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
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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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