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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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In general, there is still a big gap in the resea

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
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Example Embodiment

[0026] Example 1 Extraction of Buffalograss Genomic DNA

[0027] (1) Take about 1g of fresh bison grass leaves, wash with clean water, wipe dry, cut into mortar, pour into liquid nitrogen to cool and grind;

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

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

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

[0031] (5) Take the supernatant;

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

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

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

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

Example Embodiment

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

[0037] According to the principle of orthogonal design, L 9 (3 4 ) Orthogonal design table, first of all dNTPs, Taq enzyme, primers and Mg 2+ Exploratory orthogonal experiment with 4 factors and 3 levels was performed for the concentration. 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. The orthogonal design is shown in Table 3. The selected ISSR-PCR primer sequence is 5'-AGAGAGAGAGAGAGAGYG-3'[Y=(C,T)], and the experiment is set to repeat twice. ISSR-PCR amplification program: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 2 minutes, 35 cycles, extension at 72°C for 4 minutes, and finally storage at 4°C.

[0038] 2.1 Establishment of ...

Example Embodiment

[0053] Example 3 Detection of the stability of ISSR-PCR reaction system

[0054] Use other template DNAs, and perform ISSR-PCR with the best combination of the 9 treatment combinations in the fine-tuning orthogonal test (treatment No. 2) and the best treatment combination in statistical theory to test the stability of the two systems. Compare the amplification efficiency of the two systems.

[0055] The result is image 3 Shown. The bands of these two amplification systems are basically the same, but some of the bands amplified by the best theoretical combination are obviously brighter, and there are more amplified bands, so the best theoretical combination is selected as the ISSR-PCR reaction of the formal experiment system. Therefore, the best theoretical combination is selected as the ISSR-PCR reaction system for the formal experiment, that is, a 25μL reaction system contains 1×PCR buffer, dNTPs 0.3mM, Taq enzyme 1.0U, primers 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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