CN110273023B - Method for identifying or distinguishing cinnamon and related plants thereof, and primers and kit used by method - Google Patents

Method for identifying or distinguishing cinnamon and related plants thereof, and primers and kit used by method Download PDF

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CN110273023B
CN110273023B CN201910731615.4A CN201910731615A CN110273023B CN 110273023 B CN110273023 B CN 110273023B CN 201910731615 A CN201910731615 A CN 201910731615A CN 110273023 B CN110273023 B CN 110273023B
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cinnamomum
camphora
zeylanicum
micranthum
cassia
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CN110273023A (en
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丁平
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Guangzhou University of Traditional Chinese Medicine
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Abstract

The invention aims to provide a method for identifying cinnamon (cinnamon cassia) and related plants thereof with high accuracy, and primers and a kit used by the method. The kindred plants are cinnamon (Cinnamomum zeylanicum), cinnamomum camphora (Cinnamomum camphora) and Cinnamomum camphora (Cinnamomum micranthum) plants. The method finds the difference of the chlorophyll genomes of the four plants such as cinnamon by detecting and comparing the sequences of the chloroplast genomes of the four plants, and provides a scientific basis for scientifically identifying the four plants.

Description

Method for identifying or distinguishing cinnamon and related plants thereof, and primers and kit used by method
Technical Field
The invention relates to the technical field of biology, in particular to a method for identifying or distinguishing cinnamon and related plants thereof, and primers and a kit used by the method.
Background
The DNA molecular identification technology is to analyze and identify specific DNA sequences in different species of traditional Chinese medicinal materials or plants by utilizing a molecular biology technology, so as to achieve the aim of identifying the species and individuals on a molecular level. It has been widely used for classification and identification of various species as a vital method for the development of modern science and technology and new technologies are continuously applied in this field. The 2010 version of Chinese pharmacopoeia includes molecular identification methods of bulbus fritillariae cirrhosae, zaocys dhumnade and agkistrodon, and the 2015 version of Chinese pharmacopoeia establishes a guiding principle of a Chinese medicinal material DNA bar code molecular identification method, so that the molecular identification of the traditional Chinese medicine gradually becomes an advanced and feasible Chinese medicinal material identification method. Among these techniques, many PCR techniques are used as the basis, and according to the difference of the principle, methods such as Random Amplification Polymorphic DNA (RAPD), simple sequence repeat (ISSR), simple Sequence Repeat (SSR), amplified Fragment Length Polymorphism (AFLP), single Nucleotide Polymorphism (SNP), etc. are widely used for identifying traditional Chinese medicinal materials. With the development of sequencing technology, polymorphism identification has been developed into DNA barcode identification.
In recent years, traditional Chinese medicine DNA barcode identification, namely a method for obtaining a gene short segment which can be used for identifying, finding, distinguishing species and typing an undetermined individual from a genome standardization interval, is rapidly developed in traditional Chinese medicine identification.
The cinnamon is dry bark of cinnamon (cinnamon cassia) of Lauraceae, cinnamomum, has wide medicinal value and food flavoring, has effects of supplementing fire, tonifying yang, guiding fire to origin, dispelling cold, relieving pain, and warming and dredging channels, and is mainly used for treating sexual impotence, cold womb, lumbago and knee pain, dysmenorrhea, amenorrhea, etc. The market demand is large, the variety is complicated, wherein the Cinnamomum zeylanicum (Cinnamomum zeylanicum) is a common foreign variety, the main production place is Srilanka, the Cinnamomum zeylanicum is widely distributed in China market in recent years, and the Cinnamomum zeylanicum can be used as cinnamon medicine. Cinnamomum camphora (Cinnamomum camphora) is a medicinal plant widely distributed in southern and southwest areas of China, and camphor oil can be extracted from wood, roots, branches and leaves, and are used in the pharmaceutical and perfume industries, and the bark of Cinnamomum camphora is often confused with cinnamon bark. The submerged camphor (Cinnamomum micranthum) is an important fast-growing economic tree species of Cinnamomum in Lauraceae in China, and the specific gravity of camphor oil contained in the submerged camphor leaves is greater than that of water, so that the name is that the plants can extract aromatic oil, mainly contain safrole, and are important raw materials for industry and medicine, and the bark of the trees is also mixed with cinnamon. Because the four similar medicinal plants are similar in appearance, shape, color and smell, the condition that the four medicinal plants are mixed in the prior art is often existed, which is not favorable for regulating the medication and ensuring the safety of cinnamon foods.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for identifying cinnamon and kindred plants with high accuracy, and primers and a kit used by the method.
Detecting the sequence of cinnamon chloroplast genome, finding the difference of the chlorophyll genome of four plants such as cinnamon and the like by comparing the common closely related plants of cinnamon (cinnamon cassia), cinnamomum zeylanicum (cinnamon zeylanicum), cinnamomum camphora (cinnamon camphora) and Cinnamomum camphora (cinnamon miranthum), and providing scientific basis for scientifically identifying the four plants.
Specifically, the invention relates to a primer combination product, which comprises the following primer pairs:
primer pair P1: the amino acid sequence of SEQ ID NO:1-2;
a primer pair P2: the amino acid sequence of SEQ ID NO:3-4;
primer pair P3: SEQ ID NO:5-6; and
primer pair P4: SEQ ID NO:7-8.
The primer pair can be used for amplifying 4 sections of specific sections of cinnamon, cinnamomum zeylanicum, cinnamomum camphora and cinnamomum camphora plants, and then the plants can be distinguished through SNP sites positioned on the specific sections.
The invention also relates to a kit containing the primer combination product.
The invention also relates to a method for identifying or differentiating cinnamon and its related plants, namely cinnamon sienna, cinnamomum camphora and cinnamomum camphora plants, so that the method comprises:
a) Obtaining genome DNA or mitochondrial DNA of a plant to be detected;
b) Amplifying the DNA obtained in a) using a primer combination product as described above or a kit as described above;
c) Sequencing the amplified fragments, comparing the sequencing result with a cinnamon specific DNA molecular marker matrix, and judging that the plant to be detected is the species if the total similarity of the sequencing result and the species in the matrix is more than 95%;
the matrix is:
TABLE I SNP contained in the region amplified by primer pair P1
Figure BDA0002160781610000031
TABLE II SNPs contained in the region amplified by primer pair P2
Figure BDA0002160781610000041
TABLE II
Figure BDA0002160781610000042
TABLE III SNP contained in the region amplified by primer pair P3
Figure BDA0002160781610000043
TABLE III
Figure BDA0002160781610000044
TABLE IV SNP contained in the region amplified by primer pair P4
Figure BDA0002160781610000045
The applicant of the present invention has obtained primers and methods that can be used to identify or differentiate cinnamon, cinnamomum zeylanicum, cinnamomum camphora, and cinnamomum camphora through creative labor and a large number of experiments. The invention provides guidance for the identification of cinnamon and kindred plants and provides powerful technical support for the standard medication and the safety guarantee of cinnamon foods.
Detailed Description
The invention relates to a primer combination product, which comprises the following primer pairs:
a primer pair P1: SEQ ID NO:1-2;
a primer pair P2: the amino acid sequence of SEQ ID NO:3-4;
primer pair P3: SEQ ID NO:5-6; and
a primer pair P4: SEQ ID NO:7-8.
The primer pair may be synthesized according to the prior art in the field, or may be prepared by a professional company.
The combination product can convert SEQ ID NO:1-8 are stored separately in different containers, e.g.EP tubes, and may be stored in mixtures for each pair, preferably in the proportions used.
According to one aspect of the invention, the invention also relates to a kit comprising a primer combination as described above.
In some embodiments, the kit further comprises one or more of a DNA extraction system, PCR reaction buffer, water, DNA polymerase, and molecular weight marker.
In some embodiments, the DNA polymerase is selected from any one of Taq, bst, vent, phi29, pfu, tru, tth, tl1, tac, tne, tma, tih, tf1, pwo, kod, sac, sso, poc, pab, mth, pho, ES4DNA polymerase, and Klenow fragment.
In some embodiments, the Water is nuclease-free Water, such as Distilled Water (Distilled Water), deionized Water (deinized Water), or Reverse osmosis Water (Reverse osmosis Water).
In some embodiments, the kit further comprises reagents required for sequencing.
According to one aspect of the invention, the invention also relates to a method for identifying or differentiating cinnamon and its related plants, namely cinnamon cinnamomum zeylanicum, cinnamomum camphora and cinnamomum camphora plants, so that the method comprises:
a) Obtaining genome DNA or mitochondrial DNA of a plant to be detected;
b) Amplifying the DNA obtained in a) by using the primer combination product or the kit;
c) Sequencing the amplified fragments, comparing the sequencing result with a cinnamon specific DNA molecular marker matrix, and judging the plant to be detected as the species if the total similarity of the amplified fragments and the species in the matrix is more than 95% (such as 96%, 97%, 98%, 99%, 99.9%);
the matrix is shown in tables I-IV.
The method can be used for identifying cortex Cinnamomi, cinnamomum zeylanicum, cinnamomum camphora and Cinnamomum camphora; it can also be used for identification of any two or more of the above four plants.
The sample to be tested is not particularly limited as long as it may contain cinnamon and a closely related plant. For example, crushed, pulverized, dried or extracted materials of cinnamon and related plants, crude drugs and chinese drugs containing the same, and pharmaceuticals, foods, beverages and cosmetics containing the same.
In some embodiments, the annealing temperature is 49 ℃ to 53 ℃, e.g., 50 ℃,51 ℃, 52 ℃ for each primer pair.
In some embodiments, the sequencing is selected from high throughput sequencing.
In some embodiments, the sequencing is selected from Sanger sequencing.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
The embodiment is realized by the following technical scheme:
s1, collecting cinnamon leaves;
s2, extracting DNA of cinnamon leaves;
s3, constructing an Illumina DNA library for the DNA of cinnamon leaves;
s4, illumina high-throughput sequencing;
s5, assembling a cinnamon chloroplast genome sequence;
s6, searching and downloading cinnamon related species sequences, such as Cinnamomum zeylanicum, cinnamomum camphora and Cinnamomum camphora chloroplast genome sequence;
s7, carrying out software comparison analysis on the chloroplast genome sequences of cinnamon, cinnamomum zeylanicum, cinnamomum camphora and cinnamomum camphora;
s8, designing a specific primer;
s9, constructing a cinnamon specific DNA molecular marker matrix and a scoring standard;
s10, verification: collecting the closely related species of Cinnamomum zeylanicum, cinnamomum camphora and Cinnamomum camphora leaves;
s11, extracting total DNA of Cinnamomum zeylanicum, cinnamomum camphora and Cinnamomum camphora leaves;
and S12, carrying out PCR amplification and sequence sequencing, and carrying out species identification comparison.
S10-S12 are sequence verifications.
The method for constructing the Illumina DNA library in the step S3 comprises the following steps: according to the construction process of an Illumina library, constructing the total DNA in the step S2 into a double-end high-throughput sequencing library with the fragment size of 400-600 bp;
the method for comparatively analyzing the chloroplast sequence in the step S7 comprises the following steps: and (3) comparing the cinnamon chloroplast genome DNA sequence obtained in the step (S5) with the chloroplast genome sequence of the closely related species by using sequence alignment analysis software (such as clustal, multiscale, blast and the like) to find out the regions with differences. In the following sequence alignment results, the positions without the "+" mark are regions of difference.
Specific segment 1
Cinnamomum_camphora ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomum_micranthum ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomum cassia ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
Cinnamomumzeylanicum ATTTCAATGAATTAGGTCCATAAGAACCAGAAGCCCTAGCTTTTCAATCAAAAATGAATC
************************************************************
Cinnamomum_camphora ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomum_micranthum ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomum cassia ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
Cinnamomumzeylanicum ACTTAGGACTCAGATTTATAGTCCATTCTGGTAGTTTGACCGTGGAATTCCGTTGTTTCG
************************************************************
Cinnamomum_camphora GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum_micranthum GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum cassia GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
Cinnamomum zeylanicum GTATTTCCGGAATATGAGTGTGCGACTTGTTATAATTGATCCTATTGATAGTACAGAGAA
************************************************************
Cinnamomum_camphora TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum_micranthum TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum cassia TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
Cinnamomum_zeylanicum TGGGTCTGTCATCTCGACAGAGATGGTTCTGCCTCGTCGGATATTCATCCTAGTATCTGG
************************************************************
Cinnamomum_camphora AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum_micranthum AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum cassia AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
Cinnamomum zeylanicum AGCACGGAATATATGGAATAGATCAAGAAATATTTGAACTATGATTCATACCTACTATTC
************************************************************
Cinnamomum_camphora AGACCTCGTGACTGGACTTCCAAAAATTTTCAAACAAAGAGGTATTTGATAAATTGAACG
Cinnamomum_micranthum AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGAGAAATTGAACG
Cinnamomum cassia AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGATAAATTGAACG
Cinnamomum_zeylanicum AGACCTCGTGACTGGACTTCAAAAAATTTTCAAACAAAGAGGTATTTGCTAAATTGAACG
*********************************************************
Cinnamomum_camphora ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum_micranthum ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum cassia ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
Cinnamomum zeylanicum ATTTTTCTTCCTTTAGAATCATGCTTATTTTGACCGAAGGACAAATCTTTCTCTGGATTT
************************************************************
Cinnamomum_camphora TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum_micranthum TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum cassia TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
Cinnamomum zeylanicum TTAGTCATTACATCTATGAATAAGTGATGATCAAATAGTTCTTACTCATAGAACCCTTGG
************************************************************
Cinnamomum_camphora TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum_micranthum TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum cassia TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
Cinnamomum zeylanicum TCTTAGTTTTTGGGTTTTATTGAATCATCGTGGTTCTAGTATGAATCTGAGGTTTCAATC
************************************************************
Cinnamomum_camphora GATTCATAGGCTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum_micranthum GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum cassia GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
Cinnamomum_zeylanicum GATTCATAGGGTCTCAACAAGAGAATTCCTATCAAAAAAAAATAGTAAACAATAGTCAAT
***********************************************************
Cinnamomum_camphora CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum_micranthum CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum cassia CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
Cinnamomum_zeylanicum CTGCATTACGCACAAACAAAAACAACAAATCAAATAACAAATAAATAGGGGAATAGAAGA
************************************************************
Cinnamomum_camphora TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum_micranthum TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum cassia TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
Cinnamomum_zeylanicum TTCAAGAGGCCTGTAACGATCAACATAAAGACAGATGAGCTAACTTGATATTTTGGCATT
************************************************************
Cinnamomum_camphora CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum_micranthum CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum cassia CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
Cinnamomum_zeylanicum CTCATCACAACAAAGAAGAGAGTTCGGATTTTGGTTCCTTCGTATCTTCAGAGACGATTG
************************************************************
Cinnamomum_camphora AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum_micranthum AATCAAATGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum cassia AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
Cinnamomum_verum AATCAAGTGGATAAATAAGAAATTTCAAATTTTCTATTACATATCCATTGTAATCAGTAT
***********************************************************
Cinnamomum_camphora TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum_micranthum TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum cassia TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
Cinnamomum zeylanicum TTGGGTGTTTCTGCTTGAGCCGTACGAGATGAAATTCTCATATACGGTTCTCAGAGGGGG
************************************************************
Cinnamomum_camphora AGTCCCCTTGGTTTACCTATATGAGTAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum_micranthum AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum cassia AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
Cinnamomum zeylanicum AGTCCCCTTGGTTTACCTATCTCAATAAAGTATATGATTGGTTCGAGGAGCGTCTCGAGA
*********************************************************
Cinnamomum_camphora TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum_micranthum TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum cassia TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
Cinnamomum zeylanicum TTCAGGCGATTGCAGATGATATAACTAGTAAATATGTTCCTCCTCATGTCAATATATTTT
************************************************************
Cinnamomum_camphora ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum_micranthum ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum cassia ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
Cinnamomum zeylanicum ATTGTCTAGGAGGGATCACACTTACTTGTTTTTTAGTACAAGTAGCTACGGGTTTTGCTA
************************************************************
Cinnamomum_camphora TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum_micranthum TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum cassia TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
Cinnamomum zeylanicum TGACTTTTTACTATCGTCCGACCGTTACGGAGGCTTTTGCCTCTGTTCAATACATAATGA
************************************************************
Cinnamomum_camphora CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum_micranthum CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum cassia CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
Cinnamomum zeylanicum CTGAAGCCAACTTTGGTTGGTTAATCCGATCAGTTCATCGATGGTCAGCAAGTATGATGG
************************************************************
Cinnamomum_camphora TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum_micranthum TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum cassia TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
Cinnamomum zeylanicum TCCTAATGATGATCCTACACGTATTTCGTGTGTATCTCACGGGCGGATTTAAAAAACCTC
************************************************************
Specific segment 2
Cinnamomum_micranthum TTATAGGGATAGCAAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum zeylanicum TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum cassia TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
Cinnamomum_camphora TTATAGGGATAGCGAATGCTGGGCGAACGACGGGAATTGAACCCGCGCATGGTGGATTCA
***********************************************************
Cinnamomum_micranthum CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum zeylanicum CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum cassia CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
Cinnamomum_camphora CAATCCACTGCCTTGATCCACTTGGCTACATCCGCCCCTCCTCTCTCAAAAGGATTCCAT
************************************************************
Cinnamomum_micranthum TTTCACCATTCATTATTTTTTTATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum zeylanicum TTTCACCATTCATTATTTTTTAATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum cassia TTTCACCATTCATTATTTTTTGATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
Cinnamomum_camphora TTTCACCATTCATTATTTTTTGATTTAGTCTTTATTACTTCACTCTCCTTCCTGCTGAAA
***********************************************************
Cinnamomum_micranthum TACAGATATTGTACATAAAACAAAATGTTGTACGTAAAATAAAAAAAAAAAAAGAAAAAT
Cinnamomum zeylanicum TACAGATATTGTACATAAAACAAAATGTTGTACGTAA---AAAAAAAAAAAAAGAAAAAT
Cinnamomum cassia TACAGATATTGTACATAAAACAAAATGTTGTACGTAAA--AA AAAAAAAAAAAGAAAAAT
Cinnamomum_camphora TACAGATATTGTACATAAGACAAAATGTTGTACGT-----AA AAAAAAAAAAAGAAAAAT
******************************************************
Cinnamomum_micranthum GCTTTGATTTTTTCCTAAAATCAAATTCTTTTGAAGAATAAGAGTATATAAATTGCAGGT
Cinnamomum zeylanicum GCTTTGATTTTTTCCTAAAATCAAATTATTTTGAAGAATAAGAGTATATAAAATGCAGGT
Cinnamomum cassia GCTTTGATTTTTTCAAAAAATCAAATTATTTTGAAGAATAAGAGTATATAAAATGCAGGT
Cinnamomum_camphora GCTTTGATTTTTTCAAAAAATCAAATTATTTTGAAGAATAAGAATATATAAAATGCAGGT
*******************************************************
Cinnamomum_micranthum TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum zeylanicum TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum cassia TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
Cinnamomum_camphora TGGTACAGAAGAAACTACGATATTCGATCATGAAATAACCAGCGGTTTTCATAAGTTGAA
************************************************************
Cinnamomum_micranthum TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATAGATC
Cinnamomum zeylanicum TAAAAGAAATTAAAATGAAAAACGATTATGTGAATAAAACACTACTTAACCAAATGGATC
Cinnamomum cassia TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATAGATC
Cinnamomum_camphora TAAAAGAAATGAAAATGAAAAACGATTATGTGAATAAAACACTACTGAACCAAATGGATC
*********************************************************
Cinnamomum_micranthum AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum zeylanicum AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum cassia AATACCAAACTTCTTGTTCTATTAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
Cinnamomum_camphora AATACCAAACTTCTTAATAGAACAAGAAGTTTGGTATTGATCCTTCAACGACTCGTATAC
******************************************************
Cinnamomum_micranthum ACTAATACCAAAGTATTATCCATTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum zeylanicum ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum cassia ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
Cinnamomum_camphora ACTAATACCAAAGTATTATCCGTTTGTAGATGGAACTTCGACAGCAGCTAGGTCTAGAGG
***********************************************************
Cinnamomum_micranthum GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum zeylanicum GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum cassia GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
Cinnamomum_camphora GAAGTTGTGAGCATTACGTTCATGCATAACTTCCATACCAAGGTTAGCACGATTGATGAT
************************************************************
Cinnamomum_micranthum ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum zeylanicum ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum cassia ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
Cinnamomum_camphora ATCAGCCCAAGTGTTAATAACACGACCTTGACTGTCAACTACAGATTGGTTGAAATTGAA
************************************************************
Cinnamomum_micranthum ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTGAACCAGATACCTACTAC
Cinnamomum zeylanicum ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTGAACCAGATACCTACTAC
Cinnamomum_cassia ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTAAACCAGATACCTACTAC
Cinnamomum_camphora ACCATTTAGGTTGAAAGCCATGGTGCTGATACCTAAAGCAGTAAACCAGATACCTACTAC
***********************************************************
Cinnamomum_micranthum AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum zeylanicum AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGAGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum cassia AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
Cinnamomum_camphora AGGCCAAGCTGCTAGGAAGAAATGTAAGGAACGGGAGTTGTTAAAACTAGCATATTGGAA
***********************************************************
Specific segment 3
Cinnamomum_micranthum TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum zeylanicum TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum cassia TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
Cinnamomum_camphora TACAGAAGCGACCCCATAGGCTTGTGCTTTCGCGTCTCTCTAAAATTGCAGTCATGGTAA
************************************************************
Cinnamomum_micranthum AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum zeylanicum AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum cassia AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
Cinnamomum_camphora AATCTTGGTTTATTCAATTCTCAGGGACTCCCAAGCACACAGATTATCTATAAATAGAAA
************************************************************
Cinnamomum_micranthum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum zeylanicum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum_aromaticum TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
Cinnamomum_camphora TAGACAACGGAAGGCTTGTTATTCAACAGTATAACATGACTTATATGTCCGTGTCAACCA
************************************************************
Cinnamomum_micranthum ATAAGAGAGATATCTATCTGGATAGATCCATCAGAACGATTTGTAAATTAAGTGAGTAGG
Cinnamomum zeylanicum ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
Cinnamomum cassia ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
Cinnamomum_camphora ATAAGAGAGATATCTATCTGGATAGATCCATCCGAACGATTTGTAAATTAAATGAGTAGG
**********************************************************
Cinnamomum_micranthum GATTTATCCAATAACAAATATTTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum zeylanicum GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum cassia GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
Cinnamomum_camphora GATTTATCCAATAACAAATA-TTTTTTCGTATTTTCCGTACGATTGGTAATGGGTTGCCC
***********************************************************
Cinnamomum_micranthum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum zeylanicum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum_aromaticum GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
Cinnamomum_camphora GGGACTCGAACCCGGAACTAGTCGGATGGAGTAGATAATTTCCTTCTTGCAATATAATAG
************************************************************
Cinnamomum_micranthum ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCATTGCACAGGGCTT
Cinnamomum zeylanicum ATATAGAGTAAAAAGACCCCCCCAAAAAGCCGTGCTTGCATTTTTCATTGCACAGGGCTT
Cinnamomum cassia ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCAGTGCACAGGGCTT
Cinnamomum_camphora ATATAGAGTAAAAAGACCCCCCAAAAAAGCCGTGCTTGCATTTTTCAGTGCACAGGGCTT
**********************************************************
Cinnamomum_micranthum TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum_zeylanicum TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum cassia TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
Cinnamomum_camphora TACCTATGTATACATCCAAAACTAAGTTCCCTAAAAGGGGACCTAAGAAACTTGAAGACT
************************************************************
Cinnamomum_micranthum CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATCAAATTTTTTTG
Cinnamomum zeylanicum CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
Cinnamomum cassia CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
Cinnamomum_camphora CAGTTGATTCAACCACTACTGTATGAACATTTCAGAATTCAAATGAATAAAATGATTTTG
*********************************************************
Cinnamomum_micranthum TGATTTTCTCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum zeylanicum TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum cassia TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
Cinnamomum_camphora TGATTTTATCTCTTCATCATTTAGGGATCCTTTCTATTTACATGACCTCATGACCAATCA
***********************************************************
Cinnamomum_micranthum -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
Cinnamomum zeylanicum -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAACCCGTTCTCTAT
Cinnamomum cassia -TAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
Cinnamomum_camphora TTAAATGACTGACTAGGTCATTGATACGGATAATATCCAAATACCAAATCCGTTCTCTAT
**********************************************************
Cinnamomum_micranthum GTGACCTATGAGAAGGAGAAGAGGTTGTTGGAAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum zeylanicum GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum cassia GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
Cinnamomum_camphora GTGACCTATGAGAAGGAGAAGAGGTTGTTGGGAAGATCAAAGAAAGAGCTTGTTCTTCCT
***********************************************************
Cinnamomum_micranthum CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum zeylanicum CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum cassia CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
Cinnamomum_camphora CCGTAAAGAATTCTTCCAAGAACTCCGAACCTAATCTTTTCAAAAAAGCACGTATCGTAC
************************************************************
Cinnamomum_micranthum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum zeylanicum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum_aromaticum TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
Cinnamomum_camphora TTTTATGTTTACGAGACAAAGTTCTAGCACATGAAAGTCGAAGTATATACTTTATACGAT
************************************************************
Cinnamomum_micranthum ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum zeylanicum ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum cassia ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
Cinnamomum_camphora ACAAACTCTGTTTTTTTGAGGATCCACTGTGATAATGAGAAAGATTTCTGCATATCCGCC
************************************************************
Cinnamomum_micranthum CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum zeylanicum CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum cassia CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
Cinnamomum_camphora CAAATCGATTGAGAATCTCAGAATCTGACAAATCGGCCCGAAACGGCTTACTAATGGGAT
************************************************************
Cinnamomum_micranthum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum zeylanicum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum_aromaticum GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
Cinnamomum_camphora GCCCTGATACATTACAAAATTTTGCTTTAGCCAATGATCCAATCAGAGGAATAATTGGGA
************************************************************
Cinnamomum_micranthum CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum zeylanicum CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum cassia CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
Cinnamomum_camphora CTACGGTCTCGAATTTCTTAATAGCAGTATCTATTCGAAACGAATTCTCTAGCATTTGAC
************************************************************
Cinnamomum_micranthum TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum zeylanicum TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum cassia TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
Cinnamomum_camphora TCCTTATCACCGAAGAGTTTAGTCGTACACTTGAAAGATAGCCCAGAAAATAGAAGGGAT
************************************************************
Cinnamomum_micranthum GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGACATT
Cinnamomum zeylanicum GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGAGATT
Cinnamomum cassia GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTCAAAATGACATT
Cinnamomum_camphora GATTATATAATTGCTTTATATGGATCCTGGCCGGTTGAGACCACAAGTAAAAATGACATT
**********************************************************
Cinnamomum_micranthum GCCAAAAGTTGATAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum zeylanicum GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum cassia GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
Cinnamomum_camphora GCCAAAAGTTGACAAGGTGAGATTTCCATTTCTTTATCAGAAGACGAGCCCCCCTTGAAG
***********************************************************
Cinnamomum_micranthum CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum zeylanicum CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum cassia CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
Cinnamomum_camphora CCAGAATCGATTTTCCTTGATATCTGACATAATGCATAAAAGGGTCTTTGAACAACCATA
************************************************************
Cinnamomum_micranthum GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum zeylanicum GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum cassia GGGTTTTTTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
Cinnamomum_camphora GGGTTTTCTGAAAATCGTTACAAAGCACTACTACAAGATATTCTATTTTTGCATAGAAAT
***********************************************************
Cinnamomum_micranthum GTGTTCGCTCAAGAAAGGATCCAAAAGATTTGGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum zeylanicum GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum cassia GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
Cinnamomum_camphora GTGTTCGCTCAAGAAAGGATCCAAAAGATTTTGATCGTAAATGAAAGGGTTGTTTACGGA
***********************************************************
Cinnamomum_micranthum GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum zeylanicum GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum cassia GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
Cinnamomum_camphora GAAAAATGAATATGAATTCACATTCATATACATGAGAATTAGAGAGGAACA
***************************************************
Specific segment 4
Cinnamomum cassia TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum_camphora TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum zeylanicum TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
Cinnamomum_micranthum TTTCACCTCTCCCATCAATGGGTTTAGCCAGAGCATTTATAACACGACCCAAATAAGCCT
************************************************************
Cinnamomum cassia CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum_camphora CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum zeylanicum CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
Cinnamomum_micranthum CACTAACGGGTATCTGAGCAATTCTTCCTGTTGCTTTTACAGAACTTCCCTCTTGTATCA
************************************************************
Cinnamomum cassia TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum_camphora TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum zeylanicum TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
Cinnamomum_micranthum TCAAACCGTCACCCATTAATACAACGCCAACATTATTTGATTCCAAATTCAGAGCAATGC
************************************************************
Cinnamomum cassia CTATTGTACCTTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum_camphora CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum zeylanicum CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
Cinnamomum_micranthum CTATTGTACCCTCTTCAAATTCTACTAATTCCCCTGCCATTACTTCATCAAGACCATGAA
***********************************************************
Cinnamomum cassia TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_camphora TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_verum TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
Cinnamomum_micranthum TACGAGCAATGCCGTCGCCTACCTGAAGTACTGTGCCGGTATTCACAATCTTGACTTCTC
************************************************************
Cinnamomum cassia TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum_camphora TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum zeylanicum TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
Cinnamomum_micranthum TATTATATTGTTCAATACGTTCACGGATAATATTACTAATTTCGTCGGCTCGAATGGTTA
************************************************************
Cinnamomum cassia CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG--AAAAA AAAAAAAAAAAATAATG
Cinnamomum_camphora CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG--AAAAAA AAAAAAAAAAATAATG
Cinnamomum zeylanicum CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAGAAAAAAAAAAAAAAAAAAATAATG
Cinnamomum_micranthum CCATTAGTGTCTCTTAATTCTTTTTCGGAAACAAAG-AAAAAAAAAAAAAAAAAATAATG
**********************************************************
Cinnamomum_cassia CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum_camphora CCTTCAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum zeylanicum CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
Cinnamomum_micranthum CCTACAGTAGAAGGGCTAATCAGTTACTTCTTTCATGGCCCCGAGCATGCCAATATTAGC
***********************************************************
Cinnamomum_cassia ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum_camphora ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum zeylanicum ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
Cinnamomum_micranthum ACCGATGGTGCGTAAATGTAACTCGCTGTTTGAACAACTATTCAGAGTTCCTAGAGCTCC
************************************************************
Cinnamomum cassia TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum_camphora TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum zeylanicum TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
Cinnamomum_micranthum TTGTAAGGCTTGTTGGAAAACTCGTTGTCGCACCTGATTAATTGCTCTTTGTTGTTCAAA
************************************************************
Cinnamomum cassia ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum_camphora ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum zeylanicum ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
Cinnamomum_micranthum ATGAATGGTTTCATTTTTGTAATTTTCTAATCGTTCCAAATTCTCATAAGTGGCATTAAT
************************************************************
Cinnamomum cassia CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum_camphora CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum zeylanicum CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
Cinnamomum_micranthum CAAATTCCATTTTTCTCGTTCTATCTCAGAGTATCCATTCACTCGAAACTCATCTGCTTC
************************************************************
Cinnamomum cassia TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum_camphora TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum zeylanicum TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
Cinnamomum_micranthum TATTTCCACTTTCCGTAAGCGAGCCCGGGCTTCTTCGAGCTGCTCAATGGCTCCTTCACG
************************************************************
Cinnamomum cassia TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_camphora TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_verum TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
Cinnamomum_micranthum TAGTTCTTCTGAATTTCGAATAGTACTCAAGATCCTCTGTTTTCGATTATCTAATAAATC
************************************************************
Cinnamomum cassia ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum_camphora ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum zeylanicum ACTTAATGAAAGTAGATTATCTTCCCATTC
Cinnamomum_micranthum ACTTAATGAAAGTAGATTATCTTCCCATTC
****************************************************
The method for designing the specific primer in the step S8 comprises the following steps: primers are designed in regions without difference using Primer design software (e.g., primer3, primer5, primer blast, etc.), and the Primer sequences are required to amplify the regions of difference.
Universal PCR primer List
Figure BDA0002160781610000181
The method for constructing the cinnamon specific DNA molecular marker matrix in the step S9 comprises the following steps: and (3) selecting SNP sites different from cinnamon and allied species according to the different SNP site information, and constructing a DNA molecular marker matrix (shown in tables I-IV) according to the sequence in the genome. According to the base sites of the DNA matrix, the following scoring principle is established, wherein the base of the species to be detected is the same as the base of the matrix, the species in the matrix is scored for 1, and the species in the matrix are not scored or not subtracted due to difference. And finally, calculating to obtain the identification similarity by calculating the total score and the proportion of the total score to the total score of the SNP loci. The higher the similarity, the greater the likelihood of being a species in the matrix, the greater the similarity is 95%, and the species can be judged to be.
Example 2
Collecting fresh leaves of cinnamon, and extracting total DNA of the leaves; the total DNA extracted was stored at-20 ℃. The designed primer sequences are shown in Table 1. Carrying out PCR amplification by taking the total DNA as a template; the PCR reaction system is shown in the following table.
PCR reaction System (40. Mu.L)
Figure BDA0002160781610000191
PCR procedure: pre-denaturation at 94 ℃ for 5min; 30s at 94 ℃, 30s at 51 ℃, 1min20s at 72 ℃ and 35 cycles; 10min at 72 ℃.
mu.L of the PCR amplification product was detected by electrophoresis on 1.2% agarose gel (EB staining). PCR product fragments with corresponding sizes were recovered by agarose gel electrophoresis and sequenced by the Sanger method.
Performing Sanger sequencing on the recovered fragments; then, comparing and identifying the sequencing result SNP locus and the difference result, and calculating the similarity of each sequence.
And (4) counting the scores of all the sequences in the DNA molecular marker matrix according to the segment sites, wherein the scores are one score when the bases are the same, and the scores are not different when the bases are different. The matrix contains 65 DNA molecular markers in total, and the total score is 65. In the examples, the identification rate of cinnamon was 100%.
Figure BDA0002160781610000192
Example 3
Collecting fresh leaves of cinnamomum camphora, and extracting total DNA of the leaves; the total DNA extracted was stored at-20 ℃. The designed primer sequences are shown in Table 2. Performing PCR amplification by using the total DNA of the cinnamomum camphora as a template; the PCR reaction system is shown in the following table.
PCR reaction System (40. Mu.L)
Figure BDA0002160781610000201
PCR procedure: pre-denaturation at 94 ℃ for 5min; 30s at 94 ℃, 30s at 51 ℃, 1min20s at 72 ℃ and 35 cycles; 10min at 72 ℃.
mu.L of the PCR amplification product was detected by electrophoresis on 1.2% agarose gel (EB staining). The PCR product fragments corresponding in size were recovered by agarose gel electrophoresis.
And (4) counting the scores of all the sequences in the DNA molecular marker matrix according to the segment sites, wherein the scores are one score when the bases are the same, and the scores are not different when the bases are different. The matrix contains 65 DNA molecular markers in total, and the total score is 65. In the examples, the identification rate of cinnamon was 52.31%, and the identification rate of cinnamomum camphora was 95.38%. The species identified was cinnamomum camphora, not cinnamomum cassia.
Figure BDA0002160781610000202
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Guangzhou college of traditional Chinese medicine (Guangzhou institute of traditional Chinese medicine)
<120> method for identifying or distinguishing cinnamon and its related plants, and primer and kit used in the method
<160> 8
<170> PatentIn version 3.3
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Claims (10)

1. A primer combination comprising the following primer pairs:
a primer pair P1: SEQ ID NO:1-2;
a primer pair P2: SEQ ID NO:3-4;
a primer pair P3: SEQ ID NO:5-6; and
primer pair P4: SEQ ID NO:7-8.
2. A kit comprising the primer combination of claim 1.
3. The kit of claim 2, wherein the kit further comprises one or more of a DNA extraction system, PCR reaction buffer, water, DNA polymerase, and molecular weight marker.
4. The kit of claim 3, wherein the DNA polymerase is selected from any of Taq, bst, vent, phi29, pfu, tru, tth, tl1, tac, tne, tma, tih, tf1, pwo, kod, sac, sso, poc, pab, mth, pho, ES4DNA polymerase, and Klenow fragment.
5. The kit of claim 3, wherein the water is nuclease-free water.
6. The kit according to any one of claims 2 to 5, wherein the kit further contains reagents required for sequencing.
7. Cinnamon bark (cinnamon)Cinnamomumcassia) And a method for identifying or differentiating a related plant thereof, wherein the related plant is Cinnamomum zeylanicum: (A)Cinnamomum zeylanicum) Cinnamomum camphora (I) and (II)Cinnamomum camphora) And Cinnamomum camphora (A) andCinnamomummicranthum) A plant, the method comprising:
a) Obtaining genome DNA or mitochondrial DNA of a plant to be detected;
b) Amplifying the DNA obtained in the step a) by using the primer combination product of claim 1 or the kit of any one of claims 2 to 6;
c) Sequencing the amplified fragments, comparing the sequencing result with a cinnamon specific DNA molecular marker matrix, and judging that the plant to be detected is the species if the total similarity of the sequencing result and the species in the matrix is more than 95%;
according to the base sites of the DNA matrix, the following scoring principles are established: the base of the species to be detected is the same as the base of the matrix, the species in the matrix is scored with 1, the species in the matrix are not scored or not scored with different base, and finally the identification similarity is obtained through calculation by calculating the total score and the proportion of the total score to the total score of the SNP sites;
the matrix is:
TABLE I SNP contained in the region amplified by primer pair P1
Position of 321 349 350 551 787 921 923 925 Cinnamomum cassia A A T G G C C A Cinnamomum zeylanicum A C T G G C C A Cinnamomum micranthum A A G G A C C A Cinnamomum camphora C A T C G A G G
TABLE II SNPs contained in the region amplified by primer pair P2
Position of 14 142 199 229 230 231 253 254 266 282 291 Cinnamomum cassia G G A A A A A A A G A Cinnamomum zeylanicum G A A A A - C T A G A Cinnamomum micranthum A T A A A A C T C G T Cinnamomum camphora G G G - - - A A A A A
TABLE II
Position of 369 405 414 434 435 437 438 440 441 500 701 752 Cinnamomum cassia G G A G T C T T T G A G Cinnamomum zeylanicum T T G A A A G A C G G A Cinnamomum micranthum G G A A A A G A C A G G Cinnamomum camphora G G G A A A G A C G A G
TABLE III SNP contained in the region amplified by primer pair P3
Position of 213 232 382 407 528 533 534 547 647 690 Cinnamomum cassia C A A G A G A A T G Cinnamomum zeylanicum C A C T A G A A C G Cinnamomum micranthum A G A T C T T C T A Cinnamomum camphora C A A G A G A A T G
TABLE III
Position of 1187 1195 1211 1326 1410 Cinnamomum cassia C C C T T Cinnamomum zeylanicum C G C C T Cinnamomum micranthum C C T C G Cinnamomum camphora A C C C T
TABLE IV SNP contained in the region amplified by primer pair P4
Position of 191 422 Cinnamomum cassia T A Cinnamomum zeylanicum C A Cinnamomum micranthum C A Cinnamomum camphora C T
。
8. The method of claim 7, wherein the annealing temperature is 49 ℃ to 53 ℃ for each primer pair.
9. The method of claim 7, wherein the sequencing is selected from high throughput sequencing.
10. The method of claim 7, wherein the sequencing is selected from Sanger sequencing.
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