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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1612-1620.doi: 10.3724/SP.J.1006.2018.01612

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻稻瘟病抗性基因Bsr-d1功能标记的开发和利用

王军1,2,赵婕宇3,许扬1,2,范方军1,2,朱金燕1,2,李文奇1,2,王芳权1,2,费云燕1,仲维功1,杨杰1,2,*()   

  1. 1 江苏省农业科学院粮食作物研究所 / 国家水稻改良中心南京分中心 / 江苏省优质水稻工程技术研究中心, 江苏南京 210014
    2 扬州大学 / 江苏省粮食作物现代产业技术协同创新中心, 江苏扬州 225009
    3 扬州大学生物科学与技术学院, 江苏扬州 225009
  • 收稿日期:2018-03-28 接受日期:2018-07-20 出版日期:2018-11-12 网络出版日期:2018-07-25
  • 通讯作者: 杨杰
  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0100400-3);江苏省农业科学院探索性项目(ZX(17)2014);江苏省重点研发计划项目(BE2017368)

Development and Application of Functional Markers for Rice Blast Resistance Gene Bsr-d1 in Rice

Jun WANG1,2,Jie-Yu ZHAO3,Yang XU1,2,Fang-Jun FAN1,2,Jin-Yan ZHU1,2,Wen-Qi LI1,2,Fang-Quan WANG1,2,Yun-Yan FEI1,Wei-Gong ZHONG1,Jie YANG1,2,*()   

  1. 1 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Branch of Chinese National Center for Rice Improvement / Jiangsu High Quality Rice R&D Center, Nanjing 210014, Jiangsu, China;
    2 Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
    3 College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Jiangsu, China
  • Received:2018-03-28 Accepted:2018-07-20 Published:2018-11-12 Published online:2018-07-25
  • Contact: Jie YANG
  • Supported by:
    This study was supported by the National Key Research and Development Program(2017YFD0100400-3);the Exploratory Project of the Jiangsu Academy of Agricultural Sciences(ZX(17)2014);Jiangsu Province Key Research and Development Program Project(BE2017368)

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摘要：

稻瘟病是危害水稻最严重的病害之一, 选育抗病品种是防治该病害最有效的方法。Bsr-d1是对稻瘟病菌具有广谱抗性的一个重要基因。为提高Bsr-d1基因在育种中的选择效率, 根据Bsr-d1与其感病等位基因bsr-d1在功能区域存在的单核苷酸差异, 设计和筛选出Bsr-d1基因不同类型的基因功能标记CAPs5-1和3Bsr-d1/3bsr-d1, 结合测序分析验证, 可准确鉴定出Bsr-d1的不同基因型。利用3Bsr-d1/3bsr-d1对34份籼稻品种、江苏历年来主要推广的110份粳稻品种、其他省份的13份粳稻品种、148份太湖流域地方粳稻资源和19份太湖流域地方籼稻资源进行了Bsr-d1基因型检测, 筛选到携带Bsr-d1基因的籼型资源11份, 271份粳型资源中均不携带Bsr-d1基因, 这也说明Bsr-d1主要分布在籼型水稻资源中, 在粳型资源中几乎不存在。本研究为Bsr-d1基因的育种利用和分子标记辅助选择奠定了基础。

关键词: 水稻, 稻瘟病, Bsr-d1, 功能标记, CAPs, 等位基因特异PCR

Abstract:

Rice blast is one of the most serious rice diseases, breeding resistant cultivars is the most effective way to prevent this disease. Bsr-d1 is one vital non race-specific resistance gene for rice blast disease. To improve the selection efficiency of rice blast resistance gene Bsr-d1 in breeding, we selected different types of gene marker, CAPs5-1 and 3Bsr-d1/3bsr-d1, based on the single nucleotide polymorphism in the functional region of alleles Bsr-d1/bsr-d1. Furtherly confirmed by sequencing, we found both CAPs5-1 and 3Bsr-d1/3bsr-d1 could obviously distinguish different genetypes of Bsr-d1 locus. 3Bsr-d1/3bsr-d1 was employed to genotype 34 indica rice varieties, 110 japonica varieties certificated in Jiangsu Province, 13 japonica varieties certificated in different provinces, 148 local japonica cultivars and 19 local indica cultivars from Taihu Basin. There were only 11 indica varieties carrying Bsr-d1 gene, while Bsr-d1 gene was not detected in 271 japonica rice samples, indicating that Bsr-d1 mainly exists in indica germplasm, and is rarely found in japonica rice. This study facilitates the use of Bsr-d1 gene in rice blast resistance breeding and marker assisted selection.

Key words: rice (Oryza sativa L.), rice blast, Bsr-d1, functional marker, CAPs, allele specific PCR

表1

基于变异位点设计的功能标记和测序引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 片段长度
Expected size (bp)
CAPs1F AGTCTAGCATCCACCGTTCCAC 313
CAPs1R GTAGGCAGGCAGTGGGATGA
CAPs2F TTTTATAGGACAGAGGGAATATGTA 368
CAPs2R GCAGTGGGATGAACCTGTAC
Bsr-d1-F AGTCTAGCATCCACCGTTCCAC
1Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAGC 241
1bsr-d1-R CTTTTCGCTTATACTTATATTTATCAGT 241
2Bsr-d1-R CTTTTCGCTTATACTTATATTTATCgGC 241
2bsr-d1-R CTTTTCGCTTATACTTATATTTATCgGT 241
3Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAaC 241
3Bsr-d1-R CTTTTCGCTTATACTTATATTTATCAaT 241
C1F AGTCTAGCATCCACCGTTCCAC 755
C1R ATGATTTGATGGGATTGATTGC
C2F TTTTATAGGACAGAGGGAATATGTA 983
C2R GCGAGGTACTCCTCCTTGTTGAT

图1

2对引物梯度PCR扩增产物在1.5%的琼脂糖胶电泳结果M: DL2000; 1~12退火温度分别为: 45.1°C, 45.3°C, 45.9°C, 46.8°C, 47.9°C, 49.1°C, 50.4°C, 51.7°C, 52.8°C, 53.8°C, 54.5°C, 54.8°C。"

图2

CAPs5-1 PCR扩增产物和酶切结果M左侧为PCR扩增产物; M右侧为酶切后的产物; M: DL2000; 1~6: 地谷, Nipponbare, 南粳9108, 广陆矮4号,武育粳3号, 南粳45。"

图3

部分水稻品种Bsr-d1基因位点功能区的序列比对结果"

图4

CAPs5-1对部分水稻品种的分子检测结果M: DL2000; 1~24: 地谷, Nipponbare, 南京1号, 绵恢501, 浙恢7954, 盐恢559, 镇恢084, BG90-2, 圭630, 明恢63, 青四矮16B, 辐恢718, 扬稻6号, 冈46B, 陆财号, 明恢78, IRBB21, 蜀恢527, 多恢1号, 桂朝2号, 绵恢725, 乐恢188, 广恢128, 珍汕97B。"

图5

Bsr-d1基因等位基因特异标记的梯度PCR扩增结果 M: DL2000; A: 1Bsr-d1引物梯度PCR扩增产物; B: 1bsr-d1引物梯度PCR扩增产物; C: 2Bsr-d1引物梯度PCR扩增产物; D: 2bsr-d1引物梯度PCR扩增产物; E: 3Bsr-d1引物梯度PCR扩增产物; F: 3bsr-d1引物梯度PCR扩增产物; 1~12为地谷的不同引物对在退火温度分别为45.1°C、45.3°C、45.9°C、46.8°C、47.9°C、49.1°C、50.4°C、51.7°C、52.8°C、53.8°C、54.5°C、54.8°C时的扩增产物; 13~24为Nipponbare的不同引物对在退火温度分别为45.1°C、45.3°C、45.9°C、46.8°C、47.9°C、49.1°C、50.4°C、51.7°C、52.8°C、53.8°C、54.5°C、54.8°C时的扩增产物。"

图6

Bsr-d1基因等位基因特异标记3Bsr-d1和3bsr-d1扩增结果M: DL2000; 1~6: 地谷, Nipponbare, 南粳9108, 广陆矮4号, 武育粳3号, 南粳45。"

图7

3Bsr-d1和3bsr-d对部分水稻品种的分子检测结果 M: DL2000; 1~24: 地谷, Nipponbare, 南京1号, 绵恢501, 浙恢7954, 盐恢559, 镇恢084, BG90-2, 圭630, 明恢63, 青四矮16B, 辐恢718, 扬稻6号, 冈46B, 陆财号, 明恢78, IRBB21, 蜀恢527, 多恢1号, 桂朝2号, 绵恢725, 乐恢188, 广恢128, 珍汕97B。"

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