CN107287300A - A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application - Google Patents
A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application Download PDFInfo
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- CN107287300A CN107287300A CN201710500410.6A CN201710500410A CN107287300A CN 107287300 A CN107287300 A CN 107287300A CN 201710500410 A CN201710500410 A CN 201710500410A CN 107287300 A CN107287300 A CN 107287300A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
Bar code is combined the invention discloses a kind of DNA for differentiating 9 kinds of Dalbergia timber, and the DNA combinations bar code is any two or more or DNA combination bar codes that all combination is formed in bar code sequence trnL, trnH psbA, trnV trnM or ITS2.The invention also discloses a kind of method for differentiating 9 kinds of Dalbergia timber.The DNA combination bar codes and its method that the present invention is provided can realize the accurate discriminating of 9 kinds of Dalbergia timber, solving traditional timber recognition methods can not differentiate Dalbergia timber to the problem of " kind " level, it is that the timber-trade law enforcement agencies such as customs, quality inspection quarantine and wood management trade personnel provide a kind of discrimination method of high efficient and reliable, execution for CITS (CITES) provides technical support, with higher application value.
Description
Technical field
The invention belongs to the molecular Biological Detection field of wood material species identification, and in particular to one kind differentiates 9 kinds of Dalbergias
The DNA combination bar codes of timber and its discrimination method and application.
Background technology
Dalbergia (Dalbergia L.f.) is subordinate to pulse family (Leguminosae), and about more than 300 plant, and are distributed mainly on the torrid zone
And subtropical zone.Dalbergia wood quality is hard, texture is fine and smooth, and color and luster is soft and graceful, is loved by consumers.Vigorous market is needed
Driving for summation great number interests, makes the behaviors such as non-causative fault and illegal trading grow in intensity, and has ultimately resulted in Dalbergia timber money
The critical shortage in source and the destruction for exacerbating global wildwood resource.The present situation of Dalbergia timber resources causes international community
Highly give more sustained attention.86 kinds of Dalbergia timber are had at present is put into International Union for Conservation of Nature and Natural Resources (IUCN) Red List.2016
Endangered species of wild fauna and flora kind international trade protection pact (CITES) the 17th conference of states parties that year holds in South Africa, it is consistent logical
Cross and all seeds of Dalbergia are included in annex II.
Dalbergia timber varieties of trees enriches, and distribution is wider.The Dalbergia timber of different tree species, different sources, its market
Value also tends to far from each other.Therefore, scientific, rapid and accurate identification is carried out to Dalbergia timber with very high application valency
Value.Traditional anatomy of wood identification technology can only differentiate Dalbergia timber to " category " or " class ", it is impossible to realize " kind " level
Discriminating, and emerging DNA bar code technology provides possibility for the identification of Dalbergia timber " kind " level.
DNA bar code be a segment standard, with enough variations, easily amplification and relatively short DNA fragmentation.In recent years
Come, DNA bar code has been developed as the important tool of species identification.But unique DNA bar code for evolutionary relationship it is complicated and
The recognition capability of the nearlyer species of affiliation is relatively limited.And DNA combination bar codes to a plurality of unique DNA bar code by carrying out
Combination, with more rich sequence information site, and then improves species identification ability, is a kind of effective species discrimination method.
On the whole, Dalbergia wood species are enriched, and the conventional identification techniques based on the anatomy of wood are difficult to timber
The identification of " kind " level, and emerging DNA bar code technology, particularly DNA combination bar code progressively develop into timber identification
Effective tool.
The content of the invention
The technical problem to be solved in the present invention is can not accurately to differentiate 9 kinds of Dalbergia timber for traditional timber identification technology
To kind, and one kind is provided and is based on nucleus nrDNA ITS such as ITS2 sequences and chloroplast gene spacer region such as
The DNA combination bar codes of trnH-psbA sequences and a kind of accurate method for differentiating 9 kinds of Dalbergia timber.
So, the first object of the present invention is to provide a kind of DNA combination bar codes for differentiating 9 kinds of Dalbergia timber.
The second object of the present invention is to provide a kind of method for differentiating 9 kinds of Dalbergia timber.
The third object of the present invention is to provide the method that the DNA combines 9 kinds of Dalbergia timber of bar code and the discriminating
Application in the discriminating of Dalbergia timber.
To achieve these goals, the technical solution adopted by the present invention is as follows：
Bar code is combined present invention firstly provides a kind of DNA for differentiating 9 kinds of Dalbergia timber, the DNA combines bar shaped
Code be in bar code sequence trnL, trnH-psbA, trnV-trnM or ITS2 it is any two or more or all combination formed
DNA combines bar code.
Specifically, the DNA combinations bar code includes：ITS2+trnL、ITS2+trnH-psbA、ITS2+trnV-trnM、
trnH-psbA+trnL、trnH-psbA+trnV-trnM、trnV-trnM+trnL、ITS2+trnH-psbA+trnL、ITS2+
TrnH-psbA+trnV-trnM, ITS2+trnL+trnV-trnM, trnH-psbA+trnL+trnV-trnM and ITS2+trnH-
psbA+trnL+trnV-trnM。
It is preferred that, 9 kinds of Dalbergia timber is respectively dalbergia odorifera Dalbergia odorifera T.C.Chen, Huang
Wingceltis D.hupeana Hance, Hainan yellow wingceltis D.hainanensis Merr.＆Chun, toe yellow wingceltis D.cochinchinensis
Pierre, broad-leaved yellow wingceltis D.latifolia Roxb., East Africa rosewood D.melanoxylon Guill.＆Perr., Ovshinsky are yellow
Wingceltis D.oliveri Prain, nick yellow wingceltis D.retusa Hemsl. and Belize yellow wingceltis D.stevensonii Standl..
In view of factors such as expense cost and experimental periods, compared to three and the above bar code combination, ITS2+
Two bar code combinations such as trnH-psbA, trnH-psbA+trnL, trnL+trnV-trnM, ITS2+trnL are more preferable choosings
Select；In addition, for timber DNA identifications, DNA cloning success rate is a very important condition of screening DNA bar code.Table
4 displays, ITS2+trnH-psbA combinations have highest amplification success rate (ITS2 and trnH-psbA sequences more than in 4 combinations
Column-slice section amplification success rate is all higher than 90%), being more suitable for the discriminating that the wood sample seriously degraded occurs for DNA.
11 bar codes combination is screened according to DNA bar code optimum principle, optimal DNA combobars are determined
Shape code is ITS2+trnH-psbA.The ITS2+trnH-psbA combines bar code respectively by the nucleus of 9 kinds of Dalbergia timber
NrDNA ITS ITS2 sequences are formed with chloroplast gene spacer region trnH-psbA combined sequences.
It is preferred that, the ITS2+trnH-psbA sequences are as shown in SEQ ID No.9-17.
Further, present invention firstly provides a kind of method for differentiating 9 kinds of Dalbergia timber, this method utilizes DNA groups
Bar code is closed, and differentiates by molecular biology method 9 kinds of Dalbergia timber；The DNA combinations bar code is bar code sequence
Arrange trnL, trnH-psbA, trnV-trnM or ITS2 in it is any two or more or all combination formed DNA combination bar code.
Specifically, it the described method comprises the following steps：
(1) by wood sample milled processed into wood powder；
(2) DNA of the wood powder sample obtained by extraction step (1)；
(3) obtained DNA is extracted as template using step (2), PCR expands ITS2 the and trnH-psbA bar code sequences；
(4) pcr amplification product for obtaining step (3) is sequenced, and obtains ITS2 and trnH-psbA sequences, and to it
It is combined；
(5) 9 kinds of Huangs are differentiated with the ITS2+trnH-psbA bar codes combination constructing system development chadogram obtained by step (4)
Wingceltis belongs to timber.
It is preferred that, in the step (1) wood powder sample be ground to 200 mesh and more than.
It is preferred that, DNA need to be purified in the step (2), the final concentration of 1-200ng/ μ L of DNA.
It is preferred that, the positive anti-primer of ITS2 sequences is expanded as shown in SEQ ID No.1-2, amplification trnH-psbA sequences
Positive anti-primer such as SEQ ID No.3-4.
It is preferred that, pcr amplification reaction system is 0.5-3U archaeal dna polymerases, 1.0-2.0mM in the step (3)
MgCl2, 200 μM of single dNTP, 0.1-5.0 μM of single primers, pH7.0-8.0 0.1-1.5mg/mL bovine serum albumin(BSA)s and
10-1000ng template DNAs；
It is preferred that, PCR reaction conditions are 94-96 DEG C of pre-degeneration 0.5-10min in the step (3)；94-96 DEG C of denaturation
0.05-2min, 40-65 DEG C of annealing 0.5-2min, 72 DEG C of extension 0.3-2min, are circulated 20-50 times；72 DEG C extend 2-15min eventually.
It is preferred that, the method that constructing system development chadogram uses in the step (5) is adjacent method.
Further, bar code, or the side for differentiating 9 kinds of Dalbergia timber are combined present invention also offers the DNA
Application of the method in the discriminating of Dalbergia timber.
Beneficial effects of the present invention are as follows：
1st, the present invention combines bar code based on a kind of DNA, it is possible to achieve the accurate discriminating of 9 kinds of Dalbergia timber, breaches
The limitation of traditional timber identification technology；
2nd, the diagnostic primerses specificity that the present invention is screened is good, and amplification and sequencing success rate are high；
3rd, currently preferred ITS2+trnH-psbA combined sequences DNA bar code exists bright in 9 kinds of timber of Dalbergia
Aobvious difference site, with very strong distinguishing ability；
4th, currently preferred adjacent method constructing system development chadogram is simple to operate, stronger to seeds separating capacity；
5th, sampling amount of the present invention is few, and not by materials position (sapwood, heart sapwood transition region and heartwood), the original shape of sample
State (wooden unit, wood powder) factor influences；
6th, experiment condition of the present invention relies on few, and general Molecular Biology Lab can meet；
7th, the present invention accurately identifying there is provided a kind of new approach and thinking for Dalbergia timber.
In a word, the DNA combination bar codes and its method that the present invention is provided can realize the accurate mirror of 9 kinds of Dalbergia timber
Not, Dalbergia timber can not be differentiated to the problem of " kind " level by solving traditional timber recognition methods, be customs, quality inspection inspection
Yi Deng timber-trades law enforcement agency and wood management trade personnel provide a kind of discrimination method of high efficient and reliable, are in imminent danger wild dynamic
The execution of plant species international trade pact (CITES) provides technical support, with higher application value.
Brief description of the drawings
Fig. 1 is that the present invention combines the systematic growth adjoining tree that bar code is built based on DNA；
In figure：(a)ITS2+trnH-psbA,(b)trnH-psbA+trnL,(c)trnL+trnV-trnM,(d)ITS2+
trnL,(e)ITS2+trnL+trnV-trnM,(f)ITS2+trnH-psbA+trnL,(g)ITS2+trnH-psbA+trnV-
TrnM, (h) trnH-psbA+trnL+trnV-trnM and (i) ITS2+trnH-psbA+trnL+trnV-trnM.
Fig. 2 is the systematic growth that the DNA combination bar codes ITS+trnH-psbA built based on the present invention differentiates unknown sample
Adjacent tree.
Embodiment
Following examples are merely to illustrate the present invention, but are not limited to the invention scope of the present invention.The technical field
Technician can make the modifications and adaptations of some non-intrinsically safes according to the content of foregoing invention.
Embodiment 1：Dalbergia tree lumber specific primer design
(1) chloroplast DNA sequence trnL, trnH-psbA, trnV-trnM and cell of Dalbergia are downloaded from GenBank
Core DNA sequence dna ITS2 (table 1)；
The Dalbergia DNA bar code sequence downloaded in the GenBank of table 1
(2) the software aligned sequences of application Clustal X 1.81, search and determine 4 kinds of DNA bar code sequences between Dalbergia
Difference site；
(3) softwares of application Primer Premier 5 are to tetra- bar codes of trnL, trnH-psbA, trnV-trnM and ITS2
Design primer.Primer is synthesized by Shanghai bioengineering Co., Ltd, and the primer sequence is as shown in table 2.
2 four kinds of DNA bar code primer information of table
Embodiment 2：The DNA combinations bar code of 9 kinds of Dalbergia standard of wood samples preferably with determination
(1) standard sample collection
9 kinds of Dalbergia standard samples (are respectively dalbergia odorifera, yellow wingceltis, Hainan yellow wingceltis, toe yellow wingceltis, broad-leaved yellow wingceltis, East Africa
Rosewood, Ovshinsky yellow wingceltis, nick yellow wingceltis and Belize yellow wingceltis) it is total 50, it is taken from China Forestry Science Research Institute's timber mark
This shop.
(2) sample preparation
Wood sample is chosen, is cut off wood sample outer surface using the scalpel blade after 70% alcohol disinfecting, to avoid
External source pollutes；Wood sample is cut into some wood chips, low temperature precooling 3min in cryogenic freezing beveller is placed in, 3min is ground,
Running frequency 10cps；After grinding terminates, cross in 200 eye mesh screens, the microcentrifugal tube that thin wood powder is dispensed into some 50mL, often
Pipe wood powder amount 500mg, is placed in -80 DEG C of low temperature refrigerators and saves backup.
(3) DNA is extracted
In the ultra-clean working environment disinfected, with reference to the articles such as Jiao (Jiao L, Yin Y, Cheng Y, Jiang
X.DNA barcoding for identification of the endangered species Aquilaria
sinensis:comparison of data from heated or aged wood
samples.Holzforschung.2014；68(4):DNA extraction method 487-494.) carries out DNA to 9 kinds of Dalbergia timber
Extract.
(4) pcr amplification reaction and sequencing
Enter performing PCR amplification using four kinds of DNA bar code aligning primers in embodiment 1,
Pcr amplification reaction system is 30 μ L：The wherein μ L of Premix Ex Taq 15 (including l.25U Ex Taq DNA polymerize
Enzyme, 2mM MgCl2, 200 μM of single dNTP), 0.2 μM of single primer and about 20ng template DNAs.
PCR reactions are carried out in PCR amplification instrument.Response procedures are：94 DEG C of pre-degeneration 2min；94 DEG C are denatured 15s, 40 DEG C
(trnL), 61 DEG C (trnH-psbA), 53 DEG C (trnV-trnM) and 52 DEG C (ITS2) annealing 30s, 72 DEG C of extension 20s, circulation 40
It is secondary；72 DEG C extend 7min eventually, you can obtain the DNA purpose fragments of efficient amplification.Amplified production is sent by biotech firm after purification
Carry out two-way direct Sequencing (all primers of sequencing are identical with all primers of PCR).
(5) sequence alignment analysis and combined sequence.
Sequencing quality assessment is carried out to sequencing result using ContigExpress softwares, two ends low quality part is removed, and
Quality evaluation is carried out to remainder, if meeting quality requirement, sequence assembly and check and correction can be used for.9 kinds of Dalbergias
Tetra- kinds of bar code sequence GenBank accession number of trnL, trnH-psbA, trnV-trnM and ITS2 and sequence signature information of timber
Respectively as shown in Table 3 and Table 4.
Four kinds of bar codes of 9 kinds of Dalbergia wood species are combined respectively using Editseq softwares, obtained
ITS2+trnL、ITS2+trnH-psbA、ITS2+trnV-trnM、trnH-psbA+trnL、trnH-psbA+trnV-trnM、
trnV-trnM+trnL、ITS2+trnH-psbA+trnL、ITS2+trnH-psbA+trnV-trnM、ITS2+trnL+trnV-
11 DNA bar code groups such as trnM, trnH-psbA+trnL+trnV-trnM and ITS2+trnH-psbA+trnL+trnV-trnM
Close.
Four kinds of bar code sequence GenBank accession number of 9 kinds of Dalbergia timber that 3 research sequencings of table are obtained
4 four kinds of DNA bar code sequence signature information of table
(6) combined sequence DNA bar code preferably with determination
It is single to above-mentioned 4 kinds and 11 DNA combine distance in the kind of bar code by K2P distance models using MEGA softwares
Calculating analysis is carried out with inter-species distance, and using adjacent method constructing system development chadogram.
As a result show, the kind of single and DNA combination bar codes is interior and inter-species distance is as shown in table 5；Phylogenetic tree result
As shown in figure 1, single bar code cannot distinguish between 9 kinds of whole Dalbergia timber, and 9 pairs are combined bar code ITS2+trnH-
psbA、trnH-psbA+trnL、trnL+trnV-trnM、ITS2+trnL、ITS2+trnL+trnV-trnM、ITS2+trnH-
PsbA+trnL, ITS2+trnH-psbA+trnV-trnM, trnH-psbA+trnL+trnV-trnM and ITS2+trnH-psbA+
TrnL+trnV-trnM can accurately identify 9 kinds of Dalbergia timber of the above.
In view of factors such as expense cost and experimental periods, compared to three and the above bar code combination, ITS2+
Two bar code combinations such as trnH-psbA, trnH-psbA+trnL, trnL+trnV-trnM, ITS2+trnL are more preferable choosings
Select；In addition, for timber DNA identifications, DNA cloning success rate is a very important condition of screening DNA bar code.Table
4 displays, ITS2+trnH-psbA is combined as shown in SEQ ID No.7-19, in 4 combinations there is highest to expand into more than
Power (ITS2 and trnH-psbA sequence fragment amplification success rates are all higher than 90%), is more suitable for DNA and occurs the wood seriously degraded
The discriminating of material sample.
11 bar codes combination is screened according to above principle, determines that optimal DNA combination bar codes are
ITS2+trnH-psbA。
Table 5 is single and DNA is combined in the kind of bar code and inter-species distance
Embodiment 3：Unknown wood sample DNA differentiates
(1) wood sample derives from lumber market, by carrying out anatomic construction identification to it, judges category red acid branch class wood
Material, but None- identified to kind.
(2) prepare wood powder and be ground to 200 mesh and more than.
(3) timber DNA is extracted.
In the ultra-clean working environment disinfected, timber progress DNA is carried using DNA kits are extracted in embodiment 2
Take.The DNA also needs to be purified, the final concentration of 1-200ng/ μ L of DNA.
(4) pcr amplification reaction and sequencing.
Enter performing PCR amplification, amplimer such as SEQ ID NO by template of above-mentioned timber DNA：Shown in 1-4.PCR amplifications are anti-
It is 30 μ L to answer system：The wherein μ L of Premix Ex Taq 15 (including l.25U Ex Taq archaeal dna polymerases, 2mM MgCl2, 200 μ
The single dNTP of M), 0.2 μM of single primer and about 20ng template DNAs.
PCR reactions are carried out in PCR amplification instrument.Response procedures are：94 DEG C of pre-degeneration 2min；94 DEG C are denatured 15s, 40 DEG C
(trnL), 61 DEG C (trnH-psbA), 53 DEG C (trnV-trnM) and 52 DEG C (ITS2) annealing 30s, 72 DEG C of extension 20s, circulation 40
It is secondary；72 DEG C of extensions eventually.
(5) sequence alignment analysis differentiates with seeds.
The sequencing result of two sequences of ITS2 and trnH-psbA is combined, using composite sequence ITS2+trnH-
PsbA constructing systems develop chadogram.Analysis result as shown in Fig. 2 the sample can with toe yellow wingceltis sample clustering, and and its
He distinguishes Dalbergia seeds.Cluster result proves that the sample is toe yellow wingceltis.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Chinese Academy Of Forestry Research Institute Of Wood Industry
<120>A kind of DNA for differentiating 9 kinds of Dalbergia timber combines bar code and its discrimination method and application
<130> 011701704
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gcgcccgaag ccattaggcc aagggcacgc ctgcctgggt gtcaccaatc gctgccccaa 120
cccgtgcgcc tcaggccacg gagcggggcg aatgctggct tcccgtgagc acagcctcgc 180
ggttggctga aaatcgggtt cgtggtggat tcggcgccat gacagacggt ggttgagcat 240
gttctcgagg ccagtcatgc gcgcgacctc cgccagctcc gtacccagtg acccgcgagc 300
gacgtcgatc gcccatgacg cgacctcagg ttcaggcggg gctacttccc tctagaccta 360
gcttcggtcg aggctccatc tctaaatgga taatattttt gtcttaaagg atacgagttt 420
ttgaaagtaa aggagcaata tcaacagagt ttctattgct cctttacttt ttttttacat 480
tgcaaagtca tatgttaaaa aaaagcaaaa aaatgaatgc ttccattctt ttgctttttg 540
tatcccatcc tatcttatct taggaaacga gtaaaaacta aagttagaga agaaacagaa 600
aaaataataa caaaagaaaa gagtataaat ggtttagtct aggagatttt tattaagggc 660
ggatgtagcc aa 672
<210> 13
<211> 665
<212> DNA
<213>East Africa rosewood ITS2+trnH-psbA composite sequences
<400> 13
atgcgatact tggtgtgaat tgcagaatcc cgtgaaccat cgagtctttg aacgcaagtt 60
gcgcccgagg ccatccggct aagggcacgc ctgcctgggt gtcaccaatc gccgccccaa 120
cccccgcgcc tccgggcacg gagcggggcg aatgatggct tcccgtgagc accgcctcgc 180
ggctggctga aaatcgggtc cgtggcggaa gcagcgccac gacagatggt ggttgagcgt 240
gttctcgagg ccagtcgtgc gcgcggcctc cgccagctcc gtacccagtg acccgcgagc 300
gacgtcgatc gcccatgacg cgacctcagg ttcaggcggg gctacttccc tctagaccta 360
gcttcggtcg aggctccatc tctaaatgga taatattttt gtcttaaagg atacgagttt 420
ttgaaagtaa aggagcaata tcaacagagt ttctattgct cctttacttt tttttttttt 480
acattgcaaa gtcatatgtt aaaaaaaaca aatgaatgct tccattcttt tgctttttgt 540
atcccatcct atcttaagaa acgagtaaaa actaaagtta gagaagaaac agaaaaataa 600
taacaaaaga aaagagtata aataggttag tctaggagat ttttattaag ggcggatgta 660
gccaa 665
<210> 14
<211> 669
<212> DNA
<213>Ovshinsky yellow wingceltis ITS2+trnH-psbA composite sequences
<400> 14
atgcgatact tggtgtgaat tgcagaatcc cgcgaaccat cgagtctttg aacgcaagtt 60
gcgcccgaag ccattaggcc aagggcacgc ctgcctgggt gtcgccaatc gttgccccaa 120
ccccctgtgc ccgtggccac ggggtggggc gaatgctggc ctcccgtgag caccgcctcg 180
cggttggctg aaaatcgggt tcgtggtgga ttcagcgcca tgacggatgg tggttgagta 240
tgttctcgag gccagtcatg cgcgcgacct ccaccagttc cgtgccctgt gacccacggg 300
cgacgtcgat cgcccatgat gcgacctcag gttcaggcgg ggctacttcc ctctagacct 360
agcttcggtc gaggctccat ctctaaatgg ataatatttt tgtcttaaag gatacgagtt 420
tttgaaagta aaggagcaat atcaacagag tttctattgc tcctttactt tttttttaca 480
ttgcaaattc atatgttaaa aaaaaaaaaa aacaaatgaa tgcttccatt cttttgcttt 540
ttgtatccca tcctatctta tcttaggaaa cgagtaaaaa ctaaagttag agaagaaaca 600
gaaaaataat aacaaaagag tataaatggt ttagtctagg agatttttat taagggcgga 660
tgtagccaa 669
<210> 15
<211> 665
<212> DNA
<213>Nick yellow wingceltis ITS2+trnH-psbA composite sequences
<400> 15
atgcgatact tggtgtgaat tgcagaatcc cgcgaaccat cgagtctttg aacgcaagtt 60
gcgcccgagg ccattaggcc aagggcacgc ctgcctgcgt gtcaccaatc cccgccccaa 120
cccctgtgcc tccggccacg gagcggggcg aatgctggcc tcccgtgagc accgcctcgc 180
ggctggctga aaatcgggat cgtggtggat gcagcgtcat gtcagacggt ggttgagcgt 240
gttctcgagg ccagtcatga gggcagcctc caccagctcc gtacccagcg acccgcgagc 300
gatgccgatc gcccacgacg cggcctcagg ttcaggcggg gctacttccc tttagaccta 360
gcttcggtcg aggctccatc tctaaatgga taatattttt gtcttaaagg atacgagttt 420
ttgaaagtaa aggagcaata gaaactctgt tgatattgct cctttacttt tttttttcca 480
ttgcaaagtc atatgttaaa aaaaaacaca aatgaatgct tccattcttt tgctttttgt 540
atcccatcct atcttaggaa acgagtaaaa actaaagtta gagaagaaac agaaaaataa 600
taacaaaaga aaagagtata aatggtttag tctaggagat ttttattaag ggcggatgta 660
gccaa 665
<210> 16
<211> 670
<212> DNA
<213>Broad-leaved yellow wingceltis ITS2+trnH-psbA composite sequences
<400> 16
atgcgatact tggtgtgaat tgcagaatcc cgtgaaccat cgagtctttg aacgcaagtt 60
gcgcccgaag ccattaggcc aagggcacgc ctgcctgggt gtcaccaatc gttgccccaa 120
ccccctgtgc ccgtggccac ggggtggggc gaatgctggc ctcccgtgag caccgcctcg 180
cggttggctg aaaatcgggt tcgtggtgga ttcagcgcca tgacggacgg tggttgagta 240
tgttctcgag gccagtcatg cgcgcgacct ccaccagttc cgtgccctgt gacccgcggg 300
cgacgtcgat cgcccatgat gcgacctcag gttcaggcgg ggctacttcc ctctagacct 360
agcttcggtc gaggctccat ctctaaatgg ataatatttt tgtcttaaag gatacgagtt 420
tttgaaagta aaggagcaat atcaacagag tttctattgc tcctttactt ttttttttac 480
attgcaaagt catatgttaa aaaaaaagca aaaaaatgaa tgcttccatt cttttgcttt 540
ttgtatccca tcctatctta tcttaggaaa cgagtaaaaa ctaaagttag agaagaaaca 600
gaaaaaataa taacaaaaga gtataaatgg tttagtctag gagattttta ttaagggcgg 660
atgtagccaa 670
<210> 17
<211> 671
<212> DNA
<213>Belize yellow wingceltis ITS2+trnH-psbA composite sequences
<400> 17
atgcgatact tggtgtgaat tgcaggatcc cgcgaaccgt cgagtctttg agcgcaagtt 60
gcccccggag ccattaggcc aagggcacgc ctgccagggt gtcgccaatc gttgccccaa 120
cccgctgtgc ccgtggccac ggggtggggc gaatgctggc ctcccgtgag caccgcctcg 180
cggttgcctg aaaatcgggt tcttggtgga ttcagcgcca tgagggatgg tggttgagta 240
tcttctcgag gccagtgatg cgcgcgacct ctaccagttc cgtgccccgt gacccgcggg 300
cgacgtcggt cgcccatgat gcgacctcag gttcaggcgg ggctacttcc ctctagacct 360
agcttcggtc gaggctccat ctctaaatgg ataatatttt tgtcttaaag gatacgagtt 420
tttgaaagta aaggagcaat atcaacagag tttctattgc tcctttactt tttttttttt 480
tacattgcaa agtcatatgt taaaaaaaaa aaaacaaatg aatgcttcca ttcttttgct 540
ttttgtatcc catcctatct taagaaacga gtaaaaacta aagttagaga agaaacagaa 600
aaataataac aaaagaaaag agtataaata ggttagtcta ggagattttt attaagggcg 660
gatgtagcca a 671
Claims (10)
1. a kind of DNA combination bar codes for differentiating 9 kinds of Dalbergia timber, it is characterised in that the DNA combinations bar code is bar shaped
Any two or more or DNA combobars that all combination is formed in code sequence trnL, trnH-psbA, trnV-trnM or ITS2
Shape code.
2. DNA as claimed in claim 1 combines bar code, it is characterised in that 9 kinds of Dalbergia timber is respectively that dalbergia wood is yellow
Wingceltis Dalbergia odorifera T.C.Chen, yellow wingceltis D.hupeana Hance, Hainan yellow wingceltis D.hainanensis
Merr.＆Chun, toe yellow wingceltis D.cochinchinensis Pierre, broad-leaved yellow wingceltis D.latifolia Roxb., East Africa are black
Yellow wingceltis D.melanoxylon Guill.＆Perr., Ovshinsky yellow wingceltis D.oliveri Prain, nick yellow wingceltis D.retusa
Hemsl. with Belize yellow wingceltis D.stevensonii Standl..
3. DNA as claimed in claim 1 or 2 combines bar code, it is characterised in that the DNA combinations bar code is ITS2+
TrnH-psbA combines bar code.
4. DNA as claimed in claim 3 combines bar code, it is characterised in that the ITS2+trnH-psbA sequences such as SEQ
Shown in ID No.9-17.
5. a kind of method for differentiating 9 kinds of Dalbergia timber, it is characterised in that this method combines bar code using DNA, and by dividing
Sub- biological means differentiate 9 kinds of Dalbergia timber；DNA combination bar code be bar code sequence trnL, trnH-psbA,
In trnV-trnM or ITS2 it is any two or more or all combination formed DNA combination bar code.
6. method as claimed in claim 5, it is characterised in that comprise the following steps：
(1) by wood sample milled processed into wood powder；
(2) DNA of the wood powder sample obtained by extraction step (1)；
(3) obtained DNA is extracted as template using step (2), PCR expands ITS2 the and trnH-psbA bar code sequences；
(4) pcr amplification product for obtaining step (3) is sequenced, and obtains ITS2 and trnH-psbA sequences, and it is carried out
Combination；
(5) 9 kinds of Dalbergias are differentiated with the ITS2+trnH-psbA bar codes combination constructing system development chadogram obtained by step (4)
Timber.
7. method as claimed in claim 6, it is characterised in that the positive anti-primer such as SEQ ID No.1-2 of amplification ITS2 sequences
It is shown, expand the positive anti-primer such as SEQ ID No.3-4 of trnH-psbA sequences.
8. method as claimed in claim 6, it is characterised in that pcr amplification reaction system is 0.5-3U in the step (3)
Archaeal dna polymerase, 1.0-2.0mM MgCl2, 200 μM of single dNTP, 0.1-5.0 μM of single primer, pH7.0-8.0 0.1-
1.5mg/mL bovine serum albumin(BSA)s and 10-1000ng template DNAs；
PCR reaction conditions are 94-96 DEG C of pre-degeneration 0.5-10min in the step (3)；94-96 DEG C of denaturation 0.05-2min, 40-
65 DEG C of annealing 0.5-2min, 72 DEG C of extension 0.3-2min, are circulated 20-50 times；72 DEG C extend 2-15min eventually.
9. method as claimed in claim 6, it is characterised in that constructing system development chadogram uses in the step (5)
Method is adjacent method.
10. DNA described in Claims 1 to 4 any one combines bar code, or differentiates 9 described in claim 5~9 any one
Plant application of the method for Dalbergia timber in the discriminating of Dalbergia timber.
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| CN108169373A (en) * | 2017-12-22 | 2018-06-15 | 中国林业科学研究院热带林业研究所 | A kind of quick discriminating dalbergia odorifera and the gas chromatography mass spectrometry method of cochin yellow wingceltis |
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| CN108169373A (en) * | 2017-12-22 | 2018-06-15 | 中国林业科学研究院热带林业研究所 | A kind of quick discriminating dalbergia odorifera and the gas chromatography mass spectrometry method of cochin yellow wingceltis |
| CN109486984A (en) * | 2018-08-03 | 2019-03-19 | 广东天保参茸有限公司 | A kind of combination bar code sequence and its accurate identification method identified for ginseng and American Ginseng |
| CN110241243A (en) * | 2019-06-14 | 2019-09-17 | 浙江省检验检疫科学技术研究院 | The method for identifying molecules and its primer and probe of Ovshinsky yellow wingceltis |
| CN110241243B (en) * | 2019-06-14 | 2024-02-27 | 浙江省检验检疫科学技术研究院 | Molecular identification method of Pterocarpus of Orthosiphon and primer and probe thereof |
| CN112899389A (en) * | 2020-12-29 | 2021-06-04 | 中国科学院华南植物园 | Identifying primer and molecular identifying method for dalbergia odorifera |
| CN112899389B (en) * | 2020-12-29 | 2022-03-22 | 中国科学院华南植物园 | Identifying primer and molecular identifying method for dalbergia odorifera |
| CN114540532A (en) * | 2022-03-04 | 2022-05-27 | 中国林业科学研究院木材工业研究所 | DNA bar code and method for identifying multiple wood in Machilus and Machilus |
| CN114736978A (en) * | 2022-03-11 | 2022-07-12 | 中国林业科学研究院热带林业研究所 | Method for identifying tree species of Hainan Huanghua pear and related easily-confused species thereof and application |
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