KR101760066B1 - Primer set for Kwangpyeongok identification and method for identification - Google Patents
Primer set for Kwangpyeongok identification and method for identification Download PDFInfo
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Abstract
More particularly, the present invention relates to a primer set for discrimination of Gwangpyeong Okpyeong, which is capable of effectively discriminating an effective variety of Gwangpyeong, protecting Gwangpyeong species and purity of F1 seeds, The present invention relates to a method for distinguishing a Gwangpyeong oak variety by using a primer set for discriminating the Gwangpyeong product.
Description
More particularly, the present invention relates to a primer set for discriminating an effective variety of Gwangpyeong jung, a variety of Gwangpyeong jade, and a purity of F1 seed, The present invention relates to a discrimination method useful for discrimination of Gwangpyeong Okdong using a primer set for discriminating the above-mentioned Gwangpyeong Okpyeong.
Maize ( Zea mays L.) is one of the three major crops cultivated globally and is an important crop in the world, but the feed corn consumed in our country depends on imports from foreign countries. Since the feed corn has a high productivity per unit area and high total digestible nutrients, it is important to cultivate corn that has better yield and quality than any other crop in Korea where the land area is narrow.
In recent years, as the price of imported corn and grains has risen, the import prices of feed and forage have soared, and the difficulties of livestock farmers have been increasing. Therefore, the cultivation of forage crops such as corn has been on the increase. Domestic corn for silage cultivation is more than 70% imported paper such as P3394. Domestic cultivated varieties such as Gwangpyeong Okhwa occupy about 29% of total 265 tons of grain supplied in 2010, 78 tons. In the meantime, domestic corn for feed has been widely distributed in livestock farmers because of its low yield and poor quality. However, in recent years, domestic cultivated varieties such as Gwangpyeong Ok and Gangdaok have been found to be more resistant to collapse than conventional corn for imported silage, and the number of cultivated areas of cultivated cultivars has been increasing as it has been found that not only the quantity of the building but also the feed value are high. However, there has been a difficulty in measuring the purity of F1 seeds and preserving and improving the varieties because a method for easily and quickly distinguishing the new variety Kwangpyeong Ok, which was developed as feed corn, has not been developed.
On the other hand, DNA-based molecular marker technology today provides useful information for genetic diversity and phylogenetic analysis of plant genetic-breeding studies and conservation and management of plant genetic resources. Among them, SNP (Single Nucleotide Polymorphism) analysis is widely used to analyze the genetic diversity of corn. Molecular markers by SNPs can be effectively used to protect crop varieties in corn breeding studies and many researchers have sought to use corn genetic diversity for evaluation and discrimination.
The present invention relates to a specific primer, a probe and a standard plasmid for qualitative and quantitative analysis of genetically modified agricultural products and a test method using the specific primer, A probe and a standard plasmid that amplify all or a part of the target gene or a region including all or a part of the DNA region of the other expression control region by using the recombinant gene of various strains And a PCR primer set which can be detected and detected.
Conventionally, there has not been developed a method for easily and quickly discriminating the new variety Kwangpyeong Ok, which has been developed, and it has been difficult to measure the purity of F1 seed and to preserve and improve the breed.
In order to solve the above problems, a primer set for discrimination of Gwangpyeong Okpyeong, which can effectively identify the variety of Gwangpyeong Okpyeong, protection of Gwangpyeong Okpyeong and the purity of F1 seeds, and a primer set for the Gwangpyeong Okdong The purpose of this study is to provide a useful method of discrimination of Gapyeong jade.
In order to accomplish the above object, the present invention relates to an IBM 471 primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12; and a set of at least one primer selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
The present invention also relates to an IBM 471 primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
According to another preferred embodiment of the present invention, the IBM 471 primer set, the IBM 516 primer set and the IBM 535 primer set are complementary to the nucleotide sequence of chromosome 2 of the corn B73 standard genome, and the IBM 911 primer set And the IBM 1492 primer set are complementary to the nucleotide sequence of chromosome 4 of the maize B73 standard genome and the IBM 1492 primer set is complementary to the nucleotide sequence of chromosome 8 of the maize B73 standard genome have.
The present invention also provides a method for producing a recombinant maize comprising: (a) isolating genomic DNA from maize; (b) an IBM 471 primer set comprising the genomic DNA isolated in step (a) as a template and comprising SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12; amplifying the target sequence by performing amplification reaction; And (c) identifying the product amplified in the step (b).
According to a preferred embodiment of the present invention, the step (c) may be a step of discriminating the cultivar when the following conditions are all satisfied:
I) detection of a product of size 390 bp amplified using an IBM 471 primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2;
Ii) detection of a product of size 319 bp in the amplified product using the IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4;
Iii) detection of a 251 bp product in the amplified product using the IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6;
Iv) the product 227 bp in size amplified using the IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8 was not detected;
V) detection of a 352 bp product in the amplified product using the IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And
Vi) The product amplified using the IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12 should not be detected to be 271 bp in size.
According to a preferred embodiment of the present invention, the amplification reaction may include annealing at a temperature of 44 to 70 ° C.
According to a preferred embodiment of the present invention, the IBM 471 primer set, the IBM 516 primer set and the IBM 535 primer set are complementary to the nucleotide sequence of chromosome 2 of the maize B73 standard genome, The IBM 1492 primer set is complementary to the nucleotide sequence of chromosome 4 of the maize B73 standard genome and the IBM 1492 primer set may be complementary to the nucleotide sequence of chromosome 8 of the maize B73 standard genome .
Hereinafter, terms of the present invention will be described.
The term "primer" of the present invention is a base sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template and is used as a starting point for template strand copy Quot; short sequence " The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. At this time, the PCR conditions, the lengths of the sense and antisense primers can be modified based on those known in the art.
In addition, the term "nucleotide " of the present invention is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley , New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).
The present invention relates to a method for effectively discriminating the Gwangpyeong species by using a primer set for discriminating the Gwangpyeong species and a primer set for discriminating the Gwangpyeong species, There is an effect of providing a method.
FIG. 1 is a schematic view showing the preparation of a 3 'end mismatch primer in Example 1. FIG.
Fig. 2 shows the result of searching for a specific reaction primer in Example 1. Fig.
FIG. 3 shows the result of checking the degree of correspondence between the varieties through the selected primers and their parents.
Fig. 4 is an electrophoresis image for selecting the markers for discrimination of Gwangpyeong japonese species, which does not show nonspecific reaction among the 16 primer combinations and the amplification reaction between the transgenic plants and the cultivars is identical.
Fig. 5 shows the cultivars of 50 varieties of corn for fodder.
FIG. 6 is a photograph showing electrophoresis of primers IBM 471, IBM 516, IBM 535, IBM 911, IBM 960, and IBM 1492, which can identify Gwangpyeong species, for 50 kinds of corn cultivars for feed use.
Hereinafter, the present invention will be described in more detail.
As described above, there has been a difficulty in measuring the purity of F1 seed, preserving and improving the breed, since a method for easily and quickly discriminating the new variety Kwangpyeong Ok, which was developed as the feed corn, has not been developed.
Accordingly, the present invention relates to an IBM 471 primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12, to solve the above-mentioned problem.
Thus, a discrimination primer set for discriminating the Gwangpyeong Okpyeong, which can effectively test the variety of Gwangpyeong jade, the protection of the Gwangpyeong jade variety and the purity of the F1 seed, and the primer set for discriminating the Gwangpyeong jade variety, There is an effect of providing a method.
Preferably, the IBM 471 primer set, the IBM 516 primer set, and the IBM 535 primer set are selected from the group consisting of two of the maize B73 standard genomes, although the nucleotides are not particularly limited as long as they are complementary to some or all of the maize B73 standard genome. Wherein the IBM 911 primer set and the IBM 1492 primer set are complementary to the nucleotide sequence of chromosome 4 of the maize B73 standard genome and the IBM 1492 primer set is complementary to the maize B73 standard genome genome) of the chromosome 8 sequence can be complementary.
The corn B73 standard genome is described in 2011 by Ganal et al. (Ganal et al., 2011. PloS ONE 6: e28334) and is described on the MaizeGDB website (http://www.maizegdb.org/sequence.php) Lt; / RTI >
The "complement" means that the nucleotide and all or some of the chromosome are specifically bound.
The "corn B73 standard genome" is a polymorphic sequence comprising a polymorphic site. A polymorphic sequence means a sequence comprising a polymorphic site comprising a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.
The primers include an IBM 471 primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12, it is possible to use an appropriate primer set in an appropriate buffer (for example, four different nucleoside triphosphates and DNA, RNA polymerase or reverse transcriptase , And a single-stranded oligonucleotide capable of acting as a starting point of template-directed DNA synthesis under appropriate temperature. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15-30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template.
The primer sequence need not be completely complementary to the SNP marker, but should be sufficiently complementary to hybridize with the maize B73 standard genome.
The primers 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 ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
(A) isolating genomic DNA from maize; (b) an IBM 471 primer set comprising the genomic DNA isolated in step (a) as a template and comprising SEQ ID NO: 1 and SEQ ID NO: 2; An IBM 516 primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4; An IBM 535 primer set consisting of SEQ ID NO: 5 and SEQ ID NO: 6; An IBM 911 primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8; An IBM 960 primer set consisting of SEQ ID NO: 9 and SEQ ID NO: 10; And an IBM 1492 primer set consisting of SEQ ID NO: 11 and SEQ ID NO: 12; amplifying the target sequence by performing amplification reaction; And (c) identifying the product amplified in the step (b).
First, genomic DNA is isolated from corn.
As a method for isolating genomic DNA from corn, a method known in the art can be used. For example, a CTAB method can be used, a wizard prep kit (Promega) can be used, and Dellaporta et al. , 1983. Plant Mol. Biol., Rep. 1: 19-211983).
Further, the corn is not particularly limited as long as it uses some or all of the corn plants, but preferably at least one of the roots, stems, leaves and fruits of the corn can be used.
Next, using the separated genomic DNA as a template, the
The method of amplifying the target nucleic acid is not particularly limited as long as it is a method for amplifying a nucleic acid sequence, and examples thereof include a polymerase chain reaction (PCR), a ligase chain reaction, a nucleic acid sequence- acid sequence-based amplification, a transcription-based amplification system, a strand displacement amplification or an amplification through a Q [beta] replicase, or any amplification of nucleic acid molecules known in the art Other methods can be used, and preferably PCR can be performed.
The 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.
The amplification reaction is not particularly limited as long as the amplification reaction is usually used for nucleic acid sequence amplification. Preferably, the amplification reaction may include annealing at a temperature of 44 to 70 ° C, more preferably 47 to 67 ° C RTI ID = 0.0 > of < / RTI >
The annealing temperature can be determined specifically for the primer set used, which can affect the target sequence amplification effect of the isolated genomic DNA.
The amplified target sequence may be labeled with a detectable labeling substance.
The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-terminal of the primer with Cy-5 or Cy-3, so that the target sequence can be labeled with a detectable fluorescent labeling substance.
When the radioactive isotope such as 32P or 35S is added to the PCR reaction solution in the PCR using the radioactive material, the amplification product is synthesized, and the radioactive substance is incorporated into the amplification product and the amplification product can be labeled as radioactive.
Preferably, the
The corn B73 standard genome is described in 2011 by Ganal et al. (Ganal et al., 2011. PloS ONE 6: e28334) and is described on the MaizeGDB website (http://www.maizegdb.org/sequence.php) Lt; / RTI >
The "complement" means that the nucleotide and all or some of the chromosome are specifically bound.
The "corn B73 standard genome" is a polymorphic sequence comprising a polymorphic site. A polymorphic sequence means a sequence comprising a polymorphic site comprising a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.
The primer set is not particularly limited as long as it is represented by two or more nucleotides of the group consisting of SEQ ID NOS: 1 to 12, but may be selected from the group consisting of DNA, RNA polymerase, Or a reverse transcriptase) and a single strand oligonucleotide capable of serving as a starting point of template-directed DNA synthesis under a suitable temperature. The appropriate length of the primer may vary depending on the purpose of use, but is usually 15-30 Nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template.
The primer sequence need not be completely complementary to the SNP marker, but should be sufficiently complementary to hybridize with the maize B73 standard genome.
The primer set may 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 ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
Next, the amplification product is detected.
The detection is not particularly limited as long as it is a method used for detecting an amplification product containing a target sequence amplified, for example, by capillary electrophoresis, DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement But is not limited thereto.
For example, the capillary electrophoresis can use an ABi Sequencer, and gel electrophoresis can use agarose gel electrophoresis or acrylamide gel electrophoresis according to the size of the amplification product. 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 32P or 35S is added to the PCR reaction solution to mark the amplification product, and then the radioactivity is measured using a radioactive measuring device such as a Geiger counter or a liquid scintillation counter A liquid scintillation counter can be used to measure radioactivity.
Further, the detection is not particularly limited as long as it is for detecting an amplification product containing the amplified target sequence, but preferably it may be determined that the amplified product is a lightpenicum species when all of the following conditions are satisfied.
I) detection of a product of size 390 bp amplified using an
The "amplification product size" differs depending on the position and size of the target sequence, and may vary depending on the primer set used for amplification.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Preparation of Gwangpyeong Jade variety
Gongpyeong in the following example was purchased from the Agricultural Technology Application Foundation and the sample of the Gwangpyeong Ore was cultivated at the National Institute of Food Science and Technology in 2005 by using the seeds maintained by the National Institute of Rural Development in the Rural Development Administration The leaves were collected and used.
2-1: DNA extraction
The maize genomic DNA was extracted from the young leaves of Gapyeong Okhwa corn obtained in Preparative Example 1 by slightly modifying the method of Dellaporta et al. (1983 Plant Mol. Biol. Rep. 1: 19-211983) Respectively.
Specifically, 0.1 g of the leaf tissue of Gwangpyeong jade was cut out, added to the induced liquid nitrogen, completely pulverized with a pestle, and then placed in a 2.0 ml tube. Thereafter, 0.16 g of sodium bisulfate was added to an extraction buffer (1 M Tris-HCl pH 8.0, 0.5 M EDTA, 5 M NaCl, 20% SDS) prepared in a water bath at 65 ° C and then 600 μl of an extraction buffer containing sodium bisulfate And tapping was performed. Then, 600 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v) was added to the tube and the tube was incubated for 15 minutes in a water bath at 65 ° C. and then immediately incubated at 13,000 rpm for 20 minutes After centrifugation, the supernatant was transferred to a new tube.
Then, 600 μl of chloroform: isoamyl alcohol (24: 1 = v: v) solution was added to the supernatant, followed by tapping and centrifugation at 14,500 rpm for 20 minutes. The supernatant was then transferred to a new tube, 600 μl of 95% ethanol and 60 μl of 3M sodium acetate were added, incubated at -20 ° C. for 2 hours and centrifuged at 14,500 rpm for 20 minutes.
Subsequently, all the solution was discarded so that the precipitated pellets did not fall off, and then 600 μl of 70% ethanol was added thereto, followed by light tapping, centrifugation at 10,000 rpm, discarding ethanol, and drying in air. Thereafter, 30 쨉 l of distilled water was added to dissolve, and 1 ㎕ of Ribonuclease (Rnase) (product name of RNase I, manufactured by Thermo Scientific) was added to completely remove RNA to obtain maize DNA.
2-2: Primer production
(Ganal et al., 2011. PloS ONE 6: e28334) SNP array information using the corn B73 standard genome in order to select the primer for discrimination of Gongpyong jade varieties. We found 200 SNP sites (20 SNP sites / chromosome) per chromosome through this information. Sequence information was obtained using the selected SNP site using the MaizeGDB website (http://www.maizegdb.org/).
After that, 200 primers were prepared by using 3 'end-inconsistent primer production method published by Professor Lee Seok-ha of Seoul National University using the principle that the PCR product is not generated due to inconsistency of the forward primer 3' al., 2005. Theor. AppL. Genet. 110: 1003-1010) (see Figure 1).
Specifically, if the second nucleotide sequence at the 3 'end of the SNP site is inconsistent to generate a PCR product in the presence of the SNP site and a SNP site is not present in the SNP site, the presence or absence of the SNP site , And it was tried to discriminate the cultivars easily. The Primer3 program was used and the PCR product was constructed to be generated at 200 ~ 500bp.
2-3: Specific reaction primer selection
In order to select the primer combination that specifically reacts with the DNA of Gwangpyeong japon among the combination of 200 primer sets with the second base at the 3 'end of the forward primer prepared in Example 2-2, After cultivating DNA of 50 cultivars including cultivars and imported varieties, primer combinations which were not amplified or amplified by a single band were firstly selected. The first selection process is shown in Fig.
Thereafter, in the case of hybridization of Gapyeong japonica and Gwangpyeong japonica, primers that do not match with the amplification reaction were removed secondarily, and primers that can be specifically selected for Gapyeong japonica were selected (see Fig. 3).
As a result, 20 primers per chromosome were amplified by PCR using the primers prepared in Example 2-2, and 16 primers with clear amplification patterns were selected and used for analysis (see FIG. 4).
The composition of the solution in the PCR amplification was 20 μl in total, and 20 ng of Gwangpyung Ok DNA of Example 2-1, 0.3 M of the primer prepared in Example 1-2 and 2 × Polymerase (manufactured by Promega) were used.
The PCR reaction was carried out at 95 ° C for 5 minutes, denatured at 95 ° C for 1 minute, annealed at 45 ° C to 65 ° C for 1 minute, and extended at 72 ° C for 2 minutes. This process was repeated 29 cycles. For 10 min and the PCR amplification was terminated at 4 ° C. Then, 18 μl of the amplified PCR product was loaded on 1% agarose gel and electrophoresed at 100 V (volts) for 40 minutes. After electrophoresis, EtBr (Ethidium bromide ), And the band was confirmed using UV.
As can be seen in Figure 2, only the primer combinations that specifically reacted to the SNP site were selected, except for primer combinations that resulted in unexpected PCR bands.
Then, in order to remove the primer combination in which the primer combination is inconsistent between the offspring and the cultivars, DNA was extracted by using the same method as in Example 2-1, and the possibility of discrimination between the DNAs was examined.
For the specific test method, the DNA of Gwangpyeong-yang harvested in 2015 was used as F1, the hybrid sample (KS124) was used and the replicate (KS85) was used (sample and duplicate requested to the National Institute of Food Science and Technology , And the cultivar was the same as that obtained in Example 2-1. In addition, the DNA extraction method was performed in the same manner as in Example 2-1, and the PCR amplification method was performed as described above, and the results are shown in FIG.
As can be seen in FIG. 3, primer combinations that were not generated in the varieties but not in the PCR bands were excluded from the selection because they were not suitable as a primer combination capable of discriminating the varieties. The following six primer sets are listed in Table 1 below.
number
number
(° C)
As shown in Table 1, the primers of the present invention selected three primers on chromosome 2, two primers on chromosome 4, and one primer on chromosome 8. The selected primer combination had an annealing temperature of 50-64 ° C.
In order to determine whether the combination of the two primers of the present invention can selectively screen Gwangpyong Ok from among the fifty kinds of corn for fodder, the corn variety shown in Fig. 5 was analyzed by PCR using the above six primer sets.
As a result, as shown in FIG. 6, a product having a size of 390 bp was detected in the amplified product using the
As can be seen from the above examples, it was found that the primer set of the present invention can effectively discriminate the Gwangpyeong species from various cultivars, effectively protect the Gwangpyung species and the purity of F1 seeds. Further, the present invention provides a useful discriminating method for the Gapyeong jade by using the primer set for the Gapyeong jade.
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Primer set for Kwangpyeongok identification and method for identification <130> 1042418 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IBM 471 Forward sequence <400> 1 ttctgctgtg gtaaatgcaa tt 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 471 Reverse sequence <400> 2 gctgtccaac accttgctta 20 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IBM 516 Forward sequence <400> 3 aacgagcctt tcattcttgc agatt 25 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 516 Reverse sequence <400> 4 acaagccacc gctcttggta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 535 Forward sequence <400> 5 tgctggattc gctcatccgg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IBM 535 Reverse sequence <400> 6 tgcgtcggca gcaaggaaca t 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IBM 911 Forward sequence <400> 7 ttggtacttg gtatcacaat gtta 24 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 911 Reverse sequence <400> 8 gcattgtgtc cagtgttctc 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IIBM 960 Forward sequence <400> 9 gcaaatgtgt gaacaataac ccag 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 960 Reverse sequence <400> 10 gaggcaagag catcactgtc 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IBM 1492 Forward sequence <400> 11 ggctgacgag tgacagtatt ttac 24 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBM 1492 Reverse sequence <400> 12 tgttgaatgt ggcggcactt 20
Claims (7)
(b) a primer set consisting of a forward primer represented by SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO: 4;
(c) a primer set consisting of a forward primer represented by SEQ ID NO: 5 and a reverse primer represented by SEQ ID NO: 6;
(d) a primer set consisting of a forward primer represented by SEQ ID NO: 7 and a reverse primer represented by SEQ ID NO: 8;
(e) a primer set consisting of a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10; And
(f) a primer set comprising a forward primer represented by SEQ ID NO: 11 and a reverse primer represented by SEQ ID NO: 12.
(ii) isolating the genomic DNA isolated in step (i)
(a) a primer set consisting of a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2;
(b) a primer set consisting of a forward primer represented by SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO: 4;
(c) a primer set consisting of a forward primer represented by SEQ ID NO: 5 and a reverse primer represented by SEQ ID NO: 6;
(d) a primer set consisting of a forward primer represented by SEQ ID NO: 7 and a reverse primer represented by SEQ ID NO: 8;
(e) a primer set consisting of a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10; And
(f) amplifying the target sequence by performing amplification reaction using a primer set comprising a forward primer represented by SEQ ID NO: 11 and a reverse primer represented by SEQ ID NO: 12; And
(iii) identifying the amplified product in step (ii);
Wherein said method comprises the steps of:
(a) detecting a product having a size of 390 bp among amplified products using a primer set consisting of a forward primer represented by SEQ ID NO: 1 and a reverse primer represented by SEQ ID NO: 2;
(b) detecting a product having a size of 319 bp among amplified products using a primer set consisting of a forward primer represented by SEQ ID NO: 3 and a reverse primer represented by SEQ ID NO: 4;
(c) detecting a 251 bp product in the amplified product using a primer set consisting of a forward primer represented by SEQ ID NO: 5 and a reverse primer represented by SEQ ID NO: 6;
(d) a product having a size of 227 bp among amplified products using a primer set consisting of a forward primer represented by SEQ ID NO: 7 and a reverse primer represented by SEQ ID NO: 8;
(e) detection of a 352 bp product in the amplified product using a primer set consisting of a forward primer represented by SEQ ID NO: 9 and a reverse primer represented by SEQ ID NO: 10; And
(f) A primer set consisting of a forward primer represented by SEQ ID NO: 11 and a reverse primer represented by SEQ ID NO: 12 should be used so that a product having a size of 271 bp among amplified products should not be detected.
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Non-Patent Citations (4)
| Title |
|---|
| Moon et al. Korean J. Breed. 33(4) : 350-351 (2001) |
| NCBI Reference Sequence: NW_007617758.1, NW_007617730.1, NW_007617764.1 |
| Park et al. Korean J. Breed. Sci. 40(3) : 250-527 (2008) |
| Tian et al. Mol Breeding (2015) 35 :136 |
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