KR20130010172A - A method for identifying lettuce varieties using microsatellite primer set - Google Patents

Abstract

PURPOSE: A method for identifying the breed of lettuce using a microsatellite primer is provided to protect the breed of lettuce. CONSTITUTION: A primer set for identifying the breed of lettuce contains a primer set containing two base sequences selected from sequence numbers 1-48. The primer further contains a DNA intercalating fluorescent, phosphorescent, or radioactive substance. A kit for identifying the breed of lettuce contains the primer set and PCR or RT-PCR reagent. A method for identifying the breed of lettuce comprises: a step of isolating genome DNA from lettuce; a step of performing PCR or RT-PCR of the genome DNA using the primer set; and a step of checking the amplification of the genome DNA fragment. A genome DNA is isolated from lettuce leaves, stems, or a mixture thereof.

Description

A method for identifying lettuce varieties using microsatellite primer set}

The present invention relates to a method for identifying lettuce varieties using a supersatellite primer set, and more particularly, to selecting a variety of lettuce varieties for lettuce varieties, which show polymorphism with respect to 90 lettuce varieties in Korea. It relates to an identification method.

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 results of Amplified Fragment Length Polymorphism (AFLP) or Random Amplification of Polymorphic DNA (RAPD) analysis techniques used in the past in the use of molecular labels to protect breeders' rights are not recognized. 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 used the SSR molecular label to database the DNA profiles of crops for peppers, cabbages, watermelons, tomatoes, peaches, roses, onions, and so on. The label is used directly, and recently, the results of genetic analysis are used as a supplementary data for selection of control varieties for crops applied for protection of varieties. 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, Asteraceae crop, lettuce is directly used raw and is the most consumed and popular vegetable among Ssam vegetables. The number of domestically grown varieties and production-reported varieties of lettuce reported to the National Species Resource is 666 varieties (as of April 30, 2010), which can be said to be high in the seed market. Lettuce is influenced by the cultivation environment during the cultivation test, and it takes time to select the target trait when breeding the varieties. Therefore, the importance of breed identification by the breeding and DNA analysis method using the molecular label is increasing. Situation.

Looking at the trend of molecular label research and development for lettuce, the research is actively conducted in the US and abroad, and Simko (2008) developed SSR markers derived from the EST (Expressed Sequence Tag) to genetically adapt to each type of lettuce varieties. Jeuken (2008) conducted a search for quantitative genetic loci related to disease resistance, and most of the lettuce researches focused on the mapping of lettuce genes and development of disease resistance related markers rather than the identification of lettuce varieties. .

In Korea, Yang (2010) studied the genetic flexibility of lettuce varieties using RAPD, Inter-SSR and AFLP markers. However, the selection of appropriate marker combinations to identify lettuce varieties using a set of supersatellite markers for domestic varieties and precise genotyping techniques using automatic sequencing have not been established in Korea. . Although lettuce is an important vegetable crop, it lacks basic molecular biology research and molecular genetic data compared to crops such as eggplant (pepper and tomato) and gourd (watermelon, cucumber, etc.) and lacks basic molecular genetic research. It is understood that this is because.

On the other hand, EST-derived SSR markers are based on the expressed nucleotide sequences and directly affect the phenotype of plants compared to genomic library-derived SSR markers. However, in the case of EST-derived markers, it is known that selection of markers showing polymorphism between varieties is more difficult than genomic SSR markers. This is because there is a conserved region between species based on the expressed nucleotide sequence, so it is harder to find polymorphism than genomic markers.

However, if EST-derived markers show distinct polymorphisms, cultivation efficiency will be increased compared to genomic SSR markers that are not related to phenotype expression. In particular, if the development of genomic SSR markers, such as lettuce, is poor, and the crops that have not been studied for molecular identification using molecular markers are selected, the markers (genotypes) and phenotypes may be correlated if the EST-derived marker sets are selected first. I think it will contribute to the increase.

: IVAN SIMKO, Journal of Heredity, 2008.09,15 : Tae-Jin Yang et al., RESEARCH ARTICLES J. Crop Sci. Biotech. 10: 29-34

The inventors of the present invention have been continuing to study the lettuce variety identification method that can significantly save time and cost compared to the method of developing a SSR marker by making a genomic library directly from the lettuce genome as a method for solving the above problems, From the EST-derived SSR markers used for mapping the lettuce genes or searching for disease-resistant loci, we selected the first supersomal markers that show polymorphism for 90 lettuce varieties in Korea and used these primers as primers to make domestic lettuce varieties. By amplifying the genomic DNA of the amplification product generated after amplification, it was confirmed that it can be practically used for scientific verification of the authenticity of lettuce varieties, and completed the present invention.

An object of the present invention is to provide a method for identifying lettuce varieties using a primer set for identifying lettuce varieties as a supersatellite marker showing polymorphism for 90 lettuce varieties for the first time in Korea.

Still another object of the present invention is to provide a kit for identifying a lettuce variety comprising the primer set.

Still another object of the present invention is to provide a method for identifying the authenticity of lettuce varieties and identifying the varieties of lettuce for the selection of the control varieties of the lettuce varieties protection application varieties using the primer set.

The present invention provides a primer set for identifying lettuce varieties exhibiting polymorphism with respect to 90 lettuce varieties distributed in Korea.

The present invention provides a kit for identifying a lettuce variety comprising the primer set.

The present invention provides a method of identifying the variety of lettuce for identifying the authenticity of lettuce varieties, selection of the control varieties of lettuce varieties protection application varieties using the primer set.

Hereinafter, the present invention will be described in detail.

The present invention

SML-001 primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and 2;

SML-002 primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and 4;

An SML-003 primer set consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6;

SML-007 primer set consisting of the nucleotide sequence of SEQ ID NO: 7 and 8;

An SML-015 primer set consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10;

SML-019 primer set consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12;

SML-038 primer set consisting of the nucleotide sequences of SEQ ID NOs: 13 and 14;

SML-039 primer set consisting of the nucleotide sequences of SEQ ID NOs: 15 and 16;

SML-042 primer set consisting of the nucleotide sequences of SEQ ID NOs: 17 and 18;

SML-043 primer set consisting of the nucleotide sequences of SEQ ID NOs: 19 and 20;

SML-045 primer set consisting of the nucleotide sequences of SEQ ID NOs: 21 and 22;

SML-048 primer set consisting of the nucleotide sequences of SEQ ID NOs: 23 and 24;

SML-051 primer set consisting of the nucleotide sequences of SEQ ID NOs: 25 and 26;

A SML-054 primer set consisting of the nucleotide sequences of SEQ ID NOs: 27 and 28;

SML-055 primer set consisting of the nucleotide sequences of SEQ ID NOs: 29 and 30;

SML-056 primer set consisting of the nucleotide sequences of SEQ ID NOs: 31 and 32;

SML-057 primer set consisting of the nucleotide sequences of SEQ ID NOs: 33 and 34;

A SML-061 primer set consisting of the nucleotide sequences of SEQ ID NOs: 35 and 36;

SML-021 primer set consisting of the nucleotide sequences of SEQ ID NOs: 37 and 38;

SML-022 primer set consisting of the nucleotide sequences of SEQ ID NOs: 39 and 40;

SML-026 primer set consisting of the nucleotide sequences of SEQ ID NOs: 41 and 42;

SML-028 primer set consisting of the nucleotide sequences of SEQ ID NOs: 43 and 44;

A SML-032 primer set consisting of the nucleotide sequences of SEQ ID NOs: 45 and 46; And

SML-037 primer set consisting of the nucleotide sequences of SEQ ID NOs: 47 and 48;

Provided is a primer set for lettuce variety identification, comprising one or more primer sets selected from the group consisting of:

More specifically, after amplifying genomic DNA of 90 domestic varieties of lettuce distributed from lettuce EST-derived SSR markers, gene mapping, or disease resistance-related markers, it is possible to identify lettuce varieties using the size of the resulting amplification products. High primer combinations were selected. From the combination of the selected primers, it was possible to select a primer set for identifying a lettuce variety having excellent PIC (Polymorphic Information Content) value of the allele, and confirm that the primer set for identifying a lettuce variety is suitable for the lettuce breed identification method. Could.

The primer set for identifying lettuce varieties according to the present invention is particularly strong among 90 distribution varieties distributed in Korea, such as strong wind apron, most cheongsam, Samsung green, Ss 33, red apple, high green, top red, whirlwind pork red noodles, galle It characterized in that the polymorphism for the lettuce variety is excellent for one or more distribution varieties selected from (d), Java black skirt, and improved tobacco.

A primer set for identifying a lettuce variety according to the present invention is, in particular, SEQ ID NO: 1 and 2, SEQ ID NO: 7 and 8, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 27 and 28, SEQ ID NO: 33 and 34, sequence No. 37 and 38, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44 characterized in that it exhibits an excellent PIC value of 0.45 or more.

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. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of oligonucleotide primers used to amplify the target sequence are as described above.

The present invention provides a primer set for identifying the lettuce variety (SEQ ID NOS: 1 to 48); And reagents required for polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR); It includes, provides a kit for identifying lettuce 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 lettuce;

b) using the isolated genomic DNA as a template and performing PCR (Polymerase chain reaction) or RT-PCR (Reverse-transcriptase polymerase chain reaction) using the primer set for identifying the lettuce variety (SEQ ID NOS: 1 to 48). step; And

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

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

In the present invention, the genomic DNA may be isolated from seeds, leaves, stems or mixtures thereof of lettuce. More specifically, genomic DNA may be separated from leaves or stems of young plants after sowing and seeding lettuce seeds. In the present invention, genomic DNA is isolated from lettuce 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 the lettuce sample 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.

The primer set for identifying a lettuce variety of the present invention is 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 NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, and SEQ ID NO: One or more primer sets selected from primer sets consisting of 47 and 48;

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 can be performed using 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, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

In addition, in the present invention, the step of identifying the lettuce varieties from the amplified product can be identified by using a computer program to determine the size of the alleles (band) by using an automatic sequencer, and then to identify each variety. Specifically, for each lettuce variety, the presence or absence of an amplification product by the primer set for identifying a lettuce variety of the present invention can be prepared in a table, and the lettuce variety can be identified through a comparative analysis with the amplified product. 1 is a table showing the presence or absence of amplification products by the primer set for identifying each variety for each lettuce variety. Specifically, FIG. 1 illustrates the presence or absence of a band appearing when amplifying each of 90 lettuce varieties using a primer set for identifying 90 lettuce varieties of the present invention. If is not present, it is indicated as “0”.

Therefore, after separating genomic DNA from the lettuce to be identified cultivars, it is possible to identify the lettuce cultivars by amplifying using the marker of the present invention and comparing the result of the amplified product with the result of FIG.

The primer set for identifying lettuce varieties according to the present invention has excellent identification ability against 90 varieties of lettuce distributed in Korea, identifying the authenticity of lettuce varieties, arranging seed disputes between farmers and companies, and companies and companies, and protecting varieties. It will be able to contribute greatly in various fields such as selection of control varieties.

1 is a code of the presence or absence of the band appearing when amplified by using a primer set for identifying lettuce varieties according to the present invention targeting 90 varieties of lettuce, represented by "1" when the band is present and the band does not exist Is represented by “0”
Figure 2 codes each lettuce variety analyzed by the primer set for lettuce variety identification according to the present invention, and shows the genetic similarity of lettuce variety identification and varieties by using NTSYS pc computer program.

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 Lettuce Varieties Identification Method

(1) lettuce sample

In the present invention, the 90 varieties of lettuce described in Table 1 were used for the selection of lettuce varieties primer set identification and lettuce varieties identifiability assay.

(2) Isolation of Lettuce Genomic DNA

Seed 15-20 grains of the seed lettuce varieties of Table 1 were put in two tungsten beads in a 2ml 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) Preparation of primer set

Candidate sequencer databases for primer sets of the invention include SSR markers (Simko et al. 2008) and disease resistance related quantitative traits derived from Expressed Sequence Tags (EST) developed and published in lettuce ( Lactucasativa L.). A total of 81 primer sets were used based on the sequencing of the EST marker (Jeuken et al. 2008) for locus search.

To select markers that can distinguish domestic distribution varieties, DNA from strong wind skirts, Most Blue Ssam, Samsung Green, Ss 33, Jeokdan, High Green, Top Red, Whirlwind Puffs, Galera, Java Black Skirt and improved tobacco varieties Was isolated and gene amplified using the 81 primer sets. Then, 30 markers having high reproducibility between repeats, clear band patterns, and high polymorphism were selected. In order to drive the molecular labeling of these 30 markers through an automatic sequence analysis, oligonucleotides fluorescently labeled with one of the fluorescent generating materials VIC, NED, FAM and PET were prepared in the forward primers. Using the 30 markers, the final 24 markers with high varieties were selected when they were extended to 90 varieties of lettuce in Korea. The base sequences of primer sets showing polymorphism in the lettuce lettuce varieties are shown in Table 2 above.

(4) PCR amplification and electrophoresis

PCR was performed using the prepared genomic DNA and the prepared markers as materials. The composition of the PCR reaction solution was prepared 20 ng of lettuce genomic DNA, 10 pmol of the marker, 2.0 μl dNTP mixture (2.5 mM), Taq DNA polymerase 1U (GeNet bio, Korea), 2.5 μl of 10 × PCR Buffer (50 Distilled water was added to 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 lettuce 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 45 cycles of 45 seconds, 10 minutes of stretching was performed at 72 ° C. for final stretching of the PCR product. After the PCR was completed, the amplification was confirmed by electrophoresis on 2% agarose gel.

The amplified samples were stored in a 4 ° C. refrigerator for the next experiment. Samples amplified by PCR were diluted to 150 μl of sterile distilled water to an appropriate concentration, and then 10 μl of deionized formamide and allele size of 1 to 3 μl of the diluted PCR product (adjusted according to the amplification amount). 0.25 μl of a size marker (LIZ500 size standard) was mixed and mixed, and then denatured at 94 ° C. for 2 minutes.

The denatured gene amplification product was electrophoresed using an automatic sequencing analyzer (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) Test for Identifying Lettuce Varieties of Markers According to the Present Invention

The allelic size identified by the automatic sequencing was examined for the possibility of identifying the lettuce variety of the marker according to the present invention. Polymorphism information content (PIC) values were calculated using the following equation (1).

In Equation 1, K is the total number of bands of each allele and Pi 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, SEQ ID NO: 1 and 2 (marker name: SML-001), SEQ ID NO: 7 and 8 (marker name: SML-007), SEQ ID NO: 17 of the primer set for identifying lettuce varieties according to the present invention And 18 (marker name: SML-042), SEQ ID NOs: 19 and 20 (marker name: SML-043), SEQ ID NO: 27 and 28 (marker name: SML-054), SEQ ID NO: 33 and 34 (marker name: SML- 057), SEQ ID NOs: 37 and 38 (marker name: SML-021), SEQ ID NOs: 39 and 40 (marker name: SML-022), SEQ ID NOs: 41 and 42 (marker name: SML-026), and SEQ ID NOs: 43 and 44 (Marker name: SML-028) was confirmed to exhibit an excellent PIC value of 0.45 or more.

In addition, the primer set of SEQ ID NOs: 25 and 26 showed the lowest PIC value of 0.310, and the primer set of SEQ ID NOs: 1 and 2 showed the highest PIC value of 0.598. The average PIC value of each marker is 0.432, which is also a result of confirming that the marker of the present invention can sufficiently identify lettuce 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, it is coded as the number 1 The results of the presence or absence of bands for 24 supersatellite markers for all 90 varieties from 90 to 90 are shown in FIG. 1.

1 is isolated from genomic DNA from the lettuce to be identified varieties, amplified using the primer set of the present invention can be used as data to identify the lettuce varieties by comparing the results of Figure 1 with the amplified product have.

Specifically, the boutique No. 61 and the No. 63 U-lake varieties were coded as “1” because of the presence of the band in the second allele of SML-022 marker. Coded. Among the markers for the identification of varieties, there was no difference between the boutique and the U-lake varieties.

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, 'Summer true taste skirt' and 'heart red skirt', 'Hongpung skirt' and 'Asian deep blue skirt', 'boutique' and 'U Lake', 'Saint Green' and 'Gold Green' are hereditary It was confirmed that the varieties with a very high similarity, and from the above results, the primer set of the present invention was confirmed that it is possible to distinguish all varieties.

In addition, a lettuce seed dispute between a lettuce farmer and a nursery grows in Hapcheon-gun, Gyeongnam, Korea. Analyzed using 24 sets.

As a result, three of the 24 primer sets (marker names: SML-045, SML-022, SML-026) were able to identify sensitized varieties and varieties with different allele sizes, and also the primers of the present invention. The set saved the effort and time required to grow the lettuce variety and investigate its properties.

<110> The republic of Korea (Korea Seed & Variety Service) <120> A method for identifying lettuce varieties using microsatellite          primer set <160> 48 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 ccatggatcc tgtgtgaaga 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 caccatgttc cacttccact t 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gtgattgcat gccaaatgaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 ttagtagccc gcatgctttt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 cgggctggtt ttgattttta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 tgtcaaatcg tcacgtggtt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 acacttgccg attccttcac 20 <210> 8 <211> 20 <212> DNA 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gctgctccat 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 catgaagtgt tttggggtga 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 ggcctttcat ttcttcctca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 ttctttcgcc catctgaaac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 aaacaggggg ctaacgatct 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 21 acaaaaccgt ttcacccaaa 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 22 agccctgtcc tcttcaggat 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 tttgaggcag gtgttagctg 20 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 gaacaaagtt acaacgagag gaaa 24 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 ctcaaggcgg catctacttc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 gaccccttgc ttgtttcaga 20 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 27 ccatgccatg cagtatacct t 21 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 28 aaaaatgact gcatactttg tgaa 24 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 29 ctgcgtgttt taagccgttt 20 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 30 tccataataa tataatcgca ccaa 24 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 31 gcctagtcca attgctttgc 20 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 32 cagcttaaca tacttttgtt cattca 26 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 33 tcccatgatg gagagactca 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 34 cccaaaaggg aatagcaacc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 35 gagtgctgag aaagcccaag 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 36 taagctgctc ttccctcctg 20 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 37 ttgggagaat tttcatttcc a 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 38 agtcatcttt ttcaccccac a 21 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 39 gggcctcaaa tcctctctg 19 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 40 tgttcttccc ctctttggaa 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 41 gggttctcat tggctgacat 20 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 42 tgtcttccaa ccaaaacata ca 22 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 43 tgatccaggc tctccagaat 20 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 44 cacgaccatg aatgataagt gc 22 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 45 ttggatttgg ggtgatgaat 20 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 46 gcatagtaat ttgacatttt ggcata 26 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 47 tttttcccga tctttgcatc 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 48 agcgaatctt tgctttttcg 20

Claims (6)

SML-001 primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and 2;
SML-002 primer set consisting of the nucleotide sequence of SEQ ID NO: 3 and 4;
An SML-003 primer set consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6;
SML-007 primer set consisting of the nucleotide sequence of SEQ ID NO: 7 and 8;
An SML-015 primer set consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10;
SML-019 primer set consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12;
SML-038 primer set consisting of the nucleotide sequences of SEQ ID NOs: 13 and 14;
SML-039 primer set consisting of the nucleotide sequences of SEQ ID NOs: 15 and 16;
SML-042 primer set consisting of the nucleotide sequences of SEQ ID NOs: 17 and 18;
SML-043 primer set consisting of the nucleotide sequences of SEQ ID NOs: 19 and 20;
SML-045 primer set consisting of the nucleotide sequences of SEQ ID NOs: 21 and 22;
SML-048 primer set consisting of the nucleotide sequences of SEQ ID NOs: 23 and 24;
SML-051 primer set consisting of the nucleotide sequences of SEQ ID NOs: 25 and 26;
A SML-054 primer set consisting of the nucleotide sequences of SEQ ID NOs: 27 and 28;
SML-055 primer set consisting of the nucleotide sequences of SEQ ID NOs: 29 and 30;
SML-056 primer set consisting of the nucleotide sequences of SEQ ID NOs: 31 and 32;
SML-057 primer set consisting of the nucleotide sequences of SEQ ID NOs: 33 and 34;
A SML-061 primer set consisting of the nucleotide sequences of SEQ ID NOs: 35 and 36;
SML-021 primer set consisting of the nucleotide sequences of SEQ ID NOs: 37 and 38;
SML-022 primer set consisting of the nucleotide sequences of SEQ ID NOs: 39 and 40;
SML-026 primer set consisting of the nucleotide sequences of SEQ ID NOs: 41 and 42;
SML-028 primer set consisting of the nucleotide sequences of SEQ ID NOs: 43 and 44;
A SML-032 primer set consisting of the nucleotide sequences of SEQ ID NOs: 45 and 46; And
SML-037 primer set consisting of the nucleotide sequences of SEQ ID NOs: 47 and 48;
A primer set for identifying lettuce varieties, comprising one or more primer sets selected from the group consisting of: The method of claim 1,
Said primer is a primer set for identifying lettuce varieties, characterized in that it further comprises DNA intercalating fluorescence, phosphorescence 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, lettuce breed identification kit. a) separating genomic DNA from lettuce;
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 the lettuce variety according to claim 1 or 2 Performing; And
c) confirming whether said genomic DNA fragment has been amplified;
A method of identifying a variety of lettuce, including. 5. The method of claim 4,
Wherein said genomic DNA is isolated from seeds, leaves, stems or mixtures of lettuce. 5. The method of claim 4,
The method of identifying the varieties of lettuce is characterized by analyzing the gene amplification using gel electrophoresis, DNA intercalating fluorescence, phosphorescence or radioactive material.

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