南方水稻黑条矮缩病抗性的遗传分析及主效QTL的精细定位

doi:10.16819/j.1001-7216.2019.8057 135

中国水稻科学 ›› 2019, Vol. 33 ›› Issue (2): 135-143.DOI: 10.16819/j.1001-7216.2019.8057 135

南方水稻黑条矮缩病抗性的遗传分析及主效QTL的精细定位

农保选^1,^2,³, 秦碧霞⁴, 夏秀忠^2,³, 杨行海^2,³, 张宗琼^2,³, 曾宇^2,³, 邓国富^2,³, 蔡健和⁴, 李战彪⁴, 刘丕庆^1,^*(), 李丹婷^2,^3,^*()

¹广西大学农学院, 南宁 530004
²广西农业科学院水稻研究所, 南宁 530007
³广西水稻遗传育种重点实验室, 南宁 530007
⁴广西农业科学院植物保护研究所, 南宁 530007

收稿日期:2018-05-16 修回日期:2018-12-20 出版日期:2019-03-10 发布日期:2019-03-10
通讯作者: 刘丕庆,李丹婷
基金资助:
国家重点研发计划资助项目(2016YFD0100100);广西科技计划资助项目(桂科AB16380117);水稻生物学国家重点实验室开放课题(160105;170102);广西农业科学院基本科研业务专项(2015YT15, 桂农科2018YM16)

Genetic Analysis and Fine Mapping of a Major QTL for the Resistance to Southern Rice Black-Streaked Dwarf Disease

Baoxuan NONG^1,^2,³, Bixia QIN⁴, Xiuzhong XIA^2,³, Xinghai YANG^2,³, Zongqiong ZHANG^2,³, Yu ZENG^2,³, Guofu DENG^2,³, Jianhe CAI⁴, Zhanbiao LI⁴, Piqing LIU^1,^*(), Danting LI^2,^3,^*()

¹Agricultural College, Guangxi University, Nanning 530004, China
²Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
³Guangxi Key Laboratory of Rice Genetics and Breeding, Nanning 530007, China
⁴Plant Protection Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China

Received:2018-05-16 Revised:2018-12-20 Online:2019-03-10 Published:2019-03-10
Contact: Piqing LIU, Danting LI

摘要/Abstract

摘要：

【目的】近年来由白背飞虱传播的南方水稻黑条矮缩病给水稻生产造成了巨大损失,开展该病的抗性遗传分析和基因精细定位,将为抗性育种提供材料和理论依据。【方法】分析了抗性材料D4对南方水稻黑条矮缩病的抗性特征,并通过广恢998/D4 F₂群体分析该病抗性的遗传规律,利用QTL-seq技术联合遗传连锁分析定位主效抗性QTL。【结果】D4对南方水稻黑条矮缩病的抗性表现为抗病毒性而非抗虫性,且受主效基因和微效基因共同控制。QTL-seq和连锁分析将南方水稻黑条矮缩病主效抗性QTL定位于第9染色体上,命名为qSRBSDV9。利用代换作图法进一步将qSRBSDV9定位在102.3 kb的区间内,该区间包含21个预测基因,其中9个基因与赤霉素信号传导相关。【结论】揭示了D4对南方水稻黑条矮缩病的抗性特征及遗传规律,精细定位了南方水稻黑条矮缩病主效抗性QTL qSRBSDV9。这为该QTL的图位克隆及育种利用奠定了基础。

关键词: 南方水稻黑条矮缩病, 白背飞虱, 遗传分析, QTL, 精细定位

Abstract:

【Objective】Southern rice black-streaked dwarf virus(SRBSDV), which is transmitted by white-backed planthopper (Sogatella furcifera Horváth, WBPH), has caused severe yield loss in epidemic years in rice production. We analyzed the resistance mechanism of SRBSDV and identified the related gene locus by fine gene mapping to lay a scientific base for effective prevention and control of SRBSDV. 【Method】The resistance of rice material D4 against white-backed planthoppers were analyzed, and the genetic basis for the resistance to SRBSDV was analyzed by using F₂ population of Guanghui 998×D4. The major QTL for resistance to SRBSDV was identified using QTL-seq, and further verified by genetic linkage analysis. 【Result】D4 was resistant to SRBSDV but not to WBPH. Genetic analysis showed that the resistance of D4 was controlled by major gene and minor genes. QTL-seq and linkage analysis revealed that the major QTL for resistance to SRBSDV in rice was located within the 1.40 M region on chromosome 9, which was termed as qSRBSDV9. Then qSRBSDV9 was fine mapped on chromosome 9 with a 102.3 kb physical distance by substitution mapping. This region contained 21 predicted genes, nine of which were related to gibberellin signaling. 【Conclusion】The results reveals the characteristics and inheritance of resistance to SRBSDV in D4. The fine mapping of qSRBSDV9 lays a base for map-based cloning and utilization.

Key words: Southern rice black-streaked dwarf disease, white-backed planthopper, genetic analysis, QTL, fine mapping

中图分类号:

农保选, 秦碧霞, 夏秀忠, 杨行海, 张宗琼, 曾宇, 邓国富, 蔡健和, 李战彪, 刘丕庆, 李丹婷. 南方水稻黑条矮缩病抗性的遗传分析及主效QTL的精细定位[J]. 中国水稻科学, 2019, 33(2): 135-143.

Baoxuan NONG, Bixia QIN, Xiuzhong XIA, Xinghai YANG, Zongqiong ZHANG, Yu ZENG, Guofu DENG, Jianhe CAI, Zhanbiao LI, Piqing LIU, Danting LI. Genetic Analysis and Fine Mapping of a Major QTL for the Resistance to Southern Rice Black-Streaked Dwarf Disease[J]. Chinese Journal OF Rice Science, 2019, 33(2): 135-143.

图/表 7

图1 人工接种南方水稻黑条矮缩病后水稻D4和广恢998的表型 A-D4接种南方水稻黑条矮缩病毒的表型;B-广恢998接种南方水稻黑条矮缩病毒的表型。N-未接种对照;I-接种。

Fig. 1. Phenotype of D4 and Guanghui 998 by artificial inoculation of SRBSDV. A, Phenotype of D4 after artificial inoculation of SRBSDV; B, Phenotype of Guanghui 998 after artificial inoculation of SRBSDV. N, Control; I, Inoculation.

图2 D4和广恢998对白背飞虱的抗性分析 A-D4和广恢998(GH998)及两个对照品种对白背飞虱的排驱性表现;B-白背飞虱在D4和广恢998及两个对照品种中的存活率。数据用平均值±标准误表示, n=3。柱上相同字母表示差异未达0.01显著水平(t测验)。

Fig. 2. Resistance to white-backed planthoppers(WBPH) in D4 and Guanghui 998. A, Feeding preference of D4, Guanghui 998(GH998) and two control rice materials; B, Survival rates of D4, Guanghui 998(GH998) and two control rice materials. The error bars represent the SE(n=3). Bars superscripted by the same letter are not significantly different at the 0.01 level, by the Student’s t-test.

图3 广恢998/D4 F2:3株系接种南方水稻黑条矮缩病后的发病率频率分布

Fig. 3. Frequency distribution of the F2:3 lines derived from the cross between Guanghui 998(GH998) and D4 with southern rice black-streaked dwarf disease as determined by artificial inoculation identification.

表1 D4、广恢998(GH998)、抗池(R-pool)及感池(S-pool)测序结果数据统计

Table 1 Sequencing data statistics of D4, Guanghui 998(GH998), R-pool and S-pool.

样品Sample	Clean数据 Clean data/bp	HQ clean数据 HQ clean data/bp	平均测序深度 Average depth/×	Read总数 Total reads	比对read数 Mapped reads	覆盖度 Coverage/%	GC含量 GC content/%	Q30含量 Q30 content/%
D4	22 783 003 500	21 308 456 571	55.88	142 913 376	138 406 793	92.66	44.78	84.36
GH998	30 069 649 500	29 089 431 251	76.28	195 193 248	189 390 713	93.26	43.45	94.56
R-pool	30 313 518 000	29 287 082 085	76.80	196 696 800	190 730 909	93.65	43.18	94.87
S-pool	27 220 437 600	26 367 983 261	69.14	177 553 354	171 577 580	93.35	45.11	95.02

图4 单核苷酸多态性(SNP)指数增量在染色体上的分布横坐标表示染色体位置, 纵坐标表示SNP指数增量(突变型减去野生型)。图中每个点代表每个SNP指数增量, 相邻的染色体分别用橙色和绿色的点来表示, 黑线代表各个窗口SNP指数的平均值拟合线。蓝线代表95%置信水平的阈值线。

Fig. 4. Δ(SNP-index) value distribution in rice chromosomes. X-axis represents the chromosome position, and the y-axis represents the Δ(SNP-index) values. Dot on the map represents Δ(SNP-index) values, which were dyed orange and green of the neighboring chromosome, respectively. Black line represents the average SNP index values, as determined by sliding window analysis. Blue lines represents the threshold value(P<0.05).

表2 本研究中定位qSRBSDV9所用的InDel标记

Table 2 InDel primer used for mapping of qSRBSDV9 in this study.

引物 Primer	正向引物序列 Forward primer (5'-3')	反向引物序列 Reverse primer (5'-3')
Indel 4	TGAAACTGGACAAATTACCC	AGGAGTACATGACAAGTGGG
Indel 5	AAAATGAAACCCAACATGAC	CTTTATTTGCTTGCTCGATT
Indel 6	AACCAGATATCTTGACCCCT	GAACGAAGACTCGGTCAATA
Indel 7	AATTACGTTGTGGCAATACA	CGTAGCGATAAGAATGTGTG
Indel 18	AGTCCCATCCAAACATAGTG	CAGAGCAGGTTGTCAACAG
Indel 22	CTGTCAGACACGATCAGAGA	CATGCATGTCCTCTTGCT
Indel 23	TTCTGCACACAATCTTTTTG	AGCCATGCTAGCTAGTTCAC
Indel 29	ATCCTTTGCCATATCATCAG	AACGGTTGTACGAGTGAGAT
Indel 40	AAATCCACGCAGTTCAAG	ACTAGGACCTGATTCCCATT
Indel 43	AACCACTTGTCTTAGTCAAAAA	CACTGTTTGATCATTCGTCT
M1	CAAAATAAGTTGGTTTTGGC	GTGTTTCTCCTTAATCTGCG
M2	CTTCTCCGTCCAAACTACC	CCCCAGTAGAAGGTCATGT
M3	TAATTAGTGTGTTGCACCGA	AAGCTAAACAGCACCATCAT
M4	TGTGGGGTATCCACTAGTTC	AAGACGAGTGGTCAAACATT
M5	GATAGATATAACGCGAGGCA	ATATTCCCCTATTCCACACC
M6	AGCCAAAAACAACCTTACAA	AGCTCTCTGTCACTAGCTGC
M7	TACGGTTTTGTAAACCTGCT	TCGGATCTAACAGGTGGTAA
M8	AATGAATCAGCAGAAGGAAA	GGTAGTTGTGTCAAGGGCT
M9	ACATGTTACTGGGCCTAAAA	CCCTTGAAAATGACGATAAG
M10	ACTCTGGGAGTGGAGGAT	ACCTCACCTGTTCTGTGTTC
M11	CCGTTTTTCTTATTCCTACG	GAACATATGATTGGGTTGGT
M12	GAATCATCCACACACACAGA	GTTCACCTGCAGACTCTCTC
M13	TAACGGCTGTTTCTTTCTTC	TATCTACAGTTTCCCGCTGT
M14	TCTTTCCCTTCCTCTCTCTC	AAATATGCCACTCCAGAAAA
M15	GTCGGTGCTGGAGGAACT	CCGCCTTCTTCCTCGTTAT

图5 第9染色体上南方水稻黑条矮缩病主效抗性位点qSRBSDV9的验证及确认 A—连锁分析;B—利用代换作图精细定位qSRBSDV9。代换图中黑色条状区域代表来源于广恢998的导入片段; 空白条状区域代表来源于D4的导入片段;灰色条状区域代表杂合双亲基因的片段。

Fig. 5. Identification and validation of qSRBSDV9 in rice chromosome 9. A, Linkage analysis; B, Substitution mapping of qSRBSDV9 in an interval defined by InDel markers. Black bar, Homozygous segments from Guanghui 998(GH998); Blank bar, Homozygous segments from D4; Grey bar, Heterozygous segments.

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南方水稻黑条矮缩病抗性的遗传分析及主效QTL的精细定位

Genetic Analysis and Fine Mapping of a Major QTL for the Resistance to Southern Rice Black-Streaked Dwarf Disease

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