A kind of dna combined barcode for identifying 9 kinds of Dalbergia wood and its identification method and application

A technology of Dalbergia and barcoding, applied in the field of DNA combination barcoding and identification thereof, can solve the problem of inability to accurately identify 9 species of Dalbergia wood, and achieve the effects of less dependence on experimental conditions, less sampling amount, and simple operation.

Active Publication Date: 2018-07-06
INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is that the traditional wood identification technology cannot accurately identify 9 kinds of Dalbergia wood species, and provides a method based on the nuclear ribosomal DNA internal transcriptional spacer such as ITS2 sequence and chloroplast gene spacer such as trnH-psbA DNA combinatorial barcoding of sequences and a method for accurate identification of nine species of Dalbergia wood

Method used

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  • A kind of dna combined barcode for identifying 9 kinds of Dalbergia wood and its identification method and application
  • A kind of dna combined barcode for identifying 9 kinds of Dalbergia wood and its identification method and application
  • A kind of dna combined barcode for identifying 9 kinds of Dalbergia wood and its identification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Dalbergia species wood-specific primer design

[0045] (1) Download the chloroplast DNA sequence trnL, trnH-psbA, trnV-trnM and nuclear DNA sequence ITS2 (Table 1) of Dalbergia from GenBank;

[0046] Table 1 Dalbergia DNA barcode sequences downloaded from GenBank

[0047]

[0048]

[0049]

[0050] (2) Using Clustal X 1.81 software to compare the sequences, find and determine the difference sites of the four DNA barcode sequences among Dalbergia;

[0051] (3) The Primer Premier 5 software was used to design primers for trnL, trnH-psbA, trnV-trnM and ITS2 barcodes. The primers were synthesized by Shanghai Bioengineering Co., Ltd., and the sequences of the primers are shown in Table 2.

[0052] Table 2 Information of four kinds of DNA barcode primers

[0053]

[0054]

Embodiment 2

[0055] Example 2: Optimization and determination of DNA combined barcodes of 9 kinds of Dalbergia wood standard samples

[0056] (1) Standard sample collection

[0057] Nine standard samples of Dalbergia genus (respectively Dalbergia Dalbergia, Dalbergia, Hainan Dalbergia, Cochinate Dalbergia, Broad-leaved Dalbergia, East African Dalbergia, Osmanthus Dalbergia, Dalbergia dentata and Belize Dalbergia ), a total of 50 specimens were obtained from the Timber Herbarium of the Chinese Academy of Forestry.

[0058] (2) Sample preparation

[0059] 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 the wood sample into several sawdust, and place it in a low-temperature freezer grinder for 3 minutes at low temperature, and grind 3min, operating frequency 10cps; after grinding, pass through a 200-mesh sieve, divide the fine wood powder into several 50mL microcentrifuge tubes, each tube conta...

Embodiment 3

[0084] Embodiment 3: unknown wood sample DNA identification

[0085] (1) The wood samples came from the wood market. Through the identification of its anatomical structure, it was determined that it belonged to Siamese rosewood wood, but the species could not be identified.

[0086] (2) Prepare wood flour and grind it to 200 mesh or above.

[0087] (3) Wood DNA extraction.

[0088] In a sterilized ultra-clean working environment, use the DNA extraction kit in Example 2 to extract DNA from wood. The DNA still needs to be purified, and the final concentration of the DNA is 1-200 ng / μL.

[0089] (4) PCR amplification reaction and sequencing.

[0090] The above-mentioned wood DNA is used as a template for PCR amplification, and the amplification primers are shown in SEQ ID NO: 1-4. The PCR amplification reaction system is 30 μL: Premix Ex Taq 15 μL (including 1.25U Ex Taq DNA polymerase, 2mM MgCl 2 , 200 μM single dNTP), 0.2 μM single primer and about 20 ng template DNA.

[...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) combined bar code for identifying nine types of dalbergia wood. The DNA combined bar code is formed by more than two of bar code sequences trnL, trnH-psbA, trnV-trnM or ITS2. The invention also discloses a method for identifying the nine types of dalbergia wood. According to the DNA combined bar code and the identification method of the DNA combined bar code provided by the invention, accurate identification on the nine dalbergia woods can be realized, and the problem that a species level of the dalbergia wood cannot be identified by adopting a conventional wood identification method is solved, so that a high-efficient and reliable identification method is provided for wood trade law-enforcing departments such as customs and quality inspection and quarantine and wood management trading persons, and a technical support is provided for execution of Convention on the International Trade in Endangered Species (CITES); therefore, the DNA combined bar code has a higher application value.

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 DNA combined barcode for identifying nine species of Dalbergia wood, an identification method and application thereof. Background technique [0002] Dalbergia L.f. belongs to the Leguminosae family, with more than 300 species, mainly distributed in tropical and subtropical regions. Dalbergia lumber is hard in material, delicate in texture and soft in color, and is deeply loved by consumers. Driven by strong market demand and high profits, illegal logging and illegal trade have intensified, eventually leading to a serious shortage of Dalbergia timber resources and exacerbating the destruction of global natural forest resources. The status quo of Dalbergia wood resources has aroused high and continuous attention from the international community. At present, 86 species of Dalbergia wood have been included in the Red List o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/686C12Q1/6895C12Q2563/185
Inventor 殷亚方余敏焦立超何拓郭娟张永刚姜笑梅
Owner INST OF WOOD INDUDTRY CHINESE ACAD OF FORESTRY
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