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Marker primer and detection kit for detecting SNP mutation of rape ALS gene and application thereof

A technology of detection kits and marker primers is applied in the field of molecular genetic breeding of crops, which can solve the problems of decreased resistance effect of materials, long years of resistance breeding and high production costs, and achieves elimination of aerosol pollution, high specificity and high cost. Effect

Pending Publication Date: 2019-06-14
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of breeding practice, due to the influence of various factors such as spraying period, spraying concentration, herbicide efficacy and ambient temperature, it is easy to lead to misselection.
In addition, DS3 contains two resistance gene loci, and the selection of resistant individual plants by spraying herbicides will also lead to the loss of a resistance gene locus, which will reduce the resistance effect of the material, and the resistance breeding period will be long , heavy workload, high production cost

Method used

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  • Marker primer and detection kit for detecting SNP mutation of rape ALS gene and application thereof
  • Marker primer and detection kit for detecting SNP mutation of rape ALS gene and application thereof
  • Marker primer and detection kit for detecting SNP mutation of rape ALS gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Design of SNP molecular markers for detection of mutations at the Pro182Leu site of rapeseed ALS1 gene

[0036] DS3 is a new germplasm of rapeseed that is highly resistant to sulfonylurea herbicides, which we created through two rounds of EMS mutagenesis and years of directional selection. ) (Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101, and the deposit number is CGMCC No. 14298). The germplasm has important application value in the breeding of herbicide-resistant rapeseed varieties, and has been introduced and applied by more than 20 rapeseed breeding units across the country for the breeding of herbicide-resistant rapeseed varieties. Through gene cloning and functional verification, it was found that there were two ALS gene mutations in DS3, namely Pro182Leu in the amino acid sequence of rapeseed ALS1 and Trp556Leu in the amino acid sequence of ALS3. In the process of breeding herbicide-resistant varieties usi...

Embodiment 2

[0046] Example 2: Establishment of KASP Molecular Marker Detection System for Pro182Leu Resistance Mutation Site of Rapeseed ALS1 Gene

[0047] Using the two sets of KASP primers designed above, select DS3 containing the resistance mutation site and its progeny derivatives, N131 and other rapeseed varieties (lines) without the resistance mutation site, and the F 1 A total of 30 rapeseed materials were generated, and the KASP reaction was verified on the LGCSNPline genotyping platform. The specific operation steps are as follows:

[0048] 1. Extraction of Rapeseed Genomic DNA:

[0049] Take an appropriate amount of leaf tissue of rape varieties (lines) to be tested, and use the CTAB method to extract whole-genome DNA (Murray MG, et al., Nucleic Acids Research, 1980, 8(19): 4321-4326). The quality of the extracted DNA was detected by agarose electrophoresis and Nanodrop2100 respectively, and it was found that the extracted genomic DNA all met the relevant quality requirements,...

Embodiment 3

[0082] Example 3: Validation of the detection effect of the molecular marker KBnALS1_1968545A in the isolated population

[0083] To verify the KASP molecular marker KBnALS1_1968545A in F 2 For the detection effect of the BnALS1 gene resistance mutation site (C / T) genotype of DS3 in the population, we used the MICMS double-low restorer lines N340 and N341 (Pu Huiming et al., Chinese Journal of Petroleum Science, 2011, 33 (1): 15-19) is the female parent, and the resistant germplasm DS3 is the male parent. Two hybrid combinations (N340×DS3 and N341×DS3) are arranged at the flowering stage of rape, F 1 Bagging selfing gets F 2 population, with the molecular marker KBnALS1_1968545A vs. F 2 The genotype of the BnALS1 gene resistance mutation site (C / T) of individual plants in the population was detected. Specific steps such as DNA extraction, PCR reaction, and KASP detection are the same as in Example 2 (using the detection kit of Example 2). Genotyping results showed that in ...

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Abstract

The invention discloses a marker primer and a detection kit for detecting SNP mutation of a rape ALS gene and application thereof. The marker primer can directly reflect the resistance of a plant, hasno misidentification due to genetic exchange, and can specifically distinguish and detect the C or T base of the SNP mutation site of the rape ALS1 gene. The marker primer only needs two steps of PCRand fluorescence detection, has low cost, high flux and high specificity, and is particularly suitable for classification screening and identification of different resistance genotypes in the breeding population. A molecular marking method completely eliminates aerosol pollution of a PCR product, environmental pollution of EB and harm of formaldehyde to the human body, and has the remarkable advantages of being simple, reliable, rapid, high in specificity and sensitivity and environmentally friendly. An SNP molecular marker application method is accurate and reliable, easy to operate and suitable for identification of the ALS1 resistance mutation site and assisted selection of breeding.

Description

technical field [0001] The invention relates to the technical field of crop molecular genetics and breeding, in particular to a labeled primer for detecting SNP mutations in rapeseed ALS genes, a detection kit and applications thereof. Background technique [0002] Weeds are one of the important biological disasters affecting rapeseed production. Due to the competition of weeds for water, fertilizer, air, heat and space, the growth of rapeseed reduces the rate of flowering and seed setting, and the yield loss is serious. According to statistics, the damage of weeds can reduce the yield of rapeseed by 15.8%, and the severe yield reduction can reach more than 50% (Guan Chunyun. Physiological Ecology and Modern Cultivation Techniques of High Quality Rapeseed. Beijing: China Agricultural Press, 2013: 127-135 ). There are many kinds of weeds in rapeseed fields in my country, large in number, and serious damage. The area of ​​weed damage in winter rapeseed area in the Yangtze Ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
Inventor 胡茂龙浦惠明龙卫华高建芹郭月程丽彭琦周晓婴张维张洁夫
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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