RAPD primer for distinguishing varieties of Luffa cylindrica, and application thereof

The technology of a variety, loofah, applied in the field of molecular biology, can solve the problems of primers that cannot be visually displayed to distinguish varieties, large amount of operation, lack of practicability, etc., and achieve the effect of early identification, simple operation, and wide versatility

Active Publication Date: 2018-07-20
JIANGSU ACAD OF AGRI SCI
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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 primer for distinguishing varieties of Luffa cylindrica, and application thereof
  • RAPD primer for distinguishing varieties of Luffa cylindrica, and application thereof
  • RAPD primer for distinguishing varieties of Luffa cylindrica, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: DNA extraction.

[0057] The loofah variety samples of the present invention are all common and important loofah varieties in the market, and the 20 varieties include: Changxiangyu, Lvling No. 2, Snake Luffa, Green Prince, Lvyou No. 1, Wuyexiang, Xiangfei 206, Improved Changsha loofah, fragrant long loofah, Fangzhen AB321, long fragrant loofah, Lvshuai, Saijiali, Jasper, Zhonglu, Chunxiang white loofah, Xiangyou No.1, Xiangyubai, Tiebaleng, Zaoyu No.2.

[0058] About 0.2 g of loofah leaves were used as material, ground with liquid nitrogen, and 500 μl of CTAB lysis solution (2% CTAB, 2M NaCl) was added. 2 , 20mM EDTA, 100mM Tris-HCl (PH=8.0) and 0.2% β-mercaptoethanol), transferred to a 1.5ml centrifuge tube, taken out and cooled in a water bath at 65°C for 0.5-1h, and an equal volume of chloroform:isoamyl alcohol ( 24:1) Shake the mixture gently, centrifuge the emulsion at 12000r / min for 8-10min, extract the supernatant with chloroform / isoamyl alcohol (24:1...

Embodiment 2

[0059] Example 2: RAPD-PCR reaction system and conditions.

[0060] RAPD-PCR reaction system is 20μl, 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.

[0061] The RAPD-PCR amplification procedure was pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30S, annealing for 45S, extension at 72°C for 90S, 42 cycles, extension at 72°C for 10 min, and storage at 4°C. Amplification was carried out on a PCR amplifier, and the amplified products were electrophoresed on agarose gel (containing 1.2% ethidium bromide), 1×TAE electrophoresis buffer (1L contained: 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 conduct electrophoresis at 120V constant pressure for 0.5-0.8 hours until the amplified DNA bands are clearly separated. , and then observed and photog...

Embodiment 3

[0062] Example 3: Stringent screening of specific primers.

[0063] The 4 primers of the present invention are obtained through strict screening of 52 originally designed random primers. First, 52 random primers were designed according to the genome of the existing loofah varieties, and 11 bases of RAPD random primers with good polymorphism, strong amplification and high stability were selected. Tape the same temperature as the annealing temperature. Specifically, the gradient PCR reaction system was 20 μl, containing 50 ng of genomic DNA, 2.0 mmol·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 2 min, denatured at 94 °C for 30 s, annealed for 45 s, extended at 72 °C for 90 s, 42 cycles, and extended at 72 °C for 10 min. The random primer sequence and annealing temperature of the present invention are as follows:

[0064] RP487: 5'-AGTCAGCCACG-3', annealing temperature 43.1℃;

[006...

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Abstract

The invention discloses an RAPD primer for distinguishing the varieties of Luffa cylindrica, and belongs to the field of molecular biological molecular markers. The nucleotide sequences of four RAPD random primers designed and screened through a method in the invention are represented by SEQ ID NO:1 to 4, the four RAPD random primers are used to amplify the DNA of Luffa cylindrical with an known variety, spectral bands obtained after gel electrophoresis are counted to distinguish the varieties having unique specific spectral bands, and a tree-shaped identification pattern of the distinguishedvarieties is established. The primer of the invention has the advantages of improvement of the stability of an RAPD reaction, simplicity in operation, high efficiency, accuracy and primer saving, andallows "Changxiangyu" and other 19 Luffa cylindrica varieties to be easily distinguished at the molecular level by performing the PCR four times. The obtained identification pattern of the varieties is more intuitive than a clustering tree, so the primer for distinguishing any two varieties can be quickly found according to the variety identification pattern. The method can realize the early identification of Luffa cylindrica seedlings, and also has broad versatility in 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 loofah varieties by using RAPD molecular marker technology. Background technique [0002] Loofah is a worldwide vegetable that originated in China. It has a long history of cultivation and abundant variety resources. Since 2006, the country’s annual planting area has exceeded 1,200,000 hectares, ranking among the top 3 of all vegetable crops. It is widely used in vegetable production and supply. To have great importance. In recent years, with the substantial expansion of my country's loofah production and cultivation area, more and more fine varieties have been used in loofah production. Therefore, the establishment of a fast and reliable identification technology system for loofah varieties is of great significance for the research and protection of genetic resources of loofah varieties, seedling identification, variety ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6827C12N15/11
CPCC12Q1/6827C12Q1/6895C12Q2531/113C12Q2535/139
Inventor 许园园苏小俊娄丽娜刘哲
Owner JIANGSU ACAD OF AGRI SCI
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