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A rapd primer for distinguishing loofah varieties and its application

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

Active Publication Date: 2021-09-07
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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  • A rapd primer for distinguishing loofah varieties and its application
  • A rapd primer for distinguishing loofah varieties and its application
  • A rapd primer for distinguishing loofah varieties and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Extraction of DNA.

[0057] The loofah variety samples of the present invention are common important loofah varieties in the market, and these 20 varieties include: Changxiangyu, No. Improved Changsha meat loofah, fragrant long loofah, Fangzhen AB321, long fragrant loofah, Lvshuai, Saijiali, Jasper, medium green, Chunxiang white loofah, Xiangyou No. 1, Xiangyubai, Tiebaleng, Zaoyu No. 2.

[0058] About 0.2 g of the young leaves of loofah were ground by liquid nitrogen, and 500 μl of CTAB lysate (2% CTAB, 2M NaCl 2 , 20mM EDTA, 100mM Tris-HCl (PH=8.0) and 0.2% β-mercaptoethanol), transfer to a 1.5ml centrifuge tube, take out and cool in a 65°C water bath for 0.5-1h, add an equal volume of chloroform: isoamyl alcohol ( 24:1) After shaking the mixture gently, centrifuge the emulsion at 12000r / min for 8-10min, extract the supernatant with chloroform / isoamyl alcohol (24:1) for 1-2 times, add pre-cooled isopropanol 4 ℃ refrigerator for more than 3 hours, the coll...

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 program was pre-denaturation at 94°C for 2min, denaturation at 94°C for 30S, annealing for 45S, extension at 72°C for 90S, 42 cycles, extension at 72°C for 10min, and storage at 4°C. Amplify on a PCR amplification instrument, and 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 were...

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 random primers originally designed. Firstly, 52 random primers were designed for the genome of the existing loofah varieties, and 11 RAPD random primers with good polymorphism, strong amplification and high stability were selected from them, and then the strips were selected by 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 2min, denatured at 94°C for 30S, annealed for 45S, extended at 72°C for 90S, 42 cycles, and extended at 72°C for 10min. The random primer sequence and annealing temperature of the present invention are as follows:

[0064] RP487: 5'-AGTC...

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Abstract

The invention discloses a RAPD primer for distinguishing loofah varieties, belonging to the field of molecular biology molecular markers. The method designs and screens four RAPD random primers whose nucleotide sequences are shown in SEQ ID NO: 1-4, which are used to amplify the DNA of unknown loofah varieties, and distinguish the The varieties with unique specific bands were obtained, and the dendrogram of the differentiated varieties was established. The primers of the present invention improve the stability of the RAPD reaction, are easy to operate, efficient and accurate, save primers, and can easily and quickly distinguish 20 loofah varieties such as "Changxiangyu" at the molecular level by only 4 PCRs. The obtained variety identification map is more intuitive than the clustering tree, that is, the primers that can distinguish any two varieties can be quickly found according to the variety identification map. This method can realize early identification of loofah seedlings, and it is also widely used in other species. versatility.

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. It occupies an important position. 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,...

Claims

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Application Information

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Patent Type & Authority Patents(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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