CN113481317A - Nanmu producing area tracing high-polymorphism single-copy microsatellite locus combination and related primer combination - Google Patents
Nanmu producing area tracing high-polymorphism single-copy microsatellite locus combination and related primer combination Download PDFInfo
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Abstract
The invention discloses a nanmu production area tracing high-polymorphism single-copy microsatellite locus combination and a related primer combination. The related primer combination for tracing the provenance of nanmu disclosed by the invention consists of 40 single-stranded DNAs shown as sequences 1-40 in a sequence table. By utilizing the primer combination provided by the invention, the genetic diversity of the nanmu can be correctly evaluated, the source area of the nanmu species can be quickly and accurately identified, and the primer combination has important significance for protecting the gradually-endangered species. Meanwhile, by utilizing the primer combination provided by the invention, the genetic composition of the nanmu can be conveniently determined, a nanmu fingerprint database is established, the source of the nanmu species can be rapidly and accurately identified, and the false production place of the nanmu is prevented. The primer combination provided by the invention can be developed into a kit and made into a product, has a wide application market, and obtains good economic benefits.
Description
Technical Field
The invention relates to a nanmu production area tracing high polymorphism single copy microsatellite locus combination and a related primer combination in the field of biotechnology.
Background
The nanmu is a secondary protection gradually-critical species in China, is a famous traditional precious tree species in China, and has important social, cultural and economic values. Historically, a plurality of nanmu are arranged in the palace, the camphor tree, the catalpa ovata and the tung are called as four famous trees in the south of the Yangtze river, the nanmu is located at the head of the nanmu, the materials are excellent, the nanmu is often used as raw materials for appliances, boat coffins, wood inner and outer liners, house construction and the like, and the nanmu is taken as a "royal wood" in the Ming and Qing dynasties and is seen in important buildings such as palace urban buildings, temples and the like in the Ming and Qing dynasties, for example, a plurality of nanmu trees are arranged in the Guardian and the thirteen lings. There are many views of people's call on "nanmu": one group of viewpoints considers the nanmu to be a general term of nanmu species (Chinese main name of timber, 2004; plum forest and the like, 2004), and the other group of viewpoints considers the nanmu to be nanmu species and machilus species (poplar foal, 2010), but the nanmu is generally considered to be only specific to machilus within nanmu (Chinese plant, 2019). The silkwood element has the reputation of 'wood of emperor' and the theory of 'water can not be soaked and ants can not be inserted', is mild in property, warm in winter and cool in summer, fresh and pleasant in fragrance, stable in wood property, free of warping and cracking, durable (China forestry industry, 2017), transparent and moist in texture, has the fantasy effect of moving and changing scenes under the illumination (poplar foals, 2010), is one of famous and precious wood pursued by consumers and furniture manufacturers, and has great commercial value. However, the formal name of the silkwood is not found in relevant national standards and academic monographs, and more, the silkwood is a word of verbena. The scholars (mahogany, 2012) think that the popular "golden silk nanmu" is really the old frame material with the crystallization rate of more than 80%. The golden silk nanmu has specific connotation in the traditional Chinese culture, is highly matched with the personality pursuit of the pome people, embodies the cultural paradigm of superstructure of the Chinese enclosed society, and is an ultimate representative of Chinese elegant culture (Ma Zhi Yong, 2012). The furniture and the artware made of the golden silk nanmu have mild texture and are bright like gold, so that the furniture and the artware bring beautiful artistic enjoyment to people and have high collection value.
However, because of the historical remaining problems, the nanmu suffers from a large amount of felling, the resources are rare, the growth is slow, and at present, the nanmu is basically not a group of forestation. Due to the unique collection value, illegal nanmu selling behaviors are not eliminated, and besides, genuine products are difficult to distinguish, so that fake and fake products are provided in the market. In order to attack the behaviors of illegally selling national protection plants and protect the producing area of the nanmu products, accurate traceability is urgently needed, and the development of a nanmu producing area traceability technology is realized. Therefore, people must find molecular markers which effectively reflect the genetic background of the nanmu species and can accurately identify the producing area of the nanmu species. Among the numerous molecular markers, microsatellite markers (or referred to as SSRs) are the most effective molecular markers in addition to SNPs. The method has the outstanding characteristics of high resolution, no need of a large number of sites, and simplicity and easiness in use once the development is successful. However, the microsatellite loci used for the nanmu molecular marker at present are very limited, and a single-copy core microsatellite locus combination is urgently needed in order to effectively evaluate the genetic variation of the nanmu and accurately identify the species source.
Disclosure of Invention
The invention aims to solve the technical problem of how to detect the genetic variation of nanmu and accurately detect the source area of the nanmu.
In order to solve the technical problems, the invention firstly provides a primer combination which is named as P01-F, P01-R, P02-F, P02-R, P03-F, P03-R, P04-F, P04-R, P05-F, P05-R, P06-F, P06-R, P07-F, P07-R, P08-F, P08-R, P09-F, P09-R, P10-F, P10-R, P11-F, P11-R, P12-F, P12-R, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13-F, P13, P19-R, P20-F and P20-R,
said P01-F, said P02-F, said P03-F, said P04-F, said P05-F, said P06-F, said P07-F, said P08-F, said P09-F, said P10-F, said P11-F, said P12-F, said P13-F, said P14-F, said P15-F, said P16-F, said P17-F, said P18-F, said P19-F, and said P20-F have sequences of sequences 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, respectively, in sequence; the sequences of the P01-R, the P02-R, the P03-R, the P04-R, the P05-R, the P06-R, the P07-R, the P08-R, the P09-R, the P10-R, the P11-R, the P12-R, the P13-R, the P14-R, the P15-R, the P16-R, the P17-R, the P18-R, the P19-R and the P20-R are the sequences 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 in a sequence table respectively.
In the above primer combination, the sequences of the P01-F, the P02-F, the P03-F, the P04-F, the P05-F, the P06-F, the P07-F, the P08-F, the P09-F, the P10-F, the P11-F, the P12-F, the P13-F, the P14-F, the P15-F, the P16-F, the P17-F, the P18-F, the P19-F, and the P20-F may be sequences 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 in the sequence, respectively.
Each single-stranded DNA in the primer combination can be independently packaged.
The primer combination can be applied to any one of the following applications:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
Any of the following applications of the primer combination also belong to the protection scope of the invention:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
The invention also provides a kit, which comprises the primer combination.
The kit may further comprise a fluorescent dye, which may be FAM, VIC, NED, or ROX, such as FAM, VIC, NED, ROX from Invitrogen, and/or other reagents required for PCR amplification.
The other reagents required for the PCR amplification may be DNA polymerase (e.g., DNA polymerase of Takara), Buffer (e.g., 10 XBuffer (Mg) of Takara)2+) And/or dNTPs (e.g., dNTPs from Takara).
The kit can be used for any one of the following applications:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
Any one of the following applications of the above kit also belongs to the protection scope of the present invention:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
The kit may consist of the primer combination alone, the primer combination and the fluorescent dye, the primer combination and the other reagents required for PCR amplification, and the primer combination, the fluorescent dye and the other reagents required for PCR amplification.
The invention also provides a method for detecting or assisting in detecting the source of nanmu, which comprises the following steps: performing PCR amplification on genome DNA of a nanmu to be detected by using the primer combination, and comparing the PCR product of the nanmu to be detected with the PCR product of the nanmu in a known source area to determine the genotypes of 20 microsatellite loci, wherein if at most three genotypes of the 20 microsatellite loci of the nanmu to be detected are inconsistent with the genotypes of the nanmu in the known source area, the nanmu to be detected is the same as or candidate for the nanmu source area in the known source area; if the genotypes of at least four of the 20 microsatellite loci of the nanmu to be detected are inconsistent with the genotype of the nanmu from the known source, the nanmu to be detected is different from the nanmu from the known source or is different from candidates.
The genotype may be typed with ABI3730 xl.
The invention also provides a preparation method of the primer combination, and the method comprises the step of independently packaging each single-stranded DNA.
In the invention, the source of the nanmu can be Chongqing Yunyang county cuprum Zhengshi material factory, Guizhou province south county green rod slope Zhengmu Wancun, Hubei Enshi city banana county kaokoucun or Si chuan Shujing temple.
By utilizing the primer combination provided by the invention, the genetic diversity of the nanmu can be correctly evaluated, the source area of the nanmu species can be quickly and accurately identified, and the primer combination has important significance for protecting the gradually-endangered species. Meanwhile, by utilizing the primer combination provided by the invention, the genetic composition of the nanmu can be conveniently determined, a nanmu fingerprint database is established, the source of the nanmu species can be rapidly and accurately identified, and the false production place of the nanmu is prevented. The primer combination provided by the invention can be developed into a kit and made into a product, has a wide application market, and obtains good economic benefits.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
Example 1 preparation of primer combinations
The present example provides a set of primer combinations that can be used for nanmu genetic heterogenous evaluation and origin tracing detection, the sequences of the primers are shown in table 1, the primer labeled "F" is a forward primer, the primer labeled "R" is a reverse primer, and the 5' end of each forward primer is loaded with an M13 sequence, i.e., positions 1-19 of each forward primer. In the primer combination, each primer is independently packaged, and the mole number of each primer is equal.
TABLE 1 Machilus wood 20 core microsatellite locus primer sequences
Example 2 detection of Phoebe Wood Source
1. A nanmu material:
124 parts of nanmu materials are selected from 4 communities of Guizhou, Chongqing, Hubei and Sichuan, and the materials are shown in Table 2.
TABLE 2, 124 parts of nanmu material information
2. Detection of source area of nanmu
Genomic DNAs of 124 parts of nanmu materials were extracted, and PCR amplification was performed using the primer combinations of example 1 using the genomic DNAs of each nanmu material as templates.
Preparing PCR amplification reaction systems, wherein each system comprises a pair of primers, a genome DNA and a fluorescent dye, each pair of primers selects a fluorescent dye, and each genome DNA is amplified by adopting 20 primer pairs respectively.
The PCR amplification reaction systems containing FAM, VIC and NED are as follows: 10 × Buffer (Mg)2+)1 μ L, 2mM dNTP 1 μ L, 5U/. mu.L Taq DNA polymerase 0.1 μ L, 5mM forward primer 0.4 μ L, 5mM reverse primer 0.4 μ L, FAM fluorescent dye, VIC fluorescent dye or NED fluorescent dye 0.4 μ L, 10 ng/. mu.L genomic DNA 2 μ L, ddH2O 4.7μL。
The PCR amplification reaction system containing ROX is as follows: 10 × Buffer (Mg)2+)1μL、2mM dNTP 1. mu.L, 5U/. mu.L Taq DNA polymerase 0.1. mu.L, 5mM forward primer 0.4. mu.L, 5mM reverse primer 0.8. mu. L, ROX fluorescent dye 0.6. mu.L, 10 ng/. mu.L genome DNA 2. mu. L, ddH2O 4.1μL。
Wherein 10 times Buffer (Mg)2+) dNTP, Taq DNA polymerase are all Takara reagent, FAM fluorescent dye, VIC fluorescent dye, NED fluorescent dye and ROX fluorescent dye are all Invitrogen products.
Each reaction system was subjected to PCR amplification in a PCR amplification apparatus (Master S apparatus of Eppendorf corporation), and the PCR amplification reaction conditions were: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, 31 cycles; finally, extension is carried out for 5min at 72 ℃, and the sample is stored in the dark at 4 ℃.
After the PCR amplification is finished, all PCR products are sequenced, and every four different fluorescence-labeled primer amplification products can be subjected to mixed sample sequencing.
After PCR amplification, the PCR products were electrophoresed and genotyped at ABI3730xl, and the data generated from ABI3730xl instrument was subjected to allelic discrimination using GeneMarker to form a sample-genotype data matrix (see tables 3-4).
TABLE 3 sample-genotype data matrix
TABLE 4 sample-genotype data matrix
The size of the numbers in Table 3 represent the length of the allelic fragment, and different combinations of numbers represent different genotypes, wherein the same number indicates that the sample is homozygous and the different number indicates that the sample is heterozygous.
As can be seen from table 3, the materials of the same population have substantially the same genotype at the same location in the longitudinal direction, the materials of different populations have the same or different genotypes, and the materials of the same population have different genotypes at different locations in the transverse direction, so that the different populations have different combinations of genotypes at different locations, and therefore, the 20 microsatellite locations in the present invention are sufficient to distinguish the materials from the 4 different populations, thereby achieving the purpose of tracing the origin of the origin.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of plant of Chinese academy of sciences
<120> Nanmu producing area tracing high polymorphism single copy microsatellite locus combination and primer combination
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atcccccatt cacgattcca aa 22
<210> 23
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
cacgacgttg taaaacgacc tcacaaacgc gcataagcac 40
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
tggagtactg gctgcatact tg 22
<210> 25
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
cacgacgttg taaaacgacg cgtctagtct ttgagattcg c 41
<210> 26
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
ccagcccatt gacaaccaaa at 22
<210> 27
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
cacgacgttg taaaacgaca cagaaatcgc caggacattc t 41
<210> 28
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
cattggttgg ggaaagacca tc 22
<210> 29
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
cacgacgttg taaaacgaca acctgcaact ccaagagaga g 41
<210> 30
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
attgggtgac gatattgcca ct 22
<210> 31
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
cacgacgttg taaaacgact tcctctgcaa gtctgagttc c 41
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
gcagatctcc aaaaggtagc ct 22
<210> 33
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<220>
<221> misc_feature
<222> (37)..(37)
<223> n is a, c, g, or t
<400> 33
cacgacgttg taaaacgacg tggttgcagg ggaaacncca g 41
<210> 34
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
ctcccagtga tgtaggttgt gt 22
<210> 35
<211> 43
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
cacgacgttg taaaacgact ccacaaagca aaagaaagtg tca 43
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
cttcaaaatt tgaggccctg ca 22
<210> 37
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
cacgacgttg taaaacgaca gaagtcaagg cacaatctgc t 41
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
accatgttgc ctttgctgaa aa 22
<210> 39
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
cacgacgttg taaaacgacc gcgtggatgc tctacttctt a 41
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
agagagggga aaagctagct aga 23
Claims (9)
1. The primer combination consists of single-stranded DNA with the names of P01-F, P01-R, P02-F, P02-R, P03-F, P03-R, P04-F, P04-R, P05-F, P05-R, P06-F, P06-R, P07-F, P07-R, P08-F, P08-R, P09-F, P09-R, P10-F, P10-R, P11-F, P11-R, P12-F, P12-R, P13-F, P13-R, P14-F, P14-R, P15-F, P15-R, P16-F, P16-R, P17-F, P17-R, P18-F, P18-R, P19-F, P19-R, P20-F and P20-R respectively,
said P01-F, said P02-F, said P03-F, said P04-F, said P05-F, said P06-F, said P07-F, said P08-F, said P09-F, said P10-F, said P11-F, said P12-F, said P13-F, said P14-F, said P15-F, said P16-F, said P17-F, said P18-F, said P19-F, and said P20-F have sequences of sequences 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, respectively, in sequence; the sequences of the P01-R, the P02-R, the P03-R, the P04-R, the P05-R, the P06-R, the P07-R, the P08-R, the P09-R, the P10-R, the P11-R, the P12-R, the P13-R, the P14-R, the P15-R, the P16-R, the P17-R, the P18-R, the P19-R and the P20-R are the sequences 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 in a sequence table respectively.
2. The primer combination of claim 1, wherein: the primer combination has any one of the following applications:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
3. Use of a combination of primers according to claim 1 or 2, wherein:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
4. A kit comprising the primer combination of claim 1.
5. The kit of claim 4, wherein: the kit further comprises a fluorescent dye and/or other reagents required for PCR amplification, wherein the fluorescent dye is FAM, VIC, NED or ROX; the other reagents required for PCR amplification are DNA polymerase, buffer and/or dNTP required for PCR amplification.
6. The kit according to claim 4 or 5, characterized in that: the kit has any one of the following applications:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
7. The kit of claim 4 or 5 for any one of the following uses:
x1, detecting or assisting in detecting the source of nanmu;
x2, detecting or assisting in detecting genetic variation of nanmu;
x3, detecting or assisting in detecting genetic diversity of nanmu;
x4, constructing a molecular fingerprint of the nanmu;
x5, preparing a source land product for detecting or assisting in detecting nanmu;
x6, preparing a detection or auxiliary detection nanmu genetic variation product;
x6, preparing a detection or auxiliary detection nanmu genetic diversity product;
x8, preparing and constructing or assisting to detect a nanmu molecular fingerprint product;
x9, and evaluating genetic heterogeneities of nanmu.
8. The method for detecting or assisting in detecting the source of the nanmu comprises the following steps: performing PCR amplification on genomic DNA of a nanmu to be detected by using the primer combination of claim 1, and comparing the PCR product of the nanmu to be detected with the PCR product of the nanmu from a known source to determine the genotypes of 20 microsatellite loci, wherein if the genotypes of at most three of the 20 microsatellite loci of the nanmu to be detected are inconsistent with the genotype of the nanmu from the known source, the nanmu to be detected is the same as or candidate for the nanmu from the known source; if the genotypes of at least four of the 20 microsatellite loci of the nanmu to be detected are inconsistent with the genotype of the nanmu from the known source, the nanmu to be detected is different from the nanmu from the known source or is different from candidates.
9. The method for preparing a primer set according to claim 1, comprising packaging each single-stranded DNA separately.
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| CN114921461A (en) * | 2022-06-06 | 2022-08-19 | 四川农业大学 | SSR molecular marker primer combination for germplasm identification of Phoebe fraseri and application |
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| CN114921461A (en) * | 2022-06-06 | 2022-08-19 | 四川农业大学 | SSR molecular marker primer combination for germplasm identification of Phoebe fraseri and application |
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Application publication date: 20211008 |