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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 problems such as time-consuming, complicated identification methods, quantitative detection of the purity of Nanjihong No. 6, etc.

Pending Publication Date: 2022-04-12
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Generally, the identification of Jihong No. 6 is mainly through methods such as smelling the fragrance and chewing, but these methods mainly rely on human senses to determine the grain shape and the strength of the fragrance, and the accuracy is poor, and it is even more difficult to realize the quantitative detection of the purity of Jihong No. 6.
The identification of rice varieties can be carried out by standard NY / T 1433-2014 rice variety identification technical regulations SSR markers, or NY / T2745-2015 rice variety identification SNP marker method, but these two identification methods are not only cumbersome and time-consuming, but also 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

[0078] Example 1: Materials and methods

[0079] 1. Materials

[0080] Jihong 6-rice seeds, seeds of other rice varieties and commercially available rice samples were all provided by Wilmar (Shanghai) Biotechnology R&D Center Co., Ltd.

[0081] 2. Enzymes and Reagents

[0082] 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 .

[0083] 3. Experimental method

[0084] 3.1 DNA extraction from rice seeds (rice samples)

[0085] 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 (12000rpm...

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 primer pairs JH6-C4-3F and JH6-C4-3R were detected by HRM high-resolution melting curve method for real-time fluorescent quantitative PCR amplification. The PCR reaction system is 20μL, in which SsoAdvancedTM Green Supermix 10μL, JH6-C4-3F and JH6-C4-3R 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 for HRM h...

Embodiment 3

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

[0117] Using the extracted DNA as a template, use Jihong No. 6 probe method to detect primers: JH6-3F3 and JH6-C4-3R, Jihong No. 6 specific probe: JH6H-23VIC-P3, and Feijihong No. 6 specific probe FJH6H- FAM-P1, and endogenous reference gene PLD primers PLD-F and PLD-R, endogenous reference gene PLD probe PLD-CY5-P for real-time fluorescent PCR detection. The PCR reaction system was 20 μL, including 10 μL of 2×GoldStar Best MasterMix, 1 μL of JH6-3F3 and JH6-C4-3R primers (concentration of 10 μM), 0.4 μL of each of PLD-F and PLD-R primers (concentration of 10 μM), JH6H- 23 VIC-P3 and FJH6H-FAM-P1 probes (concentration: 10 μM) each 0.25 μL, PLD-CY5-P 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-s...

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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 35th basic group from the 5'terminal of the nucleotide sequence shown in SEQ ID NO: 1, or the SNP marker is A or G of the 46th basic group from the 5 'terminal 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] Jihong No. 6 is a new rice variety bred by Jilin Province Quanhe Seed Industry Co., Ltd. in 2004 through sexual crossing and backcrossing with Jiyujing as the female parent and Nongda 3 and Xiangdao as the male parent. In January 2014, it was approved by the Jilin Provincial Crop Variety Approval Committee, and the approval number is Jishendao 2014019. [0003] Jihong No. 6 is a rice variety with excellent yield, quality, and resistance currently produced in the middle-maturing rice area of ​​Jilin Province. It has a very high innovation value. It has obvious advantages in terms of sex and other aspects. It has a pure rice fragrance taste, and its viscoelasticity and retrogradability are significantly higher than Jihong No. 6. It is currently the first new high-quality round-grain rice variety with aroma approved by Jilin Province. The breeding of Jihon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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