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RAPD (random amplified polymorphic DNA) primer for distinguishing garlic varieties and application of RAPD primer

A variety and garlic technology, applied in the field of molecular biology, can solve the problems of large operation volume, lack of practicability, and inability to visually display the primers for distinguishing varieties, so as to achieve the effect of simple operation, wide versatility and early identification.

Pending Publication Date: 2021-07-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing research work of using RAPD technology to distinguish vegetable crop varieties and identify germplasm, computer software is often used to draw digital fingerprints and combine statistical software cluster analysis to obtain cluster trees, but the cluster analysis results are not only It is impossible to visually display the primers for distinguishing varieties, and it is impossible to display the identification process, which requires a large amount of operation and lacks practicability

Method used

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  • RAPD (random amplified polymorphic DNA) primer for distinguishing garlic varieties and application of RAPD primer
  • RAPD (random amplified polymorphic DNA) primer for distinguishing garlic varieties and application of RAPD primer
  • RAPD (random amplified polymorphic DNA) primer for distinguishing garlic varieties and application of RAPD primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Extraction of DNA.

[0042] The garlic variety samples of the present invention are all common and important garlic varieties in the market, and these 16 varieties include: 'Zhongsuan No. 3', 'Zhongsuan No. 1', 'Mozi Suan', 'Sanjizao', 'Japanese Garlic' , 'Hard and Soft Leaves', 'Second Season Early', 'First Moon Early', 'Wen February', 'Grey Leaf', 'Four or Six Petals', 'Cangshan Garlic', 'Yunnan Purple Garlic', 'Er Shui Zao ', 'Jiading No. 2', 'Putuo Garlic'.

[0043] Take 0.2g of young garlic leaves, grind them with liquid nitrogen, add 500μl of CTAB lysate, transfer to a 1.5ml centrifuge tube, take out and cool in a water bath at 65°C for 0.5-1h, add an equal volume of chloroform / isoamyl alcohol mixture and gently Shake, 12000r / min, centrifuge for 8-10min, extract the supernatant with chloroform / isoamyl alcohol mixture 1-2 times, add pre-cooled isopropanol, let stand at 4°C for more than 3 hours, collect flocculent DNA Wash with ethanol with a volume fr...

Embodiment 2

[0045] Example 2: RAPD-PCR reaction system and conditions.

[0046] RAPD-PCR reaction system is 20μl, containing 50ng genomic DNA, 2.0mmol L -1 MgCl 2 , 0.15 mmol·L - 1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase.

[0047] The RAPD-PCR amplification program was pre-denaturation at 94°C for 3min; denaturation at 94°C for 40S, annealing for 50S, extension at 72°C for 90S, 40 cycles; extension at 72°C for 8min, and then stored at 4°C. Amplify on the PCR amplification instrument, the amplified product adopts agarose gel electrophoresis (containing 1.2% ethidium bromide), 1 × TAE electrophoresis buffer (contains in 1L: 0.242g Tris, 0.057ml glacial acetic acid, 0.2 ml 0.25mol / L EDTA pH 8.0). Add 4 μl of 6× loading buffer (containing 0.25% bromophenol blue, 30% sucrose) to the amplified DNA sample, and perform electrophoresis at a constant voltage of 120V for 0.5-0.8 hours until the amplified DNA bands are clearly separated After that, the amplified bands we...

Embodiment 3

[0048] Example 3: Stringent screening of specific primers.

[0049] The three primers of the present invention are obtained through strict screening of 38 random primers originally designed. First, 38 random primers were designed for the genome of the existing garlic varieties, and 13 bases of RAPD random primers with good polymorphism, strong amplification and high stability were selected, and then the strips were selected through two consecutive gradient PCRs. Take a consistent temperature as the annealing temperature. In detail, the gradient PCR reaction system is 20 μl, containing 50ng genomic DNA, 2.0mmol·L -1 MgCl 2 , 0.15mmol·L - 1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase. The reaction was pre-denatured at 94°C for 3min; denatured at 94°C for 40S, annealed for 50S, and extended at 72°C for 90S, 40 cycles; extended at 72°C for 8min, and then stored at 4°C. The random primer sequence and annealing temperature of the present invention are as fo...

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Abstract

The invention discloses an RAPD primer for distinguishing garlic varieties, and belongs to the field of molecular biology molecular markers. According to the method, three RAPD random primers are designed and screened, the nucleotide sequences of the three RAPD random primers are shown as SEQ ID NO: 1-3, the three RAPD random primers are used for amplifying DNA of unknown garlic varieties, the varieties with unique specific bands are distinguished by counting the bands after gel electrophoresis, and a tree-shaped identification graph of the distinguished varieties is established. According to the primers, the stability of RAPD reaction is improved, operation is easy and convenient, high efficiency and accuracy are achieved, the primers are saved, 16 garlic varieties such as 'Zhong garlic No.3' and the like can be easily and rapidly distinguished on the molecular level only through three times of PCR, and the obtained variety identification graph is more intuitive than a clustering tree; and the primer capable of distinguishing any two varieties can be quickly found out according to the variety identification graph, the early identification of the garlic seedlings can be realized, and the method also has wide universality on other species.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for quickly distinguishing garlic varieties by using RAPD molecular marker technology. Background technique [0002] Garlic is a spherical bulb of the Allium plant in the Liliaceae family. It originated in Central Asia. It has the functions of food, health care and medicine. It has been loved by people for a long time. my country has the largest garlic planting area in the world, accounting for about the world's total planting area. 30% more. There are abundant species resources, which are cultivated in both the north and the south, and the output accounts for more than 1 / 4 of the world's total. Therefore, garlic is one of the important foreign exchange earning vegetables for export in my country. The environment is very different, so the cultivars in different regions generally have significant regional adaptability, forming different ecotypes, prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 曾爱松许园园宋立晓邢苗苗严继勇
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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