CN113308567B - Bar code prepared based on rhubarb chloroplast genome polymorphism region and application thereof - Google Patents

Bar code prepared based on rhubarb chloroplast genome polymorphism region and application thereof Download PDF

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CN113308567B
CN113308567B CN202110827065.3A CN202110827065A CN113308567B CN 113308567 B CN113308567 B CN 113308567B CN 202110827065 A CN202110827065 A CN 202110827065A CN 113308567 B CN113308567 B CN 113308567B
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gug
trnh
psba
haplotype
rhubarb
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CN113308567A (en
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魏胜利
王晓晖
满金辉
刘凤波
石玥
张靖晗
尹光耀
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Beijing University of Chinese Medicine
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a group of chloroplast genome polymorphism regions and DNA bar codes prepared by the polymorphism regions, wherein the polymorphism regions can be used for identifying rhubarb germplasm resources, and the DNA bar codes can be used for plant seed batch anti-counterfeiting.

Description

Bar code prepared based on rhubarb chloroplast genome polymorphism region and application thereof
Technical Field
The invention relates to a method for identifying rheum officinale, in particular to a method for identifying a foundation, a production place, quality, authenticity and batch of rheum officinale by utilizing a rheum officinale chloroplast genome polymorphism region.
Background
Rhubarb (Rhei Rhizoma) is an important common bulk medicinal material and has the effects of purging pathogenic accumulation, clearing heat and purging fire, cooling blood and removing toxin, removing blood stasis and dredging channels, promoting diuresis and removing jaundice and the like. The pharmacopoeia of the people's republic of China prescribes that the certified rheum officinale is derived from the dried roots and stems of rheum palmatum (R.palmatum L.) of Liao Keda yellow (Rhem), rheum tanguticum maxim. Ex Balf) or Rheum officinale (R.franzembichi Munt). In recent years, as wild resources of rheum officinale are becoming deficient, large Huang Weipin medicinal materials such as rheum officinale (R.franzenbachii Munt), rheum palmatum (R.hotaoense C.Y. Cheng et Kao) and rheum longifolium (R.wittrockii Lundstr) of rheum officinale are always mixed and circulated in the market, and attention has been paid to the field of crude drugs on how to accurately identify the authenticity of rheum officinale medicinal materials. On the other hand, rheum officinale is a typical multi-basic and multi-functional medicinal material, and researches show that the chemical components of rheum palmatum, rheum tanguticum and rheum officinale 3 kinds of genuine rheum officinale have certain differences, and the pharmacological activities of the three are different to different extents, so that the rheum officinale is required to be selected for different functional purposes for ensuring more accurate symptomatic medication, and therefore, on the premise of identifying the authenticity of rheum officinale, the identification of Huang Jiyuan kinds of rheum officinale is also of great significance.
Compared with the traditional important identification method, the traditional Chinese medicine molecular identification technology based on DNA sequence markers has the advantages of no dependence on medicine morphology, no need of long-term experience accumulation of identified people, good repeatability, high accuracy and the like. Particularly in the aspect of identifying the related medicinal materials, the method can well solve the identification difficulty caused by the extremely similar appearance and shape of the medicinal materials. Therefore, in recent years, the molecular identification technology is widely applied to the field of traditional Chinese medicine identification. The chloroplast genome provides the plant cell with the photosynthetic organelles that must be energized. The genetic information of the chloroplast genome comes from maternal inheritance, so that the chloroplast genome is a good molecular marker of the maternal plant, and the chloroplast genome sequence is generally relatively conservative and has relatively stable genome structure, gene content, gene sequence and low mutation rate. Thus, chloroplast genome is one of the effective tools for phylogenetic studies and species identification. Recent studies have shown that chloroplast genomic mutations are not random, but rather accumulate in "hot spots" which form hypervariable region-polymorphic regions in the genome. The chloroplast genome polymorphism region not only provides a molecular marker for identification on a species level, but also can provide accurate genetic diversity and population structure for the species, and further provides valuable information for the selective breeding of the species.
Disclosure of Invention
In order to make up the blank in the prior art, the invention provides a rhubarb chloroplast genome polymorphism region and application thereof, wherein the region is a trnH-GUG_psbA gene polymorphism interregion.
In a first aspect of the invention, there is provided the use of a trnH-GUG_psbA gene polymorphic region as a rhubarb discrimination DNA barcode. In one embodiment, the identifying DNA barcodes may be used for rheum officinale germplasm identification, more specifically, the germplasm identification includes identification of sample origin, quality and authenticity.
In a second aspect of the present invention, there is provided a method for performing batch anti-counterfeiting on rhubarb seeds by using trnH-GUG_psbA gene polymorphism inter-region, the method comprising:
(1) Large Huang Shanbei selection and planting
Collecting haplotype seeds, combining haplotypes carrying different trnH-GUG_psbA intervals, planting in batches,
(2) Identification of large Huang Pici samples
Collecting the rheum officinale sample, extracting DNA, amplifying a chloroplast polymorphism region trnH-GUG_psbA of rheum officinale, sequencing, analyzing a sequencing result, and judging batch samples of the planted rheum officinale according to a haplotype sequence and a combination mode.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1trnH-GUG_psbA different haplotype sequences;
FIG. 2 is a phylogenetic tree constructed of haplotype sequences;
the standard curve of each active ingredient in rheum officinale of FIG. 3;
the content analysis of the effective components in the rhubarb medicinal materials with different haplotypes is shown in figure 4;
FIG. 5 shows the sequencing peak patterns of haplotypes 2-7, hap1 and haplotypes 2-2, hap 7;
FIG. 6 shows the sequencing peaks for haplotypes 2-7, hap1 and haplotypes 2-8, hap 9.
Detailed Description
EXAMPLE 1 screening of genomic polymorphic regions of Rheum emodi
1.1 chloroplast genomic Gene region alignment
Firstly, 7 rhubarb samples of Qinghai and Nissan, sichuan color, yichang, sichuan, ningxia long and Gansu Dangchang, shanxi county are collected, chloroplast genome is extracted, sequencing of chloroplast genome is carried out, sequencing results are adopted to carry out multi-sequence comparison analysis on the samples by using clustalw2 software, and 112 comparison results are obtained. The comparison results of 9 genes only have 1 difference site, the comparison results of 53 genes show that the sequence similarity is 100%, and the above 62 genes cannot be used for screening DNA barcodes. The remaining 50 comparison results are ranked according to the sequence similarity from small to large, and Table 1-1 shows 30 chloroplast genes with the highest mutation degree of different rheum officinale samples, and the data in the table can be seen, except trnG-The sequences of the chloroplast gene regions of rheum officinale are highly similar except the UCC and psbH genes. It can be seen from this that,identification of specific DNA of Rheum emodi core germplasm Barcoding is difficult to screen in chloroplast gene region。
TABLE 1-1 30 chloroplast genes with highest degree of rhubarb mutation
1.2 results of intergenic region alignment of chloroplast genome
The sequence of 125 intergenic regions was analyzed by using clustalw2 software for multiple alignments, respectively, and the 21 intergenic regions with only 1 site difference and 17 intergenic regions with 100% similarity were removed, and the remaining 87 intergenic regions were used for screening DNA barcodes. The sequence similarity of the alternative 87 intergenic regions is found to be lower than that of the gene regions according to the sequence similarity from small to large, but the similarity of the overall comparison results is still higher as can be seen from tables 1-2 due to the close relatedness between the species of rheum palmatum, rheum tanguticum and rheum palmatum or the slow evolution of the chloroplast of rheum officinale.
TABLE 1-2 30 chloroplast intergenic regions with highest degree of rhubarb mutation
1.3 screening of Rheum emodi chloroplast genomic polymorphic regions
For a highly efficient barcode sequence, the degree of variation is sufficient to distinguish between different sequencesIn addition to the species requirements, it is also necessary to meet the experimental requirements of PCR amplification and sequencing of PCR products, so that it is one of the screening factors for sequence length. Manually checking the screened intergenic region fragments with the comparison length of 300-700 bp and the sequence similarity of less than 97%, wherein the intergenic region of trnH-GUG_psbA gene (SEQ ID NO. 1) is inserted in the 125 th place of rheum palmatum 4-5 base, the rest is C, the 372 nd base of rheum palmatum 4-6 is C, the rest is A, and the 342-349 nd base AAATAAAAA; the positions 186 and 341 of the medicinal rhubarb 5-1 are A, T respectively; the base at positions Huang Di, 206 and 281 of Tang Gute is C, T, A respectively, the comparison result shows that the specific variation position exists in the sequence of each sample in the trnH-GUG_psbA gene interregion,i.e. trnH- The GUG_psbA intergenic region may be used as a DNA barcode for the specific identification of the rhein geodetic core germplasm.
TABLE 1-3 core germplasm specific DNA barcode fragment of rhubarb
Marker Length of consensus sequence (bp) Alignment sequence length (bp) Sequence similarity (%)
trnH-GUG_psbA 400 433 92.4
Example 2 identification of different Rheum emoral core germplasm Using trnH-GUG_psbA intergenic regions
2.1 collecting rhubarb seeds in different regions
Multiple medicinal materials (regional table 2-1) in different regions of the whole country are collected, DNA extraction is carried out, and a specific primer trnH-GUG_psbA-F is utilized: CGGGCGAACGACGGGAATTG; trnH-GUG_psbA-R: ATACTGTCGAATAACAAGCC the trnH-GUG_psbA fragment was amplified by PCR, recovered and sequenced.
TABLE 2-1 sample collection Table for different rhubarb areas
2.2 analysis of haplotypes and sequence characteristics of Rheum emodi in different regions Using trnH-GUG_psbA
The sequencing results were analyzed to co-form 13 haplotypes (FIG. 1), which were classified into two major classes based on haplotype sequence characteristics.
The first kind of mutation sites are mainly distributed in 36bp-360bp, 6 haplotypes are total, and specific bases at 179 and 218 positions of haplotype Hap2 are C and A; hap3 has an insertion sequence CTTTAAA at positions 36-42; the specific base at position of haplotype Hap4139 is A; specific bases at haplotype Hap5286 and 952 are T and A respectively; the specific bases at position G and 49 of haplotype Hap639 and 49 are G and A respectively.
The second class of mutation sites 379-404, wherein the specific bases at positions 379, 381-385, 387, 389 and 391-392, 394, 396, 399, 400-402 and 404 are C, A, T, T, T, T, T, C, T, T, G, C, G, G, G, T; in addition, other specific bases of haplotype Hap8 are a at positions 199, 200 and 952; other specific bases of haplotype Hap9 are at positions 219 and 352, T and C respectively; other specific bases of haplotype Hap10 are deleted between 274-280; other specific bases of haplotype Hap11 are T at both positions 128 and 304; the other specific bases of the haplotype Hap12 are at positions 49 and 219, which are A and C respectively; the other specific base of haplotype Hap13 is A at position 295.
2.3 phylogenetic tree
Constructing a phylogenetic tree for the haplotype sequence of rheum officinale by using MEGA7.0 software, wherein as can be seen from FIG. 2, the phylogenetic tree constructed by the haplotype sequence can be divided into two branches, which is similar to the analysis of the sequencing result; wherein Hap 1-Hap 6 are a big branch; hap7-Hap13 is distributed over the other branch.
From phylogenetic tree, it can be seen that Rheum palmatum is gathered into two branches, and one branch of Rheum palmatum and Rheum tanguticum are gathered respectively, which indicates that the two basic sources of Rheum palmatum and Rheum tanguticum have obvious pedigree division, wherein the two basic sources of Rheum palmatum and Rheum tanguticum are gathered into one branch of Rheum palmatum, henan province Luoyang (Hap 5), hubei province Yichang province (Hap 3) and Sichuan province Miaoyang (Hap 6). And Yunnan Kunming City (Hap 12) on another big branch, the obvious spectrum division exists between the medicinal rhubarb produced by Yunnan Kunming City and the rest medicinal rhubarb. The Qinghai Qilin county 2 (Hap 7) of Qinghai province, tang-Gute and the Qinghai Kagao Tibetan autonomous state (Hap 9) and the Qinghai Qilin county 1 (Hap 8) of Qinghai province are collected on a large branch. The Ganzhua county (Hap 10) in Ganzhu, sichuan province and Gansu province, ganchang county 2 (Hap 11) in Gansu province, ganchun, city, dangchang are collected into a small branch. The Sichuan Ganzhu Xiu pond county (Hap 2) and Shanxi Baoji Shang county (Hap 4) are collected into a small branch. Meanwhile, most samples (Sichuan Abba, sichuan Athens, sichuan asbestos, sichuan Mianyang) produced from Sichuan province are collected into a large branch. Most Qinghai province samples (Qilin county, north China, qinghai province, juanju nationality, qinghai province) are collected on a large branch, which indicates that rheum officinale of different producing areas has a certain geographical area division, the method also shows that the trnH-GUG_psbA haplotype of the rheum officinale has a certain tendency on the distribution of the production places, which indicates that the rheum officinale can be partially identified according to the trnH-GUG_psbA haplotype of the rheum officinale, and particularly the rheum officinale production places can be identified according to the types and the numbers of genotypes.
Example 3 quality determination of rhubarb Shan Bei
3.1 determination of standard curve
The active ingredients in rheum officinale are subjected to standard curve measurement according to pharmacopoeia standards or by referring to other published documents, and the active ingredients comprise a plurality of ingredients such as gallic acid, catechin, aloe-emodin, rhein, emodin, chrysophanol and the like. The standard curve is shown in fig. 3.
3.2 analyzing the quality characteristics of different haplotype rhubarb medicinal materials by utilizing HPLC
Analyzing 3 different haplotype rhubarb samples by HPLC, wherein Hap6 is medicinal rhubarb, hap1 is tangut rhubarb, hap4 is palmleaf rhubarb, crushing a plurality of medicinal materials of different haplotype rhubarb to extract effective components, measuring various effective components by HPLC, and calculating the content of each component according to a standard curve. The results are shown in FIG. 4.
The content measurement shows that the sennoside B% content in Hap1 is highest, the sennoside A% content in Hap6 is highest, the content of aloe-emodin in Hap4 is highest, and the content is 0.15%, which indicates thatDifferent haplotype rhubarb materials have quality difference。
Example 4 anti-counterfeiting of rhubarb seed lots Using rhubarb chloroplast polymorphic regions
4.1 selecting seeds with different mutation sites in polymorphic regions for batch anti-counterfeiting verification
The experiment adopts chloroplast gene segments as a main means for establishing batch bar codes for producing traditional Chinese medicinal materials, and the batch bar codes adopted in the experiment comprise chloroplast gene segments such as but not limited to matK segments, trnH-GUG_psbA segments and the like.
The method comprises the steps of selecting a mixture of rheum officinale seeds of which the number is 2-7 haplotypes Hap1 and the number is 2-2 haplotypes Hap7 in the figure 1 as a production batch for sowing, sowing in 4 months, collecting 10 leaves of different rheum officinale plants in 8 months for mixing, extracting DNA, amplifying trnH-GUG_psbA fragments by using a PCR instrument, and sequencing after recovery. The sequencing peak diagram is shown in FIG. 5.
The trnH-GUG_psbA fragment mutation sites are evident from FIG. 5: at 379bp, the base of Hap1 is A, the base of Hap7 is C, and the site sequencing peak diagram has an A-C set peak structure; at 3831 bp, 3832 bp,383bp and 384bp, the sequencing peak diagram shows C-A, C-T and G-T sets of peak structures; at 352bp, the Hap1 base is A, the Hap7 base is C, and the sequence peak diagram of the locus shows an A-C set peak structure.
In addition, the rheum officinale seeds of which the number is 2-8 haplotype Hap9 and the number is 2-7 haplotype Hap1 in the figure 1 are mixed to be used as a production batch for sowing, sowing is carried out in 4 months, 10 leaves of different rheum officinale plants are collected in 8 months for mixing, DNA extraction is carried out, a trnH-GUG_psbA fragment is amplified by a PCR instrument, and sequencing is carried out after recovery. The sequencing peak diagram is shown in FIG. 6.
The trnH-GUG_psbA fragment mutation sites are evident from FIG. 6: at 352bp, the Hap1 base is A, the Hap9 base is C, and the sequence peak diagram of the locus shows an A-C cover peak structure; at 219bp, the Hap1 base is A, the Hap9 base is T, and the sequence peak diagram of the locus shows an A-T cover peak structure.
The sequencing result shows that the single peak of the Huang Yangpin trnH-GUG_psbA fragment of each batch can be detected The ploidy type and variation site are obvious, a sleeve peak structure exists, and the fragment can be used as a batch bar code anti-counterfeiting method.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> university of Beijing Chinese medicine
<120> a barcode prepared based on rhubarb chloroplast genome polymorphic region and application thereof
<130> CP210212
<141> 2021-07-21
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 1702
<212> DNA
<213> Rhei Rhizoma
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cgggcgaacg acgggaattg aacccgcgca tggtggattc acaatccact gccttgatcc 60
acttggctac atccgcccct acgttcctta cttgaaaatg attcaaatta cactcactaa 120
ctcactattc atcatttttt tacttaattt tttatattca tttttttctt ttacaaaaat 180
ttgaaatctt tctctctatg aatataaatg actatgaaac aaaaaatttc cccaaaaacc 240
aaaacaacaa agagggcgta tgggtagaaa ttaaaaaaat gcgaatgcgt ccataaatat 300
aaaaaaatac aacattcaat catcactcat cacttaaaac atttttttct tttttttaat 360
ggggaagaaa acttatgact cataacataa aatatatata taataaaaat tttaattaaa 420
ggagcaatac caacccgctc gatagaacaa gaaattgggt attgctcctt taattaaaaa 480
aactcgacta cactaagacc aaaatcttat ccattgatag atggagcttg gatagcagct 540
aagtctagag ggaagttatg agcattacgt tcatgcataa cttccatacc aaggttagcg 600
cggttaataa tatcagccca agtattaatt acacgacctt gactatcaac tacagattgg 660
ttgaaattga agccgttcag attaaacgcc atagtactaa tacctaaagc agtaaaccag 720
atacctacta caggccaagc agctaagaag aaatgtaaag aacgagaatt gttgaaacta 780
gcatattgga agattaatcg gccaaaataa ccatgagcgg ctacgatatt ataagtttct 840
tcctcttgac caaatctgta accttcatta gcagattcat tttctgtggt ttccctgatc 900
aaactagagg ttaccaaaga accatgcata gcactgaata gggagccgcc aaatacacca 960
gctacgccta acatgtgaaa tgggtgcata aggatgttgt gctcggcctg aaatacaatc 1020
atgaagttga aagtaccaga gattcctaga ggcataccat cagaaaagct tccttgacca 1080
attgggtaga tcaagaaaac agcagtagca gccgcaacag gagctgaata cgcaacagca 1140
atccaagggc gcatacccag acggaaacta agttcccact cacgacccat gtaacaagct 1200
acaccaagta agaagtgtag aacaattagc tcataaggac caccgttgta taaccattca 1260
tcaacagatg cagcttccca tattggataa aagtgcaaac cgatagctgc agaagtagga 1320
ataatggcac cagagataat attgtttccg taaagaagag atccagaaac aggctcacga 1380
ataccatcaa tatctactgg aggagcagca atgaaggcta taataaatac agaagttgcg 1440
gtcaataagg tagggatcat caaaacgcca aaccatccaa tgtaaagacg gttttcagta 1500
ctggttatcc agttacaaaa acgaccccat aggctttcgc tttcgcgtct ctctaaaatt 1560
gcagtcatgg tcaaaaatct tggtctattt catttaatca tcagggactc ccaagcgcac 1620
aaattctata taatctatac tcgattaaaa actcgattaa aaatagataa ttataattga 1680
aggcttgtta ttcgacagta ta 1702

Claims (2)

1. An application of a rhubarb chloroplast genome polymorphism region as a DNA bar code for identifying germplasm of a near-source variety of rhubarb, which is characterized in that the polymorphism region is a trnH-GUG_psbA gene polymorphism interval, the germplasm comprises identification of a large Huang Yangpin base stock, a production place, quality and authenticity, the application is that the germplasm of a sample is confirmed according to trnH-GUG_psbA gene haplotypes of different near-source varieties of rhubarb by detecting the trnH-GUG_psbA gene polymorphism interval of the near-source variety, and the trnH-GUG_psbA gene haplotypes are distinguished as follows:
the first kind of mutation sites are mainly distributed on 36bp-360bp of trnH-GUG_psbA amplified fragments, 6 haplotypes are total, and specific bases of 179 and 218 sites of haplotype Hap2 on trnH-GUG_psbA amplified fragments are C and A; the inserted sequence CTTTAAA exists at 36-42 positions on the trnH-GUG_psbA amplified fragment of Hap 3; the specific base at 139 th site of the haplotype Hap4 on the trnH-GUG_psbA amplified fragment is A; specific bases of haplotype Hap5 at positions 286 and 952 on the trnH-GUG_psbA amplified fragment are respectively positioned T and A; the specific bases at 39 and 49 positions of the haplotype Hap6 on the trnH-GUG_psbA amplified fragment are G and A respectively; the trnH-GUG_psbA amplified fragment adopts a specific primer trnH-GUG_psbA-F: CGGGCGAACGACGGGAATTG; trnH-GUG_psbA-R: ATACTGTCGAATAACAAGCC, obtaining trnH-GUG_psbA fragments through PCR amplification;
a second class of mutation sites 379-404, wherein the specific bases at positions 379, 381-385, 387, 389 and 391-392, 394, 396, 399, 400-402 and 404 on the trnH-GUG_psbA amplified fragment are C, A, T, T, T, T, T, C, T, T, G, C, G, G, G, T; in addition, other specific bases of haplotype Hap8 on trnH-GUG_psbA amplified fragment are A at 199, 200 and 952; other specific bases of haplotype Hap9 on trnH-GUG_psbA amplified fragments are at positions 219 and 352, which are respectively T and C; other specific bases of haplotype Hap10 on trnH-GUG_psbA amplified fragment are deleted between 274-280; other specific bases of haplotype Hap11 on trnH-GUG_psbA amplified fragment are T at positions 128 and 304; other specific bases of the haplotype Hap12 on the trnH-GUG_psbA amplified fragment are at positions 49 and 219, which are A and C respectively; other specific bases of the haplotype Hap13 on the trnH-GUG_psbA amplified fragment are A at 295;
the haplotypes of each production place are as follows:
the rheum palmatum is gathered into two branches, and the medicinal rheum palmatum and the tangutot rhubarb are respectively gathered into one branch;
wherein, the medicinal rhubarb in Henan province of Luoyang is haplotype type Hap5, the medicinal rhubarb in Yichang province of Hubei province of Mianyang is Hap3, the medicinal rhubarb in Mianyang province of Sichuan province is Hap6, the medicinal rhubarb in Kunming province of Yunnan province is Hap12, the medicinal rhubarb in Qilin county of Qinghai, qinghai province of Qinghai, qinghai province of Luo Tibetan autonomous state is Hap9;
the rheum palmatum in the Shao county of Ganzhu, sichuan province is Hap10, the rheum palmatum in the Dangchang county of Gansu province is Hap11, the rheum palmatum in the Ganzhu county of Sichuan province is Hap2, and the rheum palmatum in the Ganzhu county of Shanxi province is Hap4;
further, the origin of rhubarb is identified according to the type and number of genotypes.
2. A method for carrying out batch anti-counterfeiting on seeds of near-source varieties of rheum officinale by utilizing polymorphic interlineages of the trnH-GUG_psbA genes of rheum officinale, comprising the following steps:
(1) Large Huang Shanbei selection and planting
Collecting haplotype seeds, combining haplotypes carrying different trnH-GUG_psbA intervals, and planting in batches;
(2) Identification of large Huang Pici samples
Collecting samples of planted rheum officinale, extracting DNA, and adopting a specific primer trnH-GUG_psbA-F for chloroplast polymorphic region trnH-GUG_psbA of the samples: CGGGCGAACGACGGGAATTG; trnH-GUG_psbA-R: ATACTGTCGAATAACAAGCC and sequencing, analyzing the sequencing result, and judging batch samples of the planted rheum officinale according to the sequence and combination mode of the trnH-GUG_psbA haplotype as described in claim 1 and combining with a sequencing peak diagram; wherein the near-source varieties of rheum officinale comprise rheum palmatum, rheum tanguticum and rheum officinale which are medicinal.
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