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Application of short n import fragments in tobacco and its screening and estimation methods

A tobacco and fragment technology, applied in chemical instruments and methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of difficulty in estimating the number of gene components, inability to quickly evaluate plant yield, yellowing and drying of short N-introduced fragments. Problems such as improving the condition of roasting

Active Publication Date: 2021-07-30
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult for conventional breeding techniques to screen out chromosome-exchanged plants containing short N-introduced fragments from a large number of plants, and it is difficult to estimate the number of gene components missing from chromosome-exchanged plants with short N-introduced fragments. Improvement of traits such as yield, yellowing and roasting

Method used

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  • Application of short n import fragments in tobacco and its screening and estimation methods
  • Application of short n import fragments in tobacco and its screening and estimation methods
  • Application of short n import fragments in tobacco and its screening and estimation methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation of embodiment 1 plant material and DNA extraction

[0048] 1. Backcrossing of N import fragments

[0049] Coker176 (genotype NN) and 5F (genotype nn) were planted, Coker176 pollen was collected to pollinate emasculated 5F flowers during flowering, and F1 seeds (genotype Nn) were harvested. Plant F1 plants, collect F1 pollen to pollinate emasculated 5F flowers during flowering, and harvest BC1F1 seeds (genotype Nn:nn=1:1). Sow BC1F1, inoculate TMV at the seedling stage, and screen out TMV-resistant plants. When flowering, collect pollen and backcross with 5F to obtain backcross seeds. Through continuous backcrossing, the seeds of the fifth generation of backcrossing (BC5F1) were obtained.

[0050] 2. Screening of TMV-resistant plants

[0051] 4-5 sheets of tobacco seedlings from BC5F1 backcross populations planted in 32-hole trays, 1 plant per hole, were artificially inoculated with TMV, and 5-7 days after inoculation, it was investigated whether the in...

Embodiment 2

[0060] Example 2 Chromosomal Plant Screening of Short N Imported Fragments

[0061] Tobacco germplasm 5F susceptible to TMV was used as a disease-resistant control, and the tobacco variety Coker176 known to contain the N gene resistant to TMV was used as a disease-resistant control; the primer pair of TN5. Genomic DNA and the tobacco genomic DNA of the disease-resistant control were used as templates for PCR amplification, and the amplified products were detected by electrophoresis;

[0062] The PCR reaction system is as follows:

[0063]

[0064] The primer pair is TN5.51 primer pair.

[0065] All reagents used were purchased from Treasure Biotechnology Co., Ltd.

[0066] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and 35 cycles; final extension at 72°C for 10 minutes. PCR amplification products can be stored at 4°C.

[0067] Ele...

Embodiment 3

[0071] Example 3 Detection of the reduction in the number of non-target gene components linked to the N gene

[0072] Tobacco germplasm 5F that is sensitive to TMV is a disease-resistant control, and the tobacco variety Coker176 that is known to contain N gene resistance to TMV is a disease-resistant control; N1N2 positive and 3 plant DNAs without 845bp amplification products obtained in Example 2, The tobacco genomic DNA of the susceptible control and the tobacco genomic DNA of the disease-resistant control are used as templates for PCR amplification, and the amplified products are detected by electrophoresis;

[0073] The PCR reaction system is as follows:

[0074]

[0075]

[0076] The primer pair is TN5.51 primer pair, TN5.34 primer pair, TN5.20 primer pair and TN4.99 primer pair.

[0077] All reagents used were purchased from Treasure Biotechnology Co., Ltd.

[0078] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 9...

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Abstract

The embodiment of the present invention discloses the application of a short N-introduced fragment in tobacco and its screening and estimation method. Compared with the N-introduced fragment of Coker176 type tobacco, the short N-introduced fragment reduces the right end of the N gene by at least 0.93Mb, preferably at least 1.56 The redundant gene component of Mb, the tobacco plants containing this short N-introduced fragment are highly resistant to TMV, and have no obvious yield and quality disadvantages, and the traits such as yield, yellowing and curing have been improved to the greatest extent. The method for estimating the number of missing redundant gene components in tobacco plants containing short N-imported fragments provided by the present invention can quickly and accurately select the plant with the shortest N-introduced fragment from plants with multiple short N-introduced fragments, and select one N-introduced The plants with the shortest fragments enter the breeding program, which can significantly reduce the breeding workload and improve the breeding efficiency.

Description

technical field [0001] The invention relates to the technical field of tobacco breeding, in particular to the application of a short N import segment in tobacco and its screening and estimation method. Background technique [0002] Tobacco mosaic virus (TMV) is an important disease of tobacco in my country, and its annual loss ranks at the top of the list of top ten tobacco infectious diseases. Planting TMV-resistant varieties is still the most fundamental and cost-effective means of preventing and controlling TMV. The promotion of disease-resistant varieties requires varieties with high resistance and no yield and agronomic trait disadvantages. [0003] At present, the TMV resistance source of cultivated tobacco mainly comes from Nicotiana.glutinosa, a wild species of tobacco, and its resistance is controlled by a dominant single gene (N). The genome sequence size of N gene is 6656bp, including 5 exons and 4 introns, belonging to TIR-NBS-LRR type disease resistance gene. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/11
CPCC07K14/415C12N15/8283C12Q1/6895C12Q2600/13
Inventor 刘勇李永平黄昌军于海芹方敦煌肖炳光
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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