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Establishing method for MSAP technical system of apocarya

A thin-shell hickory nut and a technology for establishing methods, which are applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve problems that have not been reported in the research, and achieve the effect of low silver staining background and clear target bands

Inactive Publication Date: 2019-03-19
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although relevant work has been carried out on the genetic improvement of hickory nuts, there have been no reports on the application of DNA methylation, an epigenetic regulation level, to molecular marker-assisted breeding.

Method used

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  • Establishing method for MSAP technical system of apocarya
  • Establishing method for MSAP technical system of apocarya
  • Establishing method for MSAP technical system of apocarya

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 extracts the extraction of the pecan genome DNA

[0058] Such as figure 1 As shown, samples of male flowers and leaves (the fifth pair from bottom to top) of pecan 'Pony' and 'Shaoxing' varieties were collected respectively, and genomic DNA was extracted using a kit (the extraction kit was purchased from Shanghai Shenggong Plant Genome) , wherein lanes 1, 2, 3 and 4 are the DNA samples of poni leaves, poni male flowers, Shaoxing leaves and Shaoxing male flowers, respectively. Then the quality of the extracted pecan genomic DNA was mixed, and the subsequent system optimization was carried out.

Embodiment 2

[0059] Embodiment 2 double-enzyme digestion of pecan genome DNA

[0060] Using EcoRI and MspⅠ, the double enzyme digestion reaction was carried out on the genomic DNA samples of Carya chinensis respectively. Take 6 parts of mixed DNA samples obtained in Example 1, and the enzyme digestion system is: 400ng DNA, 0.3 μL EcoR I-HF (20u / μL), 0.3 μL Msp I (20u / μL) or 0.3 μL HpaⅡ (20u / μL) , 2μL Cut smart Buffer, and make up to 20μL with sterile water.

[0061] Carry out enzyme digestion in a water bath at 37°C. The enzyme digestion time is divided into 6 time gradients, namely 1h, 2h, 5h, 8h, 11h, and 14h. After each time gradient enzyme digestion is completed, put it into a PCR instrument at 65°C for 20 minutes to inactivate the enzyme. live.

[0062] figure 2 The electrophoresis of pecan genomic DNA after double digestion with Msp I. Such as figure 2 As shown, 1h, 2h, 5h, 8h, 11h and 14h are the states after digestion of the mixed DNA sample for 1h, 2h, 5h, 8h, 11h and 14h, ...

Embodiment 3

[0063] Example 3 Ligation of the double-digestion product and the linker primer

[0064] Take 5 DNA samples again from the mixed DNA sample in Example 1, apply the enzyme digestion method in Example 2 for 5 hours of enzyme digestion, and set 5 tubes of 20 μl sterile water as a blank control for 37 ° C water bath.

[0065] Ligation is performed after enzyme digestion. The system is: 20 μL of enzyme digestion product, 0.1 μl T4 (400u / μL), 2.5 μl ATP (10 mM), E adapter (mixed upstream and downstream adapters, both 50 μM) and H / M adapter (upper The downstream joints were mixed, all 50 μM) were 0.5 μL, and the sterile water was made up to 25 μL.

[0066] Add 0.5 μL E adapter and 0.5 μL H / M adapter to each tube of the control, and make up to 25 μL with sterile water. The ligation time was set to 5 gradients, respectively 2h, 5h, 8h, 11h and 14h, and the ligase was inactivated by heating at 65° C. for 10 minutes after each time gradient ligation.

[0067] Such as image 3 as shown...

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Abstract

The invention provides an establishing method for an MSAP technical system of apocarya and relates to the technical field of molecular markers. The establishing method for the MSAP technical system ofapocarya comprises the following steps: first, carrying out double digestion on genomic DNA of apocarya to obtain an enzyme-digested product and connecting the obtained enzyme-digested product with ajoint primer to obtain a connected product; then preamplifying the connected product to obtain a preamplfied product and selectively amplifying the preamplified product; and finally, carrying out gelelectrophoresis and silver staining development on the selectively amplified product. An objective, scientific and accurate MSAP technical system of apocarya is established to obtain a DNA methylation atlas which is low in silver staining background and clear in target strip, and has the advantage of carrying out research on methylation of whole genome and easy detection of methylated loci underthe circumstance of an unknown genomic sequence.

Description

technical field [0001] The invention relates to the technical field of molecular markers, in particular to a method for establishing a pecan MSAP technology system. Background technique [0002] Thin shell hickory (Carya illinoensis Koch), also known as American hickory or long hickory, commonly known as "pecan", is a plant of the genus Hickory in the family Juglandaceae, native to the United States and northern Mexico, and is one of the world's famous dry fruit tree species. one. Thin-shelled hickory nuts have large nuts, thin shells, high kernel yield, easy kernel extraction, and high yield. At the same time, the nuts are delicious, fragrant, non-astringent, and nutritious. They are ideal health food, bread, candy and other foods Add material. In addition, pecan is also an important woody oil plant. The oil content of the seed kernel is more than 70%, and the unsaturated fatty acid content is as high as 97%. It has good storage performance and is the best cooking oil and...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/301C12Q2525/191C12Q2531/113C12Q2565/125
Inventor 彭方仁刘壮壮梁有旺谭鹏鹏曹凡
Owner NANJING FORESTRY UNIV
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