Method for identifying porphyra yezoensis high-temperature-resistant strain TM-18, molecular marker and construction method for same

A technology of Porphyra zebra and molecular markers, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of difficult identification of high-temperature-resistant strains and other strains, and restrictions on the promotion and application of improved varieties , to achieve the effect of simple and intuitive judgment criteria, stable test results, and avoidance of interference and errors

Inactive Publication Date: 2013-10-09
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, affected by factors such as the sea environment, it is difficult to identify high-temperature-resistant strai

Method used

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  • Method for identifying porphyra yezoensis high-temperature-resistant strain TM-18, molecular marker and construction method for same
  • Method for identifying porphyra yezoensis high-temperature-resistant strain TM-18, molecular marker and construction method for same
  • Method for identifying porphyra yezoensis high-temperature-resistant strain TM-18, molecular marker and construction method for same

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The method for constructing a molecular marker for identifying the high temperature resistant strain TM-18 of Porphyra variegata comprises the following steps:

[0044] (1) Extraction of total DNA: Collect fresh thallus of the high temperature-resistant strain TM-18 of P.

[0045] (2) SRAP marker analysis: use the SRAP primer set to analyze the DNA obtained in step (1). For primer sequences, see: "New molecular markers—SRAP and TRAP and their applications" (Liu Liwang, Gong Yiqin, Huang Hao, Zhu Xianwen, "Heritage" 26 (5): 777-781, 2004)

[0046]The SRAP-PCR reaction system is: 10×PCR buffer2.5μL, 20ng template DNA, forward and reverse primers 20ng each, the reaction system contains 2.0mmol / L MgCl2 (final concentration), 0.2mmol / L dNTP (final concentration) and 1U Taq DNA polymerase, plus sterile ddH 2 0 to 25 μL;

[0047] The reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 35°C for 1 min, extension at 72°C for 1...

Embodiment 2

[0072] The heat-resistant strain TM-18 and non-heat-resistant strain of P.

[0073] (1) Extraction of total DNA: the same method as in step (1) of Example 1;

[0074] (2) SCAR marker verification: use the primers designed in step (5) of Example 1 to perform PCR amplification on the obtained total DNA;

[0075] The reaction system is: 10×PCR buffer1.5μL, 20ng template DNA, forward and reverse primers 20ng each, the reaction system contains 2.0mmol / L MgCl 2 (final concentration), 0.2mmol / L dNTP (final concentration) and 1U Taq DNA polymerase, plus sterilized ddH 2 0 to 15 μL;

[0076] The reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 52°C for 45 s, extension at 72°C for 45 s, repeating 35 cycles; extension at 72°C for 7 min.

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Abstract

The invention belongs to the fields of molecular biology DNA (deoxyribonucleic acid) marking technology and applications, and relates to a molecular marker for porphyra yezoensis high-temperature-resistant strain TM-18 and a construction method for the same. Specifically, a porphyra yezoensis strain obtained by artificial selection and breeding is analysed by virtue of an SRAP (sequence-related amplified polymorphism) marking technology, and the specific bands of the high-temperature-resistant strain TM-18 are found via PCR (polymerase chain reaction) electrophoresis detection; and specific primers are designed via recovering and sequencing, and converted to SCAR (sequence characterized amplified regions) markers for extension and strain verification. A method for identifying porphyra yezoensis high-temperature-resistant strain TM-18 by the molecular marker comprises the following steps of: extracting the total DNA of porphyra yezoensis; performing PCR amplification by the specific primers; performing electrophoresis detection on amplification products; and performing result judgement, specifically, if amplification products with a length of 341 bp are generated, then judging that the strain is a high-temperature-resistant strain TM-18. In the method, identification can be performed only by one-time simple PCR and agarose electrophoresis; and the method is simple and rapid to operate, and free from the influence of environmental factors, and is an ideal means of strain identification.

Description

technical field [0001] The invention belongs to the field of DNA marker technology and application of molecular biology, and relates to a molecular marker of a high-temperature-resistant strain of Porphyra zebra TM-18 and a construction method thereof. Background technique [0002] Porphyra zebra is the main cultivated variety of laver in the north of the Yangtze River in my country. It occupies an important position in the economic seaweed industry, with a total output value of about 3 billion yuan. [0003] Porphyra zebra is a cold-temperate seaweed. The Porphyra filaments in the seedling stage are resistant to high temperatures and can naturally survive the high temperature period in summer and autumn. The suitable growth temperature is 20-25°C; the filaments mature in early autumn and release ascospores. Conchospores germinate and grow into fronds, which grow at low temperatures in late autumn, winter, and early spring. The optimum temperature for the growth of young alg...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 严兴洪贾威黄林彬
Owner SHANGHAI OCEAN UNIV
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