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An ISSR-PCR molecular mark system for Carpinus oblongifolia (Hu) Hu et W. C. Cheng

A technology of Baohua hornbeam and molecular markers, applied in the field of genetic biology, can solve problems such as uncertain taxonomic status of Baohua hornbeam, unknown level of genetic diversity, and lack of protection plans for endangered species, achieving high-scientific Value and application value, improved accuracy and stability, effect of polymorphism enrichment

Inactive Publication Date: 2019-01-15
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the uncertain taxonomic status of Baohua hornbeam, the research work has been neglected for a long time, the expression level of genetic diversity is unknown, and there is a lack of corresponding protection plan for endangered species

Method used

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  • An ISSR-PCR molecular mark system for Carpinus oblongifolia (Hu) Hu et W. C. Cheng
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  • An ISSR-PCR molecular mark system for Carpinus oblongifolia (Hu) Hu et W. C. Cheng

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Baohua Hornbeam Genomic DNA Extraction

[0031] (1) Take about 100 mg of ungerminated buds or sprouts of Baohua hornbeam, put them into a mortar, add liquid nitrogen to grind;

[0032] (2) Extract Baohua hornbeam genomic DNA from the fully ground powder according to the operating instructions of the Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.);

[0033] (3) NanoDrop was used to detect the concentration and quality of the genomic DNA eluted by TE, and at the same time, 1% agarose gel electrophoresis was used to detect the genomic DNA extracted from the Baohua hornbeam strain. figure 1 .

Embodiment 2

[0034] Example 2 ISSR-PCR reaction system orthogonal design optimization

[0035] (1) On the basis of the conventional PCR reaction system, optimize the ISSR-PCR reaction system of Baohua hornbeam, mainly for Mg 2+ , dNTPs, Taq enzyme and primer concentration 4 factors for 3-level orthogonal design, using L 9 (3 4 ) orthogonal design table for experiment optimization, and the orthogonal design table for PCR reaction system optimization is shown in Table 1. The primer used in ISSR-PCR is No3, the sequence is 5' AGAGAGAGAGAGA GAGYC 3'[Y=(C, T)], repeated twice, amplification procedure: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 30 sec , annealed at 56°C for 45 sec, extended at 72°C for 1 min, 40 cycles, extended at 72°C for another 7 min, and stored at 4°C.

[0036] Table 1 Orthogonal optimization of PCR reaction system

[0037]

[0038] (2) Orthogonal test for the optimization of the PCR reaction system by 1% agarose gel electrophoresis separation,...

Embodiment 3

[0039] Example 3 Primer screening and optimal annealing temperature determination

[0040] (1) The primers for ISSR molecular markers in this test were synthesized by Nanjing GenScript Biotechnology Co., Ltd. using the sequence (UBC801-900) provided by the University of Montreal, Canada.

[0041] (2) On the basis of the optimal reaction system determined by the experiment, primer screening was carried out. 100 primers were amplified by PCR one by one and annealed at 56°C. The results showed that more than half of the primers could amplify products with obvious signals, and some products were amplified by 1 % agarose gel electrophoresis, the result shows that the polymorphism of the PCR product is abundant, see image 3 , the band distribution area is wide and the definition is high, and the final optimal 10 primer sequences with good polymorphism are shown in Table 2.

[0042] (3) Taking the No3 primer as an example to determine the annealing temperature, 8 annealing temperat...

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Abstract

The invention belongs to the technical field of genetics and biology, in particular to an ISSR-PCR molecular mark system for Carpinus oblongifolia (Hu) Hu et W. C. Cheng. An optimized ISSR-PCR reaction system is provided and configured in a manner that: 25 [mu]L of the reaction system contains 1*PCR buffer, 2.5 mM of Mg<2+>, 0.25 mM of dNTPs, 0.6 [mu] M of a primer, 50 ng of a DNA template, and 1.2 U of Taq enzyme. The amplification procedure is as follows: predenaturation at 95 DEG C for 5 min, denaturation at 95 DEG C for 30 sec, annealing at 56 DEG C for 45 sec, extension at 72 DEG C for 1min, extension at 72 DEG C for 7 min after 40 cycles, and agarose gel electrophoresis. The ISSR-PCR system has good stability, high clarity and abundant polymorphism.

Description

technical field [0001] The invention belongs to the technical field of genetic biology, and in particular relates to a Baohua hornbeam ISSR-PCR molecular marker method. Background technique [0002] Baohua Hornbeam ( Carpinus oblongifolia ) is a deciduous tree of the Betaceae Hornbeam genus. It is only distributed in Baohua Mountain, Jiangsu Province. The number of existing wild plants is very rare. It is listed as critically endangered (CR) by the "Red List of Biodiversity in China-Higher Plant Volume". However, due to the uncertain taxonomic status of Baohua hornbeam, the research work has been neglected for a long time, the expression level of genetic diversity is unknown, and there is a lack of corresponding protection plan for endangered species. [0003] Genetic diversity is an important part of biodiversity and the basis of ecosystem diversity and species diversity. DNA diversity is the essence of genetic diversity. DNA molecular markers are genetic markers based o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895C12Q2600/16C12Q2525/151C12Q2537/143
Inventor 李素梅王淑安汪庆高露璐王鹏李亚
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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