CN113186338B - Universal primer for identifying angiosperm plant species and application thereof - Google Patents

Universal primer for identifying angiosperm plant species and application thereof Download PDF

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CN113186338B
CN113186338B CN202110676999.1A CN202110676999A CN113186338B CN 113186338 B CN113186338 B CN 113186338B CN 202110676999 A CN202110676999 A CN 202110676999A CN 113186338 B CN113186338 B CN 113186338B
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周世良
刘艳磊
徐超
董文攀
杨雪莹
陈庆
刘萌妍
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Abstract

The invention discloses a universal primer for identifying angiosperm plant species and application thereof. The invention optimizes the primers of two core barcodes rbcL and matK by using the data of a large angiosperm system (covering 69 meshes and 541 families), divides each gene into 4 segments, has the length of about 400bp, and can be directly sequenced by using a high-throughput sequencing technology. Meanwhile, the invention develops four novel chloroplast genome hypervariable regions ndhF2, ndhF3, rpoB1 and rpoB5 by mining angiosperms large-system chloroplast genome data and designs universal primers which can be directly used for high-throughput sequencing. The invention lays a technical foundation for the development and application of DNA barcode technology, such as identification of angiosperm species, auxiliary material evidence tracing by using plant information in soil and the like.

Description

Universal primer for identifying angiosperm plant species and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a universal primer for identifying angiosperm species and application thereof.
Background
DNA barcoding (DNA barcoding) is a technique for rapid, accurate identification or characterization of species using sequence difference information from standard, sufficiently variant, moderately long, easily amplified DNA fragment or fragments. The traditional taxonomy identification method has the limitations that the subjectivity is strong, a professional taxonomy academic background is required, required morphological shapes cannot appear at the same time, and plant debris cannot be accurately identified.
The DNA barcode technology can rapidly and accurately identify species, is an important supplement to the identification of species by the traditional classification method, and has stronger application prospect as the technology develops and is increasingly applied to the fields of accurate plant identification, forensic science and criminal investigation (such as plant material evidence identification).
The concept of "DNA barcode" was first proposed by the Canadian researcher Paul Hebert in 2003 and applied to taxonomic studies of lepidopteran insects. Through the exploration and research of a large number of researchers, a part of hypervariable region (a DNA fragment of about 650 bp) in the mitochondrial COI gene (cytochrome c oxidase I gene) is finally determined as an international universal DNA barcode for researching animal classification system, rapid identification and other related researches. Unlike animals, plants are complicated due to their particularity, and it is impossible to solve all plant identification problems by using a DNA sequence, and at present, there is no rational unique plant DNA barcode theorem. The international union of bio-barcodes (CBOL) preliminary suggested matK, rpoC1, rpoB, accD, nhdJ, and ycf5 as plant DNA universal barcodes, but accD, ndhJ, and ycf5 did not contain deletions for all groups. Kress, equal to 2005, proposed three fragments of ITS, trnH-psbA, rbcL as plant universal DNA barcodes. Finally, the chloroplast gene fragments rbcL and matK are determined to be core DNA barcodes of plant DNA standard barcodes at the third international DNA barcode convention in 2009, and ITS (internal transformed spacer) and the chloroplast gene spacer trnH-psbA are suggested to be supplementary barcodes of plant DNA barcodes.
With the rapid development of the second-generation sequencing technology, the technology for acquiring DNA barcodes by using the high-throughput sequencing technology will increase the data acquisition efficiency and reduce the data acquisition cost. The need to apply high throughput sequencing technologies to the acquisition of DNA barcodes is increasing. The method is limited by the sequencing read length of the current high-throughput sequencing technology (the sequencing platform can only complete the sequencing of a 600bp fragment (Illumina PE300 and Ion Torrent S5 chip600)) which is far shorter than the length of plant core DNA barcodes such as rbcL and matK, and complete plant DNA barcode data cannot be obtained through the technology, so the development and application steps of the DNA barcode technology are seriously hindered.
Disclosure of Invention
The invention aims to provide a universal primer for identifying angiosperm plant species and application thereof.
In order to achieve the above objects, the present invention provides, in a first aspect, a specific primer pair for use in the identification of angiosperm plant species.
The specific primer pair for identifying angiosperm plant species provided by the invention is at least one of the following primer pairs: primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, primer pair 10, primer pair 11 and primer pair 12;
the primer pair 1 consists of primers 1-F and 1-R; the primer pair 2 consists of a primer 2-F and a primer 2-R; the primer pair 3 consists of a primer 3-F and a primer 3-R; the primer pair 4 consists of a primer 4-F and a primer 4-R; the primer pair 5 consists of a primer 5-F and a primer 5-R; the primer pair 6 consists of a primer 6-F and a primer 6-R; the primer pair 7 consists of a primer 7-F and a primer 7-R; the primer pair 8 consists of a primer 8-F and a primer 8-R; the primer pair 9 consists of a primer 9-F and a primer 9-R; the primer pair 10 consists of a primer 10-F and a primer 10-R; the primer pair 11 consists of a primer 11-F and a primer 11-R; the primer pair 12 consists of a primer 12-F and a primer 12-R;
the primer 1-F is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
the primers 1-R are single-stranded DNA molecules shown in a sequence 2 in a sequence table;
the primer 2-F is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;
the primers 2-R are single-stranded DNA molecules shown in a sequence 4 in a sequence table;
the primer 3-F is a single-stranded DNA molecule shown as a sequence 5 in a sequence table;
the primer 3-R is a single-stranded DNA molecule shown as a sequence 6 in a sequence table;
the primer 4-F is a single-stranded DNA molecule shown as a sequence 7 in a sequence table;
the primer 4-R is a single-stranded DNA molecule shown as a sequence 8 in a sequence table;
the primer 5-F is a single-stranded DNA molecule shown as a sequence 9 in a sequence table;
the primer 5-R is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;
the primer 6-F is a single-stranded DNA molecule shown as a sequence 11 in a sequence table;
the primer 6-R is a single-stranded DNA molecule shown as a sequence 12 in a sequence table;
the primer 7-F is a single-stranded DNA molecule shown as a sequence 13 in a sequence table;
the primer 7-R is a single-stranded DNA molecule shown as a sequence 14 in a sequence table;
the primer 8-F is a single-stranded DNA molecule shown as a sequence 15 in a sequence table;
the primer 8-R is a single-stranded DNA molecule shown as a sequence 16 in a sequence table;
the primer 9-F is a single-stranded DNA molecule shown as a sequence 17 in a sequence table;
the primer 9-R is a single-stranded DNA molecule shown as a sequence 18 in a sequence table;
the primer 10-F is a single-stranded DNA molecule shown as a sequence 19 in a sequence table;
the primer 10-R is a single-stranded DNA molecule shown in a sequence 20 of a sequence table;
the primer 11-F is a single-stranded DNA molecule shown in a sequence 21 in a sequence table;
the primer 11-R is a single-stranded DNA molecule shown in a sequence 22 in a sequence table;
the primer 12-F is a single-stranded DNA molecule shown in a sequence 23 in a sequence table;
the primer 12-R is a single-stranded DNA molecule shown in a sequence 24 in a sequence table.
In the specific primer pairs, the molar ratio of each primer in each primer pair is 1: 1.
In order to achieve the above object, the present invention further provides the use of the above specific primer pair in any one of the following m1) -m 6):
m1) preparing a product identified by the angiosperm species;
m2) preparing the product of assistant identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence source tracing.
In order to achieve the above object, the present invention also provides a kit comprising the above specific primer pair.
Further, the kit also comprises other reagents for PCR amplification and reagents for sequencing PCR amplification products.
Still further, the other reagents for PCR amplification include 10 XPCR Buffer (Mg) 2+ )、dNTP、Taq DNA polymerase。
The reagents for sequencing PCR amplification products include reagents for high throughput sequencing library construction (e.g.
Figure BDA0003121086370000031
Fast DNA Library Prep Set for Ion Torrent TM kit (reagents in new England biolabs) kit) and reagents for high throughput sequencing (such as required for high throughput sequencing based on the Illumina Hiseq series platform).
In order to achieve the purpose, the invention also provides a preparation method of the kit.
The preparation method of the kit provided by the invention comprises the step of respectively and independently packaging each primer in the specific primer pair.
The application of the kit in any one of the following m1) -m6) also belongs to the protection scope of the invention:
m1) preparing a product identified by the angiosperm species;
m2) preparing the product of assistant identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence tracing.
In order to achieve the above object, the present invention also provides a DNA fragment.
The DNA fragment provided by the invention is any one of the following n1) -n 4):
n1) using the rbcL gene of the plant to be detected as a template, and adopting the primer pair 1 or the primer pair 2 or the primer pair 3 or the primer pair 4 to amplify the obtained DNA fragment;
n2) using the matK gene of the plant to be detected as the template, and adopting the primer pair 5 or 6 or 7 or 8 to amplify the obtained DNA fragment;
n3) using ndhF gene of plant to be detected as template, and adopting the primer pair 9 or 10 to amplify the obtained DNA fragment;
n4) using rpoB gene of the test plant as a template, and amplifying the resulting DNA fragment using the above primer set 11 or 12.
The application of the DNA fragment in any one of the following m1) -m6) also belongs to the protection scope of the invention:
m1) preparing a product identified by the angiosperm species;
m2) preparing products for assisting identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence source tracing.
In any of the above applications, the material evidence tracing is material evidence tracing by using plant information in soil.
To achieve the above object, the present invention finally provides a method for identifying or assisting in identifying an angiosperm plant species.
The identification or auxiliary identification method of angiosperm plant species provided by the invention comprises the following steps: using the genome DNA of a plant (cluster) to be detected as a template, and performing PCR amplification by using the specific primer pair to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a PCR amplification product sequence; and carrying out BLAST comparison on the PCR amplification product sequence in an NCBI database, and determining the species of the plant to be detected according to the comparison result.
In the above method, the method for performing BLAST comparison on the PCR amplification product sequence in NCBI database and determining the species to which the plant to be tested belongs according to the comparison result includes the following steps: downloading data of all corresponding genes of the plant (the group) to be detected, merging the data with the sequence data of the PCR amplification product, carrying out fine comparison, constructing a phylogenetic tree after comparison, and judging the species attribution of the phylogenetic tree through the branch structure of the phylogenetic tree. In practical application, when different plants (groups) to be detected are identified, different types or numbers of specific primer pairs can be selected according to actual conditions for identification, and the more the number of the selected specific primer pairs is, the higher the success rate of species identification is.
In the above method, the sequencing platform is a high-throughput sequencing platform.
In any of the specific primer pairs or uses or methods described above, the angiosperm may be any angiosperm known to those skilled in the art. In a specific embodiment of the present invention, the angiosperm is any one of the following: securinega suffruticosa, buddleia, lilium brownii, digitalis, cardamine abrotanoides, Oryctoloma minor, clary sage, litsea cubeba, tea, semen cassiae, aster, aconitum kusnezoffii, carex brachypomum, uncaria Paniculata, radix rhododendri japonici and magnolia.
The invention has the following beneficial effects:
1. by utilizing the specific primer pair provided by the invention, a universal kit for identifying angiosperm species can be developed, and the application of plant DNA barcodes in social services is promoted.
2. According to the invention, by analyzing chloroplast genome data (comprising 492 families in narrow sense in total) of a large angiosperm system, a region where rbcL and matK are relatively conserved is found, the amplification length of the region is optimized and expanded, the amplification length of rbcL gene primers is prolonged to 1428bp from original 838bp, the amplification length of matK gene primers is prolonged to 1492bp from original 827bp, the length of available data is greatly increased, and the capability of identifying and identifying species is further increased.
3. According to the invention, 11 high-variable DNA barcodes which can be used for angiosperms are found through deep mining of angiosperms large system data, two factors of fragment resolution and primer universality are integrated through subsequent primer verification and multiple sequence adjustment, 4 barcodes ndhF2, ndhF3, rpoB1 and rpoB5 are reserved for subsequent research, and a new choice is provided for the application of subsequent DNA barcodes.
The invention optimizes the primers of two core barcodes, namely rbcL and matK by using the data of a large angiosperm system (covering 69 meshes and 541 families), splits each gene into 4 segments, and can directly sequence by using a high-throughput sequencing technology, wherein the length of each segment is about 400 bp. Meanwhile, the invention develops four novel chloroplast genome hypervariable regions ndhF2, ndhF3, rpoB1 and rpoB5 by mining angiosperm large-system chloroplast genome data and designs universal primers which can be directly used for high-throughput sequencing. The invention lays a technical foundation for the development and application of DNA barcode technology, such as identification of angiosperm plant species, assisted material evidence tracing by utilizing plant information in soil and the like.
Drawings
FIG. 1 is a process flow of the present invention.
FIG. 2 shows the results of the primer-verified amplification.
Detailed Description
The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The names of the plant sample materials used for primer verification in the following examples are described in "Chinese plant journal" (author: Chinese academy of sciences, China Committee for plant journal edition), and the website links for the relevant introduction of each plant sample material in the literature are shown in Table 1.
TABLE 1 name of sample material and its associated website links
Figure BDA0003121086370000051
Figure BDA0003121086370000061
Example 1 Universal primer design for angiosperm species identification
The design process of the universal primer for angiosperm species identification of the present invention is shown in FIG. 1.
Firstly, collecting samples
Determining a material directory of angiosperm family level according to the latest APGIII system, searching the chloroplast genome condition in the existing database in NCBI on the basis, utilizing the data in the NCBI existing database for the existing chloroplast genome family, and not repeatedly sampling; for the section without any existing chloroplast genome of the section, fresh materials are firstly acquired through field acquisition, if the acquisition of the fresh materials is difficult, corresponding species specimens of a plant institute specimen museum (PE) of the Chinese academy of sciences are selected, and a small amount of leaves are taken for subsequent DNA extraction on the basis of not influencing the overall preservation condition of the specimens.
Second, DNA extraction
And (3) performing supplementary collection on a sample needing to be supplemented with a sampling point, drying the collected sample at 65 ℃ for 2 hours, grinding the sample into powder, and extracting DNA by using an mCTAB method.
Three, ultrasonic break
For newly obtained total DNA, the size of the fragment is detected by using 2% agarose gel, and the total DNA is broken into fragments of about 400-500bp by using ultrasound according to actual conditions for subsequent construction and sequencing work of an Ilumina sequencing library.
Fourthly, constructing and sequencing high-throughput sequencing library
DNA sample utilization after ultrasonic disruption
Figure BDA0003121086370000062
Fast DNA Library Prep Set for Ion Torrent TM kit (New England Biolabs) kit for high throughput sequencing library construction. The qualified library is sent to Beijing and Kangbio-technology services Limited to obtain high-throughput data by using Illumina Hiseq2000 and 150 PE.
Five, chloroplast genome assembly and annotation
After high-throughput data is downloaded, chloroplast genome assembly is carried out by utilizing Spads default parameters, assembled samples are annotated by utilizing Plann and Sequin software, genome sequence alignment work of all samples is carried out by utilizing mafft software for preliminary alignment, and manual inspection and correction are carried out by utilizing Mega software.
Six, rbcL and matK fragment primer optimization
Specifically intercepting complete genes of two fragments of rbcL and matK and redundant regions at two end parts of the complete genes, carrying out nucleotide polymorphism estimation on data by utilizing Dnasp 5.0, and carrying out universal primer design on a region with a lower pi value according to the pi value in a polymorphism result (the higher the pi value is, the larger the sequence variation is, and the smaller the sequence variation is otherwise), wherein the design principle of the primer is the same as that of a standard primer. The sequence information of the final optimized rbcL and matK fragment universal primers is shown in Table 2.
TABLE 2 sequence information of rbcL and matK Universal primers
Gene Forward primer (3'-5') Reverse primer (3'-5') Length of amplification
rbcL1 TGGATTYAAAGCKGGTGTTAAAGA ATGRGGYGGKCCTTGGAAAGTTTT 427bp
rbcL2 TTCCATTGTRGGWAATGTATTTGG TTSATCATTTCTTCRCATGTACC 402bp
rbcL3 ATCAAAGGRCATTAYTTGAATGC AGAACACCYGGTAKAGARACCCAA 422bp
rbcL4 TTGAAAAAGAYCGAAGYCGYGGTAT GATCTCYTTCCATACYTCACAAGC 335bp
matK1 ACTTMTYTTTCRGGARTATATTTA TTTGAACCAAKATTTCYARATG 422bp
matK2 AATTATGTGTYAGATRTAYTAATACC TCCATAGAAATRTRTTCGYTCAARAA 318bp
matK3 TCCGTAAMCAATCYTCTYATTTAC GCAKTATCTATTAKAAATGMATTYTC 397bp
matK4 CTCGAYTTTYTGGGYTATYTTT TCTTCYTCCGTAARRAATTCTTC 422bp
Note: r is an A/G degenerate base, Y is a C/T degenerate base, M is an A/C degenerate base, K is a G/T degenerate base, S is a G/C degenerate base, and W is an A/T degenerate base.
Development of seven, new hypervariable regions
And (3) carrying out nucleotide polymorphism estimation on the data by using Dnasp 5.0, screening out gene segments with higher variation according to pi values in polymorphism results, and screening out the following 11 hypervariable regions: ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, ndhF6, rpoB1, rpoB2, rpoB3, rpoB4, and rpoB 5. Specifically intercepting 11 fragments of ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, ndhF6, rpoB1, rpoB2, rpoB3, rpoB4 and rpoB5 and redundant regions at both ends thereof, estimating nucleotide polymorphism by using Dnasp 5.0, and designing a universal primer in a region with a lower pi value according to the pi value in the polymorphism result, wherein the principle of designing the primer is the same as that of the universal primer. The sequence information of the newly developed hypervariable region primers is shown in Table 3.
TABLE 3 sequence information for newly developed hypervariable region primers
Figure BDA0003121086370000071
Figure BDA0003121086370000081
Note: r is an A/G degenerate base, Y is a C/T degenerate base, M is an A/C degenerate base, and K is a G/T degenerate base.
Example 2 primer general purpose verification
First, primer verification sample material
In order to verify the universality of the primers, 16 samples in total of the family species with far-away evolutionary relationships are subjected to PCR, and the universality is judged according to the success rate of PCR amplification. The information on the plant sample material used to verify the versatility of the primers is shown in Table 4.
TABLE 4 primer verification of sample Material information
Figure BDA0003121086370000082
Figure BDA0003121086370000091
Second, primer verification method
1. DNA was extracted from the plant sample material for primer commonality testing.
2. PCR amplification was carried out using the DNA solution extracted in step 1 adjusted to a concentration of 10 ng/. mu.l as a template, and the primer pairs shown in Table 2 and Table 3 in example 1, respectively, to obtain PCR amplification products.
PCR amplification System (10. mu.l): 10 × PCR Buffer (Mg) 2+ ) Mu.l of 1. mu.l of 2mM dNTP, 0.5. mu.l of 5. mu.M forward primer, 0.5. mu.l of 5. mu.M reverse primer, 1. mu.l of 10 ng/. mu.l genomic DNA, 0.1. mu.l of 5U/. mu.l Taq DNA polymerase, ddH 2 O 5.9μl。
PCR amplification instrument: master S Instrument from Eppendorf company.
PCR amplification procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 40s, extension at 72 ℃ for 1min, and 35 cycles; finally, extension is carried out for 10min at 72 ℃, and the sample is stored at 4 ℃.
3. And (3) carrying out 1.5% agarose gel electrophoresis on the PCR amplification product obtained in the step (2), and judging the universality of the primers by counting the amplification success rate of each primer pair. The amplification success rate formula is the number of samples of amplified target fragment length band/total number of samples.
The PCR validation results are shown in fig. 2 and table 5. The results show that: the 8 primer pairs in the table 2 have good universality, and the amplification success rate is over 75 percent and can reach 100 percent at most. After primer verification and multiple sequence adjustment, two major factors of fragment resolution and primer universality are integrated, and two regions with better primer universality and higher pi value in a hypervariable region are selected for 11 primer pairs in the table 3: ndhF2, ndhF3, rpoB1, and rpoB 5. The amplification success rate of the primer pairs for amplifying the ndhF2, ndhF3, rpoB1 and rpoB5 regions is more than 87.5%.
Finally, the universal primers determined by the invention and used for identifying angiosperm plant species have 12 pairs of primers, the sequence information of the primers is shown in table 6, wherein the 12 pairs of primers have higher primer universality and can be used for identifying angiosperm plant species, and the rbcL gene 4 pair, the matK gene 4 pair, the ndhF gene 2 pair and the rpoB gene 2 pair are shown in the table.
TABLE 5 primer amplification success Rate
Figure BDA0003121086370000092
Figure BDA0003121086370000101
TABLE 6 12 primer pairs for angiosperm species identification
Figure BDA0003121086370000102
Note: r is an A/G degenerate base, Y is a C/T degenerate base, M is an A/C degenerate base, K is a G/T degenerate base, S is a G/C degenerate base, and W is an A/T degenerate base.
In practical application, when species identification is carried out on angiosperms, the genomic DNA of a plant (cluster) to be detected can be used as a template, and PCR amplification is carried out by adopting at least one of 12 pairs of specific primer pairs in the table 6 to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a PCR amplification product sequence; and BLAST comparison is carried out on the PCR amplification product sequence in an NCBI database, then data of all corresponding genes of the plant (group) level to be detected are downloaded and then are combined with the PCR amplification product sequence data, fine comparison is carried out, a phylogenetic tree is constructed after comparison, and the species attribution of the phylogenetic tree is judged through the branch structure of the phylogenetic tree. When different plant groups are identified, different types or numbers of primer pairs can be selected according to actual conditions for identification, and the more the number of the selected primer pairs is, the higher the success rate of species identification is.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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<221>misc_feature
<222>(16)
<223> y = c or t
<400>8
gatctcyttc catacytcac aagc 24
<210>9
<211>24
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(5)
<223> m = a or c
<220>
<221>misc_feature
<222>(7)
<223> y = c or t
<220>
<221>misc_feature
<222>(12)
<223> r = a or g
<220>
<221>misc_feature
<222>(16)
<223> r = a or g
<400>9
acttmtyttt crggartata ttta 24
<210>10
<211>22
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(11)
<223> k = g or t
<220>
<221>misc_feature
<222>(17)
<223> y = c or t
<220>
<221>misc_feature
<222>(19)
<223> r = a or g
<400>10
tttgaaccaa katttcyara tg 22
<210>11
<211>26
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(11)
<223> y = c or t
<220>
<221>misc_feature
<222>(16)
<223> r = a or g
<220>
<221>misc_feature
<222>(19)
<223> y = c or t
<400>11
aattatgtgt yagatrtayt aatacc 26
<210>12
<211>26
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(12)
<223> r = a or g
<220>
<221>misc_feature
<222>(14)
<223> r = a or g
<220>
<221>misc_feature
<222>(19)
<223> y = c or t
<220>
<221>misc_feature
<222>(24)
<223> r = a or g
<400>12
tccatagaaa trtrttcgyt caaraa 26
<210>13
<211>24
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(8)
<223> m = a or c
<220>
<221>misc_feature
<222>(14)
<223> y = c or t
<220>
<221>misc_feature
<222>(18)
<223> y = c or t
<400>13
tccgtaamca atcytctyat ttac 24
<210>14
<211>26
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(4)
<223> k = g or t
<220>
<221>misc_feature
<222>(14)
<223> k = g or t
<220>
<221>misc_feature
<222>(20)
<223> m = a or c
<220>
<221>misc_feature
<222>(24)
<223> y = c or t
<400>14
gcaktatcta ttakaaatgm attytc 26
<210>15
<211>22
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(6)
<223> y = c or t
<220>
<221>misc_feature
<222>(10)
<223> y = c or t
<220>
<221>misc_feature
<222>(15)
<223> y = c or t
<220>
<221>misc_feature
<222>(19)
<223> y = c or t
<400>15
ctcgayttty tgggytatyt tt 22
<210>16
<211>23
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(6)
<223> y = c or t
<220>
<221>misc_feature
<222>(14)
<223> r = a or g
<220>
<221>misc_feature
<222>(15)
<223> r = a or g
<400>16
tcttcytccg taarraattc ttc 23
<210>17
<211>22
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(17)
<223> y = c or t
<400>17
tatttgagat tttttgytta ta 22
<210>18
<211>24
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(4)
<223> r = a or g
<220>
<221>misc_feature
<222>(7)
<223> r = a or g
<220>
<221>misc_feature
<222>(13)
<223> r = a or g
<400>18
catrgtrgca gcrtgtataa gagc 24
<210>19
<211>25
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(14)
<223> y = c or t
<220>
<221>misc_feature
<222>(20)
<223> y = c or t
<400>19
atgtatggtt accygatgcy atgga 25
<210>20
<211>23
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(6)
<223> r = a or g
<220>
<221>misc_feature
<222>(15)
<223> r = a or g
<220>
<221>misc_feature
<222>(18)
<223> y = c or t
<400>20
gaatargcat gagtratyaa atg 23
<210>21
<211>25
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(6)
<223> m = a or c
<220>
<221>misc_feature
<222>(13)
<223> y = c or t
<220>
<221>misc_feature
<222>(23)
<223> r = a or g
<400>21
atgggmgaaa atygagttat ttrgg 25
<210>22
<211>26
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(11)
<223> y = c or t
<220>
<221>misc_feature
<222>(18)
<223> r = a or g
<220>
<221>misc_feature
<222>(21)
<223> y = c or t
<400>22
tcccatgcaa yagtcagraa ytcttg 26
<210>23
<211>25
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(7)
<223> r = a or g
<220>
<221>misc_feature
<222>(16)
<223> y = c or t
<220>
<221>misc_feature
<222>(22)
<223> r = a or g
<400>23
tagacargat atgccytatt trcaa 25
<210>24
<211>23
<212>DNA
<213>Artificial Sequence
<220>
<221>misc_feature
<222>(3)
<223> y = c or t
<220>
<221>misc_feature
<222>(18)
<223> k = g or t
<400>24
ccyctaaggg gttgttgkgt aac 23

Claims (10)

1. The specific primer pair is used for identifying angiosperm plant species and consists of a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4, a primer pair 5, a primer pair 6, a primer pair 7, a primer pair 8, a primer pair 9, a primer pair 10, a primer pair 11 and a primer pair 12;
the primer pair 1 consists of a primer 1-F and a primer 1-R; the primer pair 2 consists of a primer 2-F and a primer 2-R; the primer pair 3 consists of a primer 3-F and a primer 3-R; the primer pair 4 consists of a primer 4-F and a primer 4-R; the primer pair 5 consists of a primer 5-F and a primer 5-R; the primer pair 6 consists of a primer 6-F and a primer 6-R; the primer pair 7 consists of a primer 7-F and a primer 7-R; the primer pair 8 consists of a primer 8-F and a primer 8-R; the primer pair 9 consists of a primer 9-F and a primer 9-R; the primer pair 10 consists of a primer 10-F and a primer 10-R; the primer pair 11 consists of a primer 11-F and a primer 11-R; the primer pair 12 consists of a primer 12-F and a primer 12-R;
the primer 1-F is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;
the primers 1-R are single-stranded DNA molecules shown in a sequence 2 in a sequence table;
the primer 2-F is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;
the primers 2-R are single-stranded DNA molecules shown in a sequence 4 in a sequence table;
the primer 3-F is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;
the primer 3-R is a single-stranded DNA molecule shown as a sequence 6 in a sequence table;
the primer 4-F is a single-stranded DNA molecule shown as a sequence 7 in a sequence table;
the primer 4-R is a single-stranded DNA molecule shown as a sequence 8 in a sequence table;
the primer 5-F is a single-stranded DNA molecule shown as a sequence 9 in a sequence table;
the primer 5-R is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;
the primer 6-F is a single-stranded DNA molecule shown as a sequence 11 in a sequence table;
the primer 6-R is a single-stranded DNA molecule shown as a sequence 12 in a sequence table;
the primer 7-F is a single-stranded DNA molecule shown as a sequence 13 in a sequence table;
the primer 7-R is a single-stranded DNA molecule shown as a sequence 14 in a sequence table;
the primer 8-F is a single-stranded DNA molecule shown as a sequence 15 in a sequence table;
the primer 8-R is a single-stranded DNA molecule shown as a sequence 16 in a sequence table;
the primer 9-F is a single-stranded DNA molecule shown as a sequence 17 in a sequence table;
the primer 9-R is a single-stranded DNA molecule shown as a sequence 18 in a sequence table;
the primer 10-F is a single-stranded DNA molecule shown as a sequence 19 in a sequence table;
the primer 10-R is a single-stranded DNA molecule shown in a sequence 20 of a sequence table;
the primer 11-F is a single-stranded DNA molecule shown in a sequence 21 in a sequence table;
the primer 11-R is a single-stranded DNA molecule shown in a sequence 22 of a sequence table;
the primer 12-F is a single-stranded DNA molecule shown in a sequence 23 in a sequence table;
the primer 12-R is a single-stranded DNA molecule shown in a sequence 24 in a sequence table.
2. The specific primer pair of claim 1, wherein: the molar ratio of each primer in each primer pair was 1: 1.
3. Use of the specific primer pair of claim 1 or 2) in any one of the following m1) -m 6):
m1) preparing a product identified by an angiosperm plant species;
m2) preparing the product of assistant identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence tracing.
4. A kit comprising a specific primer pair according to claim 1 or 2.
5. A method for preparing the kit of claim 4, comprising the step of separately packaging each primer of the specific primer pair of claim 1 or 2.
6. The kit of claim 4 for use in any one of m1) -m6) as follows:
m1) preparing a product identified by the angiosperm species;
m2) preparing the product of assistant identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence tracing.
A DNA fragment, which is n1) -n4) as follows:
n1) of plants to be testedrbcLUsing the primer pair 1, the primer pair 2, the primer pair 3 and the primer pair 4 in the claim 1 to amplify the obtained DNA fragment;
n2) of plants to be testedmatKUsing the primer pair 5, the primer pair 6, the primer pair 7 and the primer pair 8 in claim 1 as a template to amplify the obtained DNA fragment;
n3) of plants to be testedndhFUsing the primer pair 9 and the primer pair 10 in claim 1 to amplify the obtained DNA fragment with the gene as a template;
n4) of plants to be testedrpoBThe DNA fragments amplified using the primer set 11 and the primer set 12 of claim 1 as templates.
8. The use of the DNA fragment of claim 7 in any one of the following m1) -m 6):
m1) preparing a product identified by an angiosperm plant species;
m2) preparing products for assisting identification of angiosperm plant species;
m3) preparing a physical evidence traceable product;
m4) identifying the angiosperm plant species;
m5) assisted identification of angiosperm plant species;
m6) material evidence tracing.
9. A method of identifying or aiding in the identification of an angiosperm species comprising the steps of: carrying out PCR amplification by using plant genome DNA to be detected as a template and adopting the specific primer pair of claim 1 to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a PCR amplification product sequence; and carrying out BLAST comparison on the PCR amplification product sequence in an NCBI database, and determining the species of the plant to be detected according to the comparison result.
10. The method of claim 9, wherein: the sequencing platform is a high-throughput sequencing platform.
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CN113621729B (en) * 2021-08-11 2024-05-28 河北师范大学 MatK primer and method suitable for identifying rhododendron species
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