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Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55

A technology for marking primers and improved varieties of camellia oleifera, applied in the direction of DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long cycle, complicated PCR amplification map, and difficult variety identification, and achieve the effect of simple method

Inactive Publication Date: 2014-07-23
ZHEJIANG FORESTRY ACAD
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Problems solved by technology

[0004] The identification of camellia oleifera varieties is mainly based on appearance characteristics, but due to the long period of identification of many morphological characters, the great influence of the environment, and the increasing number of varieties, the identification of varieties is becoming more and more difficult
Since this century, some PCR-based molecular marker technologies such as RAPD (Random Amplified Polymorphic DNA, random amplified polymorphic DNA), ISSR (Inter-Simple Sequence Repeat, simple sequence repeat interval amplified polymorphism) and SRAP ( sequence-related amplified polymorphism) have been successively used to study the genetic diversity and genetic distance of Camellia oleifera germplasm resources, but there are few studies on the linkage between molecular markers and Camellia oleifera traits, and the molecular identification between Camellia oleifera varieties. Moreover, these molecular markers are either universal random primers or a random combination of designed primers, the PCR amplification pattern is not only complex, poor repeatability, but also low specificity, so it is not suitable for variety identification
SCAR (Sequence Characterized Amplified Region) marker was proposed by Paran and Michelmore on the basis of RAPD in 1993. It is based on the sequencing of specific RAPD fragments, and a pair of 18-24 bases is designed according to the sequences at both ends The primers are used for specific amplification at a higher annealing temperature. The use of specific primers eliminates the competition between random primer binding sites, so it is a very stable molecular marker. It is widely used in applications. It has the characteristics of rapidity, simplicity and low cost. So far, there has been no research report on the application of SCAR markers in the identification of Camellia oleifera varieties

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  • Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55
  • Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55
  • Molecule specificity marker primer and identification method of improved variety of camellia oleifera Changlin Number 55

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Embodiment 1

[0045] (1) Extraction of genomic DNA of improved Camellia oleifera: Take 0.01g of young leaves of Camellia oleifera to be tested, add liquid nitrogen to grind thoroughly, and use SDS-CTAB method to extract genomic DNA. Genomic DNA crude extract. The crude DNA was purified by Magabio Nucleic Acid Purification Kit (Bioer, Hangzhou, China), and detected by 1.5% agarose gel electrophoresis and DNA / RNA UV spectrophotometer (GeneQuant Pro, GE Healthcare). Integrity, Purity and Concentration. OD 260 / OD 280 DNA samples >1.8 were used for subsequent PCR amplification. DNA extracts were stored in a -20°C refrigerator for later use.

[0046] (2) Design specific PCR amplification primers. The sequence of the primer pair is the upstream primer 5′-CATACATACACTTCCTAAGCCAAAA-3′ and the downstream primer 5′-GTTCAAGCATTGTTCAAGCACTC-3′, which were synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0047] (3) PCR amplification of SCAR molecular markers:

[0048] Composition of P...

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Abstract

The invention provides a molecule specificity marker primer of the improved variety of camellia oleifera Changlin Number 55. The sequence of the primer is that the upstream primer is 5'-CATACATACACTTCCTAAGCCAAAA-3' and the upstream primer is 5'-GTTCAAGCATTGTTCAAGCACTC-3'. The molecule specificity marker primer can be used for the early-stage identification of the improved variety of camellia oleifera Changlin Number 55. The method is simple, rapid and accurate and is a molecular method which can not be replaced by distinguishing the improved variety of camellia oleifera by appearance characteristics.

Description

(1) Technical field [0001] The invention relates to a molecular specific marker primer for the improved Camellia oleifera variety Changlin 55 and a method for quickly identifying the improved Camellia oleifera variety Changlin 55 by using the primer. (2) Background technology [0002] Camellia oleifera is the woody oil tree species with the largest planting area and wide distribution in my country. Vigorously developing the Camellia oleifera industry is of great significance to ensuring the safety of grain and oil, increasing farmers' income, promoting the comprehensive development of mountainous areas and building a new socialist countryside. Camellia oleifera industry in my country is in the ascendant with huge development potential. At present, the total area of ​​camellia oleifera forest in my country is more than 45 million mu, but the output is very low, only about 4 kilograms of camellia oil per mu. The root cause is that most varieties of camellia oleifera are poor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王丽玲李海波刘本同钱华王衍彬
Owner ZHEJIANG FORESTRY ACAD
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