SNP marker related to pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of SNP marker

A disease-resistant and specific technology, applied in the field of agricultural biology, can solve the problems of high-throughput and large-sample detection, high DNA quality requirements, unstable reaction conditions, etc., and achieve sample quality and reaction conditions. Harshness, shorten identification time, improve breeding efficiency

Active Publication Date: 2020-11-20
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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  • SNP marker related to pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of SNP marker
  • SNP marker related to pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of SNP marker
  • SNP marker related to pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of SNP marker

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 wxya 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 p...

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 to backcross for 3-4 generations, and then selfing 0-2 generations. obtained on behalf of.

[0087] With reference to the method in Example 2, the high-throughput KASP marker was used to identify the genotype of the pepper Tswv resistance gene Tsw for 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 an SNP marker related to a pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of the SNP marker. A nucleotide sequence of the SNP marker related to the pepper and tomato spotted wilt virus disease resistance gene is as shown in SEQ ID NO.1, and the SNP marker is a 21st basic group of the sequence as shown in SEQ ID NO.1. Theinvention also provides the specific primers and a corresponding test kit for identifying the SNP marker, and provides an identification method for pepper resisting the tomato spotted wilt virus disease by using the specific primers or the test kit. By adopting the SNP molecular marker disclosed by the invention, the pepper TSWV (Tomato Spotted Wilt Virus) disease resistance gene can be quickly and accurately judged only by carrying out DNA detection on the pepper plant in a seedling stage without artificial inoculation identification of pepper TSWV disease resistance, and large-batch detection can be carried out on samples, so that the breeding efficiency is improved, and the identification time is shortened.

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, and 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 control virus diseases. This mainly depends o...

Claims

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Application Information

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Patent Type & Authority Applications(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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