SNP molecular marker combination for detecting rice Wx gene and application of SNP molecular marker combination
A molecular marker, rice technology, applied in the fields of molecular biology and crop breeding, can solve the problems of unprepared judgment of test results, environmental and human harm, and low degree of automation, and achieves high-throughput rapid detection, huge social economy. Benefit, select the effect of low efficiency
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Embodiment 1
[0063] The development of the SNP molecular marker of embodiment 1 Wx gene
[0064] Previous studies have shown that Wx has a variety of allelic forms. According to the published sequence of the article, the SNPs corresponding to different haplotypes were found. According to the literature information, the position of Wx was determined to be in the 1766194-1770656 interval of rice chromosome 6. Refer to the Nipponbare Genome (MSU7.0) position , to extract the flanking sequence of the mutated SNP site. For these candidate SNP markers, using the markers designed based on the KASP reaction principle and the single base difference of materials can perform high-throughput Wx gene detection on rice materials, and select SNPs that can distinguish different material types and have good amplification effects Mark 4 markers Wx, K_060591, K_060586 and K_060585, combined to distinguish different haplotypes of Wx gene. For the flow chart of the development of the above SNP molecular marke...
Embodiment 2
[0069] The detection of the KASP reaction of embodiment 2 SNP molecular marker
[0070] 1. Design the primer combination for the molecular marker combination described in the present invention, and use the KASP reaction to detect whether the rice variety contains the Wx gene with high throughput, and use the primer combination designed in Example 1.
[0071] 2. Genomic DNA was extracted from rice leaves by simplified CTAB method.
[0072] 3. KASP reaction test
[0073] KASP response tests were performed on the LGC SNPline genotyping platform. Add 20ng DNA sample into the microwell reaction plate, add KASP reaction mixture after drying, the reaction system is shown in Table 2. PCR amplification was completed in a water bath thermal cycler. The Touchdown PCR reaction conditions were: 94°C pre-denaturation for 15 minutes; the first amplification reaction, 94°C denaturation for 20 seconds, 65°C-57°C annealing and extension for 60 seconds, 10 Cycling, the annealing and extension...
Embodiment 3
[0076] Example 3 Natural Population Verification of SNP Molecular Markers
[0077] The KASP reaction verification was carried out with 96 rice varieties marked, and the typing map of the Wx01 molecular marker is shown in figure 2 , the typing diagram of K_060591 molecular marker is shown in image 3 , the typing diagram of K_060586 molecular marker is shown in Figure 4 , the typing diagram of K_060585 molecular marker is shown in Figure 5 , and the results are shown in Table 3. Except for materials with unclear typing, 84 rice accessions were classified into wx, Wx a 、Wx HH 、Wx in and Wx b Five haplotypes, most of which are Wx b type, the japonica rice materials are all of this type, which is consistent with the previous research results, and also shows that the combination of molecular markers in Example 1 can accurately detect the Wx genotype.
[0078] Table 3 Marker typing data of Wx
[0079]
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