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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (10): 1639-1646.doi: 10.3724/SP.J.1006.2020.04056

• RESEARCH NOTES • Previous Articles    

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 Online:2020-10-12 Published:2020-06-14
  • Contact: Hui-Ming PU E-mail:humolon@163.com;puhuiming@126.com
  • 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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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

Table 1

KASP primers of the herbicide-resistant gene BnALS1R"

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

Fig. 1

Sequencing alignment of BnALS1 and BnALS3 genes and loci of polymorphic primers in rapeseed The single-site mutation is denoted by inverted triangle. The arrow lines represent primer sites and amplification directions."

Fig. 2

Allelic discrimination plot of KASP KBA1R19681913B genotyping in the small population"

Table 2

KASP KBA1R19681913B genotyping of the small population and the corresponding phenotypes"

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

Fig. 3

Allelic discrimination plot of KASP KBA1R19681913B genotyping in two F2 populations and their parents"

Table 3

Genotype detection of two F2 populations with the marker"

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

Table 4

Application of the marker KBA1R19681913B in the purity identification of hybrid rapeseed"

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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