KR20120100095A - Microsatellite primer set for identifying onion varieties - Google Patents

Abstract

PURPOSE: A microsatellite primer set for distinguishing onion species is provided to distinguish 79 species of onions distributing in the nation, thereby being usefully used for genuineness of onion species verification, control variety selection of variety protection and arbitration of seed dispute between companies, and purity test of F1. CONSTITUTION: A microsatellite primer set for distinguishing onion species comprises ACE039 primer set consisting of the base sequence described in sequence number 1(SEQ ID NO:1) and 2, ACM024 primer set consisting of the base sequence described in the sequence number 3 and 4, AFS111 primer set consisting of the base sequence described in the sequence number 5 and 6, ACE020 primer set consisting of the base sequence described in the sequence number 7 and 8, AFA08G10 primer set consisting of the base sequence described in the sequence number 9 and 10, ACM071 primer set consisting of the base sequence described in the sequence number 11 and 12, ACM154 primer set consisting of the base sequence described in the sequence number 13 and 14, AFAT05D09 primer set consisting of the base sequence described in the sequence number 15 and 16, AFA23C12 primer set consisting of the base sequence described in the sequence number 17 and 18, AFS015 primer set consisting of the base sequence described in the sequence number 19 and 20, AFAT09E05 primer set consisting of the base sequence described in the sequence number 21 and 22, or AFA11C12 primer set consisting of the base sequence described in the sequence number 23 and 24.

Description

Microsatellite primer set for identifying onion varieties}

The present invention relates to a supersatellite primer set for identifying onion varieties. More particularly, the present invention relates to a supersatellite marker showing polymorphism for 79 varieties of onions distributed in Korea, and a method for identifying onion varieties using the same.

Molecular markers include mapping of genes associated with crop resistance and functionality, development of trait markers, identification of chromosomal positions of quantitative genes associated with traits (QTL, Quantitative Trait Locus), and F1 purity assay using molecular markers. It is used in various molecular breeding fields such as Molecular labeling is also used in plant variety protection. The UPOV is discussing the use of DNA markers for characterization of new varieties and granting new plant protection rights, but it does not yet recognize simple sequence differences at the genetic level as distinctive varieties. It proposes the possibility of using molecular labeling for Specifically, the use of molecular labeling to protect breeder's rights does not recognize the results from AFLP (Amplified Fragment Length Polymorphism) or RAPD (Random Amplification of Polymorphic DNA) analysis techniques. Considering the reproducibility of the analysis and the degree of distribution on the chromosome, it is proposed to use SSR (Simple Sequence Repeat) or Microsatellite, SNP (Single Nucleotide Polymorphism) and CAPS (Cleaved Amplified Polymorphic Sequence).

In general, methods for identifying plant varieties are divided into morphological characteristics through cultivation tests, assays through isoenzyme differences between varieties, and DNA analysis methods. Among them, the DNA analysis method has an advantage of being objective and reproducible because it is not influenced by the cultivation environment and the growth stage of the crop compared to the phenotype-based identification method.

Indeed, if we look at the status of developing and utilizing DNA analysis for domestic and foreign varieties identification, Korea Rural Development Administration reported SSR molecular labeling for rice, soybean, and CAPS molecular labeling for strawberry. In addition, the National Species Resource uses SSR molecular labels to database the DNA profiles of crops for peppers, cabbages, watermelons, tomatoes, peaches, etc., and directly uses the molecular labels of some crops as a means to mediate seed disputes. Recently, genetic analysis results have been used as ancillary data for selection of control varieties for crops applied for varieties protection. On the other hand, the markers for the purity test among the SSR molecular markers for cultivation of varieties have been used for the test of the F1 purity for the fruit and crops to support breeders.

In the case of foreign countries, Spain uses SSR analysis technology to produce a database of DNA profiles for each variety of crops, such as grapes, and use them for identification of basic varieties and management of existing varieties. In China, about 4000 varieties of maize are produced by DNA database using SSR and SNP molecular labels, and the courts are actively using genetic analysis results to determine cases of infringement of varieties protection. Japan has developed a variety identification method using SSR molecular labels on peaches, pears and apples, and CAPS molecular labels on strawberries and teas to protect breeders' rights.

On the other hand, lilies crop onions are used directly as raw, and as the preference for health food has recently increased, the utilization of processed foods such as onion juice is increasing. 941 varieties (as of December 31, 2010) of domestically grown varieties of onions reported to the National Species Resource were reported in the seed market. Onion grows relatively long, so it takes time to select the target trait when breeding, and the importance of breed identification by molecular labeling and DNA analysis method is increasing, and the establishment of technology is urgently needed.

Molecular labeling research and development trends for onions Onion is known to have the largest genome size (16,415 Mbp of DNA per 1C nuclus) among the plants grown. This is a 28% larger genome size than Bunching onions of the genus Lilium Allium . Molecular genetic studies on Allium crops are based on 'wave', which is easier to study than 'onion'. Currently, research is being actively conducted in Japan, and the Japanese vegetable crop DNA marker database (http://vegmarks.nivot.affrc.go.jp) discloses markers such as SSR and AFLP of green onions. In addition, in the United States, the onion EST-SSR molecular label was developed using EST (Expressed Sequence Tag) to conduct comparative dielectric studies (Kuhl JC et al. 2004. The Plant Cell. 16: 114-125).

However, the selection of relevant marker combinations and related detailed techniques that can identify onion varieties using supersatellite marker sets for domestic varieties have not been established in Korea. Although onions are important vegetable crops, molecular biology research and molecular genetic data are insufficient compared to crops such as eggplants (pepper and tomato) and gourds (meat and cucumber, etc.) and due to their large genome size, It is understood that marker selection indicating polymorphism is very difficult.

On the other hand, SSR markers have the advantage that the transferability (transferability) of the marker is recognized even if the genetic link between the species.

Therefore, the present inventors can save time and cost significantly compared to the method of developing the SSR markers by making a genomic library directly from the onion genome, and the onion variety identification method that can overcome the difficulty of identifying the varieties with only the published onion markers. While continuing research on onions, it is possible to apply the SSR molecular label of onion, an Allium crop, which has not yet been used for onion varieties, with onion markers for practical use in scientific verification of the authenticity of onion varieties. The present invention has been completed by revealing that there is.

An object of the present invention for the first time in Korea to provide a superficial marker for polymorphism for 79 onion varieties distribution and to provide a method for identifying onion varieties using the same.

Another object of the present invention to provide a kit for identifying onion varieties comprising the primer set.

Still another object of the present invention is to provide a method of identifying the authenticity of onion varieties, selecting a control variety of onion varieties protected application varieties or identifying a variety of onions for F1 purity test using the primer set.

The present invention provides a primer set for identifying onion varieties showing polymorphism with respect to 79 onion varieties distributed in Korea.

The present invention provides a kit for identifying onion varieties comprising the primer set.

The present invention provides a method for identifying the authenticity of onion varieties, selecting a control variety of onion varieties protection application varieties or identifying a variety of onions for F1 purity test using the primer set.

Hereinafter, the present invention will be described in detail.

The present invention

ACE039 primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2;

ACM024 primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and 4;

An AFS111 primer set consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6;

ACE020 primer set consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8;

AFA08G10 primer set consisting of the nucleotide sequence of SEQ ID NO: 9 and 10;

ACM071 primer set consisting of the nucleotide sequence of SEQ ID NO: 11 and 12;

ACM154 primer set consisting of the nucleotide sequence of SEQ ID NO: 13 and 14;

AFAT05D09 primer set consisting of the nucleotide sequences of SEQ ID NOs: 15 and 16;

AFA23C12 primer set consisting of the nucleotide sequences of SEQ ID NOs: 17 and 18;

An AFS015 primer set consisting of the nucleotide sequences of SEQ ID NOs: 19 and 20;

AFAT09E05 primer set consisting of the nucleotide sequences of SEQ ID NOs: 21 and 22;

And

It provides a primer set for identifying onion varieties, comprising one or more primer sets selected from the group consisting of; AFA11C12 primer set consisting of the nucleotide sequences of SEQ ID NOs: 23 and 24.

The primer set for identifying onion varieties according to the present invention is characterized by exhibiting high polymorphism for onion varieties distributed in Korea by applying SSR molecular labels of 'pa' to onions with markers of onions.

In addition, the primer set for identifying onion varieties of the present invention significantly increases time and cost compared to a method of developing SSR markers by directly preparing a genomic library in the onion genome by simultaneously applying the 'Spar' SSR molecular label and onion marker. It is possible to save, and it is possible to overcome the difficult point identification of varieties with only the onion markers disclosed.

More specifically, in the selection of primer combinations suitable for identifying onion varieties, the genomic DNA of 79 onion varieties distributed in Korea is amplified by using the SSR molecular labels of 'wave' and 'onion' as primer sets. By confirming the amplified products, when the PIC (Polymorphic Information Content) values of the alleles were examined, it was confirmed that the selected primer set for identifying onion varieties was suitable for the onion variety identification method.

In the present invention, “primer” refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

In the present invention, the primer may further include a material that emits DNA intercalating fluorescence, phosphorescence or radioactivity, but is not limited thereto. Preferably, the labeling substance is VIC, NED, FAM or PET. When amplifying the target sequence, PCR is performed by labeling the VIC, NED, FAM or PET at the 5'-end of the primer, so that the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. The oligonucleotide primer set used to amplify the target sequence is as described above.

The present invention is a primer set for identifying the onion variety (SEQ ID NOS: 1 to 24); And

Reagents required for polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR);

It includes, provides a kit for identifying onion varieties.

In the kit of the present invention, the reagents required for the PCR include, but are not limited to, forward primers and reverse primers, an appropriate amount of DNA polymerase, dNTP mixture, KCl, Tris-HCl and MgCl ₂ . One PCR buffer and water may be included.

In addition, the reagents required for the RT-PCR may be, for example, but not limited to, forward primers and reverse primers, reverse transcriptases, ribonuclease inhibitors, heat resistant polymerases, reaction buffers, and the like, and the reaction buffers include reverse transcription buffers and Both PCR buffers are included, and the heat resistant polymerase may be EF Taq polymerase and the like, and the reverse transcriptase may be a reverse transcriptase from various sources, for example, avian myeloblastosis virus. -derived virus reverse transcriptase (AMV RTase), mouse leukemia virus-derived virus reverse transcriptase (MuLV RTase), and Rous-associated virus 2 reverse transcriptase (RAV) -2 RTase).

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The present invention

a) separating genomic DNA from onions;

b) using the isolated genomic DNA as a template, and performing a polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR) using the primer set for identifying onion varieties (SEQ ID NOS: 1 to 24). step; And

c) confirming whether said genomic DNA fragment has been amplified;

It provides a method of identifying a variety of onions, including.

In the present invention, the genomic DNA can be isolated from onion seeds, leaves, stems or mixtures thereof. More specifically, genomic DNA may be separated from leaves or stems of young plants after sowing and seeding onion seeds, but in the present invention, genomic DNA is isolated from onion seeds. Separation of genomic DNA directly from seeds eliminates the need to grow plants, which reduces the time and cost of analysis.

As a method for separating genomic DNA from onion samples of the present invention, a method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated genomic DNA as a template, a target sequence may be amplified by PCR using a primer set according to an embodiment of the present invention as a primer.

Primer for identifying onion varieties of the present invention SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22 and SEQ ID NOs: 23 and 24.

Such PCR methods are well known in the art, and commercially available kits may be used.

The method includes identifying the amplification product. Detection of the amplification product may be performed through gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed.

Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling VIC, NED, FAM, or PET at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent label, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.

In addition, in the present invention, identifying the onion varieties from the amplified product can be identified by the computer program after confirming the size of the allele (band) by using an automatic sequencing device, each of the varieties can be identified. Specifically, each of the onion varieties can be identified by the presence or absence of the amplification products by the primer set for identifying the onion varieties of the present invention in the table, and the onion varieties can be identified through a comparative analysis with the amplified products. 1 is a table showing the presence or absence of amplification products by the primer set for identification of each variety for each onion variety. Specifically, FIG. 1 codes the presence or absence of a band appearing when amplifying each of 79 onion varieties using a primer set for identifying 12 onion varieties of the present invention. If is not present, it is indicated as “0”.

Therefore, after separating genomic DNA from onions to identify the varieties, onion varieties can be identified by amplifying using the marker of the present invention and comparing the result of the amplified product with the result of FIG.

Onion varieties primer set according to the present invention can identify 79 varieties of onions circulating in the country, to identify the authenticity of onion varieties, farmers and companies, and mediation of seed disputes between the company and the company and varieties protection application varieties It may be useful as a supersatellite marker for identifying onion varieties in various fields such as selection of control varieties and F1 purity test.

1 is a code of the presence or absence of the band appearing when amplified using the primer set for identifying onion varieties according to the present invention for 79 varieties of onions, represented by "1" when the band is present and the band does not exist Is represented by “0”
Figure 2 codes each onion varieties analyzed by the onion varieties identification primer set according to the present invention, using the NTSYS pc computer program shows the onion varieties identification and genetic similarity by variety.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

EXAMPLES Onion Variety Identification Method

(1) onion sample

In the present invention, 79 onion varieties described in Table 1 were used for the selection of onion varieties primer identification set and onion varieties identification possibility assay.

(2) onion genome DNA Separation of

Seed 15-20 grains of the seed onion varieties of Table 1 were put in two tungsten beads in a 2 ml Eppendorf tube and turned into a grinder (FRITSCH, Germany) to crush the seeds evenly. Genomic DNA was then isolated using a NucleoSpinPlantII (Macherey-Nagel Cat. 740 770.250) kit in a 2 ml tube. PL2 was used as a cell lysis buffer. The extracted genomic DNA was electrophoresed on a 2% agarose gel to confirm the DNA concentration, and then quantified and stored in a freezer (-20 ° C) for PCR analysis.

(3) primer  Making of sets

Candidate material for the primer set of the present invention is onion ( Allium) cepa L.) as well as one of the crop Allium genus (Allium) onion (Allium fistulosum L.) was also used.

A total of 219 primers (204 leeks, 15 onions) primer sets were used among the SSR markers of leeks and onions published in the Japanese vegetable crop DNA marker database (http://vegmarks.nivot.affrc.go.jp). Red Prime, Powerball, Roman, Red Sun, Hanter 505, Pop, Hanter 410, Marusino 310, Marusino 330, New Mars, Changchun 2, Cho Saeng Sonic varieties Was isolated and gene amplified using a set of 219 primers. Then, 15 markers with high reproducibility between repeats, clear band patterns, and high polymorphism were selected.

In order to facilitate the detailed analysis by automatic sequencing of the molecular markers of the 15 markers selected, oligonucleotides were prepared in which the forward primer was labeled with a fluorescent factor of one of the fluorescence generating materials VIC, NED, FAM and PET. . Using the 15 markers, the final 12 markers with high cultivation ability were selected when extended to 79 varieties of onions distributed in Korea. The base sequence of the primer set showing polymorphism in the disclosure onion varieties is shown in Table 2 below.

(4) PCR  Amplification and Electrophoresis

PCR (Polymerase chain reaction) was performed using the prepared genomic DNA and the prepared markers as materials. The composition of the PCR reaction solution was prepared by 20 ng of the prepared onion genomic DNA, a marker consisting of the primer set of 10 pmol, 2.0 μl dNTP mixture (2.5 mM), Taq DNA polymerase 1U (GeNet bio, Korea), 2.5 μl 10 × Distilled water was added to PCR Buffer (50 mM KCl, 20 mM Tris-HCl, pH 8.0, 2.0 mM MgCl ₂ ) to adjust the total volume to 25 μl.

PCR (Biometra, Germany) amplification was sufficient to denature onion genomic DNA for 4 minutes at 94 ° C, then 30 seconds of denaturation at 94 ° C, 30 seconds of annealing at 55 ° C, and extension at 72 ° C. After 30 repetitions of 30 seconds, 5 minutes of elongation was performed at 72 ° C. for final elongation of the PCR product. After the PCR was completed, the amplification was confirmed by electrophoresis on 2% agarose gel.

The amplified samples were stored at -20 ° C for the next experiment. Samples amplified by PCR were diluted to 150 µl of ultrapure water at an appropriate concentration, and then 10 µl of deionized formamide and allelic size of the diluted 3 to 5 µl of the PCR product (adjusted according to the amplification amount) were measured. 0.25 μl of a size marker (LIZ500 size standard) was mixed well and denatured at 94 ° C. for 2 minutes.

The denatured gene amplification product was electrophoresed using an automatic sequencing device (Genetic Analyzer 3130XL, Applied Biosystem), and then the exact size of alleles per SSR molecule label was determined using a GeneMapper computer program (Applied Biosystem).

(5) Onion variety identification possibility test of the marker according to the present invention

Using the allele size identified by the automatic sequencing device, the possibility of identifying the onion variety of the marker according to the present invention was investigated. Polymorphism information content (PIC) values were calculated using the following equation (1).

In Equation 1 below, K is the total number of bands of each allele and P _i is the frequency of the i th common band pattern among the bands of the marker (Anderson et al. 1993).

As a result, the size (bp) of the amplification products, the number of alleles and the PIC values according to each marker are shown in Table 3 below.

As can be seen in Table 3, the ACE039 marker of the present invention showed the lowest PIC value of 0.448, the ACE020 marker showed the highest PIC value of 0.745. The average PIC value of the marker was found to be slightly higher, 0.575.

This is also a result of confirming that the marker of the present invention can sufficiently identify onion varieties.

In addition, the presence or absence of an allele (band) size for the marker according to the present invention was coded with a score of "1" in the presence of a band and "0" in the absence of a band, the result is shown in FIG. 1 is shown. 1 is isolated from the genomic DNA from the onion to be identified varieties, amplified using the primer set of the present invention can be used as data to identify onion varieties by comparing the results of the amplified product with the amplification of Figure 1 have.

According to the presence or absence of the band, it was input into the NTSYS pc (version 2.10b) (Rohlf, 2000) computer program and the genetic similarity value was calculated according to Jaccard's method (Sneath and Sokal, 1973). Genetic similarity was used to agglomerate a dendogram by aggregating by UPGMA (Unweighted Pair-group Method with Arithmetical Average) (Sneath and Sokal, 1973) and the results are shown in FIG. 2.

As can be seen in Figure 2 'Red Prime' and 'Takada', 'Marushino 310' and 'Yamamujin', 'New Mars' and 'Musso', 'Tabonghuang' and 'DB-301', 'Perfect' and 'Chunju Huang', 'Kokuwase 320' and 'Giant', 'Rapid Island 300' and 'Bora Chozoang', 'My Ball' and 'S & P 1', 'Super High Ball' and 'Quangju Used', 'Future' 'Hwang' and 'Make', 'Pop' and 'King of Kings', 'Pharaoh' and 'Super Bowl Huang', 'Earth Yellow' and 'Tsurimaru', 'Kas' and 'Daifeng' It was confirmed that, from the above results it can be seen that the primer set of the present invention enables the discrimination of all varieties.

<110> The republic of of Korea (Korea Seed & Variety Service) <120> Microsatellite primer set for identifying onion varieties <160> 24 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ACE039 forward primer <400> 1 atggtcgtcc atttcttatt gaagt 25 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> 223 ACE039 reverse primer <400> 2 ttgatgtatg aggcagcatt ctactg 26 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ACM024 forward primer <400> 3 ccccattttc ttcattttct ca 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACM024 reverse primer <400> 4 tgctgttgct gttgttgttg 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFS111 forward primer <400> 5 tgtttaatgg actttcaatg cctgt 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFS111 reverse primer <400> 6 gcattaaaat gaagaaatcc cgaag 25 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ACE020 forward primer <400> 7 agtggtcatg gttgtcttgc tt 22 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> 223 ACE020 reverse primer <400> 8 tgcacaagta cacagcgaca aac 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AFA08G10 forward primer <400> 9 tgagcatgcc agaaaatcca ctaa 24 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> AFA08G10 reverse primer <400> 10 cgagaatgag gatatgagat tcgagtg 27 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ACM071 forward primer <400> 11 tctcatttca actttctacc tatcc 25 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACM071 reverse primer <400> 12 ctgacatttg ctcgactgga 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACM154 forward primer <400> 13 cttgttttgg cagttgggat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACM154 reverse primer <400> 14 cgatgaatac accgatgacg 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AFAT05D09 forward primer <400> 15 ccggataaat ttacctgcaa atcc 24 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFAT05D09 reverse primer <400> 16 cagccattaa agaacctgat gtaag 25 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AFA23C12 forward primer <400> 17 aaactgatca aaatgtgccc cact 24 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFA23C12 reverse primer <400> 18 gagaatccga ttaaaaacga gcaag 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFS015 forward primer <400> 19 atctcactgt ccttgtacct gaaag 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> AFS015 reverse primer <400> 20 catcttgact ttgtgatatt tgtgc 25 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> AFAT09E05 forward primer <400> 21 aactgcgagt aatttctcaa caactgc 27 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> AFAT09E05 reverse primer <400> 22 ggaggaaaat ctctactccc agcatc 26 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AFA00C12 forward primer <400> 23 gggttcgagt ctccaagaca tca 23 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AFA11C12 reverse primer <400> 24 caaggggact tgatcttgcc ctat 24

Claims (6)

ACE039 primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2;
ACM024 primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and 4;
An AFS111 primer set consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6;
ACE020 primer set consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8;
AFA08G10 primer set consisting of the nucleotide sequence of SEQ ID NO: 9 and 10;
ACM071 primer set consisting of the nucleotide sequence of SEQ ID NO: 11 and 12;
ACM154 primer set consisting of the nucleotide sequence of SEQ ID NO: 13 and 14;
AFAT05D09 primer set consisting of the nucleotide sequences of SEQ ID NOs: 15 and 16;
AFA23C12 primer set consisting of the nucleotide sequences of SEQ ID NOs: 17 and 18;
An AFS015 primer set consisting of the nucleotide sequences of SEQ ID NOs: 19 and 20;
AFAT09E05 primer set consisting of the nucleotide sequences of SEQ ID NOs: 21 and 22;
And
AFA11C12 primer set consisting of the nucleotide sequences of SEQ ID NOs: 23 and 24; comprising one or more primer sets selected from the group consisting of, primer set for identifying onion varieties. The method of claim 1,
Said primer is a primer set for identifying onion varieties, characterized in that it further comprises a DNA intercalating fluorescent, phosphorescent or radioactive material. A primer set according to claim 1 or 2; And
Reagents required for polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR);
Including, onion varieties identification kit. a) separating genomic DNA from onions;
b) Using the isolated genomic DNA as a template, PCR (Polymerase chain reaction) or RT-PCR (Reverse-transcriptase polymerase chain reaction) using the primer set for identifying onion varieties according to claim 1 or 2 Performing; And
c) confirming whether said genomic DNA fragment has been amplified;
A method of identifying varieties of onions, comprising. The method of claim 4, wherein
Wherein said genomic DNA is isolated from onion seeds, leaves, stems, or mixtures thereof. The method of claim 4, wherein
The method of identifying a variety of onions is a method of identifying a variety of onions, characterized in that the analysis of gene amplification using gel electrophoresis, DNA intercalating fluorescence, phosphorescence or radioactive material.

Images