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Rice SNP (Single Nucleotide Polymorphism) marker and application thereof

A marker, rice technology, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc., can solve the problems of quantitative judgment of adulteration, prone to misjudgment, poor accuracy, etc.

Pending Publication Date: 2022-04-15
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method mainly relies on human senses to judge, and the accuracy of the results depends to a large extent on the knowledge level and experience of personnel, which is prone to misjudgment
Moreover, the accuracy of judging by the appearance and aroma is poor, and it is even more difficult to realize the quantitative detection of adulteration of Wuyoudao No. 4
Furthermore, grain type is related to maturity, and the aroma of rice is affected by external environmental factors such as climate in different years. Therefore, there are certain limitations in the detection of adulteration of Wuyoudao No. 4 through sensory organs.
[0005] The identification of rice varieties can be carried out through the standard NY / T 1433-2014 rice variety identification technical regulations SSR markers, or NY / T 2745-2015 rice variety identification SNP marker method, but these two identification methods are not only cumbersome and time-consuming, Moreover, it can only be judged whether it is consistent with the original species, and cannot be quantitatively judged for adulteration

Method used

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  • Rice SNP (Single Nucleotide Polymorphism) marker and application thereof
  • Rice SNP (Single Nucleotide Polymorphism) marker and application thereof
  • Rice SNP (Single Nucleotide Polymorphism) marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Materials and methods

[0083] 1. Materials

[0084] Wuyoudao 4-rice seeds, seeds of other rice varieties and commercially available rice samples were all provided by Wilmar (Shanghai) Biotechnology R&D Center Co., Ltd.

[0085] 2. Enzymes and Reagents

[0086] The enzymes were purchased from Kangwei Century Company and Bio-Rad Company, the reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., and the fluorescent quantitative PCR instrument Bio-Rad CFX96; the primers and probes used in the experiment were synthesized by Shanghai Sangon Bioengineering Company .

[0087] 3. Experimental method

[0088] 3.1 DNA extraction from rice seeds (rice samples)

[0089] Grind 20 grams of rice seeds (rice samples) with a grinder, weigh 50 mg of powder into a 2 mL sample lysis tube, add 500 μL of Buffer 1, vortex for 30 seconds, incubate at 52°C for 30 minutes at 1200 rpm; add 500 μL Buffer2, vortex and mix for 30s, centrifuge the mixture for 5min (12000r...

Embodiment 2

[0112] Embodiment 2: HRM high-resolution melting curve method real-time fluorescent quantitative PCR detection

[0113] Using the extracted DNA as a template, the HRM high-resolution melting curve method was used to detect the primer pairs DHX-HRM-F1 and DHX-HRM-R1 for real-time fluorescent quantitative PCR amplification. The PCR reaction system is 20μL, in which SsoAdvancedTM Green Supermix 10μL, DHX-HRM-F1 and DHX-HRM-R1 primers (concentration: 10μM) 0.5μL each, template DNA (concentration: 50-100ng / μL) 2μL, sterile water to make up to 20μL. In the blank control, sterile water was used instead of template DNA. Each reaction was repeated three times, and the PCR amplification program used a two-step method: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute, a total of 45 cycles.

[0114] After the real-time fluorescent PCR amplification is completed, the amplified product is directly applied to Bio-Rad...

Embodiment 3

[0116] Embodiment 3: Probe method real-time fluorescence quantitative PCR detects

[0117] Using the extracted DNA as a template, use the Wuyoudao No. 4 probe method to detect the primers. Real-time fluorescent PCR detection was carried out with non-Wuyoudao No. 4 specific probe FDHX-MGB-FAM-P3 and endogenous reference gene DHX-Refgene-cy5-P4 probe. The PCR reaction system was 20 μL, including 10 μL of 2×GoldStar Best MasterMix, 0.8 μL of DHX1-wholeF and DHX-HRM-R1 primers (10 μM concentration), 0.25 μL each of DHX-MGB-VIC-P2 probe (10 μM concentration), FDHX-MGB-FAM-P3 probe (concentration: 10 μM) 0.125 μL each, DHX-Refgene-cy5-P4 probe (concentration: 10 μM) 0.2 μL, template DNA 2 μL, sterile water to make up to 20 μL. In the blank control, sterile water was used instead of template DNA. Each reaction was repeated three times, and the PCR amplification program used a two-step method: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s; annealing and extensio...

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker and application thereof. Wherein the SNP marker is T or C of the 23rd basic group from the 5'end of the nucleotide sequence shown in SEQ ID NO: 1, or the SNP marker is A or G of the 43rd basic group from the 5 'end of the nucleotide sequence shown in SEQ ID NO: 1. The SNP marker is closely related to the rice variety and can be effectively used for qualitatively or quantitatively detecting the rice variety.

Description

technical field [0001] The present invention relates to SNP markers and their applications. Background technique [0002] Daohuaxiang 2 was bred by breeder Tian Yongtai from Wuyoudao 1 in the autumn of 2000. This variety has a late maturity period and low yield, but has high economic benefits. It has been planted on a large scale in Wuchang City since 2004. It is officially named "Wuyoudao No. 4", which is the representative of Wuchang rice. Daohuaxiang No. 2 not only has a pleasant aroma after threshing, but also can smell the fragrance of leaves and rice when the rice is growing in the field. [0003] Wuyoudao No. 4 is of good quality and rich in nutrition. The steamed rice is full of fragrance, and the rice grains are crystal clear like jade. It is known as "a century-old tribute rice" and is favored by consumers in the market. However, Wuyoudao No. 4 has low yield, low rice yield and high price. Therefore, there is a situation in the market where other rices with simi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
Inventor 肖玲杨华封莉牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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