KR101604832B1 - Primer set to judge tomato resitant to tomato yellow leaf curl disease, Method to judge tomato resitant to tomato yellow leaf curl disease, and Kit to judge tomato resitant to tomato yellow leaf curl disease - Google Patents

Abstract

The present invention provides a primer set for judging the resistance to yellowtail disease of tomatoes, comprising a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2. INDUSTRIAL APPLICABILITY The present invention has an advantage of being able to judge a tomato resistant to leafing sickle leaf disease and to utilize it for breeding.

Description

[0001] The present invention relates to a primer set for judging resistance to yellowtail leaf curd disease, a method for judging resistance to yellowtail leaf curd disease, and a method for judging tomato tolerance to tomato leaf curl disease, yellow leaf curl disease, and Kit to judge tomato resitant to tomato yellow leaf curl disease}

The present invention relates to a primer set for judging resistance to yellowtail leafing disease, a method for judging resistance to yellowtail leafing disease, and a tomato judging kit for resistance to yellowtail leafing disease. More particularly, A primer set for judging resistance to tomato leaf disease, a method for judging resistance to tomato leaf blight disease, and a tomato hedgehog resistance tomato judgment kit capable of judging tomato having a leaf blight resistance.

Tomato yellow leaf curd disease is a disease caused by tomato yellow leaf curly virus (TYLCV), which is transmitted by white fly.

Recently, climate warming has been spreading to USA, South America, Europe, Southeast Asia, Japan, and China. As a result, not only farm damage but also international inspection of Tomato Sulfur Leaf Blight Virus Tomato Fruit infection has been strengthened. Therefore, in order to cultivate tomato varieties having tolerance to diseases such as tomato leaf blight, it is necessary to develop a technique for judging tomatoes having tolerance to tomato blight leaf blight disease. In particular, there is a need for a study on Ty1, which is known to be a gene related to resistance to tomato yellow leaf blight virus, and development of a tomato judgment technology that possesses such a gene.

On the other hand, cleaved amplified polymorphic sequence (CAPS) analysis is a technique for genetic marker analysis, and PCR is used for faster analysis such as Restriction Fragment Length Polymorphism (RFLP). When this restriction is made or eliminated by the genetic difference between the disease-resistant individual (breed) and the diseased individual (breed), this difference is caused by a DNA fragment generated by digestion As shown in FIG.

The use of such disease resistance genes and CAPS analysis for breeding tomato varieties can maximize breeding selection efficiency and shorten breeding period.

 Verlaan MG, Hutton SF, Ibrahem RM, Kormelink R, Visser RG, Scott JW, Edwards JD, Bai Y. (2013) The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. PLoS Genet. 9 (3): e100339

One of the problems to be solved by the present invention is to provide a primer set for judging resistance to yellowtail leafroll disease.

Another problem to be solved by the present invention is to provide a method for judging resistance to yellowtail leafroll disease.

Another problem to be solved by the present invention is to provide a yellowtail leaf blight resistant tomato determination kit.

The problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a primer set for judging the resistance to yellowtail disease of tomatoes, comprising a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2.

Said leafy leafhopper-resistant tomato may be one having a gene encoding a protein exhibiting resistance to leafy leafhopper disease.

The gene may have the nucleotide sequence of SEQ ID NO: 3.

The primer set may be for amplification of a fragment amplification polymorphism sequence (CAPS).

The fragment amplification polymorphism sequence may be contained in a CAPS marker having the nucleotide sequence of SEQ ID NO: 3.

Wherein the primer set for judging the resistance to yellowtail leafing disease is a genome of a tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 on the basis of at least one base of the 226th base or 312th base in the nucleotide sequence of SEQ ID NO: Or a corresponding position in at least one of corresponding positions corresponding to the 226 < th > base or the corresponding positions corresponding to the 312 < th > base in the DNA base sequence.

The genomic DNA sequence of the tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 may be 90% or more, more preferably 98% or more homologous to the nucleotide sequence of SEQ ID NO: 3.

In addition, the present invention provides a method for detecting the presence or absence of the nucleotide sequence of the 226th base or the 312th base in the genomic DNA sequence of a tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 on the basis of at least one base among the 226th base or 312th base in the nucleotide sequence of SEQ ID NO: And determining whether or not the corresponding reference position corresponds to at least one of a corresponding position corresponding to the 312th base or a corresponding position corresponding to the 312th base, do.

The method for judging the resistance to yellowtail leafhopper disease may further include the step of judging the tomato which does not show the difference as the yellowtail leaf resistance tomato.

The difference at the 226th base corresponding position may be Insertion / Deletion polymorphism.

The difference represented at the 312 th base corresponding position may be a single base substitution polymorphism (SNP).

The inserted deletion polymorphism may be that the 226th base is adenine and the base deletion occurs at the 226th base corresponding position.

The single base substitution polymorphism may be such that the 312th base is cytosine and the base at the 312th base corresponding position is thymine.

The confirmation may be that the primer set of the present invention is used.

In addition, the present invention provides a method for judging whether or not a tomato is a leafy leafhopper-resistant tomato using the primer set of the present invention.

The method for determining the resistance to yellowtail leafhopper disease tomatoes comprises the steps of: (A) subjecting an amplified product amplified using the genomic DNA of a tomato sample as a template to a primer set of the present invention with a restriction enzyme; And (B) analyzing a pattern obtained by electrophoresis of the sample to be processed.

The analysis may be to determine whether the amplification product is cleaved by the restriction enzyme.

Also, the pattern analysis step may include a determination step of determining a tomato sample having a pattern represented by an amplification product cleaved by the restriction enzyme, as a leafy yellowtail-resistant tomato.

The pattern analysis may be a comparative analysis with a pattern of standard tomato varieties.

The standard tomato varieties may be one or more selected from the group consisting of Codonopsis, Tynthia, Daphneus, Deerose, Mamilio, TTcr, Newcastle, TY250, TY escort, or TY endorphin.

The pattern of the standard tomato variety may be obtained by using genomic DNA of a standard tomato variety as a template and electrophoresis of the sample treated by treating the amplified product amplified with the above primer set with a restriction enzyme.

In addition, the present invention provides a sulfated leaf bladder resistance tomato determination kit comprising a primer set, a restriction enzyme, and an amplification reaction performing reagent of the present invention.

The restriction enzyme may be one or more of the restriction enzymes listed in Table 2.

The reagent for carrying out the amplification reaction may include a DNA polymerase, dNTPs, and a buffer.

INDUSTRIAL APPLICABILITY The present invention has an effect of being able to judge a tomato resistant to leafing sickle leaf disease and to utilize it for breeding.

FIG. 1 is a view for explaining a method for judging resistance to tomato leaf blight disease, which is an embodiment of the present invention.
FIG. 2 and FIG. 3 are diagrams illustrating nucleotide sequences for explaining a primer set according to an embodiment of the present invention.
4 and 5 are graphs showing electrophoresis results using a primer set according to an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will be more apparent from the following detailed description taken in conjunction with the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. But is only provided to fully inform the owner of the scope of the invention, and the present invention is only defined by the scope of the claims.

Like reference numerals refer to like elements throughout the specification. Also, "and / or" include each and every combination of one or more of the components mentioned.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. It is noted that the terms "comprises" and / or "comprising" used in the specification are intended to be inclusive in a manner similar to the components, steps, operations, and / Or additions.

A "primer set" means a plurality of primers. In addition, the primer set may further include a container for receiving the primer.

In addition, "judgment" means to judge whether or not tomatoes are resistant to yellowtail leaf disease, and includes selection, selection and selection.

In addition, "resistance to yellow leaf palm disease" is meant to include resistance to tomato yellow leaf palm virus.

In addition, "resistance" means not only complete resistance but also incomplete resistance, and "incomplete resistance" means that it is not perfect to show resistance with only one resistance gene.

Also, "resistance" includes both resistance and resistance, meaning that the phenotype is resistant.

The primer set according to an embodiment of the present invention may include the primers described in Table 1. [

[Table 1] Primer

In Table 1, the primer set consists of a pair of forward primer and reverse primer, F means forward, and R means reverse.

The primer set is for the amplification of the fractional amplification polymorphism sequence (CAPS) of tomato. PCR amplified CAPS by the primer set is treated by restriction enzyme, and the enzyme treated PCR product exhibits characteristic band pattern. Such a pattern can be analyzed to determine whether the tomato is resistant to leaf blight disease. For example, it is possible to judge whether or not a tomato is resistant to leafy leafhopper disease by the band pattern difference between a disease-resistant gene (Ty) or a heterologous gene (ty) against a yellowtail leaflet disease of a tomato sample.

Thus, one embodiment of the present invention may be for judging a leafy yellowtail resistant tomato. Sulfur leaf blade disease resistance tomato may be one having a gene encoding a protein exhibiting resistance to yellow leaf blight disease. In this case, the gene may have the nucleotide sequence of SEQ ID NO: 3. The gene having the nucleotide sequence of SEQ ID NO: 3 has a cleavage site cleaved by any one or more of the restriction enzymes listed in Table 2. [ Therefore, it becomes possible to distinguish the gene from the deletion gene having no such cleavage site.

[Table 2] Electrophoretic gel concentration according to restriction enzyme and restriction enzyme

The heterologous gene differs from the resistant gene on the cleavage site. The cleavage site is associated with at least one base of the nucleotide sequence 226 or 312 of SEQ ID NO: 3.

Therefore, one embodiment of the present invention provides a method for detecting the presence or absence of the nucleotide sequence of the genomic DNA of a tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 on the basis of at least one base among the nucleotide sequence of nucleotide 226 or 312 of SEQ ID NO: To determine whether or not it represents a difference from the reference base at the corresponding position corresponding to the 226th base or the corresponding position corresponding to at least one of the corresponding positions corresponding to the 312th base. In this case, the genomic DNA base sequence of the tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 may preferably be 90% or more, more preferably 98% or more homologous to the nucleotide sequence of SEQ ID NO: 3 .

The tomato varieties (tomato samples) to be judged by the yellowtail leafhopper resistant tomatoes are not limited thereto, but may be one or more selected from the tomato varieties described in Table 3, for example.

[Table 3] Tomato

A primer is a base sequence having a short free 3 'hydroxyl group, which means a short base sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of a reagent for polymerization and four nucleoside triphosphates at the appropriate buffer solution and temperature.

The primer may be an oligonucleotide and the oligonucleotide may comprise a nucleotide analogue such as phosphorothioate, alkylphosphorothioate or peptide nucleic acid, intercalating agent.

The primer can be produced through a chemical synthesis method using a phosphoramidite method, a phosphodiester method, a diethylphosphoramidite method, or the like. It is to be understood that the primer sequence can be modified by means known in the art.

The base sequence to be amplified by the primer set may be the CAPS marker, and the CAPS marker may have the nucleotide sequence shown in Table 4 below.

The CAPS marker may have a CAPS, as shown in FIGS. That is, it may not be cleaved or cleaved by a restriction enzyme due to a polymorphism depending on whether it is a yellowtail leaf-leaf disease-resistant tomato or a yellow leaf leaf disease disease-resistant tomato. FIGS. 2 and 3 are diagrams showing nucleotide sequences for explaining a primer set. In the figure, the underlined portion indicates that the cut is not cut or uncut according to whether the restriction enzyme cleavage is resistive or non-cleavable. In the figure, TyI-IY represents a yellowtail leaf resistance gene, and tyI-IY represents a yellowtail leaf edema disease gene. As shown, the gene of the yellowtail leafhopper-resistant tomato has a cleavage site by a restriction enzyme as indicated by an underline (solid line), and the virulence gene does not have a cleavage site as indicated by an underline (dotted line). The nucleotide sequence corresponding to the 226th base or the 312th base in the genomic DNA sequence of the tomato sample corresponding to the nucleotide sequence of SEQ ID NO: And a base at a corresponding position of at least one of the 312-th base corresponding positions.

The difference may be caused by a polymorphism between the resistance gene and the dendritic gene. For example, the difference represented at the 226th base corresponding position may be insertion / deletion polymorphism, The difference may be a single base substitution polymorphism.

The inserted deletion polymorphism may be that the 226th base is adenine and appears as a base deletion at the corresponding position of the 226th base, wherein the 312th base is cytosine, and the 312th base corresponding position Lt; RTI ID = 0.0 > of thymine. ≪ / RTI > That is, the heterologous gene may be a deletion or substitution of one specific gene of the nucleotide sequence of the resistance gene. Because of this polymorphism, it is possible to judge whether or not the tomato sample is resistant to the yellow leaf palm disease by using the fact that the cleavage by the restriction enzyme changes.

In this case, the genomic DNA base sequence of the tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 may be 90% or more, more preferably 98% or more homologous with the nucleotide sequence of SEQ ID NO: 3. Thus, when the homology is high except for the part that differs by the polymorphism, the target tomato judgment becomes easier by using the difference by the polymorphism.

The nucleotide sequence shown in Table 4 may be a nucleotide sequence amplified by the primer described in Table 1 and having a site cleaved by the restriction enzyme shown in Table 2. [ Sulfate-leaf curd disease In contrast, the transgenic tomato may not have a restriction enzyme cleavage site. In other words, the CAPS marker is able to distinguish resistant and resistant varieties against leaf blight disease caused by restriction enzyme cleavage.

[Table 4] Marker sequence

In addition, the method for determining the resistance to tomato leaf blight disease tomato according to one embodiment of the present invention is a method for determining the resistance to tomato leaf blight disease on a basis of at least one base among the 226th base or the 312th base in the nucleotide sequence of SEQ ID NO: 3, Confirming whether or not the genomic DNA sequence of the tomato sample shows a difference from the reference base at the corresponding position of at least one of the position corresponding to the 226th base or the position corresponding to the 312th base. The tomatoes that do not show any difference are judged to be resistant to yellowtail leafhopper disease. The tomatoes showing the difference can be judged to be resistant to yellowtail leafhopper disease, not disease resistance.

(For example, high-resolution melting (HRM), denaturing high performance liquid chromatography (DHPLC) or the like) can be used to confirm the difference between the standard nucleotide sequence and the base sequence of the sample And the primer set of the present invention may be used.

Hereinafter, a method for judging whether or not a tomato is a leafing-resistant tomato with the use of a primer set according to an embodiment of the present invention, Explain.

Specifically, it can be judged whether or not it is a leafy leafhopper-resistant tomato by a method including (A) a sample treatment step shown in FIG. 1, and (B) a pattern analysis step.

(A) the sample processing step is a step of treating the amplified product amplified by using a primer set, which is an embodiment of the present invention, with a restriction enzyme using the genomic DNA of a tomato sample as a template, and (B) And analyzing a pattern obtained by electrophoresis of water.

As a method for isolating the genomic DNA of the tomato sample, a method known in the art can be used, and for example, a CTAB method, a commercially available wizard prep kit (Promega), and the like can be used. The target sequence can be amplified by performing amplification reaction using a separated genomic DNA as a template and using a primer set as an example of the present invention as a primer. At this time, the target sequence may be a CAPS marker. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with a Q [beta] replicase, or a method for amplifying a nucleic acid molecule known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are common in the art and commercially available kits can be used.

The restriction enzyme may be one or more of those listed in Table 2.

The pattern analysis may be to determine whether the amplification product is a pattern represented by the restriction enzyme.

In addition, the pattern analysis step may include a determination step of determining a tomato sample having a pattern represented by an amplification product cleaved by a restriction enzyme as a yellowtail leaf resistance tomato.

In addition, the pattern analysis may be a comparative analysis with a pattern of standard tomato varieties, and the standard tomato varieties may be one or more selected from the tomato varieties listed in Table 3. Preferably, the standard tomato varieties may be one or more selected from cattles, TY winner, daphne, deerose, marijuana, TT chrysanthemum, Newtonian, TY250, TY escot, or TY endorphin. Such varieties have been identified as resistant tomatoes in the specific examples.

The pattern of the standard tomato variety may be obtained by using the genomic DNA of the standard tomato variety as a template and electrophoresis of the sample treated product obtained by treating the amplified product amplified using the primer set of one embodiment of the present invention with a restriction enzyme .

After the restriction enzyme treatment, the pattern obtained by the electrophoresis shows a different pattern depending on whether it is resistant or non-resistant, and thus it can be judged whether or not it is a leafy leafhopper resistant tomato based on this pattern.

That is, by analyzing the band pattern obtained by separating the fragments obtained by the restriction enzyme treatment using a gel electrophoresis apparatus, it is possible to judge whether or not the tomatoes are resistant to the leafy leafhopper disease.

In addition, the primer set, which is one embodiment of the present invention, may be included in a yellow leaf palm disease resistant tomato determination kit.

Hereinafter, an embodiment of the yellow leaf palm disease resistant tomato determination kit according to the present invention will be described in detail.

Specifically, one embodiment of the tomato leaf judging kit according to the present invention may include a primer set, a restriction enzyme, and an amplification reaction reagent, which are embodiments of the present invention.

The restriction enzyme may be at least one of the restriction enzymes listed in Table 2.

In addition, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer, and the like.

In addition, the yellow leaf palm resistant tomato determination kit may further include instructions describing optimal reaction performance conditions. The instructions may be a recording medium that describes how to use the kit, for example, PCR buffer preparation method, reaction conditions, and the like.

As described in the embodiment of the present invention, the above-mentioned items in the primer set for yellowtail leaf resistance test, the test method for yellowtail leaf resistance test, and the yellowtail leaf resistance test tomato test kit, respectively, do.

Hereinafter, a primer set for judging the resistance of a yellowtail leaf resistance tomato, a yellowtail leaf resistance test tomato test kit, and a yellowtail leaf resistance test tomato determination method according to an embodiment of the present invention will be described in detail.

The tomatoes used in the specific examples are those shown in Table 3. Such tomatoes were obtained from the seed companies listed in Table 3, unless otherwise noted.

≪ Specific Example A > A primer set for judging tomato resistance to yellowtail leaf disease

A-1. Genomic DNA fragment amplification primer production

Based on the nucleotide sequence of Solyc06g05118 (Table 5) published in Verlaan et al., Published in 2013, a primer for genomic DNA fragment amplification of the Ty1 locus was constructed (Verlaan MG, Hutton SF, Ibrahem RM, Kormelink R, Visser RG, Scott JW, Edwards JD, Bai Y. (2013) The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allele and code for DFDGD-class RNA-dependent RNA polymerases. 9 (3): e1003399). The primers are as shown in Table 1. This primer was able to predict the full-length (about 600 bp) genomic DNA fragment from the initiation codon to the termination codon in the gene source known to be resistance to and resistance to yellow fever disease. The DNA oligomers used as primers were synthesized by Bioneer (Korea) and synthesized by applying 'phosphite triester' method, which connects phosphodiester bonds using cyanoethyl phosphoramidite developed by Koster.

[Table 5] Solyc06g05118 sequence

A-2. Genomic DNA extraction

Tomatoes listed in Table 3 were used.

For tomato DNA extraction, young leaves are collected in a 2 ml tube containing Stainless baed at 4-5 leaf stage during the growth and rapidly frozen with liquid nitrogen (LN2). After finely crushing with Vortex, add 300 μl of CTAB Buffer containing 0.2% Sodium bifulfite and mix well. After processing for 30 minutes in a water bath at 65 ° C, add 150ul of chloroform and mix well. Then centrifuge at 4 ° C for 15 minutes to induce layer separation. Transfer 200 ul of the supernatant to a new tube, add 200 ul of pre-chilled 100% Isopropanol, and slowly invert. Centrifuge for 30 seconds to leave only the DNA pellet and remove the liquid from the tube. After adding 750 ul of 70% ethanol, centrifuge for 30 seconds after inverting as before. After completely removing the liquid from the tube, dissolve in 100 μl of DNase-free water in a tube containing only the DNA pellet, and store at -20 ° C.

A-3. Amplification (PCR)

The PCR was carried out in the same manner as in Example 1 except that 1 μl of gDNA, 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl, 2 mM MgSO ₄ , 0.1% 0.2 mM each dNTP, 0.4 mM forward and reverse primers, 0.15 ul (TaKaRa, Otsu, Shiga, Janpan), totaling 25ul. PCR was performed using a T-100 Thermal Cycler (BIO-RAD, Hercules, CA, USA) for PCR reaction. The PCR conditions were denaturation at 94 ° C for 10 minutes, denaturation at 94 ° C for 1 minute, , Annealing at 72 ° C for 1 min, and reaction at 72 ° C for 10 min.

A-4. TA-cloning

The amplified PCR product was cloned using TOPO TA Cloning® Kit (Invitrogen, USA), transformed into E. coli by heat-shock method using Chemically Competent Cell (Invitrogen, USA) The experimental method was carried out according to the method provided by the manufacturer.

After cloning, the presence or absence of the insert was confirmed by colony PCR, and the clone in which the presence of the insert was confirmed was selected and utilized.

A-5. CAPS marker and primer set design

For plasmid DNA extraction, DNA was isolated using Plasmid DNA prep kit (SolGent, KOREA), and the nucleotide sequence was decoded by SolGent. SeqMan, EditSeq, and MegAlign (DNA STAR, USA) were used for sequence analysis. Using the results of the analysis, it was found that the nucleotide sequences of the varieties known to exhibit resistance to yellowtail leaf disease (dTaTaR, TYWinner, Daphne, Deerose, Mamirio, TTpur, Newtonian, TY250, TY escort, TY endorphin, etc.) From the nucleotide sequence information of Solyc06g05118 and the nucleotide sequences of the other varieties listed in Table 3, the differences in the nucleotide sequence between the sulphate leaf blight resistance factor (Ty1) and the deletion factor (ty1) were analyzed. As a result, as shown in FIGS. 2 to 3, the nucleotide sequence of the resistance factor for yellowing leaf blight and the nucleotide sequence of the virulence factor were secured, and a total of 7 SNPs and 1 insertion / deletion polymorphism were confirmed.

In the figure, the boxes represent the parts representing the difference, and both sequences were found to be 98.8% homologous. In addition, the nucleotide sequence of Solyc06g05118 (SEQ ID NO: 4) was 100% identical to the nucleotide sequence of the virulence factor. Therefore, the nucleotide sequence (SEQ ID NO: 4) shown in Table 5 corresponds to the nucleotide sequence of the deleterious factor and can be amplified by the primer shown in Table 1, but is not a nucleotide sequence having no site cleaved by the restriction enzyme shown in Table 2 . Such a tomato having the nucleotide sequence of SEQ ID NO: 4 can be judged as a yellowtail leafroll disease tomato.

CAPS markers were designed using the CAPS design tool provided by the Sol Genomics Network (http://solgenomics.net) targeting the identified polymorphic sites.

The CAPS markers are as shown in Table 4 and Figs. 2 to 3. In the figure, it is shown that the underlined part is not cut or uncut depending on whether it is resistive or immobilized on the restriction enzyme cleavage site. In the figure, TyI-IY represents a yellowtail leaf resistance gene, and tyI-IY represents a yellowtail leaf edema disease gene. As shown, the gene of the yellowtail leafhopper-resistant tomato has a cleavage site by a restriction enzyme as indicated by an underline (solid line), and the virulence gene does not have a cleavage site as indicated by an underline (dotted line). The restriction enzyme cleavage site in Fig. 2 represents a cleavage site by TaqI, and the restriction enzyme cleavage site in Fig. 3 represents a cleavage site by SspI. The presence or absence of the cleavage site is determined based on at least one base among the nucleotide sequence of nucleotide 226 or base 312 in the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: Base position or at the corresponding position of at least one of the 312-base corresponding positions.

The base corresponding position means at least 20 bases before and after the corresponding position, respectively, corresponding to the corresponding partial bases of SEQ ID NO: 3.

This difference is caused by the polymorphism between resistance gene and heterologous gene. That is, as shown, the difference at the 226th base corresponding position is the insertion / deletion polymorphism, and the difference at the 312th base corresponding position is the single base substitution polymorphism.

In this case, the insertion deletion polymorphism is that the 226th base is adenine and appears as a base deletion at the corresponding position of the 226th base, the single base substitution polymorphism is a nucleotide sequence in which the 312th base is cytosine, The base is represented by thymine. That is, the heterologous gene may be a deletion or substitution of one specific gene of the nucleotide sequence of the resistance gene. This polymorphism makes it possible to judge whether or not the tomato sample is resistant to the leaf blight disease by using the fact that the restriction enzyme cleavage is different.

Specifically, designed primers and restriction enzymes are as shown in Table 1 and Table 2. DNA oligomers used as primers were synthesized by Bioneer (Korea) and synthesized by applying 'phosphite triester' method, which connects phosphodiester bonds using cyanoethyl phosphoramidite developed by Koster.

The primer set forth in Table 1 prepared by the above method can be included to prepare a primer set for judging resistance to yellowtail leaf resistance to tomatoes or a tomato hedgehog resistance kit for yellowtail leaf resistance.

≪ Specific Example B >

B-1. Genomic DNA extraction

Genomic DNA of the tomato described in Table 3 was extracted in the same manner as in A-2.

B-2. Amplification (PCR)

Gene amplification was performed in the same manner as in A-3.

B-3. Restriction enzyme treatment

10ul of 25ul of total volume of PCR product was electrophoresed to confirm whether the PCR was amplified. 2ul of one of the restriction enzymes listed in Table 2, 2ul of buffer and 2ul of tertiary distilled water were added to 15ul PCR product and mixed. And treated at the treatment temperature according to the restriction enzyme for 1 hour.

B-4. Electrophoresis

Genomic DNA was prepared from agarose gels at the concentrations listed in Table 2 according to the restriction enzymes used, considering the expected fragment length. Gel was stained with EtBr after electrophoresis, and gel doc 2000 (BIO-RAD, Hercules, CA, USA PCR amplification and genotyping were performed. 4 and 5 show electrophoresis results.

B-5. Pattern analysis

B-5-1. Pattern analysis by application of TaqI restriction enzyme

The resistance genotypes have a truncated region (TCGA) by TaqI. Therefore, the truncated 312bp and 297bp fragments are formed after the restriction enzyme treatment, and the 608bp fragment is retained because the transgenic genotype has no TaqI cleavage site.

As shown in FIG. 4, 2% agarose gel electrophoresis revealed that the TyI-Taq CAPS Marker was divided into two types, H (heterozygous state) and S (heterozygous state). The patterns were heterozygous in H, TY Winner, Daphneus, Deerose, Mamilio, TT, Shinku, TY250, TY escort and TY endorphin. . Most commercial varieties of the tomatoes are F1 varieties. Ty1, the resistance gene, shows dominant genetic pattern. Therefore, Ty1 genotype is heterozygous and phenotype is resistant to all commercial varieties. These nine varieties may be standard tomato varieties.

B-5-2. Analysis of pattern by application of SspI restriction enzyme

Since the resistance genotype has a SspI cleavage site (AATATT), a fragment of 224 bp and 386 bp is formed after restriction enzyme treatment. The deletion genotype is not deleted due to insertion / deletion polymorphism, The intercept is maintained.

As shown in FIG. 5, the TyI-SspI CAPS Marker was distinguished from the heterozygous state H with two strains of Genoyiping, S and Lee, respectively, by electrophoresis on 2.5% agarose gel. According to the pattern analysis, 10 heterozygous states were observed in 10 different cultivars, TY Winner, Daphneus, Deerose, Mamilio, TT Chrysanthemum, TY250, TY Star, TY Escort and TY Endorphin. In the cultivar, it was identified as S - susceptible. Most commercial varieties of the tomatoes are F1 varieties. Ty1, the resistance gene, shows dominant genetic pattern. Therefore, Ty1 genotype is heterozygous and phenotype is resistant to all commercial varieties. These 10 varieties may be standard tomato varieties.

From these results, it can be concluded that the varieties of the varieties of the present invention are susceptible to yellowing of the leaf cultivars, such as TYwyner, Daphneus, Deerose, Mamilio, TTchrist, TY250, TY escort, It is possible to judge resistance to leaf disease and to judge resistance even when the corresponding varieties are not marked as disease tolerance.

As described above, the results of the electrophoresis (Figs. 4 and 5) were analyzed to identify tomato varieties (strains) showing the same pattern, and a tomato sample having a pattern represented by amplification products cleaved by restriction enzymes, Resistant tomatoes.

4 and 5 are electrophoresis results using a primer set according to an embodiment of the present invention.

In the figure, the numbers 1 to 25 are the serial numbers representing the tomatoes in Table 3, and M is the 1 Kb DNA ladder. One alphabetic capital letter indicates that the group classified by the primer set is a group (S) Group (H). Among these groups, the H group can be judged as a yellowtail leaf blight resistant tomato.

The pattern of the tomato varieties belonging to the H group was determined to be a pattern of the standard tomato varieties resistant to the leafing sickle leaf disease, and B-1. To determine whether or not the tomato sample is resistant to leaf blight disease by comparing the obtained patterns with those obtained in the same manner as in (B-4) to (B-4). It is of course possible to utilize the result of the judgment to utilize for tomato breeding.

From these results, it can be seen that the primer sets, methods, and kits for judging the resistance of the leafy leafhopper resistant tomato of the present invention can determine the leafy leafhopper resistant tomatoes.

<110> Andong National University Industry-Academic Cooperation Foundation <120> Primer set to judge tomato resitant to tomato yellow leaf curl          disease, Method to judge          curl disease, and Kit to judge tomato resitant to tomato yellow          지유 <130> GP14018 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer of Ty1-IY F <400> 1 atgaagacaa aaactgcttc 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer of Ty1-IY R <400> 2 tcagggtttc acttctatga at 22 <210> 3 <211> 609 <212> DNA <213> Solanum lycopersicum <400> 3 atgaagacaa aaactgcttc tttgaagtgc tactttgtta ggtttgagtc cattggaacc 60 tgcgatgatg gagaatccta tgtattttct accaaaacaa tcagtcaagc aaggtgtaaa 120 ttcatgcatg tgcatatggt ttctaatatg gcaaaatatg cagccaggtt ggtctccttg 180 atagtcatgt tgagtattta ttgagctttc ttggcagata gtaatattat gcatacatgc 240 aggctttcct taattctatc aaagactatt aagcttcaag tggatcttga ttctgtcacc 300 attgaaagaa tcgaagatat actttgtcgg gtagcggctc tgtttcatat atttgggctt 360 cctgttgtga ttttctgata aatatgtcta gaaaatttgc ttatcctttt atttgattaa 420 tcgtaggatg aaaatggttg tattattcaa gatgaagacg gcgaacctcg tatacatact 480 gatggtactg gtttcatatc agaagattta gctatgcatt gtcccaaaga tttttcaaaa 540 gcagaatata taaaagatga aaattatgag gtatgcataa tttctttatt catagaagtg 600 aaaccctga 609 <210> 4 <211> 608 <212> DNA <213> Solanum lycopersicum <400> 4 atgaagacaa aaactgcttc tttgaagtgc tactttgtta ggtttgagtc cattggaacc 60 tgcaatgatg gagaatccta tgtattttct accaaaacaa tcagtcaagc aaggtgtaaa 120 ttcatgcatg tgcatatggt ttctaatatg gcaaaatatg cagccaggtt ggtctccttg 180 atagtcatgt tgagtattta ttgagctttc ttggcagata gtaatttatg catacatgca 240 ggctttcctt aattctatca aagacgatta agcttcaaac agatcttgat tctgtcacca 300 ttgaaagaat tgaagatata ctttgtcggg tagcggctct gtttcatata tttggacttc 360 ctgttgtgat tttctgataa atatgtctag aaaatttgct tatcctttta tttgattaat 420 cgtaggatga aaatggttgt attattcaag atgaagacgg cgaacctcgt atacatactg 480 atggtactgg tttcatatca gaagatttag ctatgcattg tcccaaagat ttttcaaaag 540 cagaatatat aaaagatgaa aattatgagg tatgcataat ttctttattc atagaagtga 600 aaccctga 608

Claims (7)

A primer set consisting of a nucleotide sequence of SEQ ID NO: 1 and a primer consisting of a nucleotide sequence of SEQ ID NO: 2,
Wherein the primer set for judging the resistance to yellowtail leafing disease is a genome of a tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3 on the basis of at least one base of the 226th base or 312th base in the nucleotide sequence of SEQ ID NO: To confirm whether the base sequence differs from the reference base at the corresponding position corresponding to the 226th base or the corresponding position corresponding to the 312th base in the DNA base sequence, Primer set for disease resistance tomato judgment. delete Corresponding to the 226th base in the genomic DNA sequence of the tomato sample corresponding to the nucleotide sequence of SEQ ID NO: 3, based on at least one base among the 226th base or 312th base in the nucleotide sequence of SEQ ID NO: Or at a corresponding position of at least one of the corresponding positions corresponding to the 312 th base, the difference being indicative of a difference from the reference base. 4. The method according to claim 3, wherein the confirmation is carried out using a primer set consisting of a nucleotide sequence of SEQ ID NO: 1 and a primer consisting of a nucleotide sequence of SEQ ID NO: 2, Tomato judging method. The method of claim 3,
The above-described method for determining the resistance to yellowtail leafroll disease
(A) Using a genomic DNA of the tomato sample as a template, a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a primer consisting of the nucleotide sequence of SEQ ID NO: 2 was amplified A sample processing step in which one amplification product is treated with a restriction enzyme; And
(B) a pattern analysis step of analyzing a pattern obtained by electrophoresis of the sample to be processed,
Wherein said restriction enzyme is TaqI or SspI. A primer set of claim 1, a restriction enzyme, and an amplification reaction performing reagent,
The restriction enzyme is TaqI or SspI. The method according to claim 6, wherein the amplification reaction reagent comprises a DNA polymerase, dNTPs, and a buffer.

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