SNP (Single Nucleotide Polymorphism) marker for oryza sativa L.

A kind of Euris, rice technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problem of difficult to achieve quantitative detection of pure purity of wild fragrance Euris, poor accuracy, etc.

Pending Publication Date: 2022-06-24
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 Wild Fragrance Ulis is mainly through the methods of smelling fragrance and taste and chewing, but these methods mainly rely on human senses to determine the grain shape and the strength of fragrance, and the accuracy is poor, and it is even more difficult to achieve the purity of Wild Fragrance Ulis. Quantitative detection

Method used

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  • SNP (Single Nucleotide Polymorphism) marker for oryza sativa L.
  • SNP (Single Nucleotide Polymorphism) marker for oryza sativa L.
  • SNP (Single Nucleotide Polymorphism) marker for oryza sativa L.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Materials and Methods

[0077] 1. Materials

[0078] Noka Youris-rice seeds, seeds of other rice varieties, and rice samples were all commercially available.

[0079] 2. Enzymes and Reagents

[0080] 2×GoldStar Best MasterMix enzymes were purchased from Kangwei Century Company, SsoAdvancedTM Green Supermix enzyme was purchased from Bio-Rad Company, reagents were purchased from Sinopharm Chemical Reagent Co., Ltd., fluorescence quantitative PCR instrument Bio-Rad CFX96; primers and probes used in the experiment were synthesized by Shanghai Sangon Bioengineering Company.

[0081] 3. Experimental method

[0082] 3.1 DNA extraction from rice seeds (rice samples)

[0083] Use a grinder to grind 20 grams of rice seeds (rice sample), weigh 50 mg of the powder into a 2 mL sample lysis tube, and add 500 μL of Buffer 1 (0.4M NaCl, 0.1% SDS, 0.1M Tris, 0.5% high temperature amylase (purchased). from Shandong Longda Bioengineering Co., Ltd.), pH 8.0), vortex and mi...

Embodiment 2

[0109] Example 2: Real-time quantitative PCR detection by HRM high-resolution melting curve method

[0110] Using the extracted DNA as a template, the primer pairs YXYLS-HRM-F and YXYLS-HRM-R were detected by HRM high-resolution melting curve method for real-time quantitative PCR amplification. The PCR reaction system is 20 μL, in which SsoAdvancedTM Green Supermix 10μL, YXYLS-HRM-F and YXYLS-HRM-R primers (concentration of 10μM) each 0.5μL, template DNA (concentration of 50-100ng / μL) 2μL, sterile water to make up to 20μL. For blank control, sterile water was used to replace template DNA. Each reaction was repeated three times, and the PCR amplification procedure used a two-step method: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 64°C for 1 minute, a total of 45 cycles.

[0111] After the real-time fluorescent PCR amplification was completed, the amplified products were directly used for Bio-Rad to read the HRM hig...

Embodiment 3

[0113] Example 3: Real-time fluorescent quantitative PCR detection by probe method

[0114] Using the extracted DNA as a template, use the Noka Yulis probe method to detect primers: YXYLS-DF and YXYLS-DR, Noka Yulis-specific probe: YXYLS-VIC-P2, and non-Noka Yulis-specific Probe FYXYLS-FAM-P1, together with endogenous reference gene PLD primers PLD-F and PLD-R, and endogenous reference gene PLD probe PLD-CY5-P were detected by real-time fluorescent PCR. The PCR reaction system was 20 μL, including 10 μL of 2×GoldStar Best MasterMix, 1 μL of YXYLS-DF and YXYLS-DR primers (10 μM concentration), 0.4 μL each of PLD-F and PLD-R primers (10 μM concentration), YXYLS-VIC -0.25 μL each of P2 and FYXYLS-FAM-P1 probes (concentration of 10 μM), PLD-CY5-P probe (concentration of 10 μM) 0.2 μL, template DNA 2 μL, and sterile water to make up to 20 μL. For blank control, sterile water was used to replace template DNA. Each reaction was repeated three times, and the PCR amplification proced...

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker for oryza sativa var. Virens and The SNP marker is T or C of the 64th 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] Yexiang Youlis is a new type of high-quality rice variety cultivated by Guangxi Lvhai Seed Industry Co., Ltd. The approval number is Guidao Shen No. 2017045, which is a high-quality aromatic hybrid rice variety. This variety was approved in 2017, and in 2018, it won the gold medal of the first national high-quality rice (indica) variety food quality evaluation. In 2019, it won the gold medal of the first quality evaluation of high-quality rice in Jiangxi Province, and it will be approved by Jiangxi Province in 2020. It is characterized by strong adaptability, good palatability, high quality and high yield. [0003] Yexiang Youlis is a new variety of high-quality rice. The growth period is usually about 110 days, and every 667m 2 The average yield can reach 564.22kg, and each 667m in high-yield areas 2 The average yield can even reach 650kg. The compr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/13
Inventor 杨华肖玲封莉牛其文
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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