KR101464247B1 - Single nucleotide polymorphism marker for selecting fusarium wilt-resistant or sensitive cabbage cultivar and uses thereof - Google Patents

Abstract

The present invention relates to SNP markers for screening resistant varieties of cabbage wilt disease, and their use. When the SNP markers of the present invention are applied to cabbage crops, cabbage varieties resistant to wilt of cabbage wilt can be efficiently identified and cultivated. Cost and effort, and the spread of wilt resistant cabbage varieties is expected to be very useful because it enables production and supply of high quality cabbage to farmers and consumers.

Description

[0002] SNP markers for the selection of resistant or susceptible varieties of cabbage wilt, and uses thereof. [0002]

More particularly, the present invention relates to a SNP marker for isolating a resistant or susceptible varietal of cabbage wilt, and more particularly, to a method for screening a susceptible strain of cabbage wilt disease or susceptible varieties comprising at least 8 nucleotide polymorphisms (SNPs) located at the 288th base of the cabbage CYP38 ( BoCYP38 ) A primer, a probe and a microarray for discriminating a fusarium wilt resistant breed or susceptible breed comprising a polynucleotide consisting of consecutive nucleotides or a complementary polynucleotide thereof, a primer, a probe and a microarray, A kit for selecting cabbage wilt-resistant varieties containing the reagent for selecting the resistant varieties of cabbage wilt, and a method for selecting the resistant varieties of cabbage wilt.

Cabbage wilt (fusarium wilt) is a soil-borne fungal disease and is one of the main causes of inhibition of cabbage production. Cabbage breeding researchers are developing robust resistant varieties with widespread time and cost, and the development of molecular markers for wilt disease is an urgent and important issue in addressing such difficulties.

Molecular markers have the advantage that they can be detected in all tissues by differences in DNA sequence and are not affected by environmental influences and multiple expression of genes. Genomic genetic mapping of major crops is currently underway, and molecular markers are being used in the selection process of breeding. Numerous molecular markers have been developed for the study of plant breeding, and techniques and laboratory equipment for massively verifying and identifying the developed molecular markers are being developed very quickly. A variety of molecular labeling technologies such as Random Amplified Polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), Simple Sequence Repeat (SSR) and single nucleotide polymorphism (SNP) . The use of a codominant marker in the molecular markers can discriminate homozygous of the gene, and it is possible to carry out a search before the expression of the trait, thereby enabling rapid discrimination in breeding.

SNPs are single nucleotide differences that appear in DNA between individuals and exist across a wide variety of species genomes. Frequent SNPs appearing in plant genomes enable gene mapping, marker assisted breeding and map-based cloning (Batley et < RTI ID = 0.0 > al . , Plant Physiology 132: 84-91 (2003)). SNPs are the most common genomic markers, and various SNP detection methods and experimental equipments have been developed in order to detect such SNPs considering the easiness and cost of the experiment (Gupta et al . , Curr Sci. 80: 524-535 (2001)).

The present invention provides a molecular marker identification method that can be easily performed in a laboratory using PCR allele-specific amplification method, and can save cost and time. The present inventors obtained a DNA sequence having resistance to wilt of cabbage ( Brassica oleracea ) and SNPs in susceptible varieties, developed a primer set for amplifying a DNA region containing SNP, and performed genotyping assay ) Were used for the identification of resistance to cabbage wilt disease and susceptible varieties.

Korean Patent Laid-Open Publication No. 2012-0121499 discloses' Biomarker for early selection of a domestic cabbage cultivar and its use ', Korean Patent Publication No. 2008-0043164 discloses' Chinese cabbage ≪ / RTI > discloses a method for identifying a root-knot disease resistance gene. However, as in the present invention, SNP markers for selecting cabbage wilt-resistant varieties and their uses have not been disclosed.

The present invention is derived by the request as described above, the present inventors cabbage wilt pathogen on cabbage wilt resistant varieties and susceptible varieties (Fusarium oxysporum f. sp . After processing the conglutinans) to analyze protein expression BoCYP38 proteins were identified that involved the cabbage wilt resistance, to develop SNP markers to give wilt resistance through BoCYP38 genetic analysis to easily determine the cabbage wilt resistant and susceptible varieties The present invention has been completed.

In order to solve the above problems, the present invention provides a polynucleotide consisting of 8 or more consecutive nucleotides comprising SNP (single nucleotide polymorphism) located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) Primers, probes and microarrays for discriminating between fusarium wilt resistant cultivars or susceptible varieties comprising a polynucleotide of the present invention.

The present invention also provides a kit for screening cabbage wilt varieties resistant to cabbage wilt, comprising the primer and a reagent for carrying out an amplification reaction.

The present invention also provides a method for selecting a cabbage variety having resistance to cabbage wilt disease using the primer.

When the SNP marker according to the present invention is applied to cabbage crops, cabbage cultivars resistant to wilt of cabbage can be efficiently identified and cultivated, which is expected to be very useful for saving time, cost, and labor required for breeding. In addition, it is expected that the spread of the wilt resistant cabbage cultivars cultivated by the method of the present invention will enable production and supply of high quality cabbage to growers and consumers.

Fig. 1 shows the result of 2D electrophoresis of total proteins of cabbage wilt-resistant rice varieties (KR) and susceptible varieties (HY). The wilt-resistant and susceptible cabbage cultivars were germinated in soil, grown for 14 days, treated with 2 × 10 ⁷ spores / ml of cabbage wilt pathogen ( F. oxysporum f. Sp . Conglutinans ) Total protein was isolated. The separated total protein was stained with CBB (colloidal coomassie brillant blue) and scanned to obtain a gel image after 2D electrophoresis.
Fig. 2 shows the result of MALDI-TOF / TOF MS analysis after enzyme digestion of 2D gel. Proteins 20, 21, 22, 37, 38 and 39 of the 80 protein spots on the gel were identified as BoCYP38 proteins.
Figure 3 shows the BoCYP38 protein (spot 20, 21, 22) and the susceptible variant BoCYP38 protein (spot 37, 38, 39), which were confirmed as a result of analysis of 80 proteins. Image Master Program ver. 6. Comparison of the gel spot size using data analysis showed the difference in expression patterns between the two cultivars. Protein spot 21 was found to be phosphorylated in the F. oxysporum rather than in the distilled water treatment mock, and protein spot 22 showed a decrease in protein expression in the pathogens.
Fig. 4 shows the result of comparing the genomic sequence of the cabbage wilt resistant strain (BoCYP38R) and the susceptible strain (BoCYP38S). 34 SNPs (*) were identified, and SNPs at 288 bp were used as markers to determine wilt resistance and susceptibility.
Fig. 5 shows a schematic diagram of a primer designed for the BoCYP38 SNP marker capable of discriminating resistant and susceptible varieties of cabbage wilt.
FIG. 6 shows the results of 3% agarose gel after conducting genomic DNA PCR of cabbage wilt resistance and susceptible varieties using the designed primers. Lanes 1, 2, 3, 4, 9, 10, 13, and 14 were found to have a band of about 110 bp with wilt disease resistance and lanes 5, 6, 7, 8, 11 and 12 were susceptible to wilt disease, A band of bp size was identified. Two bands were identified in lanes 15 and 16, indicating a heterozygote with an allele of wilt resistance and susceptibility.
7 shows the result of pathological examination after cabbage wilt pathogen ( F. oxysporum f. Sp . Conglutinans ) treated at a concentration of 2 × 10 ⁷ spore / ml and 24 days later in cabbage germinated in soil and grown for 14 days . Pathological tests showed that cabbage lines of 518, 517 and P6-1 lines were resistant to wilt disease, while cabbage lines of 624, JK-2 and 164 lines were susceptible to wilt disease.

In order to accomplish the object of the present invention, the present invention provides a mutant CYP38 ( BoCYP38 ) gene having at least 8 nucleotide polymorphisms (SNPs) located in the 288th nucleotide of each sequence There is provided a SNP marker composition for discriminating a cabbage fusarium wilt resistant or susceptible variety comprising a polynucleotide consisting of consecutive nucleotides or a complementary polynucleotide thereof.

In one embodiment of the present invention, the contiguous nucleotides may be 8 to 100 contiguous nucleotides, but are not limited thereto.

As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless specifically stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

The SNPs of the present invention are highly stable gene markers that directly affect the phenotype. The SNP markers of the present invention can be used for the identification of resistance to cabbage wilt disease and susceptible varieties. The nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, the CYP38 gene of cabbage, C, or T. < / RTI > For example, the SNP corresponding to the 288th nucleotide of SEQ ID NO: 1 represents a base polymorphism with C or T in which the C / C homozygous genotypes are cabbage wilt resistant varieties, and the T / T homozygous genotypes And C / T heterozygous junction genotypes can be judged as susceptible varieties of cabbage wilt.

The term "homozygotic genotype " at the position of the term SNP used in the present invention means a case in which the allelic bases on the homologous chromosome corresponding to the SNP mutation positions are the same base Quot; heterozygotic genotype " in the SNP mutation position means a case where the opposite base on the homologous chromosome corresponding to the SNP mutation position is a different base.

The present invention relates to a base mutant at each SNP position in the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but when such a SNP base mutation is found in double stranded gDNA (genomic DNA) It is interpreted to include nucleotide sequences. Thus, the base at the SNP position in the complementary polynucleotide sequence is a complementary base. In this regard, all sequences provided herein are based on sequences in the sense strand of the genomic DNA unless otherwise noted.

In the present invention, the cabbage wilt disease is caused by fusarium oxysporum f. sp . Fusarium oxysporum f. sp . conglutinans ), but are not limited thereto.

The present invention also provides a polynucleotide consisting of 8 or more consecutive nucleotides comprising the SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary poly The present invention provides a primer for discriminating cabbage wilt-resistant varieties or susceptible varieties, which comprises nucleotides.

In one embodiment of the present invention, the cabbage wilt-resistant breed or susceptible variety identifying primer may be an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, but is not limited thereto. The oligonucleotide primers represented by SEQ ID NOS: 3 and 4 are forward primers, and have a sequence of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, An oligonucleotide consisting of fragments of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, and at least 21 consecutive nucleotides. For example, SEQ ID NO: 3 of the primer is at least 8 comprising a SNP in the 288th bases of the base sequence of BoCYP38 gene of SEQ ID NO: 1 of cabbage wilt resistant varieties, 9 or more, 10 or more, 11 or more, An oligonucleotide consisting of at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 contiguous nucleotides , And the primer of SEQ ID NO: 4 is at least 8, at least 9, at least 10, at least 11, at least 12, including the SNP located at the 288th base of the nucleotide sequence of SEQ ID NO: 2, which is the BoCYP38 gene of the cabbage wilt susceptible variety , At least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides and at least 9 non-specific nucleotide fragments Made up Lorca can be nucleotides.

The present invention also provides a polynucleotide capable of binding to a part of the nucleotide sequence comprising the SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2, The present invention provides a primer for discriminating a cabbage wilt-resistant variety or susceptible variety, which comprises a polynucleotide.

In one embodiment of the present invention, the cabbage wilt-resistant breed or susceptible variety-identifying primer may be an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, but is not limited thereto. The oligonucleotide primers shown in SEQ ID NOS: 5 and 6 are forward primers and can bind to a part of the base sequence including the SNP located in the 288th base of each sequence in the sequence of the BoCYP38 gene of SEQ ID NO: 1 or SEQ ID NO: 2 Lt; / RTI > oligonucleotide primer. For example, the primer of SEQ ID NO: 5 contains 8 or more, 9 or more, 10 or more, 11 or more, or more of the SNPs located in the 288th base of the nucleotide sequence of SEQ ID NO: 1, which is the BoCYP38 gene of the cabbage wilt- One or more non-specific sequences within a fragment of at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides And the primer of SEQ ID NO: 6 is 8 or more, 9 or more, 10 or more nucleotides including a SNP located in the 288th nucleotide of the nucleotide sequence of SEQ ID NO: 2, which is the BoCYP38 gene of the cabbage wilt susceptible variety, One or more nucleotides within a fragment of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 consecutive nucleotides Nonspecific sequence And oligonucleotides having at least 9 non-specific nucleotide fragments joined to the 5 'direction of the continuous nucleotide fragment. The oligonucleotide primers of SEQ ID NOS: 5 and 6, which correspond to a part of the consecutive nucleotide sequence including the 288th nucleotide sequence of each sequence in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, Can be prepared by substituting a non-specific base with one or more bases in a continuous nucleotide sequence to prevent non-specific binding by a single base difference in a site.

In the present invention, the primer of SEQ ID NO: 7 is a reverse primer for each of the primers of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 for amplifying the SNP region of the cabbage wilt resistant strain or susceptible variety, 1 or SEQ ID NO: 2, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17 Or more, and 18 or more consecutive nucleotides. PCR was performed using the oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 7 or the oligonucleotide primer set of SEQ ID NO: 5 and SEQ ID NO: 7 to obtain a 110 bp band representing the cabbage wilt- PCR using the oligonucleotide primer set of SEQ ID NO: 4 and SEQ ID NO: 7 or the oligonucleotide primer set of SEQ ID NO: 6 and SEQ ID NO: 7 can identify a 120 bp band representing the cabbage wilt susceptible variety.

Thus, the primer set of the present invention comprises an oligonucleotide primer set of SEQ ID NO: 3 and SEQ ID NO: 7, an oligonucleotide primer set of SEQ ID NO: 4 and SEQ ID NO: 7, an oligonucleotide primer set of SEQ ID NO: 5 and an oligonucleotide primer set of SEQ ID NO: And an oligonucleotide primer set of SEQ ID NO: 7. All of these primer combinations can be applied to methods for identifying varieties by constructing from a peripheral sequence at the SNP site, amplifying by PCR, and identifying the product by electrophoresis.

In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, 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. In addition, primers can incorporate additional features that do not alter the primer properties of primers that serve as a starting point for DNA synthesis. The primer nucleic acid sequence of the present invention may, if necessary, comprise a label that is detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g. horse radish peroxidase (HRP), alkaline phosphatase), radioactive isotopes (e.g., ³² P), fluorescent molecules, chemical groups (e.g. biotin) ). The appropriate length of the primer is determined by the characteristics of the primer to be used, but is usually 15 to 30 bp in length. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

The present invention also provides a polynucleotide consisting of 8 or more consecutive nucleotides comprising the SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary poly The present invention also provides a probe for discriminating cabbage wilt-resistant or cultivar-susceptible varieties, which comprises nucleotides.

The term "probe" in the present invention refers to a hybridization probe, which means a linear oligomer having a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide capable of sequence-specific binding to the complementary strand of the nucleic acid. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . Preferably, the probe may be a single strand, more preferably a deoxyribonucleotide, but is not limited to, for maximum efficiency in hybridization.

As the probe used in the present invention, a sequence complementary to the sequence including the SNP may be used, but a sequence substantially complementary to the sequence that does not interfere with the specific hybridization is used It is possible. Preferably, the probe used in the present invention comprises a sequence capable of hybridizing to a sequence comprising 8 to 100 consecutive nucleotides comprising the SNP nucleotide sequence of SEQ ID NO: 1 and the 288th nucleotide sequence of SEQ ID NO: 2. More preferably, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of a duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If not hybridized, such a duplex can be disassembled under stringent conditions. Conditions suitable for hybridization can be determined with reference to contents commonly known in the art. The stringent conditions used for hybridization should be sufficiently stringent to hybridize to only one of the alleles and may be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

The present invention also provides a polynucleotide consisting of 8 or more consecutive nucleotides comprising the SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary poly The present invention provides a microarray for discriminating cabbage wilt-resistant varieties or susceptible varieties, which comprises nucleotides.

In the present invention, the microarray for discriminating cabbage wilt-resistant disease or susceptible variety means a microarray having a substrate on which the SNP markers of the present invention are immobilized. The term "microarray" refers to a group of polynucleotides immobilized on a substrate at a high density, wherein the polynucleotide groups are immobilized in a constant region. Such microarrays are well known in the art. Microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term "substrate" refers to any substrate that has hybridization properties and to which the marker can be attached under conditions where the background level of hybridization is kept low. Typically, the substrate may be a microtiter plate, a membrane (e.g., nylon or nitrocellulose), a microsphere (bead), or a chip. Prior to application or immobilization to the membrane, nucleic acid probes can be modified to promote immobilization or improve hybridization efficiency. Such modifications may include homopolymer tailings, coupling with aliphatic groups, different reactive functional groups such as NH2, SH and carboxyl groups, or coupling with biotin, hapten, or proteins.

The present invention also relates to the aforementioned primer; And a reagent for carrying out an amplification reaction. The kit for selecting a cabbage variety resistant to cabbage wilt disease is provided. The reagents for carrying out the amplification reaction may include, but are not limited to, DNA polymerases, dNTPs, and buffers. The dNTPs include dATP, dCTP, dGTP and dTTP. The DNA polymerase can be a commercially available polymerase such as Taq DNA polymerase and Tth DNA polymerase as a heat-resistant DNA polymerase. In addition, the kit of the present invention may further include a user's manual describing optimal reaction performing conditions. In addition, in the present invention, the kit for selecting cabbage cultivars having resistance to cabbage wilt disease may be a kit including the probe or the microarray, but is not limited thereto.

The present invention also relates to a method for isolating genomic DNA from a cabbage sample,

Amplifying the target sequence by using the separated genomic DNA as a template and carrying out an amplification reaction using the primer of the present invention; And

And detecting the amplification product. The present invention also provides a method for selecting a cabbage variety having cabbage wilt resistance.

The method of the present invention comprises isolating genomic DNA from a cabbage sample. The genomic DNA may be extracted from the sample using phenol / chloroform extraction method, SDS extraction method, CTAB isolation method, or commercially available DNA extraction kit, which are conventionally used in the art.

In the method according to one embodiment of the present invention, the amplification of the target sequence is carried out using oligonucleotide primer sets of SEQ ID NOs: 3 and 7, oligonucleotide primer sets of SEQ ID NOs: 4 and 7, oligonucleotide primer sets of SEQ ID NOs: 5 and 7 Or oligonucleotide primers set forth in SEQ ID NOS: 6 and 7, but are not limited thereto. The target sequence can be amplified by performing amplification reaction using the separated genomic DNA as a template and using the oligonucleotide primer set of the present invention as a primer. 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 Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules 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 well known in the art, and commercially available kits may be used.

In the method according to one embodiment of the present invention, the amplification product has a C base at the SNP position corresponding to the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2 Or may have a T base, and preferably have a C base. When a C base is present at the corresponding SNP position, it indicates a cabbage wilt-resistant variety, and when the SNP position has a T base, it indicates a cabbage wilt susceptible variety.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of one or more oligonucleotide primers used to amplify the target sequence is as described above.

The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed by DNA chip, gel electrophoresis, sequencing, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Using the primer set according to one embodiment of the present invention, a 110 bp amplification product (a primer set of SEQ ID NOs: 3 and 7 or a primer set of SEQ ID NOs: 5 and 7) can be identified in the cabbage wilt resistant strain, In the susceptible varieties, amplification products of 120 bp (primer sets of SEQ ID Nos. 4 and 7 or primer sets of SEQ ID Nos. 6 and 7) can be identified. In addition, the sequence analysis method can be a Sanger method, micro electrophoresis analysis, pyrosequencing, and nanopore single DNA sequence analysis. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, 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 by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Cabbage Wilt  Resistant varieties and susceptible varieties Protein body  analysis

To compare the protein profiles of the cabbage-resistant and susceptible varieties, the cabbage wilt pathogens ( Fusarium oxysporum f. Sp . Conglutinans ) and distilled water (mock; control) After the day, the upper part of the tissue was collected and the total protein was separated. The separated proteins were analyzed by the 2D method reported by Kim et al. ( Journal of Plant Biotechnology , 39: 81-92 (2012)), and protein spots induced by wilt disease or differences in expression were found in cabbage wilt-resistant and susceptible varieties (Fig. 1).

Eighty protein spots with different expression depending on the cultivar or wilt disease pathogen were selected and treated with trypsin on gel. The protein peptides treated with trypsin were analyzed for their mass by MALDI-TOF / TOF MS method to identify proteins. Protein spots 20, 21, 22, 37, 38 and 39 were found to be the same protein as CYP38 (peptidyl-prolyl cis-transisomerase) and protein spots 20, 21 and 22 were expressed in wilt resistant varieties. 37, 38 and 39 were expressed (Figs. 2 and 3). Based on the above results, the present inventors selected BoCYP38 as a candidate protein for classifying cabbage wilt resistance and susceptible varieties.

Example  2. Cabbage Wilt  Identification of the nucleotide sequence of resistant and susceptible varieties

Genomic DNA was extracted from leaves of susceptible varieties of cabbage wilt disease and PCR was performed using 250 ng of extracted genomic DNA as a template to confirm the base sequence of BoCYP38 gene. (5'-ATG GCG GCG GCG TTT GCC TCT CTT C-3 '(SEQ ID NO: 8) and 5'-GGC GAT TTT GTA ACT CGG GTT AGC GA-3' based on the nucleotide sequence of the Arabidopsis derived AtCYP38 gene (SEQ ID NO: 9)) was synthesized, and PCR was performed using the synthesized primer set. PCR was performed under conditions of 5 minutes at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 32 seconds at 72 ° C for 10 seconds and 72 ° C for 5 minutes. As a result, a DNA fragment of about 1.8 kb was obtained I could. The amplified PCR product was cloned into a TA cloning vector and sequenced to obtain a base sequence of BoCYP38 (FIGS. 4A and 4B). The nucleotide sequence of BoCYP38 gene of cabbage wilt - resistant and susceptible varieties was confirmed and 34 nucleotide sequence substitutions and deletions were confirmed. Among them, nucleotides at 288 bp (C: wilt resistance / T: wilt susceptibility) were selected as SNP markers.

Example  3. BoCYP38  Gene SNP On the marker  About primer  design

A modified allele - specific primer for PCR amplification was constructed using the BoCYP38 SNP marker which can discriminate resistant and susceptible varieties of cabbage wilt. For the simple and efficient analysis of SNP genotypes, the vaginal resistance-specific gene-specific forward primer was constructed with a short allele specific primer (BoCYP38 RF and BoCYP38 ASR-F), the worm susceptibility gene specific forward primer was LAS allele-specific primers (BoCYP38 SF and BoCYP38 ASS-F). The reverse primer was prepared so as to be commonly applicable to the two forward primers (BoCYP38-R).

First, BoCYP38 RF primer (5'-AGT CTC AGT GTT GAT CTC TGG C-3 ': SEQ ID NO: 3) for cabbage wilt-resistant strain-specific gene amplification and BoCYP38 SF primer for cabbage wilt-susceptible strain- As shown in FIG. 5, in order to increase the specificity for the cabbage wilt-resistant or cultivar varieties, BoCYP38 ASR-F primer (SEQ ID NO: 4) (5'-AGT CTC AGT GTT GAT CTC TGA C-3 ': SEQ ID NO: 5) and BoCYP38 ASS-F primer (5'-CGA TTC CAC AAG TCT CAG TGT TGA TCT CTC GT- Respectively. Also, a BoCYP38-R primer (5'-GCT GTC AGT GAT GTC CTC G-3 ': SEQ ID NO: 7), which is a reverse primer commonly used for the above four forward primers, was prepared.

PCR was performed using primers set forth in SEQ ID NOS: 5 and 7 and primers set forth in SEQ ID NOS: 6 and 7, and a band of 110 bp was found in the cabbage wilt-resistant resistant varieties. In the susceptible varieties of wilt susceptible varieties, about 120 bp Of the band. In addition, we could identify breeds with two bands, which are heterozygotes with wilt resistance and susceptibility alleles (Fig. 6). The cabbage cultivars and characteristics used for the PCR were shown in Table 1 below.

Cabbage varieties used to determine resistance and susceptibility to wilt of cabbage number System name Characteristic One 518 Wilt resistance 2 518 Wilt resistance 3 517 Wilt resistance 4 517 Wilt resistance 5 624 Wilt susceptibility 6 624 Wilt susceptibility 7 164 Wilt susceptibility 8 164 Wilt susceptibility 9 518 Wilt resistance 10 517 Wilt resistance 11 164 Wilt susceptibility 12 JK-2 Wilt susceptibility 13 YCR Worm resistance F1 14 YCR Worm resistance F1 15 CT-18 Wrath susceptibility F1 16 CT-18 Wrath susceptibility F1

Example  4. SNP With a marker  Discriminated Wilt  Pathological validation of resistant and susceptible cabbage cultivars

In order to confirm whether the genotype and phenotype of the selected cabbage were matched using the SNP markers, three kinds of wilt disease resistance and susceptible varieties identified in FIG. 6 were selected and wilting disease pathology verification was performed. The pathological test was conducted by treating cabbage wilt pathogen ( F. oxysporum f. Sp . Conglutinans ) at a concentration of 2 × 10 ⁷ spores / ml in the cabbage germinated in soil and growing for 14 days and confirming the growth state of the cabbage after 24 days (Fig. 7).

Results of Pathology Test of Cabbage Varieties number System name Characteristic 101 518 Wilt resistance 102 517 Wilt resistance 103 624 Wilt susceptibility 104 JK-2 Wilt susceptibility 105 P6-1 Wilt resistance 106 164 Wilt susceptibility

As a result of the pathological test, it was confirmed that the 518, 517 and P6-1 cultivars were susceptible to wilt disease, 624, JK-2 and 164 cultivars were susceptible to wilt disease, and thus they were identical with the genotype discrimination results confirmed by the SNP markers of the present invention (Table 2).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Single nucleotide polymorphism marker for selecting fusarium          wilt-resistant or sensitive cabbage cultivar and uses thereof <130> PN13081 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 1775 <212> DNA <213> Brassica oleracea <400> 1 atggcggcgg cgtttgccac tctgcctaca ttgagtttgg tcaatacctc gagaaggaga 60 atcgattctt acagctccaa aaaacgcgtc ggagttgttc gttgttgctc cggcggcgac 120 attcccgaat ataagcaggt gcagagagga ggaggaggag ggacattgaa agaatgtgcg 180 attacattag ctttatcgtt gggtatgata gctggaggag gagcaccttc gattgcttac 240 gcggcgaacc cagcgattcc acaagtctca gtgttgatct ctggccctcc catcaaagac 300 ccaggagctt tgttgagata cgcattgccc atcgacaaca aagccatcag acaagtgcag 360 aagcctctcg aggacatcac tgacagcctc aagatcgctg gcgttaaggc tctcgattct 420 gttgaacgtg taagctgctt tatggtattg attgattgct ctgttttact tgattgttgt 480 tgcctatttg tagaatgtga ggcaagcaag tagatcactc cagcaaggga aaagcatgat 540 tgttgccggt tttgctgagt cgaagaagga tcatggtcac gaactgattg ggaaattgga 600 agctggtatg caagacatgc tacagattgt ggaagatcgc aaaagagatg cggttgctcc 660 taaacagaaa gagattctcc aatatgttgg cgggtgagtt tatacacaca gccaaatcaa 720 tggttagtaa ggaagctgtg agatattttt gctcatttta gacccccccc ccccttcttg 780 ttgattgact tgcgcgcagc tctgagatct tcctgtcctg tggcgctctc tctgcagaat 840 tgaagaggac atggttgatg gcttccctta tgatgtccct gaggagtacc gcaacatgcc 900 tcttctcaag ggaagagcca ctgtcgacat gaaggtcaag atcaaggaca accccaacct 960 cgaggattgt gttttccgcc ttgtcctcga tggctacaac gcccctgtca ccgccggcaa 1020 ctttgtggac ttggtggagc gccatttcta cgatggcatg gagatccaga gatgtaagac 1080 atcacacttg ccttaccatt tatatatatc tatgggacaa tgtttgttga tcaatggatt 1140 ggcagctgat gggtttgtgg tacaaacggg agatccagag ggtcctgcgg aaggatttat 1200 agatccaagc acagagaaag tgaggactgt tcctcttgag attatggtgg aagggaagaa 1260 gactcccttt tacggctcaa cccttgaagt aaaaaaccct tctcacaagt aagtatattc 1320 aattgattat cataatttaa catttgttca tttggttgga caggaattgg gtctgtacaa 1380 ggctcaggtg atgcttccat tcaacgcatt tgggactatg gcaatggcta gagaagtgag 1440 agttctttct cttctttcct ttcttcacaa ttgaatatat atatatatgt tgtaattata 1500 tatgtaatgc aaaacaggag tttgagaatg actcgggatc gagccaagtg ttttggctgc 1560 tgaaagagag tgagctgacg ccaagcaatt ccaacatctt ggacggtcgt tacgctgtct 1620 tcggttacgt tactcagaat gaagactttc ttgctgatct taaagtcggt gatgtcattg 1680 agtccatcca agttgtctcc ggtttagaca acctcgctaa cccgagttac aaaatcgcca 1740 agggcgaatt ccagcacact ggcggccgtt actag 1775 <210> 2 <211> 1795 <212> DNA <213> Brassica oleracea <400> 2 atggcggcgg cgtttgccac tctccctaca ttgagtttgg tcaattcctc gagaaggaga 60 atcgattctt acagctccaa aaagcgcgtc ggagttgttc gttgttgctc cggcggcgac 120 attcccgaat ataagcaggt gcagagagga ggaggaggag ggacattgaa agaatgtgcg 180 attacattag ctttatcgtt gggtttgata gctggaggag gagcaccttc gattgcttac 240 gcggcgaacc cagcgattcc acaagtctca gtgttgatct ctggtcctcc catcaaagac 300 ccaggagctt tgttgagata cgcattgccc atcgacaaca aagccatcag agaagtgcag 360 aagcctctcg aggacatcac tgacagcctc aagatcgctg gcgttaaggc tctcgattct 420 gttgaacgtg taagctgctt tatggtattg attgattgat tgctcttttt ttacttgatt 480 gttgttgcct atttgtagaa tgttaggcaa gcaagtagat cactccagca agggaaaagc 540 atgattgttg ccggttttgc tgagtcgaag aaggatcatg gtcacgaact gattgggaaa 600 ttggaagctg gtatgcaaga catgctacag attgtggaag atcgcaaaag agatgcggtt 660 gctcctaaac agaaagagat tctccaatat gttggcgggt gagtttatac acacagccaa 720 atcaatggtt agtaaggaag ctgtgagata tttttgctca ttttagaccc cccccccccc 780 ccccccccct tcttgttgat tgacttgcgc gcagctctga gatcttcctg tcctgtggcg 840 ctctctctgc agaattgaag agtacatggt tgatggcttc ccttatgatg tccctgagga 900 gtaccgcaac atgcctcttc tcaagggaag agccactgtc gacatgaagg tcaagatcaa 960 ggacaacccc aacctcgagg attgtgtttt ccgcattgtc ctcgatggct acaacgcccc 1020 tgtcaccgcc ggcaactttg tggacttggt ggagcgccat ttctacgatg gcatggagat 1080 ccagagatgt aagacatcac acttgcctta ccatttatat ttctatggga caatgtttgt 1140 tgatcaatgg atttggcagc tgatgggttt gtggtacaaa cgggagatcc agagggtcct 1200 gcggaaggat ttatagatcc aagcacagag aaagtgagga ctgttcctct tgagattatg 1260 gtggaaggga agaagactcc cttttacggc tcaacccttg aagtaaaaaa cccttctcac 1320 aagtaagtat attcaattga ttatcataat ttaacatttg ttcatttggt tggacaggaa 1380 ttgggtctgt acaaggctca ggtgatgctt ccattcaacg catttgggac tatggcaatg 1440 gctagagaag tgagagttct ttctcttctt tcctttcttc acaattgaat atatatatat 1500 atgttgtaat tatatatgta atgcaaaaca ggagtttgag aatgactcgg gatcgagcca 1560 agtgttttgg ctgctgaaag agagtgagct gacgccaagc aattccaaca tcaagggctt 1620 ggacggtcgt tacgctgtct tcggttacgt tactcagaat gaagactttc ttgctgatct 1680 taaagtcggt gatgtcattg agtccatcca agttgtctcc ggtttagaca acctcgctaa 1740 cccgagttac aaaatcgcca agggcgaatt ccagcacact ggcggccgtt actag 1795 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 agtctcagtg ttgatctctg gc 22 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 cgattccaca agtctcagtg ttgatctctg gt 32 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 agtctcagtg ttgatctctg ac 22 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 cgattccaca agtctcagtg ttgatctctc gt 32 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 gctgtcagtg atgtcctcg 19 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 atggcggcgg cgtttgcctc tcttc 25 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ggcgattttg taactcgggt tagcga 26

Claims (14)

A polynucleotide consisting of 8 or more consecutive nucleotides comprising a single nucleotide polymorphism (SNP) located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2, A SNP marker composition for identifying a fusarium wilt resistant or susceptible varieties, including nucleotides. 2. The SNP marker composition according to claim 1, wherein the continuous nucleotides are 8 to 100 consecutive nucleotides. A polynucleotide consisting of 8 or more consecutive nucleotides comprising a SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary polynucleotide thereof, A primer for the identification of susceptible varieties or susceptible varieties. 4. The primer according to claim 3, which is an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a susceptible breed-discriminating variant of cabbage wilt. Or an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a primer for discriminating a susceptible breed of cabbage wilt disease. delete A polynucleotide consisting of 8 or more consecutive nucleotides comprising a SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary polynucleotide thereof, Probe for the detection of wilt resistant or susceptible varieties. A polynucleotide consisting of 8 or more consecutive nucleotides comprising a SNP located in the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary polynucleotide thereof. Microarray for resistance to cabbage wilt disease or susceptible variety. A primer according to claim 3 or 5; And a reagent for carrying out an amplification reaction. &Lt; RTI ID = 0.0 &gt; 15. &lt; / RTI &gt; 10. The kit according to claim 9, wherein the reagent for carrying out the amplification reaction comprises a DNA polymerase, dNTPs and a buffer, wherein the kit is resistant to cabbage wilt disease. Isolating the genomic DNA from the cabbage sample;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using the primer of claim 3 or 5; And
And detecting said amplification product. &Lt; Desc / Clms Page number 20 &gt; 12. The method of claim 11, wherein the amplification of the target sequence comprises the oligonucleotide primer set of SEQ ID NOs: 3 and 7, the oligonucleotide primer set of SEQ ID NOs: 4 and 7, the oligonucleotide primer set of SEQ ID NOs: 5 and 7, &Lt; / RTI &gt; of a cabbage wilt-resistant bacterium. 12. The method according to claim 11, wherein the amplification product has a C base at the SNP position corresponding to the 288th base of each sequence in the nucleotide sequence of the cabbage CYP38 ( BoCYP38 ) gene of SEQ ID NO: 1 or SEQ ID NO: A method for screening resistant cabbage varieties. The cabbage wilt-resistant cabbage cultivar according to claim 11, wherein the detection of the amplification product is performed by capillary electrophoresis, DNA chip, gel electrophoresis, sequencing, radioactivity measurement, fluorescence measurement or phosphorescence measurement. Methods for screening.

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