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Buchloe dactyloides ISSR-PCR molecular marker system

A bison grass and system technology is applied in the field of the best system of bison grass ISSR-PCR reaction to achieve the effects of improving accuracy and stability, strong signal, and shortening breeding years

Inactive Publication Date: 2011-03-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In general, there is still a big gap in the research on bison grass resources compared with other pastures

Method used

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  • Buchloe dactyloides ISSR-PCR molecular marker system
  • Buchloe dactyloides ISSR-PCR molecular marker system
  • Buchloe dactyloides ISSR-PCR molecular marker system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Extraction of bison grass genome DNA

[0027] (1) Take about 1g of fresh bison grass leaves, wash them with water, dry them, cut them into pieces and put them in a mortar, then pour them into liquid nitrogen to cool and grind them;

[0028] (2) Add the ground powder to 900 μL of 2×CTAB extract solution at 65°C, and bathe in water at 65°C for 30 minutes;

[0029] (3) After cooling, add 500 μL of chloroform / isoamyl alcohol (volume ratio 24:1);

[0030] (4) Centrifuge at 8000rpm for 10min;

[0031] (5) get the supernatant;

[0032] (6) Repeat (4), (5) once;

[0033] (7) Add 1 / 10 volume of 3M sodium acetate and an equal volume of isopropanol, shake well, and place on ice for more than 5 minutes until flocculent precipitation appears;

[0034] (8) Centrifuge at 12000rpm for 15min, and discard the supernatant;

[0035] (9) Wash with 75% alcohol, dry at room temperature for 1 hour, dissolve in TE buffer and store at 4°C for later use.

Embodiment 2

[0036] Example 2 Establishment and optimization of ISSR-PCR orthogonal system (25 μL)

[0037] According to the principle of orthogonal design, the L 9 (3 4 ) Orthogonal design table, first for dNTPs, Taq enzyme, primers and Mg 2+ Concentration of 4 factors and 3 levels of exploratory orthogonal experiment, the exploratory orthogonal design table is shown in Table 1; then according to the results of the exploratory orthogonal experiment, the concentration gradient of each primer was reduced for fine-tuning orthogonal experiment, fine-tuning Orthogonal design is shown in Table 3. The selected ISSR-PCR primer sequence was 5'-AGAGAGAGAGAGAGAGYG-3'[Y=(C, T)], and the experiment was repeated twice. ISSR-PCR amplification program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 35 cycles, extension at 72°C for 4 min, and finally storage at 4°C.

[0038] 2.1 Establishment of exploratory ISSR-PCR reaction ...

Embodiment 3

[0053] Example 3 ISSR-PCR reaction system stability detection

[0054] Select other template DNA, use the best combination (No. 2 treatment) of the 9 treatment combinations in the fine-tuning orthogonal test and the best treatment combination in statistical theory to carry out ISSR-PCR to detect the stability of the two systems and The amplification efficiencies of the two systems were compared.

[0055] The result is as image 3 shown. The bands of the two amplification systems are basically the same, but some bands amplified by the best theoretical combination are obviously brighter, and the number of amplified bands is larger, so the best theoretical combination was selected as the ISSR-PCR reaction for the formal test system. Therefore, the best theoretical combination was selected as the ISSR-PCR reaction system for the formal test, that is, the 25 μL reaction system contained 1×PCR buffer, dNTPs 0.3mM, Taq enzyme 1.0U, primer 0.6μM, Mg 2+ 2.0mM, DNA template 50ng. ...

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Abstract

The invention belongs to the technical field of buchloe dactyloides molecular breeding, and particularly relates to an optimal buchloe dactyloides ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) reaction system. The invention provides the following ISSR-PCR reaction system that: each 25muL of reaction system contains 1*PCR buffer solution, 0.3mM of dNTPs, 1.0U of Taq enzyme, 0.6muM of any one of primers shown as SEQ ID 1-7, 2.0mM of Mg2+ and 50ng of DNA template; and the reaction mixed solution is amplified by the following procedures of: pre-denaturing for 3 minutes at 94 DEG C, denaturing for 30 seconds at 94 DEG C, annealing for 30 seconds at 50 to 60 DEG C, stretching for 2 minutes at 72 DEG C, circulating for 35 times, and stretching for 4 minutes at 72 DEG C. The buchloe dactyloides ISSR-PCR provided by the invention has good stability and high definition, and increases the richness of strips.

Description

technical field [0001] The invention belongs to the technical field of bison grass molecular breeding, and in particular relates to a set of bison grass ISSR-PCR reaction optimal system. Background technique [0002] Buffalo grass (Buchloe dactyloides) is a perennial turfgrass of the genus Buffalo in the Poaceae Teffae subfamily. It has been mainly planted as forage grass in the past one hundred years. Drought, low plant, slender and soft leaves, etc.) and gradually used for lawn planting, and compared with other turfgrass species, there is no obvious disease. With the increasing shortage of water resources and the public's increasing attention to environmental requirements, bison grass lawns are gradually being valued by people because of their strong drought tolerance. Due to its superior drought resistance and adaptability, it has become one of the main grass species for landscaping, environmental protection, and soil and water conservation in North China, Northeast Chin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张蕴薇杨富裕魏小兰邓波康俊梅沈紫微邓由飞
Owner CHINA AGRI UNIV
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