利用BSA-Seq方法鉴定谷丰B抗稻瘟病基因

陈子强; 陈松彪; 郭新睿; 颜静宛; 田大刚; 李刚; 王锋

doi:10.19303/j.issn.1008-0384.2021.01.005

利用BSA-Seq方法鉴定谷丰B抗稻瘟病基因

doi: 10.19303/j.issn.1008-0384.2021.01.005

1.
福建省农业遗传工程重点实验室/福建省农业科学院生物技术研究所，福建　福州　350003
2.
闽江学院海洋研究所海洋与农业生物技术实验室，福建　福州　350108

基金项目: 福建省科技计划公益类专项（2018R1019-9）；福建省自然科学基金项目（2017J01054）

详细信息

作者简介:
陈子强（1988−），男，硕士，助理研究员，主要从事水稻病理研究（E-mail：czq@fjage.org）

通讯作者:
王锋（1963−），男，博士，研究员，主要从事水稻分子育种研究（E-mail：wf@fjage.org）

中图分类号: S 435
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出版历程
- 收稿日期: 2020-11-03
- 修回日期: 2020-11-18
- 网络出版日期: 2020-11-24
- 刊出日期: 2021-01-31

BSA-Seq Identification of Blast-resistance Genes in Gufeng B Rice

1.
Fujian Key Laboratory of Genetic Engineering for Agriculture/Biotechnology Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China
2.
Marine and Agricultural Biotechnology Laboratory, Institute of Oceanography, Minjiang University, Fuzhou, Fujian　350108, China

摘要

摘要: 目的挖掘和鉴定谷丰B稻瘟病抗性基因，了解谷丰B稻瘟病抗性遗传模式。方法以谷丰B和日本晴杂交获得F1和F2代遗传群体，接种稻瘟菌不同生理小种并分析抗病遗传模式；在F2群体中挑选极端抗/感单株构建DNA混合池，利用群体分离分析法（BSA）定位关联区域。结果谷丰B对KJ201、RB22、CHNOS、RB6、2Y838-1、501-3和IR16-1等菌株均表现高抗性，表明谷丰B基因组可能携带了广谱高抗稻瘟病基因。谷丰B和日本晴杂交，F1群体表现抗501-3和IR16-1，F2群体的抗病/感病分离比不符合3 ∶ 1，推测谷丰B基因组存在多个位点影响稻瘟病抗性。对F2群体的极端抗病、感病混合池及亲本DNA进行全基因组测序，鉴定了1 756 964个单核苷酸多态性（SNPs）标记。分析子代△SNP-index，定位到2个与抗病性显著关联区间，分别为Chr.6: 10 082-11 397 kb和Chr.11: 120-266 kb。其中，6号染色体的关联区间与Pi2/9抗病位点等位，区间内含有4006个SNPs和623个插入缺失（InDels）标记；11号染色体的关联区间含有752个SNPs和195个InDels标记。结论谷丰B对强致病力501-3菌株抗性可能是由第6号和11号染色体上的基因共同控制。研究结果为谷丰B抗性基因的精细定位及基因克隆奠定了基础，并为水稻抗稻瘟病分子标记辅助选择提供标记资源。
- 水稻 /
- 稻瘟病 /
- 抗性基因 /
- 谷丰B /
- BSA
Abstract: Objective The rice cultivar Gufeng B confers strong, broad-spectrum, durable resistance against various rice blast isolates. The present study was aim to identify and map the blast resistance gene(s) in Gufeng B. Method The F1 and F2 population were obtained by crossing Gufeng B and Nipponbare, and the genetic model of blast resistance was analyzed after inoculating with 7 strains of Magnaporthe grisea. Subsequently, F2 population was used to construct a resistant pool and a sensitive pool respectively, and to map the associated loci via the method of bulked segregation analysis. Result Gufeng B exhibited high resistance to all of the tested strains, such as KJ201, RB22, CHNOS, RB6, 2Y838-1, 501-3 and IR16-1, suggesting that Gufeng B may carry the broad-spectrum and high resistance genes. The F1 progenies from the cross between Gufeng B and Nipponbare conferred resistance against the strains 501-3 and IR16-1, and the segregation ratio of resistance and susceptibility among F2 progenies does not fit 3:1, assuming that the resistance against the strains 501-3 and IR16-1 were controlled by multiple locus in Gufeng B. Whole genome re-sequencing of the two parental lines Gufeng B and Nipponbare identified 1,756,964 SNPs. Calculation results of △SNP-index showed that there were two candidate loci conferring resistance to rice blast disease, which were located at Chr.6: 10,082-11,397Kb, corresponding to the Pi2/9 locus, and Chr.11: 120-266Kb. 4006 SNPs and 623 InDels markers were searched within the interval of Chromosome 6, 752 SNPs and 195 InDels within the corresponding region of Chromosome 11, respectively. Conclusion The resistance of Gufeng B to 501-3 strain may be controlled by two resistance genes on chromosomes 6 and 11. Our results laid the foundation for finely mapping and cloning the resistance genes in Gufeng B, and provided marker resources for molecular marker-assisted selection.
- Rice /
- blast disease /
- resistance gene /
- Gufeng B /
- bulked segregation analysis

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图 1 不同水稻品种接种501-3菌株表型鉴定

Figure 1. Phenotypic identification on rice varieties inoculated with 501-3 blast strain

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图 2 第6号和第11号染色体关联区间部分区段所对应的物理位置

注: 箭头表示超过临界值水平的峰

Figure 2. Locations of corresponding regions in Chromosome 6 and Chromosome 11

Note: Arrow indicates peak above critical level.

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表 1 水稻品种稻瘟病抗性鉴定

Table 1. Blast resistance of rice cultivars

品种 Varieties	菌株 Strains
品种 Varieties	501-3	KJ201	IR16-1	RB22	CHNOS	RB6	2Y838-1
IR0462	S	MR	S	R	MR	MS	S
福恢838 Fuhui838	S	MR	S	MS	R	S	HS
甬优1号 Yongyou1	S	MR	S	MS	R	S	S
圭630 Gui630	HS	S	HS	S	R	HS	MS
福恢718 Fuhui718	MS	R	MS	R	R	S	S
谷丰B Gufeng B	HR	HR	HR	HR	HR	HR	HR
日本晴Nipponbare	HS	HS	HS	HS	S	S	HS
注: HR: 高抗; R: 抗病; MR: 中抗; MS: 中感; S: 感病; HS: 高感 Note: HR: Highly resistant; R: Resistant; MS: Moderately resistant; MS: Moderately susceptible; S: Susceptible; HS: Highly susceptible.

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表 2 谷丰B稻瘟病抗性遗传分析

Table 2. Genetic analysis on blast resistance of Gufeng B

群体 Population	菌株 Strains	总株数 Total number	抗病株数 Number of resistant plants	感病株数 Number of susceptible plants	期望比 Expected ration	χ²
F1（NPB×谷丰B）	501-3	30	30	0	—	—
F1（NPB×谷丰B）	IR16-1	30	30	0	—	—
F2（NPB×谷丰B）	501-3	407	255	152	3:1	33.09
F2（NPB×谷丰B）	IR16-1	430	275	155	3:1	27.98
注: NPB: 日本晴; χ2_（0.05）=3.84 Note: NPB: Nipponbare; χ2 _(0.05)=3.84.

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表 3 过滤后的数据统计表

Table 3. Statistics on data after screening

样本 Sample	有效数据量 Clean Base/G	准确度 Q30/%	GC含量 GC content/%	与参考基因组相同片段 Mapped reads	匹配率 Mapped ratio/%	覆盖度 Coverage/%
谷丰 B	9.88	92.44	44.3	62,811,386	95.36	87.48
NPB	ND	ND	ND	74,123,818	98.18	97.59
抗病池 R pool	12.177	92.44	47.26	77,601,474	95.59	97.69
感病池 S pool	10.417	91.52	47.59	66,943,380	96.39	96.94

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表 4 SNPs和InDels在水稻12条染色体的分布

Table 4. Distribution of identified SNPs and InDelsk in 12 chromosomes of rice

染色体 Chromosome	染色体长度 Chr. Length	SNPs		InDels
染色体 Chromosome	染色体长度 Chr. Length	数量 Number	密度 Density / （个·kb⁻¹）	数量 Number	密度 Density/ （个·kb⁻¹）
Chr.1	43270923	190273	4.397	46279	1.070
Chr.2	35937250	184278	5.128	42618	1.186
Chr.3	36413819	172550	4.739	42505	1.167
Chr.4	35502694	161349	4.545	31762	0.895
Chr.5	29958434	127664	4.261	30080	1.004
Chr.6	31248787	137688	4.406	35460	1.135
Chr.7	29697621	151268	5.094	34762	1.171
Chr.8	28443022	123316	4.336	28761	1.011
Chr.9	23012720	92893	4.037	22257	0.967
Chr.10	23207287	136960	5.902	30171	1.300
Chr.11	29021106	153785	5.299	33329	1.148
Chr.12	27531856	124940	4.538	31361	1.139
总计Total	373245519	1756964	4.707	409345	1.097

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