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Primer group, method and kit for identifying rice Badh2 genotype

A primer set and genotyping technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as experimental failure, high requirements for amplification conditions, and high labor intensity, and achieve the goal of using convenient effect

Pending Publication Date: 2022-05-13
贵州省水稻研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because this method uses four primers to amplify simultaneously in the same reaction, the labor intensity is high, and the requirements for the amplification conditions are high, such as the concentration of the primers, the annealing temperature and other conditions need to be accurately controlled, otherwise the experiment may fail (i.e. no amplified band)

Method used

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  • Primer group, method and kit for identifying rice Badh2 genotype
  • Primer group, method and kit for identifying rice Badh2 genotype
  • Primer group, method and kit for identifying rice Badh2 genotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] (1) Using the CTAB method to extract the DNA of rice:

[0069] A. After grinding 0.2g leaves with liquid nitrogen, add 500μl CTAB extract, and bathe in water at 65°C for 30min;

[0070] B. After cooling, add an equal volume of chloroform (500 μl), invert and mix well and let stand for 5 minutes;

[0071] C. Centrifuge at 12000rpm for 10min, take 300μl of the supernatant into a new centrifuge tube;

[0072] D. Add an equal volume of isopropanol (300 μl) and mix it upside down, and let it stand at room temperature for 30 minutes;

[0073] E. Centrifuge at 12000rpm for 10min, discard the supernatant, add 1ml of 70% ethanol to rinse twice;

[0074] F. After drying, add 50μl ddH 2 O dissolved;

[0075] (2) PCR reaction

[0076] The PCR reaction system (20μL) is configured as follows

[0077]

[0078]

[0079] PCR reaction program: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 40s, a total of 30 cycles; 72°C for 10min.

[0080] (3) Electrophoresis detection...

Embodiment 2

[0088] This embodiment uses the upstream primer exon7-2-F and the downstream primer intron7R, INTRON8R1 or INTRON8R2 to identify the aroma type of rice.

[0089] Sample extraction and PCR procedures are the same as in Example 1. The results are shown in image 3 , where, from the left, spotting well 1 is a marker (DL2000, the bands from bottom to top are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp), spotting well 2 is a negative control, spotting well 3 and The primer for 4 is exon7-2-F+intron7R, the primers for wells 5 and 6 are exon7-2-F+INTRON8R1, and the primers for wells 7 and 8 are exon7-2-F+INTRON8R2. The templates for spotting wells 3, 5, and 7 were from aromatic rice, and the templates for spotting wells 4, 6, and 8 were from non-aromatic rice.

Embodiment 3

[0091] This example examines the amplification results of the upstream primer exon7-2-F and the downstream primer Xiang7R at different annealing temperatures. Except for the annealing temperature, other conditions are the same as in Example 1.

[0092] Figure 4 and Figure 5 The amplification conditions of the upstream primer exon7-2-F and the downstream primer Xiang7R provided in this application at different annealing temperatures are shown. Figure 4 : The amplification template comes from indica rice QR35 (non-fragrant type), Figure 5 : The amplified template comes from the japonica rice Ningjing 7 (non-fragrance type). Figure 4 and Figure 5 In the middle, from the left, well 1 is a marker (DL2000, the bands from bottom to top are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp), the PCR amplification product is about 280bp, and the position is between marker 250-500bp The PCR annealing temperatures of wells 2-9 were 50°C, 50.7°C, 52°C, 53.9°C, 56.3°C, 58.3°C, 59.4°C, ...

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Abstract

The invention relates to a primer group for identifying the genotype of rice Badh2. The primer group comprises (1) an upstream primer exon7-2-F and downstream primers which are optionally selected from the following primers: Xiang7R, intron7R, INTRON8R1 or INTRON8R2; or (2) a primer group of BADH2G-F, BADH2G-1R and badh2G-2R, or (2) a primer group of BADH2G-F, BADH2G-1R and The specific sequence of the primer is defined in the specification. The invention also relates to a method and a kit for identifying the rice Badh2 genotype.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a primer set, a method and a kit for identifying two genotypes of the rice Badh2 gene. Background technique [0002] Along with the raising of living standard, people are more and more higher to the requirement of rice eating quality and local flavor (mouthfeel and fragrance). Fragrant rice is deeply loved by consumers because of its strong aroma and fragrant rice (Bradbury et al., 2005; Myint et al., 2012). [0003] The conventional method of identifying fragrant rice is to smell the leaves by soaking in KOH, or to chew the rice to determine the aroma. In the process of breeding fragrant rice, the number of offspring is large, and the workload is heavy. [0004] Previous studies on the genetics of rice aroma suggested that rice aroma is controlled by a single recessive gene. Fine mapping and map-based cloning studies have shown that the betaine aldehyde...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2600/13C12Q2531/113C12Q2565/125
Inventor 王忠妮王倩吴娴朱速松
Owner 贵州省水稻研究所
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