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作物学报 ›› 2020, Vol. 46 ›› Issue (10): 1639-1646.doi: 10.3724/SP.J.1006.2020.04056

• 研究简报 • 上一篇    

油菜抗咪唑啉酮类除草剂基因标记的开发与应用

胡茂龙1,2(), 程丽1, 郭月1, 龙卫华1, 高建芹1, 浦惠明1,*(), 张洁夫1,2, 陈松1   

  1. 1 江苏省农业科学院经济作物研究所 / 国家油料作物改良中心南京分中心 / 农业农村部长江下游棉花与油菜重点实验室/江苏省农业生物学重点实验室 / 江苏省现代作物生产协同创新中心, 江苏南京 210014
    2 江苏大学生命科学研究院, 江苏镇江 212023
  • 收稿日期:2020-03-02 接受日期:2020-06-02 出版日期:2020-10-12 网络出版日期:2020-06-14
  • 通讯作者: 浦惠明
  • 作者简介:E-mail: humolon@163.com, Tel: 025-84390372
  • 基金资助:
    国家重点研发计划项目(2016YFD0101300);国家自然科学基金项目(31671731);国家自然科学基金项目(31901503);国家现代农业产业技术体系建设专项(CARS-13);江苏省自然科学基金项目(BK20190267)

Development and application of the marker for imidazolinone-resistant gene in Brassica napus

HU Mao-Long1,2(), CHENG Li1, GUO Yue1, LONG Wei-Hua1, GAO Jian-Qin1, PU Hui-Ming1,*(), ZHANG Jie-Fu1,2, CHEN Song1   

  1. 1 Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Nanjing Sub-center, National Center of Oil Crops Improvement / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture and Rural Affairs / Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences / Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 210014, Jiangsu, China
    2 Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, Jiangsu, China
  • Received:2020-03-02 Accepted:2020-06-02 Published:2020-10-12 Published online:2020-06-14
  • Contact: Hui-Ming PU
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101300);National Natural Science Foundation of China(31671731);National Natural Science Foundation of China(31901503);China Agricultural Research System(CARS-13);Natural Science Foundation of Jiangsu Province(BK20190267)

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摘要：

草害已成为严重制约我国油菜生产的重要因素。种植抗除草剂品种和采用化学除草是防控草害的经济有效途径。为了开展分子标记辅助选择(marker-assisted selection, MAS)育种, 加速抗除草剂品种培育, 本研究针对已发现的抗咪唑啉酮类除草剂油菜M9中BnALS1R基因编码区第1913位点的SNP变异, 开发高通量、低成本的竞争性等位基因特异PCR (kompetitive allele specific PCR, KASP)标记。采用筛选出的KBA1R19681913B标记在2个F2群体中进行KASP反应。结果表明, 该标记能有效检测群体中存在的BnALS1R 3种基因型, 其分离比均为1︰2︰1, 遵循单基因遗传规律, 且基因型与表型完全吻合。将该标记用于BnALS1R抗性纯合基因的回交转育, 获得200多个抗咪唑啉酮油菜恢复系。该标记还可在油菜苗期鉴定抗性杂交种的纯度。KASP标记KBA1R19681913B的获得为油菜抗除草剂MAS育种以及抗性新种质的培育提供了技术支撑。

关键词: 草害, 油菜, 标记辅助选择, 咪唑啉酮类除草剂, KASP标记

Abstract:

Weed damage is one of the most important factors restricting the development of rapeseed production in China. Cultivation of herbicide-resistant varieties combined with chemical herbicides was an extremely effective help to control field weed in rapeseed production. To accelerate the breeding of cultivars with resistance imidazolinone herbicides through molecular marker-assisted selection in Brassica napus, the simplest, most cost-effective and high-throughput KASP markers were developed by using a SNP mutation at 1913 bp position of ALS (acetolactate synthase) gene in the mutant M9. A polymorphic marker designed KBA1R19681913B was obtained to effectively distinguish the homozygous resistant M9 from susceptible rapeseeds. Meanwhile, the KASP marker was evaluated in two F2 populations and could effectively discriminate three genotypes. F2 populations gave a good fit to the expected 1:2:1 ratio, confirming a single gene Mendel model with the perfect matched between phenotyping and genotyping. About more than two hundred homozygous restoring lines containing BnALS1R were developed through KASP marker selection in multi-generation backcross and showed stable imidazolinone herbicide resistance. This functional marker can also be used to identify the purity of hybrid seeds at the seedling stage. In conclusion, the validated KASP marker KBA1R19681913B would provide a technical support for developing herbicide-resistant rapeseed in marker-assisted selection and identification of resistant germplasm.

Key words: weed damage, rapeseed (Brassica napus L.), marker-assisted selection, imidazolinone herbicides, KASP marker

表1

抗性基因BnALS1R的KASP分子标记引物序列"

KASP名称KASP ID 引物Primer (5°-3°)
KBA1R19681913A X-allele: AACATGTGTTACCGATGATCCCAAG
Y-allele: GAACATGTGTTACCGATGATCCCAAA
Common: CTTAGTGCGACCATCCCCTTCT
KBA1R19681913B X-allele: TATTACATCTTTGAAAGTGCCACCAC
Y-allele: GTTATTACATCTTTGAAAGTGCCACCAT
Common: CAGGACCATACCTGTTGGATGTGATA

图1

油菜BnALS1与BnALS3基因序列比对与多态性引物位置 倒三角形表示突变位点。箭头线表示引物位置和扩增方向。"

图2

KASP标记KBA1R19681913B在小群体中分型图"

表2

KASP标记KALS1R19681913B在小群体中的分型结果与抗性表型"

材料
Material		 标记分型
Genotyping		 测序结果
Sequecing		 抗性特性
Resistance		 材料来源
Source
M9-2 A A R M9后代株系 M9 progeny line
M9-14 A A R M9后代株系 M9 progeny line
N341 G G S MICMS恢复系 MICMS restorer line
3075R G G S MICMS恢复系 MICMS restorer line
F1-2 G/A G/A R F1(N341×M9-2) F1 hybrid
F1-3 G/A G/A R F1(3075R×M9-14) F1 hybrid
M318-1 G G S F2(N341×M9-2)单株 F2 plant
M318-2 A A R F2(N341×M9-2)单株 F2 plant
M318-3 G G S F2(N341×M9-2)单株 F2 plant
M318-4 A A R F2(N341×M9-2)单株 F2 plant
M318-5 G/A G/A R F2(N341×M9-2)单株 F2 plant
M318-6 A A R F2(N341×M9-2)单株 F2 plant
M318-7 G/A G/A R F2(N341×M9-2)单株 F2 plant
M318-8 A A R F2(N341×M9-2)单株 F2 plant
M318-9 G/A G/A R F2(N341×M9-2)单株 F2 plant
M318-10 A A R F2(N341×M9-2)单株 F2 plant
M318-11 A A R F2(N341×M9-2)单株 F2 plant
M318-12 G/A G/A R F2(N341×M9-2)单株 F2 plant

图3

标记KBA1R19681913B在2个F2群体及亲本中的基因分型"

表3

标记在F2群体中基因型分型"

杂交组合
Cross		 基因型Genotype 总株数
Total		 卡平方值
χ2 -value		 P值
P- value
G/G G/A A/A
F2 (3075R/M9-14) 8 14 7 29 0.012 0.994
F2 (N341/M9-2) 11 23 16 50 0.085 0.958

表4

标记KBA1R19681913B在鉴定杂交种子纯度的应用"

授粉方式
Different pollination conditions		 除草剂处理
IMI treatment		 鉴定株数
Numbers of plants		 标记鉴定纯度
Seed purity
identification by the markers (%)		 田间鉴定纯度
Seed purity
identification in the field (%)		 杂种纯度提高
Increased seed purity after IMI treatment (%)
人工杂交授粉
Artificial hybridization		 喷药Treated 117 100 100 —
不喷药Untreated 90 100 100 —
网罩隔离授粉
Hybridization in tents		 喷药Treated 130 97 97 13
不喷药Untreated 194 84 84 —
天然授粉
Natural pollination		 喷药Treated 165 96 96 16
不喷药Untreated 155 80 80 —
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