KR20180049711A - SCAR marker for identification of blueberries and use thereof - Google Patents

Abstract

The present invention relates to a sequence characterized amplified region (SCAR) marker for identifying blueberry varieties, and a use thereof. More specifically, the present invention relates to an SCAR primer set for identifying blueberry varieties, a method for identifying blueberry varieties using the same, and a kit for identifying blueberry varieties including same.

Description

[0002] SCARMA MARKER FOR DISSEMINATION OF BLUEBERRY AND USE THEREOF {

The present invention relates to SCAR (Sequence Chracterized Amplified Region) markers for discriminating blueberries and their use, and more particularly, to a SCAR primer set for discriminating blueberry varieties, a method for discriminating blueberry varieties using the SCAR primer set, And a kit for discriminating a breed.

Blueberry (Vaccinium spp.) Is a shrubby crop belonging to the genus Vaccinium (Cyanoccous) section of the family Ericaceae. It is one of the world's top 10 super foods of the Time magazine. It is anthocyanin, It is also known as the 21st-century fruit tree because its efficacy has been acknowledged by its effective ingredients. Blueberries are also known to have effects such as vision recovery and enhancement, retinal degeneration and cataract prevention, carcinogenesis inhibition, and antioxidant effects.

Blueberries, known as the origin of Europe, such as North America and Finland, have been widely known for their taste and efficacy. Since the 1950s, cultivars have been developed and about 200 varieties of blueberries are known. Currently, about 100 varieties of blueberries are cultivated in Korea, but it is difficult to distinguish the correct varieties and the characteristics of purchased varieties are different.

Molecular markers, which are one of the methods to identify fruit varieties such as blueberries, can be useful because they can quickly distinguish cultivars without being affected by the cultivation environment or growing season of the crop. Molecular markers are a method of expressing individual polymorphisms in the nucleotide sequence differences of DNA, which is the essence of genetic phenomena, and include microsatellite or nucleotide sequence-based markers consisting mainly of repeating simple nucleotide sequences, or RFLP (Restriction Fragment Length Polymorphism) probe or SCAR (Sequence Characterized Amplified Region) marker.

In particular, the Random Amplified Polymorphic DNA (RAPD) method is most commonly used because it is easy and simple to analyze among various analysis methods. However, the RAPD method has disadvantages in that it is difficult to expect reproducible experimental results because the results may vary depending on the reaction conditions. In order to solve this problem, a method of performing PCR by preparing a new SCAR (Sequence Characterized Amplified Region) primer through cloning and sequencing of specific DNA bands generated from RAPD analysis results can be used. SCAR primers prepared in this manner can be used as more precise and finer primers. Korean Patent Registration No. 10-1457944, for example, discloses a scam marker for discriminating persimmon varieties and uses thereof. The need for the development of marker markers for the identification of blueberry varieties to prevent the incorporation of varieties of distribution seedlings using such molecular marker technology is required in order to develop molecular marking technology for blueberry varieties identification .

The purpose of this study is to develop molecular markers for the identification of blueberry varieties which can be used as a breeding certification technology in domestic blueberry distribution for the purpose of preventing the inclusion of variety in the distribution seedlings.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

The first aspect of the present invention provides a SCAR primer set for discriminating blueberry cultivars comprising a set of primers selected from the group consisting of SEQ ID NOS: 1-32.

According to a second aspect of the present invention, there is provided a method for producing a plant, comprising the steps of: a) extracting genomic DNA from a blueberry to be cultivated; b) amplifying the DNA using the extracted genomic DNA as a template using the SCAR primer set according to the first aspect of the present invention; And c) analyzing the amplified DNA product.

The third aspect of the present invention can provide a kit for discriminating blueberry cultivars comprising a SCAR primer set, a DNA polymerase, dNTPs and a buffer solution for PCR reaction according to the first aspect of the present invention.

Using the SCAR marker for discriminating blueberry varieties according to the present invention, it is possible to accurately identify the varieties of more than 45 kinds of blueberries, so that it can be utilized as a breed identification technology for prevention of the inclusion of varieties of distribution seedlings and distribution of domestic seedlings. In addition, SCAR markers are not affected by environmental conditions such as reaction conditions as compared with other PCR markers, and thus can be used as more accurate and detailed molecular markers.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the development process of a SCAR marker for discriminating blueberry cultivars of the present invention; FIG.
Figure 2 is a list of 45 blueberry varieties used in one embodiment of the invention.
FIG. 3 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 4 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 5 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 6 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 7 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 8 is a result of discrimination of blueberry cultivars using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention.
FIG. 9 is an image showing gel electrophoresis results using a SCAR marker for discriminating blueberry cultivars according to an embodiment of the present invention; FIG.
10 is information on the SCAR primer set for discriminating blueberry cultivars of the present application.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily carry out the present invention.

It should be understood, however, that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein. In order to clearly explain the present invention in the drawings, portions not related to the description are omitted.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

As used herein, the terms "about," " substantially, "and the like are used herein to refer to or approximate the numerical value of manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to prevent unauthorized exploitation by unauthorized intruders of the mentioned disclosure. For example, the term "about" is used to refer to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of 30, 25, 20, 25, 10, 9, Level, value, number, frequency, percentage, dimension, size, quantity, weight or length of a form, level, value varying from 1 to 6, 5, 4, 3,

Also, throughout the present specification, the phrase " step "or" step "does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout the present specification, the "SCAR (Sequence Characterized Amplified Region) Marker" is produced by analyzing the nucleotide sequence of the marker band of RAPD (Random Amplified Polymorphic DNA) or AFLP (Amplification Fragment Length Polymorphism) ) Method to check the presence or absence of a band. Because SCAR markers are relatively insensitive to amplification environments and can readily read results compared to other molecular markers, they are recognized as efficient marker markers for breed identification with reproducibility, universality and simplicity, and as a result, more precise and finer primers It is possible to use.

Throughout this specification, a "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be copied and can serve as a starting point for the 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.

Hereinafter, the present invention will be described in detail with reference to embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

According to a first aspect of the present invention there is provided a primer set comprising a BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 1 and 2; A B268_262 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 3 and 4; A BA19_288 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 5 and 6; A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8; A B504_416 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 9 and 10; A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12; A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14; A B168_520 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 15 and 16; A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18; A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20; A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22; A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24; A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26; A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; A B297_754 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 29 and 30; And a set of BE17_831 primers consisting of the nucleic acid sequences of SEQ ID NOs: 31 and 32. The set of SCAR (Sequence Characterized Amplified Region) primers for discriminating blueberry varieties can be provided.

SCAR primer sets for discriminating blueberry cultivars according to the present application are used as a primer set for DNA amplification, and can be preferably used as the set described in FIG. In addition, one or more primer sets can be used in combination to more accurately distinguish the variety of blueberries. In addition, the primer may also include a sequence in which a base sequence is added, deleted or substituted in SEQ ID NO: 1 to SEQ ID NO: 32. In Fig. 10, F represents a forward primer and R represents a reverse primer.

More preferably, at least 11 kinds of primer sets out of the listed primer sets can be used in combination.

According to one embodiment of the invention, the SCAR primer set comprises a BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 1 and 2; A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8; A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12; A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14; A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18; A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20; A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22; A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24; A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26; A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And a set of BE17_831 primers consisting of the nucleic acid sequences of SEQ ID NOs: 31 and 32. [

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a process of developing a SCAR marker for discriminating blueberry cultivars according to the present invention; FIG. Specifically, the marker of the present invention extracts genomic DNA from 45 varieties of blueberry, searches for polymorphic sites through analysis using the RAPD method, compares the polymorphic sites on the basis of the genotype, selects effective DNA-specific fragments, , Sequencing the nucleotide sequence, and re-synthesizing the primer specific for the label. The SCAR markers thus developed are simple, rapid, highly reproducible and reliable molecular markers.

The Random Amplified Polymorphic DNA (RAPD) method amplifies only the genomic region aligned by two base sequences using a short random primer of usually 9 to 11 bp (base pair). This method is very simple because the PCR products are electrophoresed on agarose gel to analyze the morphology of the fragments. However, since it is difficult to use the PCR products as selection markers of breed sarcoma in terms of efficiency and accuracy, DNA marker SCAR marker.

The blueberry varieties identified by the SCAR primer set for discriminating blueberry varieties according to the present invention were 31 varieties introduced by the US Center for Genetic Resources Research and 14 varieties preserved in the National Institute of Horticultural Science, Eight southern high bushes, four species of rabbit eyes).

According to one embodiment of the present disclosure, the blueberry varieties that can be distinguished by the SCAR primer set include, but are not limited to, Aron, Ashworth, Berkeley, Bluejay, Blueray, Bluetta, Brigitta Blue, Burlington, Cabot, Concord, Dixi, Duke, Elliott, Evelyn, Georgiagem, Hardyblue, Herbert, Ivanhoe, June, Laniera, Legacy, Northland, Olympia, Pacific, Pacific, Patriot, Pemberton, Pink Lemonade, Pioneer, Rubel, Sierra, Spartan, Alapaha, Aurora, Aurora, Camellia, Chandler, Draper, Emerald, Huron, Liberty, Meadowlark, Misty, ), Rebel, Star, T-959 (Titan), Vernon, and the like.

According to a second aspect of the present invention, there is provided a method of extracting genomic DNA, comprising the steps of: a) extracting genomic DNA from a blueberry to be cultivated; b) amplifying the DNA using the extracted genomic DNA as a template and using the SCAR primer set according to claim 1; And c) analyzing the amplified DNA product. The present invention also provides a method for identifying a blueberry variety.

The method of extracting the genomic DNA of blueberry in the step a) may be carried out by a conventional method known in the art. For example, a CTE (Cetyltrimethyelmonium Bromide) method, a phenol / chloroform extraction method, an SDS extraction method, or the like may be used, or a commercially available DNA extraction kit may be used, but the present invention is not limited thereto.

The SCAR primer set in the step b) includes at least one selected from the group consisting of primer sets each having a nucleic acid sequence of SEQ ID NOS: 1 to 32, which is capable of distinguishing 45 kinds of blueberry varieties shown in Fig. 2 It is a SCAR marker. More than one set of primers can be used as the SCAR markers for discriminating the variety of blueberries, and when a plurality of molecular markers are simultaneously used, the variety can be more accurately distinguished.

Also, the amplification of the genomic DNA in step b) may be performed by PCR (Polymerase Chain Reaction). PCR may be performed using PCR reaction mixtures containing components known to be required for PCR reactions in the art or using commercially available kits. The PCR reaction mixture may include a genomic DNA extracted from blueberry and a SCAR primer set of the present invention, an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water. The PCR buffer solution may include, but is not limited to, Tris-HCl, MgCl ₂ , KCl, and the like.

The amplified target sequence may be labeled with a detectable labeling substance. For example, the labeling material may be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance may be 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 in the PCR using the radioactive material, the radioactive isotope may be incorporated into the amplification product by radioactivity as the amplification product is synthesized. Alternatively, the amplification products can be visualized using a silver chloride kit (Bioneer, Daejeon, Korea).

The DNA product amplified in the step c) may be analyzed by any conventional method known in the art, for example, by capillary electrophoresis, DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement But may not be limited thereto.

For example, capillary electrophoresis may be performed to detect the amplification product, and may be performed using an ABi Sequencer, but this is not so limited. In addition, gel electrophoresis can be performed, and gel electrophoresis can utilize agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. For example, 6% acrylamide gel electrophoresis can be used. For example, 1 to 5%, preferably 1.4% agarose gel electrophoresis can be used. 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.

When the amplified DNA product is analyzed using gel electrophoresis, amplification products are electrophoresed on an agarose gel or an acrylamide gel, and bands can be identified by ethidium bromide (EtBr), silver staining, etc. have.

The 45 kinds of blueberry cultivars which can be distinguished by the molecular markers according to the present invention differ in their amplification by the SCAR molecular markers used and whether or not they are amplified by the respective molecular markers are as shown in FIGS. Therefore, the result of amplification from the genomic DNA of blueberry to discriminate the cultivars can be compared with the results shown in Figs. 3 to 8 to distinguish the cultivars.

According to one embodiment of the invention, the SCAR primer set comprises a BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 1 and 2; A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8; A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12; A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14; A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18; A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20; A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22; A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24; A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26; A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And a set of BE17_831 primers consisting of the nucleic acid sequences of SEQ ID NOS: 31 and 32, but not always limited thereto.

According to one embodiment of the present invention, the analysis of the amplified product may be performed by a DNA chip, electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but the present invention is not limited thereto.

According to one embodiment of the present invention, the method for distinguishing the blueberry variety may further include a step of comparing the amplified product analyzed with the amplified product of the blueberry variety standard to determine the variety, but the present invention is not limited thereto have.

According to one embodiment of the present invention, the discrimination method of the blueberry variety is selected from the group consisting of Aron, Ashworth, Berkeley, Bluejay, Blueray, Bluetta, Brigitta Blue, Burlington, Cabot, Concord, Dixi, Duke, Elliott, Evelyn, Georgiagem, Hardyblue, Herbert, Ivanhoe, June, Laniera, Legacy, Northland, Olympia, Pacific, Patriot, Patriot, Pemberton, Pink Lemonade, Pioneer, Rubel, Sierra, Spartan, Alapaha, Aurora, Camellia, ), Chandler, Draper, Emerald, Huron, Liberty, Meadowlark, Misty, Rebel, Star, T -959 (T itan, Vernon, and the like, but the present invention is not limited thereto.

According to a third aspect of the present invention, there is provided a kit for discriminating blueberry cultivars comprising a SCAR primer set, DNA polymerase, dNTPs and a buffer solution for PCR reaction according to the first aspect of the present invention.

In the kit according to the present invention, the SCAR primer set for discriminating blueberry variety may be a SCAR primer set comprising at least one selected from the group consisting of primer sets each having a nucleic acid sequence of SEQ ID NOS: 1 to 32 described in Fig. 10 . The buffer solution for PCR reaction has a conventionally known composition in the art and may include, but is not limited to, Tris-HCl, MgCl ₂ , KCl, and the like.

The blueberry variety identification kit according to the present invention may further include components necessary for electrophoresis to confirm whether the PCR product is amplified.

According to one embodiment of the invention, the SCAR primer set comprises a BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 1 and 2; A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8; A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12; A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14; A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18; A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20; A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22; A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24; A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26; A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And a set of BE17_831 primers consisting of the nucleic acid sequences of SEQ ID NOS: 31 and 32, but not always limited thereto.

The present invention may be better understood by reference to the following examples, which are for the purpose of illustration only and are not intended to limit the scope of protection defined by the appended claims.

[Example]

Test materials and DNA extraction

In this example, 31 varieties such as blueberry "Aron" and 14 varieties such as "Alapaha" which are preserved at the National Institute of Horticultural Science, Rural Development Administration were used. Among them, 33 species of Northern Highbush, 8 species of Southern Highbush, and 4 species of Rabbit. A list of varieties of blueberries used is shown in FIG.

The young leaves of blueberries were harvested and genomic DNA was extracted using DNeasy plant mini kit (Qiagen, Valencia, CA, USA). The extracted DNA was confirmed by electrophoresis on 0.8% agarose gel. The amount of DNA was quantified with a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA) and then diluted to a concentration of 5 ng · μL ^-1 and used for PCR analysis.

Random Amplified Polymorphic DNA (RAPD) analysis

A selection of 420 primers consisting of 10 bases prepared from Operon (Operon Technologies, Alameda, CA, USA) and the University of British Columbia (UBC, Vancouver, BC, Canada) U, U, W, Y, UBC conifer kit) were tested. A total of 43 random primers selected in the primer assay were used for RAPD marker selection. The PCR conditions were 20 ng of genomic DNA, 1 × PCR buffer (Genetbio, Daejeon, Korea), 5 pmol random primer, 200 μM dNTP, 3 mM MgCl ₂ and 0.5 unit of Taq DNA polymerase ). The PCR reaction was carried out using a thermocycler (C-1000, Bio-Rad Laboratories, Hercules, Calif., USA) with the following program: initial denaturation at 94 ° C for 5 min, Denaturation, annealing and extension were repeated 10 times at 94 ° C for 45 seconds and 72 ° C for 2 minutes, followed by 45 seconds at 94 ° C, 45 seconds at 42 ° C, 2 minutes at 72 ° C And repeated 30 times. Finally, the reaction was terminated after treatment at 72 ° C for 5 minutes. The amplified PCR product was stained with ethidium bromide (EtBr) and electrophoresed on 1.4% agarose gel at 150 V for 3 hrs.

A total of 420 Operon and UBC conifer kit primers were analyzed for 4 blueberries ('Bluejay', 'Duke', 'Rebel' and 'Titan' 43 primers with a high number of bands and clear bands were selected. A total of 210 polymorphic bands were selected by RAPD analysis of 45 blueberry varieties using selected primers.

Variety-specific selection RAPD Marker  SCAR (Sequence Chracterized  Amplified Region Marker Conversion

Eighty - five species of the selected blueberry variety - specific RAPD bands of 1,000 bp or less were transformed into SCAR markers by cloning and sequencing. The DNA bands were cut out and sequenced using QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif., USA) for sequence specificity of RAPD markers. The DNA was inserted into pCR2.1-TOPO vector using TOPO-TA cloning system (Invitrogen, Carlsbad, CA, USA) and transformed into E. coli TOP10. Plasmid DNA was isolated from this strain and the inserted DNA base sequence was analyzed using an automatic DNA analyzer (ABI 3730xl, Applied Biosysyems, CA, USA). Using the Primer3 (http://frodo.wi.mit.edu) program based on the results of sequencing of the RAPD markers, a SCAR primer of 20-24 mer size with or without 10 bases of the primer was prepared . At least four SCAR primers per RAPD marker were tested. PCR reaction was performed by adding 40 ng of genomic DNA, 200 μM dNTP, 5 pmol of SCAR primer, 1 × PCR buffer (Genetbio) and 0.5 units of Hot-start Taq DNA polymerase to minimize nonspecific DNA amplification (Genetbio). After the denaturation of the template DNA at 95 ° C for 10 minutes, the PCR was repeated 30 times at 95 ° C for 30 seconds, at 63-65 ° C for 30 seconds, and at 72 ° C for 1 minute, followed by treatment at 72 ° C for 5 minutes, . The amplified PCR products were confirmed by electrophoresis on 1.4% agarose gel. As a result, 16 kinds of SCAR markers were developed and it was possible to identify blueberry varieties by PCR amplification product and amplified band size.

The results of discrimination of 45 varieties of blueberries using 16 types of SCAR markers are shown in Figs. 3 to 5. Fig. The results marked with 1 indicate that specific markers were observed, and the results marked with 0 indicate that no specific markers were observed.

It was not necessary to use all 16 markers to distinguish 45 varieties of blueberries. 1 and 2, 7 and 8, 11 and 12, 13 and 14, 17 and 18, 19 and 20 (respectively SEQ ID NOS: 1 and 2, 7 and 8, 11 and 12, 13 and 14, respectively) were used as the primer sets BE12_175, BE17_354, BR09_465, BF07_502, B333_561, BR09_612, BN03_637, B268_668, BN07_727, B550_750 and BE17_831 , 21 and 22, 23 and 24, 25 and 26, 27 and 28, and 31 and 32). The results of discrimination of 45 varieties of blueberries are shown in FIGS.

FIG. 9 is an electrophoresis image of a 561 bp B333_561 SCAR marker, which is found only in some varieties of 45 blueberry varieties identified using the primer sets of SEQ ID NOS: 17 and 18.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed in a similar fashion may also be implemented.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> SCAR marker for identification of blueberries and use          the <130> 0 <160> 32 <170> KoPatentin 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BE12_175_F <400> 1 gctgaaggcc ttataaacat tgaa 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BE12_175_R <400> 2 tatgttatct ggactcgttc tgga 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B268_262_F <400> 3 aggccgctta cttggtttac atag 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B268_262_R <400> 4 aggccgctta tgaagtatta ttgc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BA19_288_F <400> 5 ggtctcgatt ctcgagctta tatc 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BA19_288_R <400> 6 tcgggttaca tgaagcaaat ttat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BE17_354_F <400> 7 acagtactcc aacaacagtc gaag 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BE17_354_R <400> 8 tcagttggaa atgacccata ttag 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B504_416_F <400> 9 accgtgcgtc atcaataaaa gata 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B504_416_R <400> 10 tttactaaac ctttgggtgt tgct 24 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BR09_465_F <400> 11 gatatagctg ccacatcagc ag 22 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BR09_465_R <400> 12 acgagatttt aagaaggttt cgtg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BF07_502_F <400> 13 ccgatatccc ttggacccat cagt 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BF07_502_R <400> 14 ccgatatccc cgagcaagtc attt 24 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B168_520_F <400> 15 gccaaatagt actccgcaag taag 24 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> B168_520_R <400> 16 gatgtgctaa tcgttaacaa ataatg 26 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> B333_561_F <400> 17 gaatgcgacg tcgtgaaggt atacata 27 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> B333_561_R <400> 18 gaatgcgacg gtataggggg atcactg 27 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BR09_612_F <400> 19 gcgagatggt gataaaagaa aaga 24 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BR09_612_R <400> 20 gcacgagtac acaacggagt aata 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BN03_637_F <400> 21 ggtgagaata gagttgaggt tggt 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BN03_637_R <400> 22 gggtgggtat aataagcata aagc 24 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B268_668_F <400> 23 aggccgctta tattgtagca tttt 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B268_668_R <400> 24 aggataaagg agaagaaaca aacg 24 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BN07_727_F <400> 25 cagcccagag taatactcca aaat 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BN07_727_R <400> 26 taggggaaga ttgataaaga gacg 24 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B550_750_F <400> 27 gtcgcctgag cagaaaaaaa tgta 24 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> B550_750_R <400> 28 cctgagaaag gtacgcaata ctct 24 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> B297_754_F <400> 29 gcgcattaga ggactgagca cctggtc 27 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> B297_754_R <400> 30 gcgcattaga tctaactgga aaggttg 27 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BE17_831_F <400> 31 ttggatgatc aatcagttca ctct 24 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BE17_831_R <400> 32 gatgtgctaa tcgttaacaa ataatg 26

Claims (10)

A BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 1 and 2;
A B268_262 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 3 and 4;
A BA19_288 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 5 and 6;
A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8;
A B504_416 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 9 and 10;
A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12;
A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14;
A B168_520 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 15 and 16;
A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18;
A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20;
A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22;
A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24;
A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26;
A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28;
A B297_754 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 29 and 30; And
A Sequence Characterized Amplified Region (SCAR) primer set for discriminating blueberry varieties, comprising at least one primer set selected from the group consisting of BE17_831 primer sets consisting of the nucleic acid sequences of SEQ ID NOs: 31 and 32;
The method according to claim 1,
A BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 1 and 2;
A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8;
A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12;
A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14;
A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18;
A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20;
A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22;
A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24;
A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26;
A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And
31. A set of SCAR primers comprising a set of BE17_831 primers consisting of nucleic acid sequences of SEQ ID NOS: 31 and 32.
The method according to claim 1,
Aron, Ashworth, Berkeley, Bluejay, Blueray, Bluetta, Brigitta Blue, Burlington, Cabot, Concord, Dixi, Duke, Elliott, Evelyn, Georgiagem, Hardyblue, Herbert, Ivanhoe, Ivanhoe, June, Laniera, Legacy, Northland, Olympia, Pacific, Patriot, Pemberton, Pink Lemonade, Pioneer, Rubel, Sierra, Spartan, Alapaha, Aurora, Camellia, Chandler, Draper, Emerald, ), Huron, Liberty, Meadowlark, Misty, Rebel, Star, T-959 (Titan), Vernon Blueberries SCAR primer set which can discriminate cultivars.
a) extracting genomic DNA from a blueberry to identify a variety;
b) amplifying the DNA using the extracted genomic DNA as a template and using the SCAR primer set according to claim 1; And
and c) analyzing the amplified DNA product.
5. The method of claim 4,
The SCAR primer set
A BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 1 and 2;
A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8;
A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12;
A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14;
A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18;
A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20;
A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22;
A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24;
A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26;
A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And
A BE17_831 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 31 and 32
Of the blueberry variety.
5. The method of claim 4,
Wherein the analysis of the amplified product is performed by a DNA chip, electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement method.
5. The method of claim 4,
Further comprising the step of comparing the amplified product analyzed with the amplified product of the blueberry variety standard to determine the variety.
5. The method of claim 4,
Aron, Ashworth, Berkeley, Bluejay, Blueray, Bluetta, Brigitta Blue, Burlington, Cabot, Concord, Dixi, Duke, Elliott, Evelyn, Georgiagem, Hardyblue, Herbert, Ivanhoe, Ivanhoe, June, Laniera, Legacy, Northland, Olympia, Pacific, Patriot, Pemberton, Pink Lemonade, Pioneer, Rubel, Sierra, Spartan, Alapaha, Aurora, Camellia, Chandler, Draper, Emerald, ), Huron, Liberty, Meadowlark, Misty, Rebel, Star, T-959 (Titan), Vernon Blueberries A method for distinguishing a variety of blueberries for discriminating a variety.
A kit for discriminating blueberry cultivars, comprising a SCAR primer set according to claim 1, a DNA polymerase, dNTPs and a buffer solution for PCR reaction.
10. The method of claim 9,
The SCAR primer set
A BE12_175 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 1 and 2;
A BE17_354 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 7 and 8;
A BR09_465 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 11 and 12;
A BF07_502 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 13 and 14;
A B333_561 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 17 and 18;
A BR09_612 primer set consisting of the nucleic acid sequences of SEQ ID NOs: 19 and 20;
A BN03_637 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 21 and 22;
A B268_668 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 23 and 24;
A BN07_727 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 25 and 26;
A B550_750 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 27 and 28; And
A BE17_831 primer set consisting of the nucleic acid sequences of SEQ ID NOS: 31 and 32
&Lt; / RTI &gt;

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