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Molecular biology method for identifying purity of tobacco varieties

A molecular biology and variety purity technology, applied in the field of plant variety purity identification, can solve problems such as the inability to detect base differences, and achieve the effects of reducing production risks, not being affected by the environment, preventing and ensuring safety

Inactive Publication Date: 2016-07-06
HUBEI TOBACCO SCI RES INST
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AI Technical Summary

Problems solved by technology

The first three purity identification methods above belong to macroscopic phenotypic identification, and the accuracy of the results is greatly affected by environmental factors such as people, seasons, and climates. The fourth method belongs to microscopic internal genetic material identification, which is not affected by environmental factors and is accurate. The accuracy is greatly improved, but this method can only judge the variety difference and purity level based on the length of the DNA fragment amplified by PCR with molecular markers or whether there is a polymorphic difference, but it cannot detect the difference between the base as the smallest unit of genetic material In other words, varieties with individual base differences often cannot be detected by the above molecular markers. Therefore, it is very necessary to develop a method that can identify the purity of varieties at the level of the smallest genetic unit of bases

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the identification of the purity of flue-cured tobacco main variety Yunyan 87, its specific steps are as follows:

[0019] A. Sow the seeds of the control and the Yunyan 87 species to be tested in the marked nutrient pots respectively, sow 5 pots respectively, and place them on the light culture rack for cultivation. The auxiliary lights are designed to turn on the lights for 8 hours and turn off the lights for 16 hours within 24 hours. , keep the indoor temperature at 25~28℃, add water according to the dryness and wetness of the nutrient soil in the nutrient bowl, and generally observe it every 2~3 days;

[0020] B. After the tobacco seedlings grow 7-10 true leaves, randomly select the same number of tobacco seedlings of the control and Yunyan 87 varieties to be tested, 10 plants each, and 1-3 true leaves from each tobacco seedling, and use CTAB sample The total DNA of the genome was extracted and purified by the method, and the concentration of each DNA ...

example 2

[0024] The purity identification of example 2, Burley tobacco kind Burley37, its concrete steps are as follows successively:

[0025] A. Sow the seeds of the control and to-be-tested Burley37 varieties in marked nutrient bowls, respectively sow 7 bowls, and place them on the light culture rack for cultivation. The auxiliary lights are designed to turn on the lights for 10 hours and turn off the lights for 14 hours within 24 hours. The indoor temperature is kept at 25~28°C, and the water should be supplemented according to the dryness and wetness of the nutrient soil in the nutrient bowl. Generally, it should be observed every 2~3 days;

[0026] B. After the tobacco seedlings grow 7-10 true leaves, randomly select the same number of control and Burley37 varieties of tobacco seedlings to be inspected, 15 plants each, and collect 1-3 true leaves from each tobacco seedling, using the CTAB small sample method Extract and purify the total genome DNA, adjust the DNA concentration of ...

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Abstract

The invention relates to a molecular biology method for identifying purity of tobacco varieties. The method comprises the following main steps: respectively extracting total genome DNA of a control group and a to-be-detected tobacco variety, performing whole genome sequencing of proper depth by adopting the latest sequencing technology, performing sequence splicing, assembling and whole genome sequence comparison on the control group and the to-be-detected tobacco variety based on a tobacco genome reference sequence by utilizing bioinformatics means, counting base differences of the two groups, and calculating the purity percentage of the to-be-detected tobacco variety relative to the control tobacco variety. The method disclosed by the invention is not influenced by environmental conditions and seasons, is accurate and reliable in result, can accurately identify the purity of the tobacco varieties from a single base variation level of the smallest hereditary unit, can be used for parent purification and authenticity identification of the tobacco varieties and has great significances for guaranteeing safety of tobacco production varieties, maintaining economic benefits of tobacco growers and effectively solving intellectual property disputes of the tobacco varieties.

Description

technical field [0001] The invention relates to a method for identifying the purity of plant varieties, in particular to a molecular biology method for accurately identifying the purity of tobacco varieties by genome sequencing and sequence comparison, and belongs to the technical field of tobacco molecular biology. Background technique [0002] Variety purity (varietalpurity) refers to the degree of typical consistency between individuals in a batch of seeds (or improved breeding fields) in terms of characteristics and characteristics, that is, the number of seeds (strains and ears) of this variety accounts for The percentage of the number of crop (species) samples. The purity requirements stipulated in the Tobacco Seed Inspection Regulations of the Tobacco Industry of the People's Republic of China (YC / T20-1994), the original seed requires a purity of not less than 99.9%, the first-class improved variety is not less than 99.5%, and the second-grade improved variety is not ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869
Inventor 蔡长春
Owner HUBEI TOBACCO SCI RES INST
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