CN110951912A - Method for identifying dendrobium huoshanense based on DNA bar code - Google Patents
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Abstract
Compared with the prior art, the invention utilizes an ITS sequence as a DNA bar code, wherein the ITS sequence is a gene sequence for coding ribosomal RNA (nr DNA) in a nuclear genome and contains more variation sites to provide a basis for identifying various species. The invention also provides a primer for identifying the varieties of dendrobium huoshanense and dendrobium henryi, has the advantages of clear amplified fragments, high resolution, good sensitivity, reliable and accurate result, high efficiency, high speed and the like, can identify dendrobium huoshanense and related species dendrobium henryi, realizes the identification of the germplasm resources of dendrobium huoshanense with high efficiency, low cost and convenient operation, and ensures the quality of dendrobium huoshanense provenance in the dendrobium huoshanense industry.
Description
Technical Field
The invention belongs to the field of species identification, and particularly relates to a method for identifying dendrobium huoshanense based on a DNA (deoxyribonucleic acid) bar code.
Background
Dendrobium huoshanense (Dendrobium huoshanense CZ. Tang et S.J.Chen) belongs to perennial herb of Dendrobium of Orchidaceae, also known as Huohu and Mihuhu. The dendrobium huoshanense is a common and famous traditional Chinese medicinal material, is mainly distributed in the large hills and the adjacent areas in China, has extremely high ornamental and medicinal values, is regarded as a treasure in medicinal dendrobium since ancient times, and enjoys the reputation of the best Chinese mesona.
Modern pharmacological studies show that: the dendrobium huoshanense has the effects of resisting oxidation, aging, tumors and the like, has good effects on treating cataract, reducing blood sugar and protecting liver, but has extremely strict requirements on living environment, and is slow in growth, and due to excessive mining, wild dendrobium huoshanense resources are endangered to be extinct.
In the genuine land area of dendrobium huoshanense, tissue culture rapid propagation technology is adopted to quickly obtain test-tube seedlings or artificially induce seeds to germinate to generate seedlings, and the cultured dendrobium huoshanense is cultivated in a large-scale field, and gradually replaces wild resources to become the main source of medicinal dendrobium huoshanense. At present, dendrobium medicinal materials are mainly circulated in the market in the form of deep-processed maple pipes or decoction pieces.
However, the dendrobium candidum has complicated and various basic sources. The geographical distribution of the Dendrobium huinan (Dendrobium hennense J. L. Liu et L. X. Gao) is basically consistent with that of the Dendrobium huoshanense, and the Dendrobium huinan and the Dendrobium huoshanense are very similar in appearance, so the Dendrobium huinan becomes a main source of Dendrobium huoshanense counterfeit products in the current market.
The existing identification method takes whether the petals of the petals have purple plaques or not as a main basis for distinguishing dendrobium huoshanense and dendrobium henna, but plants which are not in the flowering period or dendrobium huoshanense medicinal materials which are used as medicines by stems are difficult to distinguish from dendrobium henna. Until now, no complete identification system is used for identifying the authenticity of dendrobium huoshanense and dendrobium hennanense, so that an identification method for the authenticity of dendrobium huoshanense needs to be established urgently to ensure the seed source quality of dendrobium huoshanense in the dendrobium huoshanense industry.
The DNA molecular marker technology has the advantages of stable heredity, no environmental influence and rapid and accurate detection, and can rapidly and accurately identify some plant populations with shapes which are difficult to distinguish. In recent years, DNA barcode technology is widely used in species identification, DNA barcode (DNA barcode)) is a molecular technology for rapidly identifying species by using a relatively short, standard and acknowledged DNA fragment in a gene, the DNA barcode enables automation and standardization of a specimen identification process, the identification success rate is high, and identification of any qualified DNA sample can be basically achieved, such as morphological original plant leaves, flowers, seeds, specimens, and amorphous powder.
With the development of the second-generation sequencing technology, the identification of the Chinese patent medicine components can be realized by combining the DNA bar code with the second-generation sequencing technology. The international bio-barcode consortium proposed the chloroplast gene mat K + rbc L as a universal DNA barcode for plants.
However, due to dendrobium huoshanense and its kindred species dendrobium huoshanense, the existing technology can not distinguish dendrobium huoshanense from dendrobium huoshanense.
Disclosure of Invention
The invention aims to provide a method for identifying dendrobium huoshanense based on a DNA bar code, wherein the DNA bar code is an ITS sequence.
The specific technical scheme of the invention is as follows:
the invention provides a method for identifying dendrobium huoshanense based on a DNA barcode, wherein the DNA barcode is an ITS sequence and is used for identifying the dendrobium huoshanense.
Further, dendrobium huoshanense and dendrobium huinanense are identified.
The method for identifying dendrobium huoshanense based on the DNA bar code comprises the following steps:
1) extracting DNA of a sample;
2) PCR amplification;
3) sequencing the PCR amplification product;
4) and (5) constructing a phylogenetic tree and identifying the dendrobium huoshanense.
Further, the method for extracting DNA from the sample in the step 1) comprises the following steps: the sample was dried with silica gel, and after drying, genomic DNA was extracted by the CTAB method.
The specific DNA extraction method is as follows:
1-1) weighing 0.020g of dried dendrobium leaves, putting the dendrobium leaves into a marked EP tube, adding sterilized steel balls, vibrating and grinding the dendrobium leaves for 60s by using a nucleic acid extractor, and taking out the steel balls;
1-2) adding 1.4ml of CTABfree solution, shaking up for 30min at a constant temperature of 37 ℃ by a shaking table, centrifuging for 15min under the condition of 10000rpm, and removing supernatant;
1-3) adding 700 μ l of 2% CTAB solution preheated to 65 ℃ into an EP tube, uniformly mixing, carrying out water bath at 65 ℃ for 1h, and shaking up by hand once every 10 min;
1-4) after the water bath is finished, cooling the solution to room temperature, centrifuging for 15min at 10000rpm, sucking 700 mu l of supernatant, transferring the supernatant into another 1.5ml centrifuge tube, adding 70 mu l of pre-preheated 10% CTAB solution, and shaking up;
1-5) adding 700 mu l of chloroform/isoamylol with the volume ratio of 24:1 into the centrifuge tube 1-4), uniformly mixing, placing into a constant temperature shaking table, shaking for 10min at 37 ℃, centrifuging for 15min at 12000rpm, and taking supernatant to another centrifuge tube;
1-6) repeating the operation of 1-5) twice;
1-7) adding 200 mul NaCl solution and 400 mul isoamyl alcohol precooled at-20 ℃ into the obtained supernatant, mixing uniformly, and placing in a refrigerator at-20 ℃ for freezing for 2 h; taking out the centrifuge tube, centrifuging at 12000rpm for 15min, and removing supernatant;
1-8) washing the precipitate obtained in the step 1-7) twice by using 1000 mu l of 75% absolute ethyl alcohol, then washing once by using 1000 mu l of absolute ethyl alcohol, and naturally drying in the air;
1-9) adding 100 mul of TE buffer solution, standing for 2h at room temperature, detecting whether the obtained DNA stock solution has bright and clear bands in 1.0% agarose gel electrophoresis, detecting the DNA concentration by using a spectrophotometer, diluting to 20-30 ng/mul, and placing in a refrigerator at-20 ℃ for later use.
In the extraction process, the traditional method uses liquid nitrogen to quickly grind the blades into powder, the FastpreprFP 120 quick nucleic acid extractor is used for processing the blades, and compared with other existing sample preparation methods, the sample processing system can simultaneously process 24 samples, and a quick 8-shaped oscillation mode is adopted, and the steel balls are matched, so that the sample can be efficiently and fully crushed, and the fast 8-shaped oscillation method has the advantages of wide universality, high efficiency and flexibility. In addition, the invention adds CTABfree liquid firstly to provide a buffer environment, prevent the nucleic acid from being damaged and inhibit the activity of DNase at the same time. Also, the invention adds preheated 10% CTAB solution, can make DNA combine with CTAB in high salt solution to form complex and dissolve in high salt solution, for the next step of DNA complete elution preparation.
The step 2) PCR amplification specifically comprises the following steps: and (3) carrying out PCR amplification by using a primer, wherein the PCR amplification adopts a 30 mu L reaction system: 15 μ L of 2 XTSINGKE Master Mix (New Biotech Co., Ltd., Okins, Beijing), 1.5 μ L of upstream and downstream primers (10 μmol/L), 2 μ L of DNA template, ddH2O is complemented to 30 mu l;
further, the primers used were: ITS sequence upstream primer F: 5'-CGTAACAAGGTTTCCGTAGGTGAAC-3', respectively;
ITS sequence downstream primer R: 5'-TTATTGATATGCTTAAACTCAGCGGG-3', respectively;
target band length: 605 + 715bp, and an annealing temperature of 56 ℃.
Further, the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 30s for 35 cycles; 8min at 72 ℃; storing at 4 ℃.
Step 3) sequencing the PCR amplification;
the PCR amplification product is sent to the general biological System (Anhui) limited company for sequencing, the ITS sequence is subjected to forward sequencing, and the length of the ITS sequence band is in the interval of 605-715 bp.
Step 4), constructing a phylogenetic tree, and identifying dendrobium huoshanense specifically as follows:
after sequencing, software Contig Express is used for proofreading and splicing, MEGA7 software is used for sequence alignment, and a phylogenetic tree is constructed and used for identifying dendrobium huoshanense.
Compared with the prior art, the invention utilizes the ITS sequence as the DNA bar code, the ITS sequence is the gene sequence of coding ribosomal RNA (nr DNA) in the nuclear genome, and the ITS sequence contains more variation sites to provide a basis for identifying various species. The invention also provides a primer for identifying the varieties of dendrobium huoshanense and dendrobium henryi, has the advantages of clear amplified fragments, high resolution, good sensitivity, reliable and accurate result, high efficiency, high speed and the like, can identify dendrobium huoshanense and related species dendrobium henryi, realizes the identification of the germplasm resources of dendrobium huoshanense with high efficiency, low cost and convenient operation, and ensures the quality of dendrobium huoshanense provenance in the dendrobium huoshanense industry.
Drawings
FIG. 1 is an agarose gel electrophoresis of 19 genomic DNAs of Dendrobium huoshanense;
FIG. 2 is agarose gel electrophoresis of 10 Henan Dendrobii caulis and 1 Dendrobium huoshanense genome DNA;
FIG. 3 is a gel electrophoresis diagram of the ITS2 sequence PCR amplification product;
FIG. 4 is a gel electrophoresis diagram of a PCR amplification product of a MatK sequence;
FIG. 5 is a gel electrophoresis image of a PCR amplification product of rbcL sequences;
FIG. 6 is a gel electrophoresis of the PCR amplification product of ITS sequence;
FIG. 7 is a block diagram of an ITS sequence construction phylogenetic NJ tree according to the method of the present invention;
FIG. 8 is an ITS2 sequence building phylogenetic NJ tree;
FIG. 9 is a MatK sequence construction phylogenetic NJ tree;
FIG. 10 is a phylogenetic NJ tree constructed from rbcL sequences.
Detailed Description
The required materials of the invention are dendrobium huoshanense and dendrobium huoshanense, which are mainly collected from Dabieshan mountain area of Anhui province, and because wild species resources of dendrobium huoshanense are deficient, 5 wild dendrobium huoshanense resources are used as dendrobium huoshanense samples, and the rest are dendrobium huoshanense cultivation resources.
The nucleic acid extractor used in the present invention is model fastprepFP 120.
The preparation method of the reagent used for extracting the DNA comprises the following steps:
Tris-HCl solution (pH8.0), Tris (12.11g), concentrated hydrochloric acid (4.2ml) were added thereto, and distilled water was added to the solution to a volume of 100 ml.
NaCl solution NaCl (23.376), made up to 100ml with distilled water.
EDTA (pH8.0), EDTA (18.61g), NaOH (about 2g), distilled water to 100 ml.
CTAB free solution β -mercaptoethanol (2ml), Tris-HCl solution (100. mu. mol/L) (10ml),20mmol/LEDTA (10ml), NaCl (1.46g), PVP (2g), distilled water to 100 ml.
2% CTAB solution CTAB (2g), Tris-HCl solution (10ml), EDTA (4ml), NaCL (8.19g), PVP (2g), β -mercaptoethanol (2ml), distilled water to 100 ml.
10% CTAB solution: CTAB (10g), NaCl (4.10g) and distilled water to 100 ml.
TE buffer solution: Tris-HCl solution (1ml), EDTA (0.2ml), distilled water to 100 ml.
The above solutions are mixed and sterilized for use, wherein the CTAB free solution and the 2% CTAB solution are prepared by mixing and sterilizing other raw materials and then adding mercaptoethanol.
Chloroform/isoamyl alcohol: chloroform and isoamylol according to a volume ratio of 24:1, and storing in a brown bottle.
Example 1
A method for identifying dendrobium huoshanense based on DNA bar codes comprises the following steps:
the samples identified by the method are dendrobium huoshanense and dendrobium heonanthum, wild resources of dendrobium huoshanense are 5, cultivation resources are 15, and the number of dendrobium heonanthum samples is 10, and the specific information is shown in table 1.
The identification method comprises the following steps:
1) DNA extraction of the sample:
1-1) weighing 0.020g of dried dendrobium leaves, putting the dendrobium leaves into a marked EP tube, adding sterilized steel balls, vibrating and grinding the dendrobium leaves for 60s by using a nucleic acid extractor, and taking out the steel balls;
1-2) adding 1.4ml of CTAB free solution, shaking uniformly at constant temperature of 37 ℃ for 30min, centrifuging for 15min under the condition of 10000rpm, and removing supernatant;
1-3) adding 700 μ l of 2% CTAB solution preheated to 65 ℃ into an EP tube, uniformly mixing, carrying out water bath at 65 ℃ for 1h, and shaking up by hand once every 10 min;
1-4) after the water bath is finished, cooling the solution to room temperature, centrifuging at normal temperature, separating for 15min at 10000rpm, sucking 700 mu l of supernatant, transferring the supernatant into another 1.5ml centrifugal tube, adding 70 mu l of pre-preheated 10% CTAB solution, and shaking up;
1-5) adding 700 mu l of chloroform/isoamylol with the volume ratio of 24:1 into the centrifuge tube 1-4), uniformly mixing, placing into a constant temperature shaking table, shaking for 10min at 37 ℃, centrifuging for 15min at 12000rpm, and taking supernatant to another centrifuge tube;
1-6) repeating the steps 1-5) twice;
1-7) adding 200 mul NaCl solution and 400 mul isoamyl alcohol precooled at-20 ℃ into the obtained supernatant, uniformly mixing, and placing in a refrigerator at-20 ℃ for freezing for 2 h; taking out the centrifuge tube, centrifuging at 12000rpm for 15min, and removing supernatant;
1-8) washing the precipitate obtained in the step 1-7) twice by using 1000 mu l of 75% absolute ethyl alcohol, then washing once by using 1000 mu l of absolute ethyl alcohol, and naturally drying in the air;
1-9) adding 100 mu l of TE buffer solution, standing for 2h at room temperature, detecting whether the obtained DNA stock solution has bright and clear bands in 1.0% agarose gel electrophoresis, and the result shows that the extracted genomic DNA bands are clear, bright and not dispersed, and can be used for subsequent PCR experiments. And detecting the DNA concentration by using a spectrophotometer, diluting to 20-30 ng/mu l, and placing in a refrigerator at the temperature of-20 ℃ for later use. The detection result of the dendrobium huoshanense genome electrophoresis is shown in figure 1, wherein numbers 1-3 in figure 1 are HS-WJD1, HS-WJD2, HS-WJD4, numbers 4-6 are HS-KS5, HS-KS6, HS-KS7, number 7 is HS-HSZ3, numbers 8-9 are HS-FK5, HS-FK7, numbers 10-12 are HS-PYT1, HS-PYT2, HS-PYT3, numbers 13 and 14 are HS-TPF3, HS-TPF4, numbers 15 and 16 are HS-JZ-1-7, HS-JZ-1-8, numbers 17 and 18 are HS-JXZ-1-3, HS-JXZ-1-5, and number 19 is HS-SKJ 3. Number 11 in FIG. 2 is Dendrobium huoshanense HS-YK1, numbers 1-10 in FIG. 2 are Dendrobium huinanense genome HN1-HN10, wherein M is MarKer III (Tiangen Biochemical technology, Beijing) Co., Ltd.).
TABLE 1 information on samples identified according to the invention
2) And (3) carrying out germplasm resource amplification on the sample by using a nuclear gene ITS sequence primer. The specific operation steps are as follows: using the ITS sequence upstream primer F: 5'-CGTAACAAGGTTTCCGTAGGTGAAC-3', respectively; ITS sequence downstream primer R: 5'-TTATTGATATGCTTAAACTCAGCGGG-3' are provided. Performing PCR amplification on dendrobium huoshanense and dendrobium huoshanense, wherein the PCR amplification adopts a 30 mu L reaction system: 15 μ L of 2 XTSINGKE Master Mix (New Biotechnology Co., Ltd., Okinsoniaceae, Beijing), 1.5 μ L of upstream and downstream primers (10 μmol/L), 2 μ L of DNA template, ddH2O complement to 30 μ l, PCR amplification program: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 30s for 35 cycles; extending for 8min at 72 ℃; storing at 4 ℃.
After the PCR product is subjected to 1.0% agarose gel electrophoresis detection, the amplification result is observed under an ultraviolet gel imaging system, the length of a target fragment amplified by the ITS sequence is about 700bp, no non-specific band exists, and the target band is clear and bright. The ITS PCR amplification agarose gel electrophoresis detection result is shown in FIG. 6, wherein the numbers 1-8 are Dendrobium huoshanense samples, the numbers 1-3 are HS-WJD1, HS-WJD2 and HS-WJD4, the numbers 4 and 5 are HS-TPF3, HS-TPF4, the numbers 6,7 and 8 are HS-KS5, HS-KS6 and HS-KS7 respectively. Numbers 9-12 are Henan dendrobe samples, numbers 9-12 are HN1, HN2, HN3, HN4, respectively, and M is Marker II (Tiangen Biochemical technology, Beijing, Ltd.).
3) The PCR amplification product is sent to the general biological System (Anhui) limited company for sequencing, the ITS sequence is subjected to forward sequencing, and the length of the ITS sequence band is in the interval of 605-715 bp.
4) After sequencing, software contighexpress is used for proofreading and splicing, an ' Assemblem Selected Fragments ' command under the Assemblem ' column is used for obtaining a contig1 result under the Assemblem 1, if the sequencing results of the two sequences have errors, a green vertical bar in a splicing picture frame represents different places, a specific base is finally judged through a peak shape, and meanwhile, corresponding sequences of the ITS, namely dendrobium huoshanense and the Henan dendrobium nobile are downloaded from NCBI (https:// www.ncbi.nlm.nih.gov). The sequence alignment is carried out by using MEGA7 software, a phylogenetic tree is constructed by adopting a neighbor-join algorithm, No. of Bootstrap replies are 1000, Kimura2-parameter Model is adopted by a Model, the constructed phylogenetic tree is shown in figure 7, and the dendrobii henryi and dendrobii huoshanense are obviously divided into two branches by using the ITS sequence, so that the dendrobii henryi and dendrobii huoshanense can be efficiently distinguished by using the DNA barcode ITS sequence.
Comparative example 1
The DNA barcodes are ITS2, MatK and rbcL sequences respectively for identification, and the sample identification and the method are the same as the example 1, and specifically comprise the following steps:
1) the sample DNA was extracted as in example 1;
2) PCR amplification was performed in the same manner as in example 1, except for the primer sequences and the annealing temperature, as shown in Table 2 below.
TABLE 2 DNA Bar code Universal primer sequences and related information
After the PCR product obtained in comparative example 1 was detected by 1.0% agarose gel electrophoresis, the amplification result was observed under an ultraviolet gel imaging system, as shown in FIGS. 3 to 5. FIG. 3 shows the result of PCR amplification electrophoresis detection of ITS2, where the numbers 1-8 are Dendrobium huoshanense samples, the numbers 1-3 are HS-WJD1, HS-WJD2 and HS-WJD4, the numbers 4 and 5 are HS-TPF3 and HS-TPF4, and the numbers 6,7 and 8 are HS-KS5, HS-KS6 and HS-KS7, respectively; numbers 9-12 are Henan dendrobe samples, and numbers 9-12 are Henan dendrobe samples, which are HN1, HN2, HN3 and HN4 respectively. FIG. 4 shows the results of MatK PCR amplification electrophoresis, wherein the numbers 1-14 are Dendrobium huoshanense samples, wherein the numbers 1-3 are HS-WJD1, HS-WJD2, HS-WJD4, the numbers 4 and 5 are HS-TPF3, HS-TPF4, the numbers 6,7 and 8 are HS-KS5, HS-KS6, HS-KS7, the numbers 9 are HS-HSZ3, the numbers 10 and 11 are HS-FK5, HS-FK7, the numbers 12,13 and 14 are HS-PYT1, HS-PYT2 and HS-PYT3, respectively. Numbers 15-24 are Henan dendrobe samples, HN1, HN2, HN3, HN4, HN5, HN6, HN7, HN8, HN9, and HN 10. FIG. 5 shows rbcL PCR amplification electrophoresis detection results, wherein numbers 1-8 are Dendrobium huoshanense samples, wherein numbers 1-3 are HS-WJD1, HS-WJD2, HS-WJD4, numbers 4 and 5 are HS-TPF3, HS-TPF4, numbers 6,7 and 8 are HS-KS5, HS-KS6, HS-KS7, and numbers 9-10 are Dendrobium huinanense samples, and are HN1 and HN2, respectively. Wherein M in FIG. 3 is Marker II (Tiangen Biochemical technology (Beijing) Co., Ltd.) and M in FIG. 5 is DL2000 DNA Marker (Tiangen Biochemical technology (Beijing) Co., Ltd.).
The length of the target fragment amplified by the ITS2 sequence is between 400-500bp, the lengths of the target fragments amplified by the chloroplast genes MatK and rbcL are respectively between 700-900bp and 1000-1200bp, no non-specific band exists, and the target band is clear and bright.
3) Sending the PCR amplification product to a general biological system (Anhui) limited company for sequencing, carrying out forward sequencing on ITS2 and MatK sequences, carrying out bidirectional sequencing on the rbcL amplification product, wherein the strip length of the ITS2 sequence is 446-462bp, the strip length of the MatK sequence is 771-834bp, and the strip length of the rbcL sequence is 1241-1291 bp.
4) The sequenced DNA barcode sequence is proofread and spliced by using software ContigExpress, and the obtained sequence is as follows: wherein the ITS SEQUENCE of the dendrobium huoshanense with the number of HS-TPF4 is shown as SEQUENCE Listing NO. 1. The ITS SEQUENCE of Dendrobium huinanense with number HN5 is shown as SEQUENCE Listing NO. 2. Meanwhile, the corresponding sequences of Dendrobium huoshanense, Dendrobium huoshanense and Dendrobium Henan are downloaded from NCBI (https:// www.ncbi.nlm.nih.gov) to ITS2, MatK and rbcL. The sequences were aligned and phylogenetic trees were constructed using MEGA7 software. The phylogenetic tree is constructed by adopting neighbor-join algorithm, No. of Bootstrap replicons is 1000, Model adopts Kimura2-parameter Model, and the constructed phylogenetic tree is shown in FIG. 8-FIG. 10.
Due to the fact that double peaks exist in sequencing of ITS2, MatK and rbcL amplification products, samples with double peaks exist in sequencing are removed when an NJ tree is constructed, sequencing double peaks exist in sequencing of ITS2 sequences, HS-WJD4, HS-TPF3, HS-TPF4, HS-PYT1, HS-PYT2, HN4 and HN6, sequencing double peaks exist in sequencing of MatK sequences, sequencing double peaks exist in HS-KS7, sequencing double peaks exist in rbcL sequences, HS-WJD4, HS-FK5, HS-JZ-1-7, HS-JXZ-1-3, HS-YK1, HS-PYT1, HN5 and sequencing double peaks exist in sequencing of HN 7.
The comparison shows that the dendrobii Henan and dendrobii huoshanensis are obviously divided into two major branches in the phylogenetic tree constructed by the ITS sequences, and the phylogenetic tree constructed by the ITS2, MatK and rbcL sequences shows the phenomenon that the dendrobii Henan and dendrobii Henan overlap with each other, the interspecies difference is small, and the dendrobii Henan and dendrobii Henan cannot be well distinguished, so the invention can efficiently distinguish the dendrobii Henan and dendrobii Huoshanensis by using the DNA barcode ITS sequences.
SEQUENCE LISTING
<110> university of teacher's university in Anhui
<120> method for identifying dendrobium huoshanense based on DNA bar code
<130>1
<160>10
<170>PatentIn version 3.3
<210>1
<211>694
<212>DNA
<213> dendrobium huoshanense ITS sequence
<400>1
tattgtcgag accgaacaca acgagcgatt ttgtgaacct gtaaaaataa gcggtggctc 60
ttgctgctgc gataaaatcc acccgagtca tcgcctcatc ccctctttgg ggtggggacg 120
tgatgaagga tggatgaacc ctcaaatcgg cgcagcgttg cgccaaggga atcttgaagc 180
acaagcccat aaatgggttt cgtgggatgg ggtgctgtcg cacgccatat tgattgacac 240
gactctcggc aatggatatc tcggctctcg catcgatgaa gagcgcagcg aaatgcgata 300
tgtggtgcga attgcagaat cccgcgaacc atcgagtctt tgaacgcaag ttgcgcctga 360
ggccaaccgg ctgagggcac gtccgcctgg gcgtcaagca ttttatcact ccgtgcctac 420
tctcccatcc atggatgtgt tgctaaggct cggatgtgca cggtggctcg tcgtgcccct 480
tggtgcggcg ggctgaaggg cgggtcatct tctcgttggc tgccaacaat aaggggtgga 540
ttaaataagg cctatgctat tgtgtcaagc gcgcccgaga gatggtcata ctttttaggt 600
gatcccaatt catgcgttga tccatggatg gcgtatcgaa tgtgacccca ggatgggcga 660
ggccacccgc tgagtttaag aataaatcat aaaa 694
<210>2
<211>737
<212>DNA
<213> Dendrobium Henan ITS sequence
<400>2
ggtaagggat ccgtaggtga cctgcggaag gatcattgtc gagaccgaaa cacaacgagc 60
gattttgtga acctgtaaaa ataagcggtg gctcttgctg ctgcgataaa atccacccga 120
gtcatcgcct catcccctct ttggggtggg gacgtgatga aggatggatg aaccctcaaa 180
tcggcgcagc gtagcgccaa gggaatcttg aaacacaagc ctataaatgg gttttgtggg 240
atggggtgtt gtcgcacgcc atattgattg acacgactct cggcaatgga tatctcggct 300
ctcgcatcga tgaagagcgc agcgaaatgc gatatgtggt gcgaattgca gaatcccgcg 360
aaccatcgag tctttgaacg caagttgcgc ctgaggccaa ctggctgagg gcacgtccgc 420
ctgggcgtca agcattttat cactctgtgc ctagtctccc atccatggat gtgttgccaa 480
ggctcggatg tgcacggtgg ctcgtcgtgc cccttggtgc ggcgggctga agggcgggtc 540
atcttctcgt tggctgccaa caataagggg tggattaaat aaggcctatg ctattgtgtc 600
aagcgcgcct gagagatggt catacttttc tatgtgatcc caattcatgc gttgatccat 660
ggatggcgta tcgaatgtga ccccaggatg ggcgaggcca cccgccgagt ttaagcatat 720
caaaaggggg gagagaa 737
<210>3
<211>25
<212>DNA
<213> ITS upstream primer
<400>3
5'-cgtaacaagg tttccgtagg tgaac-3' 25
<210>4
<211>26
<212>DNA
<213> ITS downstream primer
<400>4
5'-ttattgatat gcttaaactc agcggg-3' 26
<210>5
<211>18
<212>DNA
<213> ITS2 upstream primer
<400>5
5'-gcgatacttg gtgtgaat-3' 18
<210>6
<211>21
<212>DNA
<213> ITS2 downstream primer
<400>6
5'-gacgcttctc cagactacaa t-3' 21
<210>7
<211>20
<212>DNA
<213> MatK upstream primer
<400>7
5'-cctatatccg ctactccttc-3' 20
<210>8
<211>19
<212>DNA
<213> MatK downstream primer
<400>8
5'-ctcagtgaat ttcaccacg-3' 19
<210>9
<211>23
<212>DNA
<213> rbcL upstream primer
<400>9
5'-gagagactaa agcaagtgtt gga-3' 23
<210>10
<211>22
<212>DNA
<213> rbcL downstream primer
<400>10
5'-atgatctcca ccagacatac ga-3' 22
Claims (8)
1. A method for identifying dendrobium huoshanense based on a DNA barcode is characterized in that the DNA barcode is an ITS sequence.
2. The method as claimed in claim 1, wherein the ITS SEQUENCE of Dendrobium huoshanense is represented by SEQUENCE LISTINGNO.1.
3. Method according to claim 1 or 2, characterized in that it comprises the following steps:
1) extracting DNA of a sample;
2) PCR amplification;
3) sequencing the PCR amplification product;
4) and (5) constructing a phylogenetic tree and identifying the dendrobium huoshanense.
4. The method of claim 3, wherein the DNA extraction of the sample of step 1) is performed by: the sample was dried with silica gel, and after drying, genomic DNA was extracted by the CTAB method.
5. The method according to claim 3, wherein the PCR amplification of step 2) is specifically: and (3) carrying out PCR amplification by using a primer, wherein the PCR amplification adopts a 30 mu L reaction system: 15 μ L of 2 XTSINGKE Master Mix (New Biotechnology Co., Ltd., Okinsoniaceae, Beijing), 1.5 μ L of upstream and downstream primers (10 μmol/L), 2 μ L of DNA template, ddH2Make up to 30. mu.l of O.
6. The method according to claim 5, wherein the primers used are: ITS sequence upstream primer F: 5'-CGTAACAAGGTTTCCGTAGGTGAAC-3', respectively;
ITS sequence downstream primer R: 5'-TTATTGATATGCTTAAACTCAGCGGG-3' are provided.
7. The method of claim 5, wherein the PCR amplification procedure is: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 56 ℃ for 35s, and extension at 72 ℃ for 30s for 35 cycles; and 8min at 72 ℃.
8. The method according to claim 3, wherein the sample in step 1) comprises Dendrobium huoshanense and Dendrobium hanense.
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