KR101755231B1 - Molecular marker for selecting fusarium crown root rot resistance gene in tomato - Google Patents

Abstract

The present invention relates to a molecular marker for selecting a Fusarium crown root rot (FCRR) -resistant gene, and more particularly, to a primer set for identification of resistance to anomalous tomato in a tomato root region, Provides a method for identifying an arthritic resistance. By using these DNA markers, it is expected that they can be used for seed breeding programs of seed companies by rapidly and accurately cultivating muscle - resistant breeding varieties.

Description

{Molecular marker for selecting fusarium crown root rot resistance gene in tomato}

The present invention relates to a molecular marker for selecting an artificial resistance-resistant gene in a tomato root portion.

Tomato areas near manic (Fusarium crown root rot, FCRR) is Fusarium oxysporum f. sp . radicis - lycopersici (FORL), an infectious disease that afflicts or damages fruit before it becomes fruit. It is known that the gene Frl , which is resistant to FORL, is located near the long arm of chromosome 9. Fazio et al. (1999) The genetic map by creating markers using RAPD markers looking for a location close to the Frl, but said that these markers are positioned UBC655-UBC116-UBC194- Frl net, tomatoes muscle area manic-resistant plants Further studies on marker-phenotype association analysis are needed for marker-assisted selection (MAS) and cloning of resistance genes.

Korean Patent Laid-Open No. 10-2012-0121639 (published November 6, 2012)

It is an object of the present invention to provide a DNA marker composition, a primer set, and a method for distinguishing resistance to manure in a tomato root by using the DNA marker composition,

In order to achieve the above object, the present invention provides a DNA marker composition for discriminating resistance to Fusarium crown root rot (FCRR) represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The present invention also provides a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4, a primer set represented by SEQ ID NO: 5 and SEQ ID NO: 6, a primer set represented by SEQ ID NO: 7 and SEQ ID NO: (FCRR) resistance discrimination primer set comprising a primer set represented by SEQ ID NO: 10.

Also, the present invention provides a tomoskeletal anthrax resistance resistance kit comprising the primer set.

In addition, the present invention provides a method for determining the resistance to manure in a tomato root region using the primer set.

The present invention relates to a molecular marker for selecting a gene for resistance to anthrax in tomato root, and has developed a DNA marker capable of identifying a trait of a root portion required for cultivating a new variety of tomato, using a gene mapping method. By using these DNA markers, it is expected that they can be used for seed breeding programs of seed companies by rapidly and accurately cultivating muscle - resistant breeding varieties.

Figure 1 shows the sequence of PCR fragments amplified from AV107-4 and L3708 via the PNU-D4 primer. Single nucleotide polymorphisms (SNPs) are shown in blue and SNPs used in RFLP analysis are shown in green rectangles.
FIG. 2 shows the results of resistance analysis against Fusarium crown root rot (FCRR).
FIG. 3 is a graph oxysporum f. sp . radicis - lycopersici And the genetic and physical location of the 14 markers used in the association analysis of FORL.
4 (A) shows the results of the FORL-resistance test on tomato commercial varieties (F1 hybrid). Fig. 4 (B) shows that PNU-D4 is highly correlated with muscle area arthritis resistance in the above varieties.

The present inventors have identified the amplified nucleotide sequence of the RAPD marker located close to Frl and identified the position in the tomato reference genome and then selected and positioned genetically closely located markers on the Tomato EXPEN-2000 map The locus of Frl was observed more precisely and the present invention was completed.

The present invention provides a DNA marker composition for discriminating resistance to Fusarium crown root rot (FCRR) represented by SEQ ID NO: 1 or SEQ ID NO: 2.

SEQ ID NO: 1 is the nucleotide sequence of the PNU-D4 marker amplified in the FORL resistance strain AV107-4 ( S. lycopersicum ), and SEQ ID NO: 2 is the nucleotide sequence of the PNU-D4 marker amplified in the 'susceptible strain L3708 ( S. pimpinellifolium ) It is the base sequence of the D4 marker.

A primer set represented by SEQ. ID. NO. 3 and 4, a primer set represented by SEQ ID NO. 5 and SEQ ID NO. 6, a primer set represented by SEQ ID NO. 7 and SEQ ID NO. 8, or a primer set represented by SEQ ID NO. (FCRR) resistance discrimination primer set comprising a primer set to be displayed.

Preferably, the primer set set forth in SEQ ID NO: 3 and SEQ ID NO: 4 or the set of primers set forth in SEQ ID NO: 5 and SEQ ID NO: 6 are for amplifying restriction fragment length polymorphism (RFLP) . The primer set of SEQ ID NO: 3 and SEQ ID NO: 4 of the present invention is for amplifying the "PNU-T1212" marker, and the primer set of SEQ ID NO: 5 and SEQ ID NO: 6 amplifies the "PNU-D4" .

Preferably, the primer set set forth in SEQ ID NO: 7 and SEQ ID NO: 8 or the set of primers set forth in SEQ ID NO: 9 and SEQ ID NO: 10 are for amplifying cleaved amplified polymorphic sequence (CAPS) markers. The primer set shown in SEQ ID NO: 7 and SEQ ID NO: 8 is for amplifying the "PNU-100431" marker, and the primer set shown in SEQ ID NO: 9 and SEQ ID NO: 10 is for amplifying the "PNU-53169" marker.

In the present invention, a " marker " refers to a nucleotide sequence used as a reference point when identifying genetically unrelated loci. The term also applies to nucleic acid sequences that are complementary to a marker sequence, such as a nucleic acid used as a primer set capable of amplifying a marker sequence. The location of the molecular marker on the gene map is referred to as the genetic locus.

In the present invention, "molecular marker" or "DNA marker" can be usefully used because it can quickly distinguish cultivars without being affected by the cultivation environment or growth period of the crop. A molecular marker or a DNA marker is a method for expressing a polymorphism of an individual based on the nucleotide sequence difference of DNA, which is the essence of genetic phenomena. The marker is a microsatellite consisting mainly of repeating simple nucleotide sequences or a marker using gene sequences Or a Restriction Fragment Length Polymorphism (RFLP) probe or a Sequence Characterized Amplified Region (SCAR) marker.

In the present invention, "restriction fragment length polymorphism (RFLP) marker" means that a PCR-amplified DNA or gemomic DNA cleaved by a restriction enzyme is electrophoresed with agarose gel or Southern blotted, is a technique for detecting polymorphism of DNA using a probe. RFLP can be used to analyze the profiles of different DNAs. This difference in profiles means that DNA rearrangement caused by base substitution mutations due to changes in single nucleotide sequence, insertion, deletion, or inversion of genes. RFLP markers show relatively high polymorphism and homology and are highly reproducible. However, when Southern blot is required, a large amount of DNA is required and it takes a long time.

In the present invention, a " cleaved amplified polymorphic sequence (CAPS) marker "is a marker capable of analyzing a change in a site where a single nucleotide sequence is changed, such as a SNP, or a restriction enzyme generated by InDel . CAPS markers are PCR-amplified primers specific to loci, followed by restriction enzyme digestion and polymorphism analysis.

In the present invention, the "simple sequence repeat (SSR) marker" is also referred to as STR (Short Tandem Repeats), and refers to a sequence in which 1 to 6 bases are repeated on DNA. In general, SSR sequences can be used as a variety of markers in a gene and are also used in studies of replication or deletion of specific genes.

In the present invention, the term "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be amplified, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.

In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.

The present invention provides a tomato tomato muscle manure resistance discrimination kit including the primer set.

In addition to the above-described primer sets, conventional components included in the PCR kit may be included. Typical components included in the kit may be reaction buffers, polymerases, dNTPs (dATP, dCTP, dGTP and dTTP), and joins such as Mg2 +, and the like. A variety of DNA polymerases can be used in the amplification step of the present invention, including the Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species. Most of these enzymes can be isolated from the bacteria itself or commercially available.

On the other hand, when the primer set includes an RFLP marker or a CAPS marker, a restriction enzyme is required in addition to a primer set and a reagent for performing an amplification reaction. That is, the RFLP marker or the CAPS marker can be amplified, and then digested with the corresponding restriction enzyme to distinguish amplified products.

(1) separating DNA from tomatoes; (2) performing PCR using the separated DNA as a template and using the primer set; And (3) digesting the PCR-amplified product with restriction enzymes and analyzing the result.

Methods for isolating DNA from tomatoes can be performed by methods known in the art. In addition, PCR is a method of amplifying a target nucleic acid from a primer set that specifically binds to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

The method of the present invention comprises the step of analyzing said amplification product. Detection of the amplification product can be performed by capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. As one method of detecting the amplification product, capillary electrophoresis can be performed. Capillary electrophoresis can, for example, use an ABI Sequencer. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acryl amide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is performed, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter 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.

Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Plant material

To investigate the genetic association between molecular markers and FORL- resistance (genetic linkage or association), an F ₂ population using FORL resistant strains of AV107-4 (S. lycopersicum) and susceptible strains of L3708 (S. pimpinellifolium) Respectively. F ₁ generation was modeled as AV107-4, L3708 was artificially pollinated with a duplicate, and F ₁ individuals were self-fertilized to develop F ₂ generation. Using these F ₂ populations, the FORL resistance locus Frl And the marker genotyping was performed.

In order to analyze the linkage between marker and FORL-resistance in tomato commercial varieties (F1 hybrid), 17 F1 hybrid varieties were obtained from seed companies and pathogenicity test and molecular marker genotyping were performed in the same manner as in F2 group. In addition, for 42 varieties, molecular markers and association analysis were carried out using the information of the near-site manure disclosed by the seed company (29 resistant varieties, 13 susceptible varieties).

2. Muscle root disease  Pathology test

To determine the root portion manic resistance of the L3708 AV107-4 345 F ₂ of the object 17 and the commercial varieties that generated by using a (10 per breed object), the hospital pathology black inoculated and the onset also including the measured circumferential University Gangneung Lt; / RTI &gt;

For inoculation of the strain, FORL strain KACC 40031 was purchased from Korean Agricultural Culture Collection (KACC) and cultured in PDA medium at 25 ° C for 5 days. The hyphae were transferred to potato dextrose broth and cultured at 25 ° C for 7 days Lt; RTI ID = 0.0 &gt; rpm &lt; / RTI &gt; Mycelia were sieved using three layers of disinfected gauze, and the spore suspension was diluted with distilled water to a final concentration of 1 x 10 ⁷ conidia / mL. Tomato seeds were planted in a 96 hole plug tray filled with horticultural soil and cultivated in a greenhouse at 25 ± 5 ℃ for 3 weeks. Each seedling was then washed to remove soil from the roots, and the root was cut and inoculated by immersing in a FORL 1 x 10 ⁷ conidia / mL spore suspension for 15 minutes. The inoculated plants were transferred to a 15 cm diameter pollen of the greenhouse and maintained at about 90% humidity, 25 ± 5 ° C for 4 weeks. After 4 weeks, the roots of each plant were cut with a sharp knife, and the degree of FORL disease severity index (DSI) was evaluated from 0 to 3 according to the degree of browning of the cross section of the cross section.

0 = no browning inside the root and stem, 1 = weak browning inside the root, 2 = somewhat severe internal browning in the entire root, and 3 = severe internal browning over the lower stem adjacent to the soil from the root. The FORL resistance of each F ₂ subject was determined by using the DSI value, with 0 as resistance, and 1 to 3 as susceptibility.

3. Frl Relation RAPD Marker  analysis

Random Amplified Polymorphic DNA (RAPD) markers reported to associate with the FORL resistance gene, Frl , were evaluated for their plant material using PCR. The PCR mixture contained 40 ng of genomic DNA, 1.25 uM of primer, 1X PCR buffer, 0.2 mM dNTPs and 0.6 U of Taq polymerase (Solgent ™), using UBC # 194, UBC # 116 and UBC # 655 RAPD primer sequences in Fazio's paper. The PCR conditions were 94 ° C for 30 seconds, 92 ° C for 20 seconds, 36 ° C for 50 seconds, 72 ° C for 45 minutes, 72 ° C for 7 minutes, 1 &gt; C for 10 minutes. Electrophoresis was performed in 1.5% Tris-acetate EDTA (TAE) agarose gel at 160V for 1 hour and then stained with Ethidium bromide (Et-br).

Whereas aneunde domestic resistant varieties is the resistance between Bahru four DNA fragments reported in the UBC # 194 Frl close to the PCR results using the DNA is not observed, the resistance bands was confirmed at UBC # 116. The amplified DNA fragment of UBC # 116 was collected from agarose gel and purified using Expin ^™ Gel SV (GeneAll Biotechnology, Seoul, Korea) and sequenced in Genentech using the dye-termination method. Using the Basic Local Alignment Search Tool (BLAST), we identified the physical location of UBC116 on the Tomato reference genome ( http://solgenomics.net/ , ITAG2.3 release).

4. Marker - For phenotypic association analysis Marker  Development

Markers were selected from 40 cM to 55 cM in Tomato EXPEN-2000 map ( http://solgenomics.net/ ) based on the physical location of UBC # 116 Tomato reference genome. Of these markers genetically and physically related to UBC116, three RFLP markers with polymorphism were identified by treatment with two SSR markers that showed polymorphism after PCR amplification and restriction enzymes after PCR amplification in AV107-4 and L3708 The two SSR markers were located at 42 cM and 50.37 cM on the map as SSR70 and SSR237 respectively and the three RFLP markers were located at 48cM, 50cM and 52.1cM on the map as T1212, Clet-2-D4, and CD3, Respectively.

In order to test the polymorphism between AV107-4 and L3708 of the selected markers, the two SSR markers used the primer information disclosed in the Tomato EXPEN-2000 map as they were. However, for the three RFLP markers, the nucleotide sequence of the probe disclosed in the map was used The reference sequence was blasted to the reference sequence to identify the same sequence, and a primer was prepared so as to amplify a longer base sequence including the sequence.

PCR was performed using 20 ng of genomic DNA, 20 uL of each forward and reverse primer, 0.3M, 1X PCR buffer, 0.2 mM dNTPs and 0.6 U of Taq polymerase (Solgent, Daejeon, Korea) (1) for 5 minutes, 30 seconds at 94 ° C, 30 seconds at annealing temperature (AT, Table 1), 1 minute at 72 ° C for 7 minutes, 72 ° C for 7 minutes, minute. For SSR markers after PCR, PCR amplification products were stained with ethidium bromide (Et-br) for 1 hour at 160 V in 1.5% Tris-acetate EDTA (TAE) agarose gel and confirmed under UV light. The RFLP markers , PCR amplification products were treated with various restriction enzymes (NEB, Ipswich, Suffolk, England) and then electrophoresed with 2.5% Tris-acetate EDTA (TAE) agarose gel to obtain restriction enzymes that showed polymorphism between AV107-4 and L3708 Respectively.

Genomic DNA of AV107-4 and L3708 was cloned by SNP Genetics Inc. to develop a more precise Frl locus by developing additional molecular markers for these markers. (Seoul, Korea) were requested to analyze the Infinium platform (Illumina Inc., San Diego, CA, USA) of SolCAP Tomato SNP array and the annotation information of each SolCAP SNP was downloaded from the SGN database. A total of 8 SNPs from 5Mb to 72Mb of chromosome 9 were converted into CAPS markers using the CAPS designer software ( http://solgenomics.net/ ) (Table 1) . Two SNPs (solcap_snp_sl_100431 and 53169) on the physical location are located between PNU-D4 and SSR237, two SNPs (solcap_snp_sl_100181 and 100461) are located between SSR237 and PNU-CD3, and four SNPs (solcap_snp_sl_43011, 46840, 58307 and 63662) It was located between the ends of PNU-CD3 and chromosome 9. PCR amplification, restriction enzyme digestion and electrophoresis were performed in the same manner as in the above RFLP marker.

Table 1 shows the following. ^a SSR, simple sequence repeat; RFLP, restriction fragment length polymorphism; CAPS, Cleaved amplified polymorphic sequence.

^b Contrasting genomic sequence ITAG2.4 ( http://solgenomics.net/ ) Physical location of the top primer sequence.

^C Tomato EXPEN-2000 map ( http://solgenomics.net/ ) image position; -, unknown.

^d Tm, PCR annealing temperatures; TD, touchdown PCR

^e PCR fragment sizes specific for L3708 and A107-4 are indicated by "/" in parentheses.

< Example  1> Marker - For resistance association analysis Marker  Development

According to the report of Fazio et al. (1999), Frl gene has been mapped to the long arm of chromosome No. 9 position with RAPD markers and UBC655-UBC116-UBC194- Frl net UBC194 and is closely related to Frl and 5.1 cM . In the present invention, polymorphisms of these markers between AV107-4 and L3708 were observed. As a result, a polymorphism PCR band was observed only in the UBC116 and UBC655 primers, and the nucleotide sequence of the PCR band of UBC116 , which is closer to that of Frl, was cloned. The site of the reference genome (ITAG 2.4) was confirmed by blasting the obtained nucleotide sequence. As a result, the nucleotide sequence of about 400 bp was physically located at about 45 Mb of chromosome 9 and 50 cM genetically on the Tomato EXPEN-2000 map (Fig. 1). Two SSR markers (SSR70, SSR237) and three RFLP markers (PNU-T1212, -D4) that were polymorphic between AV107-4 and L3708 at 40 cM to 55 cM, the chromosomal map region predicted to contain the nucleotide sequence of the UBC116 marker developed, -CD3) were tested for the marker genotype of the F ₂ population (Table 1). In order to predict the position of Frl in more detail, eight CAPS markers (PNU-100431, -53169, -53169) were obtained by using 8 SolCAP SNPs (solcap_snp_sl_100431, 53169, 100181, 100461, 43011, 46840, 58307, 63662) ranging from 3.6 Mb to 72 Mb , -100181, -100461, -43011, -46840, -58307, -63662) were added to the F ₂ group (Table 1).

< Example  2> F ₂  In the group Marker - Resistance association analysis

Determine whether the resistance of the strain FORL (KACC 40031) of a total of 345 F ₂ generated on the object crosses the AV107-4 and L3708 and the Frl, compare the genotype of the 13 markers present on chromosome 9 genetic location Respectively. As a result of the FORL biochemical test, AV107-4 showed a high level of resistance with an average DSI of 0.2, while L3708 showed a high sensitivity of 2.38 2). In the F ₂ group, the ratio of the resistance individuals to the susceptible individuals was 3: 1 (χ ² = 0.29, P ( P < <0.05), confirming that the FORL resistance gene was dominant dominant.

Genotyping of F ₂ entities was performed using 13 markers (two SSR markers, three RFLP markers based on the Tomato EXPEN-2000 map, and eight CAPS markers based on the SNPs between AV107-4 and L3708). The SSR70 marker located at chromosome 9 at 3.62 Mb (Tomato EXPEN-2000 at 42 cM) of the Tomato reference genome showed a relatively low marker-resistance agreement of 89.5%. PNU-T1212 and PNU-D4, which are located at 5.09Mb and 6.09Mb (48.0cM and 50.0cM), respectively, showed 93.2% and 93.0% high marker-resistance agreement rates, respectively. Interestingly, the matching rate (92.7%) was 28.02Mb, 46.42Mb, 48.78Mb, and 56.94Mb (3.62Mb ~ 56.94Mb), respectively, compared to 92.7% for the next 25.17Mb PNU-100431 Were observed in PNU-53169, SSR237 (50.37 cM), PNU-100181, and PNU-100461. The tendency to maintain marker-resistance concordance in such a large region is that this region is included in the pericentromeric region where the genetic recombination or crossing-over is extremely suppressed and the genetic distance is maintained even if the chromosomal physical distance is distant . However, PNU-CD3, PNU-43011, PNU-46840, PNU-58307 and PNU-63662 located at the sub-areas of these markers (63.10 Mb, 64.52 Mb, 67.13 Mb, 68.87 Mb, 72.02 Mb (63.10 Mb to 72.02 Mb) The markers show that the concordance rate with the phenotype is sequentially lowered from 90.8% to 60.8%, which means that as the marker is located near the telomere of the chromosome long arm, the frequency of crossing between the marker and the resistant locus gradually increases (Fig. 3).

< Example  3> In commercial varieties Marker - phenotypic association analysis

Since the FORL resistance gene, Frl, is located within the linkage block where the frequency of chromosomal crossing is limited, it is difficult to find a more precise locus by saturating the marker in the F ₂ group obtained from the parent, We tried to test the relationship between Frl and markers in the group. In the present invention, 17 commercial cultivars were subjected to mycorrhizal pathology tests. 14 of the 17 inoculation the plants are resistant (DSI = 0), 3 dogs 5 shown in Susceptible (DSI> = 1) was consistent with the phenotypes disclosure in seed companies, showing a resistance and a high match rate from the F ₂ population Genotypes of these varieties were investigated and compared with the markers (PNU-T1212, PNU-D4, PNU-100431, PNU-53169 and SSR237). In addition, the other 5 susceptible varieties of 29 resistant and 13 susceptible varieties announced by the seed company were tested with the above five markers.

PNU-T1212, PNU-D4, PNU-100431, PNU-53169 and SSR237 distributed over approximately 41 Mb (5.09 Mb to 46.42 Mb) of chromosome 9 for 60 varieties (17 susceptibility and 43 resistance) the analysis of the genotype of the marker, F PNU-T1212 showed the results with different phenotypes and the highest concordance rate in the _second group have exhibited a low agreement rate of 100% in susceptible cultivars, 46.51% in the resistant varieties (Table 2 and Fig. 4) . On the other hand, PNU-D4 marker showed the highest concordance rate of 88% in susceptible varieties and 95% in resistant varieties, Suggesting that genetic recombination in the region between D4 and D4 occurred frequently. In the PNU-100431, PNU-53169 and SSR237 markers under the PNU-D4 marker, the agreement rate was further reduced (Table 2). Therefore, when various commercial cultivars were analyzed, crossing frequency higher than that of marker-F rl was confirmed in F ₂ group, and detailed Fr l locus could be guessed. The results of the present invention Frl gene is present in the pericentromeric region of the short arm where the chromosome crossing is greatly suppressed rather than existing in the previously reported long arm of chromosome 9. PNU-D4, which was developed in the present invention, has a very high correlation with mycorrhizal resistance in the F2 group and commercial cultivar group. Thus, the marker-assisted selection for cultivating the myopathic resistance- It is expected to be very effective for MAS.

Cultivar Seed Provider Phenotype Marker Genotype T1212 D4 100431 53169 SSR237 Alexander Deluxe PPS R S H H H H All Round Nongwoobio R R R R R R AS6 * Daeyoun R S R R R R AS7 * Daeyoun R S R R R R ASD-152 * Daeyoun R S H R R R ASD-312 * Daeyoun R S H H H H B Blocking * Koregon R H H R R R Barkus R S H H H H BetaAlexander PPS R S H H H H Block * Daeyoun R S H R R R Doctor Q Nongwoobio R R R R R R DOTAERANG DIA Koregon R S H H H H Dotaerang Gourmet Takii R S H H H H Dotaerang Master Koregon R S H H H H Dotaerang Season Koregon R S H H H H Fighting Takii R S H R R R Ganbarune 11 * Daeyoun R S H H H H Greenguard * Daeyoun R R R R R R Greensaver * Daeyoun R R R R R R Harmony R R R R R R High Power * Daeyoun R R H R R R Kyupirang Nongwoobio R S H H H H Magnet Sakata Korea R H R R R R MB * R H H R R H Prime Alexander PPS R S H H H H Rafito R H H R R R Rex AD * R S R R R R Shincheonggang R S H H H H SPECIAL Koregon R H H R R R Spider R R R R R R Spike 23 * Daeyoun R H R R R R Suhosin PPS R R R R R H Super 334 R H H H H H Super sun road Sakata Korea R H H H H S Superprime PPS R S H H H H SUPPORT Sakata Korea R H R R R R Tosama * R H H H H S TY Altorang Nongwoobio R S H H H H Ultra PPS R R R R R R Zuiken Sakata Korea R S H H H H Ace Ggul S S S S S S Biolight S S S S S R Charming S S H S S S Dream 900 * Daeyoun S S S H H H Galuxy S S S S S S House Doterang S S S H H H Kary S S S H H R Koko S S S H H S Marune TY * Daeyoun S H S H H H Mascara S S S H H R Olkeepper * S S H - R R Sinhunggwang S S S H H H Super Ace S S S S S S Super Doterang S S R H H R Tenten S S S H H S Tiara PPS S S S H H H Umgi S S S H H H

<110> Pusan National University Industry-University Cooperation Foundation <120> Molecular marker for selecting fusarium crown root rot resistance          gene in tomato <130> ADP-2015-0286 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 851 <212> DNA <213> Solanum lycopersicum L. <400> 1 gttatgaaaa tactcatata tctcaaattt ctgcatattc tcaccttttg gaatctttcc 60 acaaagtcta ttataactca cgttcaaaat ttgccacgga ttcttcgata ttattttcgg 120 tatactccca taaatcttat tatgactcaa atccaacctc cataacccct tcccaaacgt 180 caattttgac atatcgaatt caaatttatt ccgatccaat aacatttcaa acgtagtctt 240 atctttacca aacaaaaacg aaatatcccc ttcaagcatg ttccttgaca aatcaattgt 300 gtcaaaactc cacccagcaa atgaaattgg tacaatccct gtaagttggt tatgcccaag 360 gtaaagatac gttaaattgg gagctaattt gctaaacgac tccggtattg gtccagtcaa 420 tttgttccta tctaatcgta aaaattccaa atatggaagt tgcgatagcg aggctgggat 480 agttccaaca agcttattat acgacaaatt aatgtacgtt aagcttttca atttactaag 540 gacttcaggt actggtcctg aaatgtccga ttcactaatt ctgaagaaat tgagattcgt 600 ggcttaaca attgttgatg gaattggacc ggtgagatta cgtacattgt ggatactgaa 660 tttcgtaagg tatgtgagat ctccgatggc gggagagaga tatccagaga gattcatttt 720 ggagaaatcg atgagattaa tccggttcga tttttcatcg cattcgagag taggtccgta 780 ccagtcaatg cagcaatcag ttttgggatt ccaattaccc aaatcatcag gattacccag 840 agcttttttg a 851 <210> 2 <211> 851 <212> DNA <213> Solanum lycopersicum L. <400> 2 gttatgaaaa tactcatata tctcaaattt ctgcatattc tcaccctttg gaatctttcc 60 acaaagtcta ttataactca cgttcaaaat ttgccacgga ttcttcgata tgattttcgg 120 tatactccca taaatcttgt tatgactcaa ttccaacctc cataacccct tcccaaacgt 180 caattttgac atatcgaatt caaatttatt ccgatccaat aacatttcat acgtcgtctt 240 atctttacca aacaaaaacg aaatatcccc ttcaagcatg ttccttgaca gatcaattgt 300 gtcaaaactc cacccagcaa atgaagttgg tacaatccct gtaagttggt tatgcccaag 360 gtaaagatac gttaaattgg gagctaattt gccaaatgac tccggtattg gtccagttaa 420 tttgttccta tctaatcgta aaaattccaa atatggaagt tgtgatagcg aggctgggat 480 agttccaaca agcttattat acgacagatt aatgtacgtt aagcttttca atcgactaag 540 gacttcaggt actggtcctg aaatgtccga ttcactaatt ctgaagaaaa tgagattcgt 600 ggcttaaca attgttgatg gaattggacc ggtgagatta cggacattgt ggaaactgaa 660 tttcgtaagg tatgtgagat ctccgatggc gggagagaga tatccagaga gattcatttt 720 ggagaagtcg atgagattaa tccgattcga tttttcatcg cattcgagag taggtccgta 780 ccagtcaatg cagcaatcag ttttgggatt ccaattactc aaatcatcag gattacctag 840 agcttttttg a 851 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggaggagtta agtgcatggc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtgaccatcg gatcctttgc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cagctgaaag atgtcaccca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tgatcattta caaggcggca 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gatcactagt cgtcctggtc aa 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttcacgatgg tctttattcg t 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agagacggta catgctttga ca 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gttcatttgc atggcttcct 20

Claims (6)

A DNA marker composition for discriminating resistance to Fusarium crown root rot (FCRR) expressed in SEQ ID NO: 1 or SEQ ID NO: 2. A set of primers set forth in SEQ ID NO: 5 and SEQ ID NO: 6, wherein the set of primers for discriminating resistance to Fusarium crown root rot (FCRR) for amplifying the DNA marker of claim 1. delete delete A tomato tomato muscle aptamia resistance discrimination kit comprising the primer set according to claim 2. (1) separating DNA from the tomato;
(2) carrying out PCR using the separated DNA as a template and using the primer set according to claim 2; And
(3) a step of cleaving the PCR amplification product with a restriction enzyme and then analyzing the tomato amplification product.

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