Miniature DNA (deoxyribonucleic acid) bar code for identifying pterocarpus santalinus and pterocarpus tinctorius, and identifying method and application of identifying method

A technology for sandalwood and red sandalwood and dye red sandalwood, which is applied in the directions of recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as inability to accurately identify sandalwood red sandalwood and dye red sandalwood, and achieves less dependence on experimental conditions, Amplification and sequencing with high success rate and good specificity

Active Publication Date: 2017-07-07
INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to provide a molecular identification method based on the sequence difference of plastid gene spacer ndhF-rpl32 micro-DNA barcode to realize the accurate distinction between the two, aiming at the inability of traditional wood identification technology to accurately identify sandalwood red sandalwood and dyed red sandalwood

Method used

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  • Miniature DNA (deoxyribonucleic acid) bar code for identifying pterocarpus santalinus and pterocarpus tinctorius, and identifying method and application of identifying method
  • Miniature DNA (deoxyribonucleic acid) bar code for identifying pterocarpus santalinus and pterocarpus tinctorius, and identifying method and application of identifying method
  • Miniature DNA (deoxyribonucleic acid) bar code for identifying pterocarpus santalinus and pterocarpus tinctorius, and identifying method and application of identifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Sandalwood pterocarpus and dye pterocarpus specific primer design

[0038] (1) Download the plastid DNA sequences rbcL, matK and ndhF-rpl32 (Table 1) of pterocarpus sandalwood and dye pterocarpus in GenBank;

[0039] Table 1 The accession numbers of the three DNA fragments of sandalwood and dyed pterostilbene in GenBank

[0040]

[0041] (2) Use Clustal X 1.81 software to compare the sequences, and find and determine the difference sites of the fragment sequences of the sandalwood and the dyed pterostilbene;

[0042] (3) The Primer Premier 5.0 software was used to design primers for the three barcodes of rbcL, matK and ndhF-rpl32. The amplification primers are shown in Table 2.

[0043] Table 2 Three barcode primer information

[0044]

Embodiment 2

[0045] Example 2: Selection and determination of miniature DNA barcode sequences of sandalwood rosewood and dye rosewood standard samples

[0046] (1) Standard sample collection.

[0047] The standard sample of sandalwood and red sandalwood is No. 9, of which wood specimens CAFW23674, CAFW23675, CAFW23676, and CAFW22653 of sandalwood and red sandalwood species No. 4 were selected from the Timber Herbarium of the Chinese Academy of Forestry; they were collected from Jianfengling, Ledong County, Hainan Province, China for botanical identification Sandalwood rosewood leaves No. 5, respectively TXL01, TXL02, TXL03, TXL04, TXL05. Dye standard sample No. 3 of red sandalwood, RLW01, RLW02, RLL01, respectively collected from the sapwood, heartwood and leaves of the Democratic Republic of the Congo.

[0048] (2) Sample preparation.

[0049]Select a wood sample, use a 70% alcohol-sterilized scalpel blade to cut off the outer surface of the wood sample to avoid external pollution; cut ...

Embodiment 3

[0060] Embodiment 3: Dye Pterocarpus fresh wood sample DNA identification

[0061] (1) Collection of fresh wood samples of dyed red sandalwood.

[0062] The dyed red sandalwood fresh wood sample RLW03 was collected from Katanga Province (S10°E28°) in the Democratic Republic of Congo on April 16, 2015. Observation of wood anatomy shows that the wood sample has the characteristics of rosewood wood in the national standard GB / T 18107-2000 of "Redwood", but the wood anatomy characteristics cannot distinguish whether the sample is sandalwood or dyed red sandalwood.

[0063] (2) Prepare wood flour and grind it to 200 mesh or above.

[0064] (3) Wood DNA extraction.

[0065] In a sterilized ultra-clean working environment, DNA extraction is performed on wood. The DNA still needs to be purified, and the final concentration of the DNA is 1-200 ng / μL.

[0066] (4) PCR amplification reaction and sequencing.

[0067] The wood DNA extract is used as a template for PCR amplification, a...

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Abstract

The invention discloses miniature DNA (deoxyribonucleic acid) bar code for identifying pterocarpus santalinus and pterocarpus tinctorius. The miniature DNA bar code is DNA sequence ndhF-rpl32 or a part of the sequence. The invention also discloses a molecular method for identifying the pterocarpus santalinus and the pterocarpus tinctorius, and application of the molecular method. The miniature DNA bar code for identifying the pterocarpus santalinus and pterocarpus tinctorius and the method provided by the invention have the advantages that the accurate identification of the pterocarpus santalinus and the pterocarpus tinctorius can be realized; the difficult problem that the pterocarpus santalinus and the pterocarpus tinctorius cannot be identified by a traditional wood anatomy means is solved; an efficient and reliable pterocarpus santalinus and pterocarpus tinctorius identifying method is provided for wood production and trade personnel and wood trade supervision and law-executing departments such as customhouse departments and quality inspection and quarantine inspection departments; high application value is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology detection for identification of wood species, and in particular relates to a miniature DNA barcode for identifying sandalwood rosewood and dyed red sandalwood and its identification method and application. Background technique [0002] Sandalwood red sandalwood (Pterocarpus santalinus L.f.), belonging to the genus Pterocarpus santalinus, is mainly produced in India and is a rare and precious mahogany tree species. Due to its huge economic value, sandalwood and red sandalwood resources are gradually scarce, and are included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In recent years, a kind of red sandalwood (Pterocarpus tinctorius Welw.), which is very similar in structure to sandalwood red sandalwood, has appeared in the timber market, commonly known as "blood sandalwood" or "Zambian blood sandalwood", which is mainly produced in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2563/185
Inventor 焦立超殷亚方余敏何拓张永刚姜笑梅
Owner INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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