CN113881803A - Bar code gene for identifying rhodiola medicinal materials and method and application thereof - Google Patents
Bar code gene for identifying rhodiola medicinal materials and method and application thereof Download PDFInfo
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Abstract
The invention discloses a barcode gene for identifying rhodiola medicinal materials, a method and application thereof, and particularly relates to a DNA combined barcode gene formed by using a ribosome DNA second internal transcribed spacer ITS2, a matK encoding gene matK and a ribulose-1, 5-diphosphoryl/oxygenase large subunit encoding gene rbcL.
Description
Technical Field
The invention relates to the field of molecular biological detection of germplasm resources of medicinal materials, in particular to a barcode gene for identifying rhodiola medicinal materials, and also relates to an identification method and application of the rhodiola medicinal materials.
Background
The Rhodiola (Rhodiola) plants of Crassulaceae have 73 varieties 2 and 7, and are distributed in Himalayan mountain, northwest Yunnan and plateau of Sichuan province. The rare species diversity and the numerous convergent morphological characteristics make the authenticity and quality identification of a plurality of medicinal materials in rhodiola very difficult, and a rapid and objective identification method is urgently needed along with the advancement of the modernization and the universalization of Chinese and Tibetan medicines. Therefore, a method for rapidly and conveniently identifying rhodiola rosea is urgently needed.
The DNA molecular marker technology has the advantages of stable heredity, no environmental influence and rapid and accurate detection, can rapidly and accurately identify some plant populations with shapes which are difficult to distinguish, and is widely used in species identification. However, no report on the identification of rhodiola rosea is found at present.
Disclosure of Invention
In view of the above, one of the objectives of the present invention is to provide a method for identifying barcode gene of rhodiola herb; the second object of the present invention is to provide a primer set for identifying the barcode gene; the third purpose of the invention is to provide a method for identifying rhodiola medicinal materials; the invention aims to provide application of the barcode gene for identifying the rhodiola medicinal material in identifying the variety of the rhodiola medicinal material.
In order to achieve the purpose, the invention provides the following technical scheme:
1. identifying a barcode gene of the rhodiola medicinal material, wherein the barcode gene is one or more of a ribosomal DNA second internal transcribed spacer ITS2, a matK encoding gene matK and a ribulose-1, 5-diphosphoryl oxygenase large subunit encoding gene rbcL.
Preferably, the barcode gene sequence is shown in SEQ ID NO. 7-SEQ ID NO. 63.
Preferably, the Rhodiola herbs are Rhodiola crenulata, Rhodiola crenulata Rhodiola fasciculata, Rhodiola tibetana Rhodiola tibetitica or Rhodiola crenulata Rhodiola bupleuroides.
Preferably, the barcode gene sequence is shown in SEQ ID NO. 7-SEQ ID NO. 33.
2. A primer pair for identifying the barcode gene according to claim 1, the primer pair comprising a ribosomal DNA second internal transcribed spacer ITS2, a matK mature enzyme encoding gene matK, and a ribulose-1, 5-bisphosphoryl oxygenase large subunit encoding gene rbcL;
the amplification primers of the second internal transcribed spacer ITS2 of the ribosomal DNA are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the amplification primers of matK encoding gene of the mature enzyme K are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the amplification primers of the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL are shown as SEQ ID NO.5 and SEQ ID NO. 6.
3. The method for identifying the rhodiola medicinal materials comprises the following steps:
1) extracting genomic DNA of a rhodiola drug to be detected;
2) performing PCR amplification by using a primer pair for amplifying a second internal transcribed spacer ITS2 of ribosomal DNA, a matK encoding gene and a rbcL encoding gene for ribulose-1, 5-diphosphoryl/oxygenase large subunit, sequencing the amplified product, comparing the sequencing result with a barcode gene of rhodiola rosea medicinal material, and constructing a phylogenetic evolution tree, wherein if the sequencing result is consistent with the sequence of the second internal transcribed spacer ITS2 of the ribosomal DNA of rhodiola rosea, the matK encoding gene for matK and the rbcL encoding gene for ribulose-1, 5-diphosphoryl/oxygenase large subunit, or the similarity is 99% or more, and the sequence can be gathered as one branch with one of the rhodiola rosea in the phylogenetic tree, the sample is one of the rhodiola rosea plants;
the amplification primers of the second internal transcribed spacer ITS2 of the ribosomal DNA are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the amplification primers of matK encoding gene of the mature enzyme K are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the amplification primers of the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL are shown as SEQ ID NO.5 and SEQ ID NO. 6.
In a preferred embodiment of the present invention, the PCR procedure for amplifying the second internal transcribed spacer ITS2 of ribosomal DNA comprises a pre-denaturation step at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s, for 32 cycles; extending for 5min at 72 ℃;
the PCR program for amplifying matK encoding gene of matK comprises pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extending for 5min at 72 ℃;
the PCR program for amplifying the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL comprises pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extension at 72 ℃ for 5 min.
Preferably, the method for constructing the phylogenetic evolution tree is an adjacency method.
4. The application of the barcode gene for identifying the rhodiola medicinal material in identifying the variety of the rhodiola medicinal material is provided.
Preferably, the Rhodiola herbs are Rhodiola crenulata, Rhodiola fastigiata, Rhodiola tibetans, or Rhodiola crenulata.
The invention has the beneficial effects that: the invention discloses a bar code gene for identifying rhodiola medicinal materials, which combines an ITS2 sequence with a matK sequence or a rbcL sequence to be used as a DNA bar code for identifying rhodiola medicinal material germplasm resources. The method has good universality in identifying 4 Rhodiola medicinal material resources of Rhodiola crenulata, Rhodiola tibetana or Rhodiola crenulata, can give stable results in the four Rhodiola medicinal material resources, and provides reference for strengthening research of Rhodiola medicinal material germplasm resources and improving the utilization rate of the Rhodiola medicinal material resources.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is an electrophoretic test chart of ITS2, in which 1-2 is rhodiola crenulata, 3-4 is rhodiola tibetana, 5-6 is rhodiola saikowii, 7-9 is rhodiola crenulata, 10 is rhodiola hexalobata, 11 is rhodiola rosea, 12 is rhodiola heterochrous, 13 is rhodiola angusta, 14 is rhodiola crenulata, 15 is rhodiola dentata, 16 is rhodiola tibetana, 17 is rhodiola isopulensis, 18 is rhodiola crenulata, 19 is rhodiola intergrowth, and M is DNA Marker III of Biosharp.
FIG. 2 is a map of the electrophoretic detection of matK gene, wherein 1-2 is rhodiola crenulata, 3-4 is rhodiola tibetana, 5-6 is rhodiola saikoides, 7-9 is rhodiola longiligularis, 10 is rhodiola hexalobata, 11 is rhodiola rosea, 12 is rhodiola heterochrous, 13 is rhodiola angusta, 14 is rhodiola crenulata, 15 is rhodiola dentata, 16 is rhodiola tibetana, 17 is rhodiola anomala, 18 is rhodiola crenulata, 19 is rhodiola intergrowsa, and M is DNA Marker III of Bioshrrp.
FIG. 3 is an electrophoretic test chart of rbcL gene, wherein 1-2 is rhodiola crenulata, 3-4 is rhodiola tibetana, 5-6 is rhodiola saikowii, 7-9 is rhodiola crenulata, 10 is rhodiola hexalobata, 11 is rhodiola rosea, 12 is rhodiola heterochrous, 13 is rhodiola angusta, 14 is rhodiola crenulata, 15 is rhodiola dentata, 16 is rhodiola tibetana, 17 is rhodiola anomala, 18 is rhodiola crenulata, 19 is rhodiola intergrowth, and M is DNA Marker III of Biosharp.
FIG. 4 is an NJ tree constructed based on the ITS2+ matK sequence, labeled as Latin names for experimental samples Nos. 1-19.
FIG. 5 is an NJ tree constructed based on the ITS2+ rbcL sequence, labeled as Latin names for experimental samples Nos. 1-19.
FIG. 6 is an NJ tree constructed based on the ITS2+ matK + rbcL sequence, labeled as Latin names for experimental samples Nos. 1-19.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
The experimental materials of the present invention are as shown in table 1:
DNA extracted from 19 rhodiola plants collected in 7 Xizang counties was selected as template DNA, the sample information is shown in Table 3, and primers of 3 fragments (ITS2, matK and rbcL) were used for PCR amplification sequencing. The primer information for each fragment is shown in Table 1.
TABLE 1 rhodiola plant samples
Example 1 PCR detection of plants of the genus rhodiola
1) DNA extraction:
after silica gel of leaves of 19 groups of plants of rhodiola collected in the field is dried, total genomic DNA is extracted by adopting a plant genomic DNA extraction kit DP320-02 of Tiangen corporation, and then 1% agarose gel electrophoresis is used for detection.
2) PCR amplification primer design
Amplification universal primers were designed based on the ribosomal DNA second internal transcribed spacer (ITS2), the matK encoding gene (matK) and the ribulose-1, 5-bisphosphoryl/oxygenase large subunit encoding gene (rbcL), with the primer sequences shown in Table 2.
Table 2, ITS2, matK and rbcL Universal primers
Using 19 groups of extracted DNA of rhodiola plants as a template and the sequence shown in Table 2 as a primer to carry out PCR amplification, wherein a 30 mu L reaction system is used for PCR reaction, wherein 1 mu L of genome DNA, 15 mu L of 2 xTaq Mastermix, 1.2 mu L of upstream primer and downstream primer respectively, and ddH2O11.6. mu.L. PCR amplification procedure:
the ITS2 fragment is pre-denatured at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s, for 32 cycles; extension at 72 ℃ for 5 min.
The matK fragment is pre-denatured at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extension at 72 ℃ for 5 min.
The rbcL fragment is pre-denatured at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extension at 72 ℃ for 5 min. After the amplification is finished, the PCR product is stored at 4 ℃.
The PCR products are separated and detected by using 1% agarose gel electrophoresis, the results are shown in figures 1-3, and the results show that the primers of ITS2, matK and rbcL can be used for amplifying the rhodiola plants. The single PCR product with clear bands was sent to Biotechnology engineering (Shanghai) GmbH for sequencing. To ensure sequence reliability, all fragments were sequenced in both directions.
The three barcode sequences GenBank accession numbers of the 19 sequenced salivia plants are shown in table 3:
TABLE 3 three barcodes for 19 rhodiola plants
The sequencing result shows that the length of the ITS2 sequence is in the interval of 442-445 bp. The matK sequence is subjected to bidirectional sequencing, and the length of the band of the matK sequence is within the interval of 820-863 bp. The rbcL sequence is subjected to bidirectional sequencing, and the length of a band of the rbcL sequence is in an interval of 738-754 bp.
Example 2 construction of phylogenetic Tree
After sequencing, software Geneius Prime is used for proofreading and splicing, MEGA-X software is used for sequence comparison, and a phylogenetic tree is constructed for identifying the rhodiola medicinal materials, and the result is shown in figures 4-6. The result shows that rhodiola crenulata, rhodiola tibetana, rhodiola chaihei and rhodiola crenulata are gathered into one, so that the sequence of ITS2, matK and rbcL genes can be amplified for identifying the rhodiola medicinal materials. If the sequencing result is consistent with or has the similarity of more than 99 percent with the barcode sequences of a second internal transcribed spacer ITS2, a matK encoding gene matK and a ribulose-1, 5-diphosphoryl/oxygenase large subunit encoding gene rbcL of the ribosome DNA of the rhodiola plants and can be gathered as one branch with one of the rhodiola plants in the phylogenetic tree, the sample is one of the rhodiola plants;
the above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> Tibet college of agriculture and animal husbandry
<120> identification of barcode gene of rhodiola medicinal material, and method and application thereof
<160> 63
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 1
atgcgatact tggtgtgaat 20
<210> 2
<211> 21
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 2
gacgcttctc cagactacaa t 21
<210> 3
<211> 25
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 3
cgtacagtac ttttgtgttt acgag 25
<210> 4
<211> 26
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 4
tcccayccat cttgaaatat tggttc 26
<210> 5
<211> 20
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 5
atgtcaccac aaacagaaac 20
<210> 6
<211> 20
<212> DNA
<213> Rhodiola rosea (Rhodiola)
<400> 6
tcgcatgtac ctgcagtagc 20
<210> 7
<211> 442
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 7
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgcgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg agtacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 8
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 8
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagatttga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacataaga attatagagg aacaagaagc atttttgatt 720
ccttttaaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 9
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 9
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 10
<211> 442
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 10
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgcgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg agtacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 11
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 11
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagatttga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacataaga attatagagg aacaagaagc atttttgatt 720
ccttttaaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 12
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola crenulata)
<400> 12
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 13
<211> 442
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 13
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggct 180
agacgtcgcg ataagtggtg gttgcaaggc cttgtgcctt tgagcgttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg agtacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcggc cttgtcgtct ga 442
<210> 14
<211> 859
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 14
gtgtttacga gctaaagctc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 15
<211> 727
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 15
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 16
<211> 442
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 16
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggct 180
agacgtcgcg ataagtggtg gttgcaaggc cttgtgcctt tgagcgttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg agtacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcggc cttgtcgtct ga 442
<210> 17
<211> 859
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 17
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 18
<211> 727
<212> DNA
<213> Rhodiola tibetica (Rhodiola tibetaca)
<400> 18
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 19
<211> 442
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 19
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 20
<211> 850
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 20
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaatg atgttgatcg taaataaaac gaccggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc atttttgatt 720
cctttttgaa atggacttct ttagtgtaat aaggctattc caattatcat acttgtaaag 780
aaagaagcgt actaaatgta acgaagaagc atctttcacc cagtagcgga gggtttgaac 840
caatatttca 850
<210> 21
<211> 727
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 21
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ttggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 22
<211> 442
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 22
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 23
<211> 850
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 23
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaatg atgttgatcg taaataaaac gaccggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc atttttgatt 720
cctttttgaa atggacttct ttagtgtaat aaggctattc caattatcat acttgtaaag 780
aaagaagcgt actaaatgta acgaagaagc atctttcacc cagtagcgga gggtttgaac 840
caatatttca 850
<210> 24
<211> 727
<212> DNA
<213> Rhodiola saikoensis (Rhodiola bupleuroides)
<400> 24
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ttggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 25
<211> 443
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 25
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacatatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct accgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagactgtt 180
agacgtcgcg ataagtggtg gttgcaagga cttgtgcctt tgagcgttgc gtgtcgctgc 240
cgtcgtccct ctctctcgaa attaataacc cgaagggagc atcctcgatg tttccagcat 300
tgggacccca ggtcaggcgg gaatacccgc tgagtttaag catatcaata agcggaggaa 360
aagaaactta caaggattcc cttagtaacg gcgagcgaac cgggatcagc ccagcttgta 420
aatcaggcga ccttgtcgtc tga 443
<210> 26
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 26
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 27
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 27
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 28
<211> 445
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 28
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacatatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct accgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagactgtt 180
agacgtcgcg ataagtggtg gttgcgagga cttgtgcctt tgagcgttgc gtgtcgctgc 240
cgtcgtccct ctctctctcg aaattaataa cccgaaggga gcatcctcga tgtttccagc 300
attgggaccc caggtcaggc gggaataccc gctgagttta agcatatcaa taagcggagg 360
aaaagaaact tacaaggatt cccttagtaa cggcgagcga accgggatca gcccagcttg 420
taaatcaggc gaccttgtcg tctga 445
<210> 29
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 29
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 30
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 30
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 31
<211> 443
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 31
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacatatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct accgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagactgtt 180
agacgtcgcg ataagtggtg gttgcaagga cttgtgcctt tgagcgttgc gtgtcgctgc 240
cgtcgtccct ctctctcgaa attaataacc cgaagggagc atcctcgatg tttccagcat 300
tgggacccca ggtcaggcgg gaatacccgc tgagtttaag catatcaata agcggaggaa 360
aagaaactta caaggattcc cttagtaacg gcgagcgaac cgggatcagc ccagcttgta 420
aatcaggcga ccttgtcgtc tga 443
<210> 32
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 32
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg ttttttgatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 33
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola fasciata)
<400> 33
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 34
<211> 442
<212> DNA
<213> Rhodiola hexalobata (Rhodiola sexifolia)
<400> 34
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttcggggcga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gtgtcgctgc 240
cgtcgattgt ctctctcgaa attataaccc gaacggagca tcctcgatgt ttccagcatt 300
gcgaccccag gtcaggcggg aatacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaacc gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 35
<211> 859
<212> DNA
<213> Rhodiola hexalobata (Rhodiola sexifolia)
<400> 35
gtgtttacga gccaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
acggtcgata atatccgaat cagcggaatc ggtccaggcg gctttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 36
<211> 727
<212> DNA
<213> Rhodiola hexalobata (Rhodiola sexifolia)
<400> 36
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgaattat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc acctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtttacgc ggtggacttg attttaccaa agatgatgaa 600
aacgttaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 37
<211> 442
<212> DNA
<213> Rhodiola rosea (Rhodiola sexifolia)
<400> 37
tgcagaatcc ggtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacatatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct accgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagactgtt 180
agacgtcgcg ataagtggtg gttgcaaggc cttgtgcctt tgagcgttgc gtgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaagggagca tcctcgatgt ttccagcatt 300
gcgaccccag gtcaggcggg aatacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 38
<211> 859
<212> DNA
<213> Rhodiola rosea (Rhodiola sexifolia)
<400> 38
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattaata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
ccttttaaaa aaaagggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 39
<211> 727
<212> DNA
<213> Rhodiola rosea (Rhodiola sexifolia)
<400> 39
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attaaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 40
<211> 442
<212> DNA
<213> Rhodiola rosea (Rhodiola discolor)
<400> 40
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 41
<211> 859
<212> DNA
<213> Rhodiola rosea (Rhodiola discolor)
<400> 41
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gaccggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc atttttgatt 720
cctttttaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 42
<211> 727
<212> DNA
<213> Rhodiola rosea (Rhodiola discolor)
<400> 42
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 43
<211> 442
<212> DNA
<213> Rhodiola kirilowii (Rhodiola kirilowii)
<400> 43
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttgcgaggc cttgtgcctt tgagcgttgc gtgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tcctcgatgt ttccagcatt 300
gcgaccccag gtcaggcggg aatacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaacc gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 44
<211> 865
<212> DNA
<213> Rhodiola kirilowii (Rhodiola kirilowii)
<400> 44
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga ttttggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatat taataccatt attaattata aatgaattgt ctaatagttg 300
actccgtaca actgaaagaa ttagtcccag attcgacaaa taacccataa agtcgaacgg 360
atgtttggat aattggttta tatgaactct tccgggttga acccacacgt aaaaatgaca 420
ttgccataaa ttgataaggt aatatttcca tttactcatc aggagaggcg tgccttttga 480
gcccaaaaag gcttttcctt gatacctaac ataatgaata aaaggatcct tgaacaaccc 540
tagcttggtt tgaatagaaa agacttctac aaaaggtttt tttatttttc catagaaatg 600
gattcgctca aaaaagagtc caaacgatgt tgatcgtaaa taaaacgact ggttacgaag 660
aaaaccgagg atgtattcgg attcacatac atgagaatta tagaggaaca agaagcattt 720
ttgattcctt tttaaaaaaa tggaaatgga cttctttagt gtaataaggc tattccaatt 780
atcatacttg taaagaaaga agcgtactaa atgtaacgaa gaagcatctt tcacccagta 840
gcggagggtt tgaaccaata tttca 865
<210> 45
<211> 727
<212> DNA
<213> Rhodiola kirilowii (Rhodiola kirilowii)
<400> 45
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 46
<211> 442
<212> DNA
<213> Rhodiola sacra (Rhodiola sacra)
<400> 46
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgggactggt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gtgtcgctgc 240
cgtcgtcctt ctctctcgaa attataaccc gaacggagca tcctcgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 47
<211> 859
<212> DNA
<213> Rhodiola sacra (Rhodiola sacra)
<400> 47
gtgtttacga gccaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
acggtcgata atatccgaat cagcggaatc ggtccaggcg gctttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat ggcattgcca 420
taaattaata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaaacgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 48
<211> 727
<212> DNA
<213> Rhodiola sacra (Rhodiola sacra)
<400> 48
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgaattat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtttacgc ggtggacttg attttaccaa agatgatgaa 600
aacgttaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 49
<211> 442
<212> DNA
<213> Rhodiola crenulata (Rhodiola serrata)
<400> 49
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatc gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggct 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gcgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 50
<211> 850
<212> DNA
<213> Rhodiola crenulata (Rhodiola serrata)
<400> 50
gtgtttacga gctaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gaccggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc atttttgatt 720
cctttttgaa atggacttct ttagtgtaat aaggctattc caattatcat acttgtaaag 780
aaagaagcgt actaaatgta acgaagaagc atctttcacc cagtagcgga gggtttgaac 840
caatatttca 850
<210> 51
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola serrata)
<400> 51
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ttggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 52
<211> 442
<212> DNA
<213> Tibetan Rhodiola sacra (Rhodiola sangpo-tibetana)
<400> 52
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggtc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttgggggcga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gtgtcgctgc 240
cgttgtccct ctctctcgaa attataaccc gaacggagca tccttgatgt ttccagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 53
<211> 859
<212> DNA
<213> Tibetan Rhodiola sacra (Rhodiola sangpo-tibetana)
<400> 53
gtgtttacga gccaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
tcggtcgata atatccgaat cagcggaatc ggtccaggcg ggtttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat gacattgcca 420
taaattgata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc atttttgatt 720
cctttttaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaagcgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 54
<211> 727
<212> DNA
<213> Tibetan Rhodiola sacra (Rhodiola sangpo-tibetana)
<400> 54
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgacttat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtctacgc ggtggacttg attttaccaa agatgatgaa 600
aacgtgaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
atttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 55
<211> 442
<212> DNA
<213> Rhodiola erythrosepala (Rhodiola smithii)
<400> 55
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agacgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gtgtcgctgc 240
agtcgtccgt ctctctcgaa attataaccc gaacggagca tcctcgatgt ttccagcatt 300
gcgaccccag gtcaggcggg attacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaacc gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 56
<211> 859
<212> DNA
<213> Rhodiola erythrosepala (Rhodiola smithii)
<400> 56
gtgtttacga gccaaasttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
acggtcgata atatccgaat cagcggaatc ggtccaggcg gctttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat ggcattgcca 420
taaattaata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaaacgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 57
<211> 727
<212> DNA
<213> Rhodiola erythrosepala (Rhodiola smithii)
<400> 57
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgaattat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtttacgc ggtggacttg attttaccaa agatgatgaa 600
aacgttaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 58
<211> 442
<212> DNA
<213> Rhodiola crenulata (Rhodiola stapfii)
<400> 58
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggtc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatgta ttgggggtga 120
agcttggcct cccgtgagcc caaactcgcg gatggcttaa aaacgagcct cgagacggtt 180
agatgtcgcg acaagtggtg gttacgaggc cttgtgcctt tgagctttgc gtgtcgctgc 240
cgtcgtccct ctctctcgaa attataaccc gaacggagca tcctcgatgt tttcagcatt 300
gcgaccccag gtcaggcggg actacccgct gagtttaagc atatcaataa gcggaggaaa 360
agaaacttac aaggattccc ttagtaacgg cgagcgaact gggatcagcc cagcttgtaa 420
atcaggcgac cttgtcgtct ga 442
<210> 59
<211> 859
<212> DNA
<213> Rhodiola crenulata (Rhodiola stapfii)
<400> 59
gtgtttacga gccaaagttc tggcacaaga aagtctcaat atatacttta ttcgatagaa 60
actttttttt tttgaagatc cactgtaata atgagaaaga tttcggcata tccacacaaa 120
acggtcgata atatccgaat cagcggaatc ggtccaggcg gctttactaa tgggatgccc 180
aaatagatta caaaatttag ctttagccaa tgatgcaata agagaaaaaa tgggaactat 240
aatatataac tgcttaatac cattattaat tataaatgaa ttgtctaata gttgactccg 300
tacaactgaa agaattagtc ccagattcga caaataaccc ataaagtcga acggatgttt 360
ggataattgg tttatatgaa ctcttccggg ttgaacccac acgtaaaaat ggcattgcca 420
taaattaata aggtaatatt tccatttact catcaggaga ggcgtgcctt ttgagcccaa 480
aaaggctttt ccttgatacc taacataatg aataaaagga tccttgaaca accctagctt 540
ggtttgaata gaaaagactt ctacaaaagg tttttttatt tttccataga aatggattcg 600
ctcaaaaaag agtccaaacg atgttgatcg taaataaaac gactggttac gaagaaaacc 660
gaggatgtat tcggattcac atacatgaga attatagagg aacaagaagc attttggatt 720
cctttttaaa aaaatggaaa tggacttctt tagtgtaata aggctattcc aattatcata 780
cttgtaaaga aagaaacgta ctaaatgtaa cgaagaagca tctttcaccc agtagcggag 840
ggtttgaacc aatatttca 859
<210> 60
<211> 727
<212> DNA
<213> Rhodiola crenulata (Rhodiola stapfii)
<400> 60
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgaattat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc gcctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtttacgc ggtggacttg attttaccaa agatgatgaa 600
aacgttaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
<210> 61
<211> 444
<212> DNA
<213> intergrowth Rhodiola rosea (Rhodiola alterna)
<400> 61
tgcagaatcc cgtgaaccat cgagtttttg aacgcaagtt gcgcccgaag ccattaggcc 60
gagggcacgt ctgcctgggc gtcacgtatt gtgttgcccc catacatatg tattgggggc 120
gaagcttggc ctcccgtgag cccaaactcg cggatggctt aaaaacgagc ctcgagacgg 180
ttagacgtcg cgacaagtgg tggttacgag gccttgtgcc tttgagcttt gcgtgtcgct 240
gccgtcgtcc gtctctctcg aaattataac ccgaacggag catcctcgat gtttccagca 300
ttgcgacccc aggtcaggcg ggaatacccg ctgagtttaa gcatatcaat aagcggagga 360
aaagaaactt acaaggattc ccttagtaac ggcgagcgaa ccgggatcag cccagcttgt 420
aaatcaggcg accttgtcgt ctga 444
<210> 62
<211> 769
<212> DNA
<213> intergrowth Rhodiola rosea (Rhodiola alterna)
<400> 62
atgagaaaga tttcggcata tccacacaaa acggtcgata atatccgaat cagcggaatc 60
ggtccaggcg gctttactaa tgggatgccc aaatagatta caaaatttag ctttagccaa 120
tgatgcaata agagaaaaaa tgggaactat aatatataac tgcttaatac cattattaat 180
tataaatgaa ttgtctaata gttgactccg tacaactgaa agaattagtc ccagattcga 240
caaataaccc ataaagtcga acggatgttt ggataattgg tttatatgaa ctcttccggg 300
ttgaacccac acgtaaaaat ggcattgcca taaattaata aggtaatatt tccatttact 360
catcaggaga ggcgtgcctt ttgagcccaa aaaggctttt ccttgatacc taacataatg 420
aataaaagga tccttgaaca accctagctt ggtttgaata gaaaagactt ctacaaaagg 480
tttttttatt tttccataga aatggattcg ctcaaaaaag agtccaaacg atgttgatcg 540
taaataaaac gactggttac gaagaaaacc gaggatgtat tcggattcac atacatgaga 600
attatagagg aacaagaagc attttggatt cctttttaaa aaaatggaaa tggacttctt 660
tagtgtaata aggctattcc aattatcata cttgtaaaga aagaaacgta ctaaatgtaa 720
cgaagaagca tctttcaccc agtagcggag ggtttgaacc aatatttca 769
<210> 63
<211> 727
<212> DNA
<213> intergrowth Rhodiola rosea (Rhodiola alterna)
<400> 63
acagaaacta aagcaagtgt tggattcaaa gccggtgtta aagattataa attgaattat 60
tatactcctg actatgaaac caaggatact gatatcttag cagcattccg agtaactcct 120
caacctggag ttccacctga ggaagcaggg gctgcggtag ctgctgaatc ttctactggt 180
acatggacaa ctgtgtggac tgatggactt accagccttg atcgttacaa aggccgatgc 240
taccacatcg agcccgttgc tggagaagaa aatcaatata ttgcttatgt agcttatcct 300
ttagaccttt ttgaagaagg ttctgttact aacatgttta cttccattgt gggtaatgtc 360
tttgggttca aagccctgcg tgctctacgt ctggaggatc tgcgaattcc tgttgcttat 420
gttaaaactt tccaaggacc acctcatggt attcaagttg aaagagataa attgaacaag 480
tatgggcgtc ctctattggg atgtactatt aaaccgaaat taggattatc tgctaagaac 540
tatggtagag cagtttatga atgtttacgc ggtggacttg attttaccaa agatgatgaa 600
aacgttaact ctcaaccatt tatgcgttgg agagaccgtt tcttattttg tgccgaagct 660
ctttataaag cacaagctga aacgggtgaa atcaaagggc attacttaaa tgctactgca 720
ggtacat 727
Claims (10)
1. The identification of the bar code gene of rhodiola medicinal materials is characterized in that: the barcode gene is one or more of ribosomal DNA second internal transcribed spacer ITS2, matK encoding gene and rbcL encoding gene for ribulose-1, 5-diphosphoryl oxygenase large subunit.
2. The method for identifying barcode gene of rhodiola herb according to claim 1, wherein: the bar code gene sequences are shown in SEQ ID NO. 7-SEQ ID NO. 63.
3. The method for identifying barcode gene of rhodiola herb according to claim 1, wherein: the Rhodiola medicinal materials are Rhodiola crenulata, Rhodiola tibetans and Rhodiola crenulata or Rhodiola crenulata.
4. The method for identifying barcode gene of rhodiola herb according to claim 1, wherein: the bar code gene sequences are shown in SEQ ID NO. 7-SEQ ID NO. 33.
5. The primer pair for identifying rhodiola medicinal materials of claim 1 or 2, which is characterized in that: the primer pair comprises a ribosomal DNA second internal transcribed spacer ITS2, a matK encoding gene matK and a ribulose-1, 5-diphosphoryl oxygenase large subunit encoding gene rbcL;
the amplification primers of the second internal transcribed spacer ITS2 of the ribosomal DNA are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the amplification primers of matK encoding gene of the mature enzyme K are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the amplification primers of the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL are shown as SEQ ID NO.5 and SEQ ID NO. 6.
6. The method for identifying the rhodiola medicinal material is characterized by comprising the following steps:
1) extracting genomic DNA of a rhodiola drug to be detected;
2) performing PCR amplification by using a primer pair for amplifying a second internal transcribed spacer ITS2 of ribosomal DNA, a matK encoding gene and a rbcL encoding gene for ribulose-1, 5-diphosphoryl/oxygenase large subunit, sequencing the amplified product, comparing the sequencing result with a barcode gene of rhodiola rosea medicinal material, and constructing a phylogenetic evolution tree, wherein if the sequencing result is consistent with the sequence of the second internal transcribed spacer ITS2 of the ribosomal DNA of rhodiola rosea, the matK encoding gene for matK and the rbcL encoding gene for ribulose-1, 5-diphosphoryl/oxygenase large subunit, or the similarity is 99% or more, and the sequence can be gathered as one branch with one of the rhodiola rosea in the phylogenetic tree, the sample is one of the rhodiola rosea plants;
the amplification primers of the second internal transcribed spacer ITS2 of the ribosomal DNA are shown as SEQ ID NO.1 and SEQ ID NO. 2;
the amplification primers of matK encoding gene of the mature enzyme K are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the amplification primers of the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL are shown as SEQ ID NO.5 and SEQ ID NO. 6.
7. The method of claim 4, wherein the method comprises the steps of: the PCR procedure for amplification of the second internal transcribed spacer ITS2 of ribosomal DNA involved pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 45s, for 32 cycles; extending for 5min at 72 ℃;
the PCR program for amplifying matK encoding gene of matK comprises pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extending for 5min at 72 ℃;
the PCR program for amplifying the ribulose-1, 5-diphosphoryl oxygenase large subunit coding gene rbcL comprises pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extension at 72 ℃ for 5 min.
8. The method of claim 4, wherein the method comprises the steps of: the method for constructing the phylogenetic evolution tree is an adjacency method.
9. The use of the barcode gene of claim 1 for identifying a variety of rhodiola species.
10. Use according to claim 7, characterized in that: the Rhodiola medicinal materials are Rhodiola crenulata, Rhodiola tibetans and Rhodiola crenulata or Rhodiola crenulata.
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Cited By (1)
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NL2031429B1 (en) * | 2022-03-29 | 2023-10-20 | Univ Guangxi Chinese Medicine | Identification method of stephania tetrandra from different regions, and dna barcode primer, dna barcode, kit and use |
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CN106191294A (en) * | 2016-08-26 | 2016-12-07 | 中山市中智药业集团有限公司 | A kind of method utilizing DGGE to identify mixing Chinese medicinal powder composition species |
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JIAN-QIANG ZHANG 等: "DNA Barcoding of Rhodiola (Crassulaceae): A Case Study on a Group of Recently Diversified Medicinal Plants from the Qinghai-Tibetan Plateau", 《PLOS ONE》, vol. 10, no. 3, pages 281 - 286 * |
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