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

Active Publication Date: 2021-06-29
HUAZHI RICE BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the molecular markers published in the literature are SSR, Indel and CAPS markers, which need to be detected by gel electrophoresis. The reagents such as nucleic acid dyes used in the detection process are harmful to the environment and human body, and the degree of automation is low and the detection throughput is low. In the case of non-specific amplification, the detection results cannot be prepared and judged, which greatly limits the detection efficiency

Method used

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  • SNP molecular marker combination for detecting rice Wx gene and application of SNP molecular marker combination
  • SNP molecular marker combination for detecting rice Wx gene and application of SNP molecular marker combination
  • SNP molecular marker combination for detecting rice Wx gene and application of SNP molecular marker combination

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker combination for detecting a rice Wx gene, and the combination contains four molecular markers, namely Wx01, K_060585, K_060586 and K_060591; and wherein the polymorphism of the Wx01 is G / T, the polymorphism of the K_060585 is A / C, the polymorphism of the K_060586 is T / C, and the polymorphism of the K_060591 is C / A. By utilizing the molecular marker provided by the invention, the Wx gene can be efficiently detected, the molecular marker can be used for different haplotypes of the Wx gene and auxiliary selective breeding, the breeding efficiency can be improved, the breeding cost can be reduced, the breeding period can be shortened, targeted multi-gene polymerization can be carried out, the breeding efficiency can be improved, and huge social and economic benefits can be brought. The marker is used for genotyping by applying a KASP technology principle, enzyme digestion, electrophoresis, sequencing and the like are not needed, the operation is simple and convenient, and high-throughput rapid detection is facilitated.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and crop breeding, and specifically relates to a combination of SNP molecular markers for detecting rice Wx gene and its application. Background technique [0002] Amylose content is a key factor affecting rice quality. The rice Wx gene encodes granule-bound starch synthase (GBSS), which is the main gene controlling amylose synthesis and directly affects the amylose content in rice endosperm and pollen. Starch content (Amylose Content, AC). The non-waxy gene (Wx) was incompletely dominant to the waxy gene (wx), and there was a more obvious dosage effect. In non-waxy varieties, the Wx gene differentiates into Wx a and Wx b Two alleles, all of which are Wx in wild rice a , indica with Wx a Mainly, the amylose content is higher; japonica rice is basically Wx b , low amylose content. In addition to the common 3 allelic types, the Wx gene locus also has Wx op 、Wx mp 、Wx in 、Wx HH 、...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/156C12Q2600/172
Inventor 彭佩李文博吴云天唐顺学肖金华田冰川
Owner HUAZHI RICE BIO TECH CO LTD
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