KR20140032060A - Rapd primer and method for selecting chinese cabbage having heat stress tolerance - Google Patents

Abstract

The present invention relates to a RAPD primer for selecting Chinese cabbages having high temperature stress tolerance and a selection method of Chinese cabbages having high temperature stress tolerance using the same. A RAPD primer of the present invention can easily select Chinese cabbages having high temperature stress tolerance and is advantageous for developing and breeding Chinese cabbage species having high temperature stress tolerance in a short time and at low costs.

Description

Technical Field [0001] The present invention relates to a RAPD primer for selecting high temperature stress resistant Chinese cabbage and a method for selecting high temperature stress resistant Chinese cabbage using the RAPD primer and method for selecting Chinese cabbage having heat stress tolerance.

The present invention relates to a RAPD primer for screening high temperature stress resistant Chinese cabbage and a method for selecting high temperature stress resistant Chinese cabbage using the same.

Cabbage is one of the four major vegetable crops in Korea and is a major economic crop estimated at about 1 trillion won in annual production of 2.3 million tons.

 The abiotic stress caused by various non-living organisms may be the greatest limiting factor in the productivity of Chinese cabbage crops. For example, damage caused by temperature lower than the optimum temperature for growth, damage due to high temperature, damage due to lack of water, damage due to excessive salts in the soil and the like are difficult to predict. Development of varieties is necessary.

 When a new breed is developed using the traditional breeding method, the desired trait is selected as a phenotyping test. However, phenotype testing can be difficult or time-consuming. In addition, unwanted traits can be introduced in the selection process. Therefore, selection and elimination in the breeding process should be done simultaneously in different traits, but simultaneous testing of many phenotypes is a difficult task. The development of molecular markers in these areas can replace the phenotyping step with marker validation. Therefore, the development of molecular markers that can easily and quickly carry out the step of phenotyping test is essential for establishing the breeding technology.

 The development of molecular markers located in or near to genes resistant to high-temperature stress in Chinese cabbage crops has the advantage of developing varieties at a low cost in a short period of time by incorporating molecular marker utilization techniques into traditional breeding techniques. It is an area that should be.

In recent years, along with the radical development of molecular biology, molecular biologic methods have been widely used to classify plants as genus, species, or species. To this end, nucleic acid fingerprint analysis methods and various DNA markers have been developed that enable genetic diversity studies at the DNA level. The discrimination of strains by DNA analysis at the level of molecular biology has the advantage of being able to grasp many characteristics along with the quantitative level of the strains and to exclude the influence of the environment. Among them, RFLP (Restriction Fragment Length Polymorphism) is a technique in which DNA fragments generated when a series of DNA chains are treated with a restriction enzyme have a number or length depending on the difference in genetic composition between individuals, that is, It is based on the fact that it appears differently and is being used variously. However, RFLP requires large amounts of high-purity DNA, is time-consuming, labor-intensive and costly, has a complex test procedure, and has a disadvantage of using radioactive isotopes. To overcome these problems, RAPD (Random Amplified Polymorphic DNA) method, which is technically simple and easy to track polymorphism, has been developed. The RAPD method provides quick results and is easy to apply and does not need to have sequence information in advance. As a technique using this, Korean Patent No. 10-0264743 discloses "quantitative trait gene map (QTL) and low temperature resistant rice varieties selection method involving rice cold seedling resistance using marker RAPD" 10-0215084 discloses " a method for discriminating the origin of Korean ginseng by RAPD marking and a primer used in the method ", and Korean Patent No. 10-0764561 discloses " a male sterile restorative gene Discrimination method "

Accordingly, the present inventors have completed the present invention by developing a RAPD primer sequence that specifically amplifies a specific region of Chinese cabbage having high-temperature stress resistance, in order to screen and breed Chinese cabbage with high-temperature stress resistance.

It is an object of the present invention to provide a random amplified polymorphic DNA (RAPD) primer for high temperature stress resistant Chinese cabbage comprising the nucleotide sequence of SEQ ID NO: 14.

Another object of the present invention is to provide a kit for screening a high temperature stress resistant Chinese cabbage comprising the primer.

It is still another object of the present invention to provide a method for screening hot stress-resistant Chinese cabbage using the primer.

The present invention provides a random amplified polymorphic DNA (RAPD) primer for high temperature stress resistant Chinese cabbage comprising the nucleotide sequence of SEQ ID NO: 14.

In addition, the present invention provides a high temperature stress resistant Chinese cabbage selection kit comprising the primer.

The present invention also provides a method for selecting a high temperature stress resistant cabbage using the primer.

The RAPD primer of the present invention has an advantage of being able to easily select high temperature stress resistant Chinese cabbage and to develop and breed cabbage varieties having high temperature stress resistance at a low cost in a short time.

FIG. 1 is a view showing the result of electrophoresis after performing PCR using a RAPD primer consisting of the nucleotide sequence of SEQ ID NO: 14.
Fig. 2 is a diagram showing a gene map of the sequence amplified by SEQ ID NOS: 1 to 14. Fig.
3 is a diagram showing the degree to which the sequence amplified by SEQ ID NOS: 1-14 is associated with high temperature stress resistance.

The present invention provides a random amplified polymorphic DNA (RAPD) primer for high temperature stress resistant Chinese cabbage comprising the nucleotide sequence of SEQ ID NO: 14.

Hereinafter, the present invention will be described in detail.

In the present invention, the "F2" group of "91" individuals, which is the self generation of F1 generation and F1, was breed by crossing the "92" series of Chinese cabbage strain known as "high temperature stress resistance" and the "93" strain known as high temperature stress sensitivity. Thereafter, PCR was performed on the 92, 93, and F2 populations using 520 RAPD primers, followed by electrophoresis. The electrophoretic bands of the Z08_420 region, which is amplified by SEQ. ID. NO. 14, which is present only in the cabbages having a high temperature stress resistance phenotype among the "92" and F2 groups of the high temperature stress resistant cabbage line, were confirmed. As a result of DNA sequence analysis of the band, it was confirmed to have the nucleotide sequence of SEQ ID NO: 15. The sequence amplified by SEQ ID NOs: 1 to 14 and the gene map of SEQ ID NO: 15 were generated and analyzed for their correlation with high temperature stress resistance. As a result, the nucleotide sequence of SEQ ID NO: 15 amplified by SEQ ID NO: And that it is associated with stress resistance. Therefore, it was confirmed that high temperature stress resistant Chinese cabbage can be selected using the RAPD primer of SEQ ID NO: 14.

The RAPD of the present invention means a randomly synthesized DNA sequence, which can be used as a primer.

The term primer used in the present invention is a nucleic acid sequence having a short free 3 'hydroxyl group, which can form a base pair with a complementary template and has a starting point for template strand copying ≪ / RTI > Primers can initiate DNA synthesis in the presence of reagents for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphosphates (dNTPs) at appropriate buffers and temperatures.

Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, comprise markers detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg ³² P), fluorescent molecules, chemical groups (eg biotin), and the like. There is this.

The sequence amplified by the RAPD primer of the present invention is characterized by being the nucleotide sequence of SEQ ID NO:

"Sorting" of the present invention means selecting a Chinese cabbage having a specific phenotype.

In addition, the present invention provides a PCR kit for screening high temperature stress resistant Chinese cabbages comprising a RAPD primer consisting of the nucleotide sequence of SEQ ID NO: 14.

The selection PCR kit for the high-temperature stress-resistant Chinese cabbage may include, in addition to the above-described primer set, conventional components included in the PCR kit. A typical composition included in the kit may joinja the like, such as a reaction buffer and polymerase, dNTP (dATP, dCTP, dGTP and dTTP) and Mg ^{+ 2.} 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, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) . Most of these enzymes can be isolated from the bacteria itself or commercially available. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase. When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component.

All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In addition,

1) performing PCR by mixing RAPD primer composed of the nucleotide sequence of SEQ ID NO: 14 with DNA sample extracted from Chinese cabbage; And

2) electrophoresis of the amplified sample of step 1).

In addition, the nucleotide sequence of SEQ ID NO: 15 amplified by the RAPD primer sequence of SEQ ID NO: 14 of the present invention can be used as a marker for identifying the high temperature stress resistant Chinese cabbage.

The base sequence of SEQ ID NO: 15 or its complementary base sequence can be used as a probe. In the present invention, the term " probe " refers to a hybridization probe, which means an oligonucleotide capable of specifically binding to a complementary strand of a nucleic acid. Using this, the genomic DNA of the Chinese cabbage can be extracted, and the base sequence of SEQ ID NO: 15 or its complementary base sequence can be hybridized with the genome DNA of the Chinese cabbage as a probe. In the case of high temperature stress resistant Chinese cabbage, the Chinese cabbage hybridized by the nucleotide sequence of SEQ ID NO: 15 can be determined by high temperature stress resistant Chinese cabbage.

A microarray comprising the nucleotide sequence of SEQ ID NO: 15 of the present invention or a complementary base sequence thereof can be provided. The microarray according to the present invention can be produced by a conventional method known to those skilled in the art.

For example, the nucleotide can be immobilized on a substrate coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde. In addition, the substrate may be selected from the group consisting of a silicon wafer, glass, quartz, metal, and plastic. Methods for immobilizing the polynucleotide on a substrate include micropipetting using a piezo electric method and a method using a pin-type spotter.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

Example  One Specific for high temperature stress resistant Chinese cabbage primer  And a sequence search amplified thereby

1. Chinese cabbage crossing

We breed F2 groups of "91" individuals, which are the F1 generations and the self-water generation of F1, by crossing the "93" line known as high temperature stress resistance "92" and the high temperature stress sensitive "93" line. The experiment was conducted using the F2 generation.

2. Each Chinese cabbage line DNA  extraction

After extracting the Chinese cabbage leaves from F2 plants of 92, 93 and 91 lines, DNA was extracted using CTAB (cetyltrimethylammonium bromide) method after lyophilization.

More specifically, the extraction method was pulverized by a pulverizer for 15 minutes and heated in a water bath at 65 ° C for 10 minutes. After adding 250 5 of 5M ammonium acetate, centrifugation was carried out for 10 minutes. After centrifugation, the supernatant was taken and 900 쨉 l of cold EtOH was added and kept at -70 째 C for 10 minutes. Thereafter, it was dried with alcohol and distilled water was added. The DNA thus extracted was measured for its concentration using a spectrophotometer, and the final concentration was diluted to 10 ng / μl.

3. Extracted DNA of PCR  Amplification

The DNA extracted in 2 of Example 1 was amplified by PCR using 520 kinds of random amplified polymorphic DNA (RAPD) primers (A01 to Z20) prepared by a conventional method in the art. PCR was carried out using a total amount of 15 μl (6.02 μl of distilled water, 0.4 μl of dNTP, 0.08 μl of Etaq polymerase, 1.5 μl of Etaq buffer, 2 μg / μl of primer and 5 μl of DNA). PCR was carried out at 95 ° C for 3 minutes, denaturation at 95 ° C for 15 seconds, annealing at 36 ° C for 30 seconds, and extension at 72 ° C for 2 minutes.

4. Electrophoretic analysis

Samples amplified using 520 RAPD primers were subjected to electrophoresis using 1.5% agarose gel containing ethidium bromide (1.5 μg / mL). Of these, primer sets with clear bands and showing polymorphism were selected and their sequences are shown in Table 1 below. FIG. 1 shows the results of electrophoresis analysis when amplified with the primer of SEQ ID NO: 14, which is associated with the highest temperature stress in the QTL analysis below.

A12 TCGGCGATAG SEQ ID NO: 1 C13 AAGCCTCGTC SEQ ID NO: 2 E11 GAGTCTCAGG SEQ ID NO: 3 F03 CCTGATCACC SEQ ID NO: 4 J05 CTCCATGGGG SEQ ID NO: 5 K03 CCAGCTTAGG SEQ ID NO: 6 N16 AAGCGACCTG SEQ ID NO: 7 M04 GGCGGTTGTC SEQ ID NO: 8 P14 CCAGCCGAAC SEQ ID NO: 9 Q14 GGACGCTTCA SEQ ID NO: 10 Q15 GGGTAACGTG SEQ ID NO: 11 R12 ACAGGTGCGT SEQ ID NO: 12 X01 CTGGGCACGA SEQ ID NO: 13 Z08 GGGTGGGTAA SEQ ID NO: 14

As shown in Fig. 1, 92 DNA probes with high-temperature stress resistance and DNA bands showing only resistance to high temperature stress among F2 generations were identified. The band region is designated as Z08_420, and its nucleotide sequence is shown in SEQ ID NO: Therefore, it was confirmed that the primer of SEQ ID NO: 14 can be used for screening high-temperature stress-resistant Chinese cabbage, whereby the amplified sequence is a sequence associated with high-temperature stress resistance.

Example  2 Genetic mapping and QTL  analysis

Genomic maps were generated by association analysis using the Joinmap 4.0 (Van Ooijen, 2006) program for the sequences amplified by the RAPD primers shown in Table 1,

MAPQTL 5.0 (Van Ooijen, 2004) program was used to analyze the relationship between Z08_420 and stress resistance to high temperature.

The gene map is shown in FIG. 2, and the degree of association with the endogenous expression pattern of Chinese cabbage is shown in FIG.

As shown in Fig. 2, Z08_420 of SEQ ID NO: 15 is present between the sequences amplified by Q14 and Q15 of SEQ ID NOs: 10 and 11 (Q14_590 and Q15_483).

In addition, as shown in Fig. 3, the correlation between SEQ ID NO: 15 and the cabbage was highest, and Q14_590 and Q15_483, which are genetically close to SEQ ID NO: 15, were highly correlated with the endogenous phenotype.

Thus, by detecting Z08_420 of SEQ ID NO: 15, it was confirmed that Chinese cabbage with high temperature stress resistance can be selected.

In addition, the nucleotide sequence of SEQ ID NO: 15 was specifically detected in hot stress-resistant Chinese cabbage, and it was confirmed that it can be used as a probe for hot stress-resistant Chinese cabbage.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> RAPD primer and method for selecting Chinese Cabbage having heat          stress tolerance <130> P-1.84 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is A 12 RAPD primer <400> 1 tcggcgatag 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is C 13 RAPD primer <400> 2 aagcctcgtc 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is E11 RAPD primer <400> 3 gagtctcagg 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is F03 RAPD primer <400> 4 cctgatcacc 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is J05 RAPD primer <400> 5 ctccatgggg 10 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is K03 RAPD primer <400> 6 ccagcttagg 10 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is N16 RAPD primer <400> 7 aagcgacctg 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is M04 RAPD primer <400> 8 ggcggttgtc 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is P14 RAPD primer <400> 9 ccagccgaac 10 <210> 10 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is Q14 RAPD primer <400> 10 ggacgcttca 10 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is Q15 RAPD primer <400> 11 gggtaacgtg 10 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is R12 RAPD primer <400> 12 acaggtgcgt 10 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is X01 RAPD primer <400> 13 ctgggcacga 10 <210> 14 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> it is Z08 RAPD primer for selecting Chinese Cabbage having heat          stress tolerance <400> 14 gggtgggtaa 10 <210> 15 <211> 397 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence amplified by Z08 RAPD primer <400> 15 cctgtcatag ttagctaccc acatggtgaa tgcgtgcttt gggattcctc ctttgaacca 60 aacgacatct acccaatctt taagctcttc ccgcggccgc agcacctccc atgtggttga 120 cgacctgaaa gtatgtaacg gagaatcacc agctatccat tcataatcat cattcacatc 180 aacaggaaga ggcaaactaa tagtagtaag ataagtatgg agttcaactt ccttttggga 240 tcttgggtga ggcagcgacc aaacagagtc atttattgcg tcagccacca cagcattctt 300 ccttatcctc agagatcttg gcccagcact accaatgtgt tcgattaact gaccgagggg 360 cgtccatacg tcgaaccaga aactggctct cctccca 397

Claims (6)

A random amplified polymorphic DNA (RAPD) primer for high temperature stress resistant Chinese cabbage comprising the nucleotide sequence of SEQ ID NO: 14. The RAPD primer according to claim 1, wherein the base sequence amplified by the nucleotide sequence of SEQ ID NO: 14 is the nucleotide sequence of SEQ ID NO: 15. A high temperature resistant Chinese cabbage selection PCR kit comprising the RAPD primer of claim 1 1) performing PCR by mixing RAPD primer composed of the nucleotide sequence of SEQ ID NO: 14 with DNA sample extracted from Chinese cabbage; And
2) electrophoresing the amplified sample of step 1). A high temperature stress resistant Chinese cabbage screening probe comprising a nucleotide sequence of SEQ ID NO: 15 or a complementary base sequence thereof. The high-temperature stress resistant Chinese cabbage screening probe according to claim 5, wherein the base sequence of SEQ ID NO: 15 is a base sequence amplified by a primer consisting of the base sequence of SEQ ID NO: 14.

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