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Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers

A cyst nematode and detection method technology, applied in the field of rapid molecular detection of upland rice cyst nematode SCAR markers, to achieve the effect of strong specificity and accurate detection technology

Inactive Publication Date: 2013-06-05
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Although there are research reports on the ITS region of upland rice cyst nematode ribosomal DNA at home and abroad, there is no report at home and abroad on the specific molecular detection of upland rice cyst nematode RAPD and SCAR markers based on genomic DNA

Method used

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  • Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers
  • Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers
  • Rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the screening of upland rice cyst nematode specific RAPD primer

[0042] 1.1 Extraction of genomic DNA from upland rice cyst nematodes

[0043] One full cyst of the cyst nematode population to be tested was picked respectively, put into a PCR tube with 10 ml of sterilized double distilled water, and frozen in liquid nitrogen. For nematode populations such as Banana perforator nematodes, sweet potato stem nematodes and cyst nematode second instar larvae, single nematodes were picked and put into 10 ml of sterilized double distilled water, which was also frozen in liquid nitrogen. After taking it out, put it on ice, turn the glass rod sterilized with 75% alcohol in the PCR tube until the ice melts, break the cyst, release eggs, or break the nematode. Add 8ml of 10× PCR buffer, 2ml of proteinase K solution (600mg / ml), freeze at -80°C for 2h, take out the PCR tube, incubate at 65°C for 1.5h, then 95°C for 10min, and finally 1000rpm After centrifugation for 1...

Embodiment 2

[0049] Embodiment 2: the determination of upland rice cyst nematode RAPD marker sequence and the design of SCAR marker primer

[0050] Using DNA of upland rice cyst nematode and other nematodes as templates, RAPD primer OPB 15 (Synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) for PCR amplification. The RAPD-PCR reaction system is 25 μl, and the ratio is as follows: 10×PCR Buffer 2.5 μl, 10 mmol / L dNTP 2.0 μl, RAPD primer OPB 15 3.0 μl, rTaq DNA polymerase 0.25 μl (5U / μl), template DNA 0.5 μl, sterilized redistilled water to make up 25 μl. Amplification was performed on an Eppendorf PCR cycler.

[0051] PCR amplification condition is the same as embodiment 1, random primer OPB 15 The RAPD-specific DNA fragment of about 500bp obtained by amplifying upland rice cyst nematode was cut from the agarose gel ( figure 2 ), the RAPD-specific DNA fragment of about 500bp was recovered and purified using the DNA recovery kit of Dalian Bao Bio Company, the pu...

Embodiment 3

[0055] Example 3: Rapid molecular detection of upland rice cyst nematode-specific SCAR markers

[0056] Upland rice cyst nematode-specific SCAR marker primers HeF1 and HeR1 constructed in the present invention were synthesized by Shanghai Sangon Biological Co., Ltd. The total volume of the specific SCAR marker amplification reaction system was 25 μl, which contained 2.5 μl 10×PCR Buffer (containing Mg++ ); 2.0μl dNTP; 1.0μl HeF1 (0.1μg / μl); 1.0μl HeR1 (0.1μg / μl); 0.25μl Taq DNA polymerase (5U / μl); 1.0μl template DNA (upland rice cyst nematode and other cyst nematodes Genomic DNA extracted from nematode populations); ddH 2 O to make up 25 μl.

[0057] The applied upland rice cyst nematode specific SCAR marker primer sequence is as follows:

[0058] HeF1: 5'-AATCGTTTTTCAGTTGAGACCCAT-3'

[0059] HeR1: 5'-GAGGGTGTTGAACAATTAAAAAA-3'

[0060] The PCR amplification program was as follows: pre-denaturation at 94°C for 5min, followed by 35 cycles of 94°C for 30S, 54°C for 30S, 72°C...

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Abstract

The invention relates to a rapid molecular detection method of heterodera elachista by specific RAPD (Randomly Amplified Polymorphic DNA) and SCAR (Sequence-characterized Amplified Region) markers and belongs to the technical field of biology. A DNA (Deoxyribonucleic Acid) segment marked by a specific RAPD of the heterodera elachista is a nucleotide sequence represented by SEQ ID NO: 1. Accordingto an amplified segment of the specific RAPD of the heterodera elachista, specific primers HeF1 and HeR1 are designed and are converted into a more stable SCAR marked segment; and a specific segment of 434bp can be amplified in the rapid molecular detection method of the heterodera elachista by the SCAR markers. According to the invention, the method improves the molecular detection flexibility and is rapid and accurate; and the method has a higher application value in the aspects of detection of the heterodera elachista and an early diagnosis of heterodera elachista diseases.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a complete sequence of upland rice cyst nematode specific RAPD markers, a specific SCAR marker primer sequence and a rapid molecular detection method for upland rice cyst nematode SCAR markers. Background technique [0002] Upland rice cyst nematode (Heterodera elachista Ohshima, 1974), first discovered in the mountainous rice fields of Tochigi Prefecture, Japan (Ohshima, 1974), is widely distributed in the Northeast and Kyushu regions of Japan, mainly infecting rice, and is currently also found in Iran and China distributed. Yield losses caused by the disease range from 7% to 19% (Bridge et al., Nematode parasites of rice, Plant parasitic nematodes in tropical and subtropical agriculture. Wallingford, UK, CABI Publishing, 1990, 69-108). The second-instar larvae infect the roots of upland rice in mid-May, eggs begin to deposit after five weeks, and cysts begin to form after 6-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 彭德良亓晓莉彭焕黄文坤丁中贺文婷
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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