KR20150110119A - Primer set to judge tomato resitant to verticillium wilt, Method to judge tomato resitant to verticillium wilt using the same, and Kit to judge tomato resitant to verticillium wilt using the same - Google Patents

Abstract

Provided is a primer set for identifying tomato resistant to Verticillium wilt, which includes a primer comprised of a base sequence in sequence number 1 and another primer comprised of a base sequence in sequence number 2. Moreover, provided are a method for identifying tomato resistant to Verticillium wilt using the same, and a kit for identifying tomato resistant to Verticillium wilt using the same. The present invention ensures the identification of tomato resistant to Verticillium wilt, thereby being applicable for plant breeding.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer set for judging half-life fungus disease, a method for judging half-life fungus disease resistance using the same, and a method for judging tomato fungus disease using the same, the same, and Kit to judge tomato resitant to verticillium wilt using the same}

The present invention relates to a primer set for judging half-falsified disease tolerance tomato, a method for judging half-falsified disease tolerance tomato using the same, and a half-counterfeit disease tolerance tomato judgment kit using the primer set, more particularly, The present invention relates to a primer set for determining the tolerance of a tomato with a half-counterfeit bottle, a half-counterfeit bottle determination method using the half-finger counterfeit bottle, and a half-counterfeit disease tolerance tomato determination kit using the same.

Verticillium wilt is a disease caused by tomato, Verticillium dahliae and Verticillium alboatrum pathogens. Fungal disease is a typical soil infectious disease.

In order to cultivate Tomato varieties having tolerance to diseases such as dwarfism, it is necessary to develop a technology for judging tomatoes having tolerance to dwarfism.

Ve1, a resistance gene for tomato dwarfism, was identified in 2001 by Kawchuk et al., And resistance-related function analysis by gene sequence differences was performed by Fradin et al. (2009). The Ve1 gene has been shown to encode a cell surface receptor protein as an extracellular leucine-rich repeat (eLRR) receptor-like protein.

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.

 Fradin, E. F., Zhang, Z., Juarez Ayala, J. C., Castroverde, C.D., Nazar, R.N., Robb, J., Liu, C.M. and Thomma, B.P. (2009) Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol. 150: 320-332  (2001) Tomato & disease resistance genes encode cell surface-like. ≪ Desc / Clms Page number 2 > receptors. Proc Natl Acad Sci U S A. 98 (11): 6511-5

One of the problems to be solved by the present invention is to provide a primer set for half-counterfeit disease tolerance tomato determination.

Another problem to be solved by the present invention is to provide a method for judging half-falsified disease tolerance tomatoes.

Another object to be solved by the present invention is to provide a half-counterfeit disease tolerance 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 half-forgery disease tolerance in a tomato comprising a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2.

The antifungal potato-resistant tomato may be one having a half-forgery-fungus resistance gene.

The antifungal resistance gene may be a gene for Lycopersicon esculentum verticillium wilt disease resistance protein (Ve1).

The tomato semicircular canine resistance resistant protein 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: 4.

In addition, the present invention provides a method for judging whether a tomato is a half-forgery disease tolerant tomato using a primer set of the present invention.

The method for determining resistance to half-counterfeit disease includes: (A) a sample processing step in which a genomic DNA of a tomato sample is used as a template and an amplification product amplified using the primer set of the present invention is treated 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 not represented by the restriction enzyme-cleaved amplification product.

In addition, the pattern analysis step may include a determination step of determining a tomato sample having a pattern represented by an amplification product not cleaved by the restriction enzyme as a half-forgery disease 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 Oyama, Dotantang, TY Winner, Daphnis, Deerose, Rhapsody, Madison, Shinkukui, Nurimari No.2, TY250, TY Star, or TY Escort.

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.

Further, the present invention provides a half-counterfeit disease tolerant 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 the restriction enzyme shown in Table 1.

The amplification reaction-performing reagent may include a DNA polymerase, dNTPs, and a buffer.

INDUSTRIAL APPLICABILITY The present invention has an effect of being able to judge a half-counterfeit disease-tolerant tomato and utilize it for breeding.

FIG. 1 is a view for explaining a method for judging half-forgery disease tolerance tomato, which is one embodiment of the present invention.
2 is a diagram showing a base sequence for illustrating a primer set which is an embodiment of the present invention.
3 is a diagram 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 anti-counterfeit disease, and includes selection, selection, and selection.

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. By analyzing such a pattern, it is possible to judge whether or not the tomato is a half-counterfeit bottle disease endemic disease.

Therefore, one embodiment of the present invention can be used to judge half-fake counterfeit tomatoes. Half-fake Disease Disease-resistant tomatoes may have a half-life counterfeit-resistant gene. In this case, the antifungal resistance gene may be a gene of Lycopersicon esculentum verticillium wilt disease resistance protein (Ve1), and the tomato antifungal resistance gene may have a sequence of SEQ ID NO: 3.

The tomato varieties subject to the half-forgery disease tolerant tomato determination are not limited thereto, but may be one or more selected from the tomato varieties described in Table 2, for example.

[Table 2] 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 a CAPS marker, and the CAPS marker can be designed from the gene sequence of Lycopersicon esculentum verticillium wilt disease resistance protein (Ve1) described in Table 3 below have. The CAPS marker may have CAPS, as shown in FIG. That is, it may not be cleaved or cleaved by a restriction enzyme due to the SNP present depending on whether it is a half-counterfeit disease resistant tomato or a half-counterfeit disease resistant tomato. 2 is a diagram showing a base sequence for explaining a primer set. In the figure, the undercuts indicate cuts or cuts (uncut) depending on whether the restriction enzyme cleavage is on top or not. In Fig. 2, Ve1 represents a half-counterfeit disease tolerance gene, and ve1 represents a half-forged disease susceptibility gene. As shown in the figure, the gene of the half-fake counterfeit resistant tomato has a restriction enzyme-cleavage site (TCTAGA) as indicated by an underline (solid line), and the disease resistance gene does not have a cleavage site as indicated by an underline (dotted line). That is, the cleavage site of the heterologous gene is a state in which C is deleted from the corresponding site of the disease-suppressing gene (TCTCAGA). Since such a site is a site that directly causes a difference in resistance and dysfunction, the primer of the present invention which can judge whether or not such a site is mutable can be used to directly judge the half-forgery disease tolerant tomato .

The CAPS marker may have the nucleotide sequence shown in Table 4. The nucleotide sequence shown in Table 4 is a part of the tomato semicircular canine resistance gene (Ve1) gene sequence, and can be a nucleotide sequence amplified by the primer described in Table 1 and having a site which is not cleaved by the restriction enzyme shown in Table 1 have. Antifungal Disease Diseased tomatoes may have a restriction enzyme cleavage site. In other words, the CAPS marker is able to distinguish between endemic and resistant breeds for the half-fake disease according to the restriction enzyme cleavage.

[Table 3] Tomato half-length fake disease resistance gene gene sequence

[Table 4] Marker sequence

In addition, a primer set, which is one embodiment of the present invention, can be used to perform a half-counterfeit-resistant tomato determination method for determining whether or not the half-counterfeit bottle is a potentially resistant tomato.

Hereinafter, an embodiment of the method for judging disease resistance of a half-counterfeit bottle according to the present invention will be described in more detail.

Specifically, it is possible to judge whether or not the tomato is a half-forgery infectious disease tomato by a method including (A) a sample processing 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 enzymes may be those listed in Table 1.

The pattern analysis may be to determine whether the amplified product is a pattern exhibited by the amplified product not cleaved 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 not cleaved by a restriction enzyme as a half-counterfeit disease resistant 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 2. Preferably, the standard tomato varieties may be one or more selected from Oyama, Dotantang, TY Winner, Daphne, Deerose, Rhapsody, Madison, New Moonwater, Nurimari No.2, TY250, TY Star, or TY Escort. These varieties have been identified as potentially 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 .

As described above, the pattern obtained by electrophoresis after the restriction enzyme treatment shows a different pattern depending on whether it is disease tolerance or dysplasia.

That is, by analyzing the band pattern obtained by separating the fragments obtained by restriction enzyme treatment using a gel electrophoresis apparatus, it is possible to judge whether the fragments are semi-falsifiable disease resistant tomatoes.

In addition, the primer set, which is one embodiment of the present invention, may be included in the antifungal pot disease tolerance tomato judgment kit.

Hereinafter, an embodiment of the antifungal disease tolerance tomato determination kit according to the present invention will be described in detail.

Specifically, one embodiment of the antifungal disease tolerance tomato determination kit according to the present invention may include a primer set, a restriction enzyme, and a reagent for performing an amplification reaction, which are one embodiment of the present invention.

The restriction enzyme may be the restriction enzyme shown in Table 1.

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

In addition, the anti-counterfeit disease tolerance 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.

In the embodiment of the present invention, the items mentioned in the primer set for judging the half-counterfeit disease, the method for judging the half-fake counterfeit tomato and the half-finger counterfeit disease tolerance tomato determination kit are applied to the same scope unless they are mutually contradictory.

Hereinafter, a primer set for half-forgery disease tolerance tomato determination, a half-fake counterfeit disease tolerance tomato determination kit, and a half-fake counterfeit disease tolerance tomato determination method, which is one embodiment of the present invention, will be described in detail below.

The tomatoes used in the specific examples are those shown in Table 2. Such tomatoes were obtained from the seed companies listed in Table 2, unless otherwise noted.

≪ Specific Example A > A primer set

The CAPS marker design was based on the nucleotide sequence information of the Ve1 resistance gene described in Fradin et al. (2009), published in 2009 (Fradin, EF, Zhang, Z., Juarez Ayala, JC, Castroverde, , RN, Robb, J., Liu, CM and Thomma, BP (2009) Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1 Plant Physiol., 150: 320-332). Among the differences in the nucleotide sequences of Ve1 (GenBank accession no. AF272367) and the heterologous gene ve1, 1bp deletion change (TCAGAG), which directly causes the difference in resistance and dysfunction, -> TAGAG) targeting CAPS marker design.

SNPs showing differences in nucleotide sequence are shown by solid lines and dotted lines in FIG. 2, and CAPS markers are as shown in Table 4 and FIG. The primer length was made with F 23mer and R 23mer, and the primer was prepared by specifying the Tm value at 55 ° C.

Specifically, designed primers and restriction enzymes are as shown in Table 1. 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 primers set forth in Table 1 prepared by the method described above may be included to prepare primer set for half-counterfeit disease resistance test for tomatoes or half-counterfeit disease resistant tomato test kit.

≪ Specific Example B >

B-1. Genomic DNA extraction

The tomatoes listed in Table 2 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 growing 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.

B-2. Amplification (PCR)

The PCR was carried out in the same manner as in Example 1 except that the PCR was carried out using 2 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 and 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.

B-3. Restriction enzyme treatment

PCR product 10ul of the total volume of 25ul was electrophoresed to confirm the PCR amplification. 2ul of the restriction enzyme listed in Table 1, 2ul of the buffer and 2ul of the third distilled water were added to 15ul of the PCR product and mixed at 37 ° C for 1 hour Respectively. That is, the presence of PCR amplification of approximately 743 bp fragment was confirmed by electrophoresis before restriction enzyme treatment, followed by electrophoresis using XbaI restriction enzyme at 37 ° C. for 1 hour.

B-4. Electrophoresis

Genomic DNA was prepared with agarose gel (2.5%) considering the expected fragment length. Gel was stained with EtBr after electrophoresis and PCR amplification and genotyping were performed using Gel Doc 2000 (BIO-RAD, Hercules, Respectively. Fig. 3 shows the results of electrophoresis.

B-5. Pattern analysis

Since the resistance genotype does not have the XbaI cleavage site (TCTAGA), the 743 bp fragment is retained after the restriction enzyme treatment and the heterozygous genotype is 431 bp and 311 bp due to the presence of XbaI cleavage site by insertion / deletion (InDel) The intercept is generated. As a result of electrophoresis on 2.5% agarose gel, the VeI-IY CAPS marker was divided into three types of heterozygous H: genotyping, S, infectious R, and heterozygous. As a result of the analysis by pattern, it was revealed that S 9, B, R 11 and H 4 were heterozygous. Most commercial varieties of tomatoes are F1 varieties, and Ve1, which is a resistance gene, shows a dominant genetic pattern. Therefore, it was confirmed that Ve1 genotype is heterozygous or homozygous and phenotype is resistant to commercial varieties. From these results, it is possible to judge the disease susceptibility of the varieties for which the corresponding varieties are indicated to be infectious disease, such as Oyama, Dotagang, TY Winner, Shinkuu, and TY250, It is also possible to judge the disease tolerance even if it is not indicated whether or not the counterfeit disease is counterfeit.

As described above, the analysis of the electrophoresis results (FIG. 3) can be used to classify tomato varieties (strains) exhibiting the same pattern, and a tomato sample having a pattern represented by amplification products not cleaved by restriction enzymes, Tomato can be judged.

FIG. 3 is a result of electrophoresis using a primer set according to an embodiment of the present invention.

In FIG. 3, the numerals 1 to 24, which are integers, denote the serial numbers representing the tomatoes in Table 2, and M denotes the 1 Kb DNA ladder. One upper case letter indicates the groups classified by the primer set as the redundant group (S) ), And a group (H) having both the disease resistance and the resistance. Among these groups, the R group can be judged as half-forgery disease resistant tomato.

The pattern of the tomato varieties belonging to the R group was determined to be a pattern of the half-counterfeit disease tolerance standard tomato variety, and the B-1. To B-4. By comparing the obtained patterns, it is possible to judge whether or not the tomato samples are anti-counterfeit disease disease resistance. 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 antifungal tomatoes can be judged by the primer sets, methods, and kits for determining the antifungal potion of the present invention.

<110> Andong National University Industry-Academic Cooperation Foundation <120> Primer set to judge tomato resitant to verticillium wilt, Method          to judge tomato resitant to verticillium wilt using the same, and          Kit to judge tomato resitant to verticillium wilt using the same <130> GP14017 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer of Ve-IY F <400> 1 cgaacttgac tacattgacc ctg 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer of Ve-IY R <400> 2 cagtcttgaa aggttgctca gcc 23 <210> 3 <211> 4875 <212> DNA <213> Solanum lycopersicum <400> 3 gtgattgatt ttttttttgg tatgtctttt tgttgactta ctcaatgttt tgcagaaatc 60 catagattaa ctaacaaaaa cttgcaattt cagcagtccc ctgtcaacat gaaaaataat 120 tcaatatacg gagttattcg ctaaagcgga ggctatgatt catctgaacc cttttcaacg 180 aaaaattaca ctatgacaaa tttattctaa accggtgtat cattttcatg aatgaagcaa 240 gaccaaagag gagacttcca caagtgttgt gcaggagtca atgaatgaga atcttcagag 300 tcttctacac aatgaaccaa aagggctaac atcaaacgag gagactagga ggtaattcaa 360 tctctacaaa tacataatta ttttttcgca ttttttatac gtgattaaaa ttatactttg 420 tgtgtatata aaaaatatta aacagtaaat ttaagttaaa ggtataaatc aaaataactt 480 ccactcatat ataggccata aatgaggtaa ttaattttca tcatatttat aaaaatatct 540 cctttcacac ttgaaagtga tcatttagtt gactgttgat agaacataag ccttacccag 600 aaaatgacag attgttgatg atcaaataca ctaaattaca ttatagtagt tatgtaaaat 660 atcgtactaa acacaacatt aattaaaagt agaaaaaaag agtggaataa caagcactcg 720 agtagaaaaa taaatcatcc ttcctataag tgttttgcta tccggttaaa taaatatata 780 tgtatattaa tttatgaacc accaataaaa ttgattgttg gcttaatggt aagtaaggac 840 ccttataaat gcttcccaca ccagtgtttt aaaaaaagga aacatgtttt gcaatatttt 900 tctctttcta tcctaaagtt agaattctca aacaacttct gtagttacaa ttacaacctt 960 tcggaaatat ccaataaaat tgaaacgata cacaaaagat tagcatgacc tttactcaaa 1020 gatgacgaca tatacaaatt gagaaatgat taaacagaga aattaaaaca aagaagaaag 1080 aaataagcct actatttttc tacacggttg aatgttcttc ttcttctaat tttttcttgt 1140 ggaatttcta catctttttt ttttaaattt ttggtctcca actaaaccag ccccagtttg 1200 gctagtgcta tttttttttt gttgactcta gtggaagtct cctaaatttt tctcttcctc 1260 tggtcttttg gtagattcaa gtttttaaaa agtcagaaat gtatcacagt agtacttaga 1320 tatgaaaatt ctaaccatac aattccaatc atattaacaa tttcataaat gaagcaagac 1380 caaagaggag acttcaaagt cttccacaga atgaaccaaa agggctaaca acaaacaagt 1440 ttttaagttc cttcgaactg cataactgag tcatattcaa gctaacaagt tgcatgaaaa 1500 tgatggcaac tctgtacttc cctatggttc tcttgattcc ctcgtttcaa atcttatcag 1560 gataccacat tttcttggtt tcctctcaat gccttgacga tcaaaagtca ttgttgctgc 1620 agtttaaggg aagcctccaa tatgattcta ctttgtcaaa gaaattggca aaatggaacg 1680 acatgacaag tgaatgttgc aattggaatg gggttacatg caatctcttt ggtcatgtga 1740 tcgctttgga actggatgat gagactattt ctagtggaat tgagaattct agtgcacttt 1800 tcagtcttca atatcttgag agcctaaatt tggctgacaa catgttcaat gttggcatac 1860 cagttggtat agacaacctc acaaacttga agtacctgaa tttatccaat gctggttttg 1920 tcgggcaaat tcctataaca ttatcaagat taacaaggct agttactctt gatctctcaa 1980 ctattctccc tttttttgat cagccactta aacttgagaa tcccaatttg agtcatttca 2040 ttgagaactc aacagagctt agagagcttt accttgatgg ggttgatctt tcgtctcaga 2100 gt; gtgattgtca aatttcaggc cctttggatg aatcactttc taagcttcac tttctctctt 2220 ttgtccaact tgaccagaac aatctctcta gcacagttcc tgaatatttt gccaatttct 2280 cgaacttgac tacattgacc ctgggctctt gtaatctaca gggaacattt cctgaaagaa 2340 tctttcaggt atcagtttta gagagtttgg acttgtcaat taacaagttg cttcgtggta 2400 gtattccaat ttttttccga aatggatctc tgaggaggat atcactaagc tacaccaact 2460 tttccggttc attaccagag tccatttcga accatcaaaa tctatccagg ttagagcttt 2520 ctaattgcaa tttctatgga tcaatacctt ccacaatggc aaaccttaga aatcttggtt 2580 atttggattt ctccttcaac aatttcactg gttctatccc atattttcga ctgtccaaga 2640 aactcaccta cttagacctt tcacgtaatg gtctaactgg tctcttgtct agagctcatt 2700 ttgaaggact ctcagagctt gtccacatta atttagggaa caatttactc agcgggagcc 2760 ttcctgcata tatatttgag ctcccctcgt tgcagcagct ttttctttac agaaatcaat 2820 ttgttggcca agtcgacgaa tttcgcaatg catcctcctc tccgttggat acagttgact 2880 tgacaaacaa ccacctgaat ggatcgattc cgaagtccat gtttgaaatt gaaaggctta 2940 aggtgctctc actttcttcc aacttcttta gagggacagt gccccttgac ctcattggga 3000 ggctgagcaa cctttcaaga ctggagcttt cttacaataa cttgactgtt gatgcaagta 3060 gcagcaattc aacctctttc acatttcccc agttgaacat attgaaatta gcgtcttgtc 3120 ggctgcaaaa gttccccgat ctcaagaatc agtcatggat gatgcactta gacctttcag 3180 acaaccaaat attgggggca ataccaaatt ggatctgggg aattggtggt ggaggtctca 3240 cccacctgaa tctttcattc aatcagctgg agtacgtgga acagccttac actgcttcca 3300 gcaatcttgt agtccttgat ttgcattcca accgtttaaa aggtgactta ctaataccac 3360 cttgcactgc catctatgtg gactactcta gcaataattt aaacaattcc atcccaacag 3420 atattggaaa gtctcttggt tttgcctcct ttttctcggt agcaaacaat ggcattactg 3480 gaataattcc tgaatccata tgcaactgca gctaccttca agttcttgat ttctctaaca 3540 atgccttgag tggaacaata ccaccatgtc tactggaata tagtacaaaa cttggagtgc 3600 tgaatcttgg gaacaataaa ctcaatggtg ttataccaga ttcattttca attggttgtg 3660 ctctacaaac attagacctc agtgcgaata acttacaagg caggctgcca aaatcgattg 3720 tgaattgtaa gttgttggag gtcctgaatg ttggaaataa cagacttgtt gatcatttcc 3780 catgcatgtt gaggaactca aacagtctga gggtcctagt cttgcgctcc aataaattct 3840 atggaaatct tatgtgtgat gtaaccagaa atagctggca gaatctccag atcatagata 3900 tagcttccaa caacttcact ggtgtgttga atgcagaatt cttttcaaat tggagaggaa 3960 tgatggttgc agatgattac gtggagacag gacgcaatca tatccagtat gagttcttac 4020 aactaagtaa attgtactat caggacacag tgacattaac catcaaaggc atggagctgg 4080 agcttgtgaa gattctcagg gtcttcacat ctattgattt ctcttccaat agatttcaag 4140 gagcgatacc agatgctatc gggaatctca gctcacttta tgttctgaat ctgtcacaca 4200 atgcccttga gggaccaatc ccaaaatcga ttgggaagct acaaatgctt gaatcactag 4260 acctgtcaac aaaccacctg tccggggaga tcccatcaga gcttgcaagt ctcacattct 4320 tagcagcttt gaacttatcg ttcaacaaat tgtttggcaa aattccatca actaatcagt 4380 ttcaaacatt ctcagcagat tcctttgaag gaaacagtgg cctatgcggg ctccctctca 4440 acaacagttg tcaaagcaat ggctcagcct cagagtccct gcctccacca actccgctac 4500 cagactcaga tgatgaatgg gagttcattt ttgcagcagt tggatacata gtaggggcag 4560 caaatactat ttcagttgtg tggttttaca agccagtgaa gaaatggttt gataagcata 4620 tggagaaatg cttgctttgg ttttcaagaa agtgattatt aaacccataa ataatgagtt 4680 tattcttgga gtgttttgtt ttaaataaac aacaggataa ggaaaatcaa gttaataagc 4740 tcgcagaaca tgattgttat ttcctttgat gaatgtatac aattttcaat attggttctt 4800 caaccataac cgcaggctaa ctgtcagttg ttggaagtcc tgaattttgg aaatgacata 4860 catttttata gtttc 4875 <210> 4 <211> 743 <212> DNA <213> Solanum lycopersicum <400> 4 cgaacttgac tacattgacc ctgggctctt gtaatctaca gggaacattt cctgaaagaa 60 tctttcaggt atcagtttta gagagtttgg acttgtcaat taacaagttg cttcgtggta 120 gtattccaat ttttttccga aatggatctc tgaggaggat atcactaagc tacaccaact 180 tttccggttc attaccagag tccatttcga accatcaaaa tctatccagg ttagagcttt 240 ctaattgcaa tttctatgga tcaatacctt ccacaatggc aaaccttaga aatcttggtt 300 atttggattt ctccttcaac aatttcactg gttctatccc atattttcga ctgtccaaga 360 aactcaccta cttagacctt tcacgtaatg gtctaactgg tctcttgtct agagctcatt 420 ttgaaggact ctcagagctt gtccacatta atttagggaa caatttactc agcgggagcc 480 ttcctgcata tatatttgag ctcccctcgt tgcagcagct ttttctttac agaaatcaat 540 ttgttggcca agtcgacgaa tttcgcaatg catcctcctc tccgttggat acagttgact 600 tgacaaacaa ccacctgaat ggatcgattc cgaagtccat gtttgaaatt gaaaggctta 660 aggtgctctc actttcttcc aacttcttta gagggacagt gccccttgac ctcattggga 720 ggctgagcaa cctttcaaga ctg 743

Claims (6)

A primer set comprising a primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2. 2. The primer set of claim 1, wherein the primer set is for amplification of a fragment amplification polymorphism sequence (CAPS). A method for judging whether or not a tomato is a semi-falsifiable disease-resistant tomato using the primer set of claim 1. The method of claim 3,
The antifungal pot disease tolerant tomato determination method
(A) a sample processing step in which a genomic DNA of a tomato sample is used as a template and an amplification product amplified using the primer set of claim 1 is treated with a restriction enzyme; And
(B) a pattern analysis step of analyzing a pattern obtained by electrophoresis of the sample to be treated. A half-counterfeit disease tolerant tomato determination kit comprising the primer set of claim 1, a restriction enzyme, and an amplification reaction-performing reagent. 6. The antifungal tomato judging kit according to claim 5, wherein the restriction enzyme is the restriction enzyme described in Table 1, and the amplification reaction reagent comprises DNA polymerase, dNTPs, and a buffer.

Images