A SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application

A disease resistance and virus disease technology, applied in the field of agricultural biology, can solve the problems of inability to achieve high-throughput large sample detection, high DNA quality requirements, unstable reaction conditions, etc., and the requirements for sample quality and reaction conditions are not strict. Harshness, shorten the identification time, improve the effect of breeding efficiency

Active Publication Date: 2022-07-01
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, CAPS-type markers often need to be combined with PCR amplification, restriction endonuclease digestion and gel electrophoresis, which not only requires high DNA quality, but also often reduces the accuracy due to unstable reaction conditions, and cannot achieve high-throughput, Detection of large samples
[0005] In the face of the above problems, KASP (KompetitiveAlleleSpecific PCR, competitive allele-specific PCR) typing technology can make accurate biallelic judgments for SNPs and InDels at specific sites in the genome, with high sensitivity and high throughput , low cost, fast, etc., but there is no SNP molecular marker specifically targeting the pepper TSWV resistance gene Tsw based on KASP typing technology.

Method used

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  • A SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application
  • A SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application
  • A SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Acquisition of Tsw-related SNP loci of pepper TSWV disease resistance gene and development of high-throughput KASP marker

[0041] 1. Test material

[0042] Susceptible materials: 0516 (CK-negative), eggplant door (202011C29);

[0043] Disease-resistant materials: PI152225 (CK-positive), 0516tsw (202011C35).

[0044] 2. Genomic DNA Extraction

[0045] Genome DNA was extracted from young leaves of pepper by CTAB method. For DNA extraction methods see Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res 8:4321-4325.

[0046] 3. Acquisition of Tsw-related SNP loci of pepper TSWV disease resistance gene

[0047] Moury et al. (Moury B, Pflieger S, Blattes A, Lefebvre V, Palloix A. 2000. A CAPSmarker to assist selection of Tomato spotted wilt virus (TSWV) resistance inpepper. Genome 43, 137–142.) developed a pair of resistance to Tsw Genetically tightly linked SCAR / CAPS marker SCAC 568 , but only the traditional C...

Embodiment 2

[0063] Example 2 Method for detecting pepper TSWV resistance gene Tsw using high-throughput KASP marker Pep(Tsw)-k497

[0064] 1. PCR amplification

[0065] The young leaves of pepper to be tested were taken as samples, and the genomic DNA was extracted from the samples by CTAB method. The above primers 1, 2 and 3 were used to pair susceptible material 0516, eggplant and disease-resistant materials PI152225, 0516tsw and their two F 1 Substitute samples for PCR amplification, and the reaction system is carried out in a 96-well plate.

[0066] PCR reaction system for KASP genotyping used for amplification (10 μl system):

[0067] Table 1 Reaction system

[0068] Element Dosage KASP V4.0 2×Master Mix 5μl KASP 72×assay mix 0.14μl genomic DNA 10ng ddH 2 O

Add to the total amount of the system to 10ul

[0069] Among them, KASP V4.0 2×Master Mix consists of fluorescent probe A, fluorescent probe B, quenching probe A and quenching pr...

Embodiment 3

[0084] Example 3 Application of high-throughput KASP marker in backcross breeding of pepper TSWV disease resistance gene Tsw

[0085] 1. Materials to be tested:

[0086] 6 pepper hybrid populations including 3 BCs 3 F 2 Population (3 generations of backcrossing, 2 generations of selfing), 3 BCs 4 colonies, each colony contains 14 to 16 individual plants (see Table 2 for details). Population construction was based on three pepper inbred lines (respectively, Lujiao M, Zhu Dachang, 0818) as recurrent parents after crossing with TSWV disease-resistant material 0516Tsw, using the recurrent parents for backcrossing for 3 to 4 generations, and then selfing for 0 to 2 generations. obtained on behalf of.

[0087] With reference to the method in Example 2, use high-throughput KASP marker to identify the genotype of pepper Tswv resistance gene Tsw on individual plants in the population (the results are shown in Table 2 and Table 2). image 3 ).

[0088] Table 2 Genotype detection r...

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Abstract

The invention provides a SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application, and the nucleotide sequence of the SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease is as shown in SEQ ID As shown in NO.1, the SNP marker is the 21st base of the sequence shown in SEQ ID NO.1. The present invention also provides specific primers and corresponding kits for identifying the SNP marker, and provides a method for identifying peppers against tomato spotted wilt virus disease using the specific primers or kits. Using the SNP molecular marker of the present invention, the TSWV disease resistance gene of pepper can be quickly and accurately judged without the need for artificial inoculation identification of pepper TSWV disease resistance. Samples are tested in large quantities to improve breeding efficiency and shorten identification time.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a SNP marker related to the resistance gene of pepper tomato spotted wilt virus disease and its specific primer and application. Background technique [0002] Tomato spotted wiltvirus (TSWV) is a typical member of the genus Tospovirus in the family Bunyaviridae. It is an emerging major threat to pepper production. It has strong host adaptability and high transmission efficiency. In recent years, there has been a trend of gradual expansion and popularity in peppers. TSWV can cause plant necrosis, verticillium, and spots on leaves, stems, and fruits. At present, TSWV has been found in vegetable producing areas in 14 regions of my country, and it has become the main virus causing the production of peppers in some areas of my country. [0003] Breeding of disease-resistant varieties is the most economical and effective way to prevent and control virus diseases. This mainl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2600/172C12Q2531/113C12Q2563/107
Inventor 于海龙王立浩张宝玺张正海曹亚从冯锡刚
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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