测序分析。结果表明,与地谷相比,80份云南地方稻种的Pi-d3编码区序列(全长2775 bp)存在39个变异位点,平均变异率为1.41%。所有材料可以归为37种单倍型,频率较高的单倍型是H8(28.8%)、H4(11.3%)、H23(5.0%),其他单倍型频率较低,显示了云南地方稻种Pi-d3基因变异丰富,单倍型类型多,但频率较低。37种单倍型中存在28个氨基酸差异位点,共编码33种蛋白,其中18种单倍型共计32份材料(包括12份籼稻品种)出现了假基因,假基因化的频率较高,可能是粳稻Pi-d3假基因化向籼稻渗透。除已发现的737位氨基酸假基因位点外,新发现32和467两个假基因位点。Pi-d3基因在籼稻、粳稻亚种,水稻、陆稻,黏稻、糯稻的单倍型类型及频率两方面均存在差异,表明亚种间或生态型间发生了一定的遗传分化。37种单倍型在云南的地理分布呈大分散、小聚居的特点,普洱、版纳、临沧的单倍型种类最丰富,并以之为中心向外扩展,单倍型种类随之减少。

关键词: 云南地方稻种, 稻瘟病, Pi-d3基因, 单核苷酸多态性

Abstract:

The sequence variations of Pi-d3 for rice blast resistance in 80 Yunnan rice landraces were analyzed by resequencing open reading frame (ORF). Compared with Pi-d3^Digu ORF, a total of 39 nucleotide variations were found in 2775-bp ORFs of 80 rice accessions with the average mutation rate of 1.41%. The WTBXPi-d3ORF of 80 rice accessions was divided into 37 haplotypes based on the nucleotide variations. H8(28.8%), H4(11.3%) and H23(5.0%) are high-frequency types, while other types occurred at low frequency. The results showed that the WTBXPi-d3 in Yunnan rice landraces was rich in genetic variations and haplotypes,but low frequency. A total of 28 amino acids variations were found in 33 translation proteins of 37 haplotypes. 18 haplotypes with 32 rice landraces including 12 indica accessions possessde the Pi-d3 pseudogenes. The frequency of pseudogene was high. It was possible due to that the Pi-d3 pseudogene permeated to indica. Terminators were newly founded at position 32 and 467 of protein except the published terminator at position 737. Pi-d3 differed in both type and frequency of haplotypes between the subspecies of indica and japonica, the ecotypes of lowland and upland rice, no-waxy and waxy rice. It indicated that genetic differentiation occurred between subspecies or ecotypes. Geographical distribution showed that 37 haplotypes spread over a large area while concentrated in small areas. Puer, Banna and Lincang were the central area with abundant haplotypes, and based on this scale, the types of haplotypes decreased outward progressively.

Key words: Yunnan rice landraces, rice blast, Pi-d3, single nucleotide polymorphism

中图分类号:

杨奕, 孙一丁, 马继琼, 王炎炎, 许明辉. 云南地方稻种抗稻瘟病基因Pi-d3序列变异分析[J]. 中国水稻科学, 2016, 30(1): 17-26.

Yi YANG, Yi-ding SUN, Ji-qiong MA, Yan-yan WANG, Ming-hui XU. Sequence Variation of Pi-d3 for Rice Blast Resistance in Yunnan Rice Landrace[J]. Chinese Journal OF Rice Science, 2016, 30(1): 17-26.

图/表 6

参考文献 20

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变异位点 Variation site	地谷碱基 Base in Pi-d3^Digu	替代碱基 Substituted base	替换类型 Substitution type	测序材料数 Sequenced accession number	替代材料数 No. of substituted accessions	频率 Frequence /%
94	G	T	颠换 Transversion	80	3	3.8
95	A	T	颠换 Transversion	80	1	1.3
130	G	C	颠换 Transversion	80	77	96.3
144	T	G	颠换 Transversion	80	77	96.3
197	C	T	转换 Transition	80	7	8.8
326	A	T	颠换 Transversion	80	4	5.0
356	G	C	颠换 Transversion	80	3	3.8
414	G	T	颠换 Transversion	80	3	3.8
458	C	T	转换 Transition	80	22	27.5
459	A	G	转换 Transition	80	77	96.3
477	A	G	转换 Transition	80	46	57.5
525	T	C	转换 Transition	80	55	68.8
537	G	A	转换 Transition	80	48	60.0
610	G	A	转换 Transition	80	24	30.0
756	T	C	转换 Transition	80	7	8.8
775	G	A	转换 Transition	80	77	96.3
785	A	G	转换 Transition	80	75	93.8
1014	C	T	转换 Transition	80	22	27.5
1128	C	A	颠换 Transversion	80	8	10.0
1197	T	C	转换 Transition	80	79	98.8
1329	T	G	颠换 Transversion	80	5	6.3
1399	A	T	颠换 Transversion	80	8	10.0
1484	G	A	转换 Transition	80	5	6.3
1544	G	A	转换 Transition	80	24	30.0
1713	T	G	颠换 Transversion	80	71	88.8
1729	G	A	转换 Transition	80	72	90.0
1766	C	T	转换 Transition	80	8	10.0
1842	C	G	颠换 Transversion	80	9	11.3
1874	A	T	颠换 Transversion	80	69	86.3
2005	G	T	颠换 Transversion	80	23	28.8
2009	G	A	转换 Transition	80	23	28.8
2209	C	T	转换 Transition	80	23	28.8
2396	A	G	转换 Transition	80	8	10.0
2444	T	A	颠换 Transversion	80	71	88.8
2566	C	G	颠换 Transversion	80	80	100.0
2671	G	T	颠换 Transversion	80	10	12.5
2680	G	A	转换 Transition	80	72	90.0
2687	G	A	转换 Transition	80	80	100.0
2699	G	A	转换 Transition	80	7	8.8

变异位点 Variation site	地谷碱基 Base in Pi-d3^Digu	替代碱基 Substituted base	替换类型 Substitution type	测序材料数 Sequenced accession number	替代材料数 No. of substituted accessions	频率 Frequence /%
94	G	T	颠换 Transversion	80	3	3.8
95	A	T	颠换 Transversion	80	1	1.3
130	G	C	颠换 Transversion	80	77	96.3
144	T	G	颠换 Transversion	80	77	96.3
197	C	T	转换 Transition	80	7	8.8
326	A	T	颠换 Transversion	80	4	5.0
356	G	C	颠换 Transversion	80	3	3.8
414	G	T	颠换 Transversion	80	3	3.8
458	C	T	转换 Transition	80	22	27.5
459	A	G	转换 Transition	80	77	96.3
477	A	G	转换 Transition	80	46	57.5
525	T	C	转换 Transition	80	55	68.8
537	G	A	转换 Transition	80	48	60.0
610	G	A	转换 Transition	80	24	30.0
756	T	C	转换 Transition	80	7	8.8
775	G	A	转换 Transition	80	77	96.3
785	A	G	转换 Transition	80	75	93.8
1014	C	T	转换 Transition	80	22	27.5
1128	C	A	颠换 Transversion	80	8	10.0
1197	T	C	转换 Transition	80	79	98.8
1329	T	G	颠换 Transversion	80	5	6.3
1399	A	T	颠换 Transversion	80	8	10.0
1484	G	A	转换 Transition	80	5	6.3
1544	G	A	转换 Transition	80	24	30.0
1713	T	G	颠换 Transversion	80	71	88.8
1729	G	A	转换 Transition	80	72	90.0
1766	C	T	转换 Transition	80	8	10.0
1842	C	G	颠换 Transversion	80	9	11.3
1874	A	T	颠换 Transversion	80	69	86.3
2005	G	T	颠换 Transversion	80	23	28.8
2009	G	A	转换 Transition	80	23	28.8
2209	C	T	转换 Transition	80	23	28.8
2396	A	G	转换 Transition	80	8	10.0
2444	T	A	颠换 Transversion	80	71	88.8
2566	C	G	颠换 Transversion	80	80	100.0
2671	G	T	颠换 Transversion	80	10	12.5
2680	G	A	转换 Transition	80	72	90.0
2687	G	A	转换 Transition	80	80	100.0
2699	G	A	转换 Transition	80	7	8.8

单倍型 Haplotype	氨基酸位点 Amino acid lous
单倍型 Haplotype	32	44	66	109	119	138	153	204	259	262	443	467	495	515	571	577	589	625	669	670	737	799	815	856	891	894	896	900
地谷Digu	E	G	A	K	R	R	T	G	V	D	N	R	G	R	H	V	S	N	V	G	Q	N	F	H	V	V	R	R
H1	-	R	-	M	-	-	M	S	I	-	K	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H2、H35	-	R	-	-	-	-	-	-	-	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H3	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H4、H17	-	R	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H5	-	R	-	M	P	S	-	-	I	-	-	-	E	-	-	-	L	-	-	-	-	S	-	D	L	-	Q	Q
H6	-	R	-	-	-	-	-	-	I	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H7	-	R	-	-	-	S	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	-	L	I	Q	-
H8、H19	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H9	-	-	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H10	-	R	-	-	-	-	-	-	I	G	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H11	-	R	-	M	P	S	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H12	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	-	-	-	-	-	Y	D	-	I	Q	-
H13	-	R	-	-	-	-	-	-	I	-	-	*	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H14、H15	-	R	-	-	-	-	M	S	I	-	K	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H16	-	R	V	-	-	-	-	-	I	-	-	*	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	Q
H18	-	R	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	L	I	Q	-
H20	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	Q
H21	-	R	-	M	P	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H22	-	R	-	-	-	-	-	-	I	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H23	-	R	-	-	-	-	-	-	I	G	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H24	-	R	-	-	-	-	M	S	I	-	-	-	-	H	-	M	-	-	-	-	*	-	Y	D	-	I	Q	-
H25	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	-
H26	-	R	-	-	-	-	-	-	-	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H27	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	L	-	Q	Q
H28	-	R	-	-	-	-	-	-	I	-	-	*	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H29	V	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H30	-	-	-	-	-	-	-	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H31	-	-	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H32	-	R	-	-	-	-	-	S	I	-	K	-	-	H	Q	M	-	I	F	D	-	-	Y	D	-	I	Q	-
H33	*	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H34	*	R	-	-	-	-	-	-	I	-	-	*	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H36	-	R	-	-	-	-	M	S	I	-	-	*	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H37	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	-	D	-	I	Q	-

单倍型 Haplotype	氨基酸位点 Amino acid lous
单倍型 Haplotype	32	44	66	109	119	138	153	204	259	262	443	467	495	515	571	577	589	625	669	670	737	799	815	856	891	894	896	900
地谷Digu	E	G	A	K	R	R	T	G	V	D	N	R	G	R	H	V	S	N	V	G	Q	N	F	H	V	V	R	R
H1	-	R	-	M	-	-	M	S	I	-	K	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H2、H35	-	R	-	-	-	-	-	-	-	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H3	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H4、H17	-	R	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H5	-	R	-	M	P	S	-	-	I	-	-	-	E	-	-	-	L	-	-	-	-	S	-	D	L	-	Q	Q
H6	-	R	-	-	-	-	-	-	I	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H7	-	R	-	-	-	S	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	-	L	I	Q	-
H8、H19	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H9	-	-	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H10	-	R	-	-	-	-	-	-	I	G	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H11	-	R	-	M	P	S	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H12	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	-	-	-	-	-	Y	D	-	I	Q	-
H13	-	R	-	-	-	-	-	-	I	-	-	*	-	-	Q	M	-	I	-	-	-	-	Y	D	L	I	Q	-
H14、H15	-	R	-	-	-	-	M	S	I	-	K	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H16	-	R	V	-	-	-	-	-	I	-	-	*	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	Q
H18	-	R	-	-	-	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	L	I	Q	-
H20	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	Q
H21	-	R	-	M	P	-	M	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H22	-	R	-	-	-	-	-	-	I	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H23	-	R	-	-	-	-	-	-	I	G	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H24	-	R	-	-	-	-	M	S	I	-	-	-	-	H	-	M	-	-	-	-	*	-	Y	D	-	I	Q	-
H25	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	-	-	Q	-
H26	-	R	-	-	-	-	-	-	-	-	-	-	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H27	-	R	V	-	-	-	-	-	I	-	-	-	-	-	-	-	L	-	-	-	-	S	-	D	L	-	Q	Q
H28	-	R	-	-	-	-	-	-	I	-	-	*	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H29	V	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H30	-	-	-	-	-	-	-	S	I	-	-	-	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H31	-	-	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H32	-	R	-	-	-	-	-	S	I	-	K	-	-	H	Q	M	-	I	F	D	-	-	Y	D	-	I	Q	-
H33	*	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H34	*	R	-	-	-	-	-	-	I	-	-	*	E	-	Q	M	-	I	-	-	-	-	Y	D	-	I	Q	-
H36	-	R	-	-	-	-	M	S	I	-	-	*	-	H	Q	M	-	I	F	D	*	-	Y	D	-	I	Q	-
H37	-	R	-	-	-	-	-	-	I	-	-	-	-	-	Q	M	-	I	-	-	-	-	-	D	-	I	Q	-

单倍型 Haplotype	材料数 No. of accessions	粳稻 japonica			籼稻 indica		陆稻 Upland rice			水稻 Lowland rice			黏稻 No-waxy rice				糯稻 Waxy rice
单倍型 Haplotype	材料数 No. of accessions	数量 No.		频率 Frequence /%		数量 No.		频率 Frequence /%		数量 No.		频率 Frequence /%	数量 No.	频率 Frequence /%	数量 No.	频率 Frequence /%	数量 No.	频率 Frequence /%
H1	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H2	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H3	2	2	100.0	0	0		1	50.0	1		50.0	1		50.0		1		50.0
H4	9	7	77.8	2	22.2		6	66.7	3		33.3	7		77.8		2		22.2
H5	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H6	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H7	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H8	23	9	39.1	14	60.9		5	21.7	18		78.3	19		82.6		4		17.4
H9	1	1	100.0	0	0		0	0	1		100.0	1		100.0		0		0
H10	1	1	100.0	0	0		0	0	1		100.0	0		0		1		100.0
H11	1	1	100.0	0	0		1	100.0	0		0	0		0		1		100.0
H12	2	1	50.0	1	50.0		2	100.0	0		0	1		50.0		1		50.0
H13	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H14	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H15	2	1	50.0	1	50.0		1	50.0	1		50.0	2		100.0		0		0
H16	2	1	50.0	1	50.0		1	50.0	1		50.0	2		100.0		0		0
H17	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H18	2	2	100.0	0	0		1	50.0	1		50.0	1		50.0		1		50.0
H19	1	1	100.0	0	0		0	0	1		100.0	0		0		1		100.0
H20	3	3	100.0	0	0		1	33.3	2		66.7	3		100.0		0		0
H21	1	1	100.0	0	0		1	100.0	0		0	0		0		1		100.0
H22	1	1	100.0	0	0		0	0	1		100.0	1		100.0		0		0
H23	4	2	50.0	2	50.0		1	25.0	3		75.0	4		100.0		0		0
H24	1	1	100.0	0	0		0	0	1		100.0	0		0		1		100.0
H25	1	1	100.0	0	0		0	0	1		100.0	0		0		1		100.0
H26	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H27	1	1	100.0	0	0		1	100.0	0		0	1		100.0		0		0
H28	3	0	0	3	100.0		0	0	3		100.0	1		33.3		2		66.7
H29	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
H30	1	0	0	1	100.0		0	0	1		100.0	0		0		1		100.0
H31	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
H32	1	0	0	1	100.0		1	100.0	0		0	1		100.0		0		0
H33	2	0	0	2	100.0		0	0	2		100.0	2		100.0		0		0
H34	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
H35	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
H36	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
H37	1	0	0	1	100.0		0	0	1		100.0	1		100.0		0		0
总数Total	80	46	57.5	34	42.5		32	40.0	48		60.0	62		77.5		18		22.5