KR20160086178A - Random Amplified Polymorphic DNA primer for discrimination of Zizyphus jujuba Miller - Google Patents

Abstract

The present invention relates to a method for discriminating the species of jujubes through a random amplified polymorphic DNA (RAPD) analysis, and a primer used for the same. More specifically, the present invention relates to an RAPD primer having a particular base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3; a method for detecting DNA polymorphism of jujubes, wherein the method comprises performing a PCR by using the same; and a method for discriminating the of jujubes. According to the present invention, an RAPD marker for discriminating the species of jujubes effectively detects DNA polymorphism of jujubes and can be very useful in writing a DNA profile of jujubes. Accordingly, a genetic source of jujubes is effectively evaluated, so doubleness of an introduced novel genetic source with a conventional retained resource can be effectively analyzed and novelty can be effectively established. Also, the species of jujubes can be discriminated. Moreover, domestically distributed native species can be clearly separated, and species which do not have titles can be registered as novel species. Accordingly, precious genetic resources can be protected, and domestic species and imported species can be identified. Thus, order in distribution can be established in the domestic jujube market.

Description

Random Amplified Polymorphic DNA Primer for Discrimination of Jujube Varieties for Discrimination of Zizyphus jujuba Miller}

The present invention relates to a method for discriminating the variety of jujubes by RAPD (Random Amplified Polymorphic DNA) analysis and a primer used in the method. More specifically, the present invention relates to a method for identifying a variety of jujubes by using a specific base sequence selected from the group consisting of SEQ ID NOS: The present invention relates to a method for detecting DNA polymorphism of jujubes and a method for discriminating jujubes from rice cultivars including PCR using the RAPD primers.

The jujube ( Zizyphus jujuba Miller ) is a species of the family Seagrass, about 40 species ranging from tropical to temperate regions in the northern hemisphere. In Korea, Japan, China and the Soviet Union, Z. jujuba, And Iran, Iraq, etc. There are two completely different ecologically grown species such as Z. mauritiana, an evergreen dairy plant of the Indian jujube species. Fruit jujube has been widely used for medicinal materials and edible use since it has excellent function to protect nourishment gland and stomach and liver from olden days, and recently it has been spotlighted as a health food as well as reporting the inhibition of cancer cell proliferation. In spite of these various uses, studies on the cultivation of jujube cultivars in Korea were poorly classified until the 1970s, and they were named not simply formally named varieties, but were classified based on the morphological characteristics of jujubes. However, phenotypic characteristics are affected by the environment and there is a problem in identifying the correct breed.

In recent years, along with the radical development of molecular biology, molecular biology 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 "

However, RAPD markers for dairy cultivars have not yet been developed in Korea. In particular, studies involving genetic resources of domesticated domestic varieties have not been studied in any other countries

Therefore, the present inventors made a total of 60 RAPD primer sets for comparative analysis of jujube varieties using RAPD analysis using molecular markers during molecular biology approach. Among them, The present inventors completed the present invention by confirming that a variety of jujube varieties cultivated domestically can be identified through the formation of specific bands in four different jujube cultivars (Kwangwang, Mizo, Sangwang, Boeun) cultivated in Korea.

Korean Patent No. 10-0215084 Korean Patent No. 10-2012-0001870 Korea Patent No. 10-2011-0135835

Accordingly, an object of the present invention is to provide a RAPD primer capable of effectively discriminating the cultivars of jujube cultivated in Korea.

Another object of the present invention is to provide a composition for discriminating jujube cultivars comprising the RAPD primer.

And a kit for discriminating the variety of jujube including the composition.

It is another object of the present invention to provide a method for efficiently discriminating jujube cultivars by performing PCR using the RAPD primer.

In order to accomplish the object of the present invention, the present invention provides a RAPD (Random Amplified Polymorphic DNA) primer for distinguishing a variety of jujube having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

In one embodiment of the present invention, the jujube cultivar may be one selected from the group consisting of ginseng, fine grains, sweet potatoes and sweet potatoes.

In one embodiment of the present invention, SEQ ID NO: 1 can discriminate the local light among the jujube cultivars.

In one embodiment of the present invention, SEQ ID NO: 2 is capable of discriminating mizuna among the dairy varieties.

In one embodiment of the present invention, SEQ ID NO: 2 or 3 is capable of discriminating a female of a jujube variety.

In one embodiment of the present invention, SEQ ID NO: 3 can discriminate complement among the jujube cultivars.

In addition, the present invention provides a composition for discriminating jujube cultivars comprising the primer.

In addition, the present invention provides a kit for discriminating jujube cultivars comprising the above composition.

In addition, the present invention provides a method for discriminating jujube cultivars, comprising the step of performing PCR using a nucleic acid sample obtained from an individual using the primer.

It can be used very useful for preparing the DNA profile of jujube according to the present invention. Thus, by efficiently evaluating the genetic resources of jujube, it is possible to effectively perform duplication analysis and novelty establishment , And there is an effect that the variety of jujube can be discriminated. In addition, it is possible to clarify the distinction between native varieties (strains) scattered in Korea, and also to register new varieties of unnamed varieties, thereby protecting our valuable genetic resources and enabling identification of domestic and imported varieties It can contribute to establishing the circulation order of the jujube market.

FIG. 1 is an electrophoresis photograph of a reaction product after PCR amplification using DNA isolated from 10 dairy varieties using primer A08 (SEQ ID NO: 1) of the present invention.
FIG. 2 is an electrophoresis photograph of a reaction product after PCR amplification using DNA isolated from 10 dairy varieties using primer A09 (SEQ ID NO: 2) of the present invention as a template.
FIG. 3 is an electrophoresis image of the reaction product after PCR amplification using DNA isolated from 10 dairy varieties using primer C08 (SEQ ID NO: 3) of the present invention as a template.

In one aspect, the present invention provides a Random Amplified Polymorphic DNA (RAPD) primer for discriminating jujube varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

In the present invention, the jujube cultivars may be one species selected from the group consisting of kwangwol, mizuho, king kyo, and boehm.

In the present invention, the 'Kwangwol' is a new product with a maximum weight of 90g or more. It is easy to cultivate because of its high adaptability to soil and low pest resistance. It has good fruit yield, high sugar content and excellent taste.

In the present invention, the 'Mizo' is a native species and has many jujube cracked on the surface of the husk. Many of the fruits are rotten, the sugar content is low and the meat quality is lower than other varieties. The harvest period is from September 30 to October, Is a widely unknown variety.

In the present invention, the 'Sangwang' is a new product with a maximum weight of 90g or more, and has an overgrowth type overgrowth. It has excellent adaptability to soil and can be cultivated all over the country. It is used as a biomass because it is dense and has high sugar content.

In the present invention, the 'Boeun' is a native species grown in Chungcheongnam-do province. Jujube is small in the bovine, has a sugar content of normal, and has no seed in the nucleus.

In the present invention, the term 'primer' refers to a short nucleic acid sequence capable of forming a base pair with a complementary template and functioning as a starting point for template strand copying. In a specific embodiment of the present invention, a primer having one base sequence selected from the group consisting of SEQ ID NOS: 1 to 3 was used.

In one embodiment of the present invention, SEQ ID NO: 1 can discriminate the local light among the jujube cultivars; Wherein SEQ ID NO: 2 is capable of discriminating flavor among the diest cultivars; The above SEQ ID NO: 2 or 3 is capable of discriminating the female of the jujube cultivars; SEQ ID NO: 3 can discriminate complement among jujube cultivars.

In another aspect, the present invention provides a composition for distinguishing jujube varieties comprising the above primer (RAPD primer for discriminating jujube varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3).

In another aspect, the present invention provides a kit for distinguishing a jujube cultivar comprising the composition.

In addition to the RAPD primer, the kit for discriminating jujube cultivars according to the present invention may further include a reaction buffer solution, a polymerase, a dNTP, a stabilizer, and all biological or chemical reagents and instructions for detecting jujube cultivars in a sample . Other configurations of such kits may be suitably selected by those skilled in the art.

In another aspect, the present invention provides a method for discriminating jujube cultivars, which comprises performing PCR using a RAPD primer with a nucleic acid sample obtained from an individual.

The subject of the present invention is a target jujube variety to be discriminated.

In the PCR of the present invention, the target sequence can be amplified by amplifying the nucleic acid sample obtained from the individual using the RAPD primer for discriminating the jujube variety having the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 3 .

In one embodiment of the present invention, the PCR is performed at 94 ° C for 15 minutes, followed by denaturation at 94 ° C for 1 minute, annealing at 35 ° C for 1 minute, extension at 72 ° C for 2 minutes, And the reaction is carried out at 72 ° C for 10 minutes in the final extension step.

The target sequence amplified by the PCR may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), 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. In addition, if the radioactive isotope such as ³² P or ³⁵ S is added to the PCT reaction solution during the PCR, the amplified product may be synthesized and the radiation may be incorporated into the amplification product and the amplification product may be labeled with the radiation.

Detection of the amplified product can be performed through DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. 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 the radioactivity is measured by a radiation measuring device such as a Geiger counter or liquid scintillation counter The radiation can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

For the identification of jujube varieties primer  making

A total of 60 RAPD primers were used for the RAPD analysis, 20 of which were Universal primers A, B and C, each consisting of 10-mers of Operon.

Jujube detection primer primer The nucleotide sequence (5'-3 ') primer The nucleotide sequence (5'-3 ') primer The nucleotide sequence (5'-3 ') A01 CAGGCCCTTC B01 GTTTCGCTCC C01 TTCGAGCCAG A02 TGCCGAGCTG B02 TGATCCCTGG CO2 GTGAGGCGTC A03 AGTCAGCCAC B03 CATCCCCCTG C03 GGGGGTCTTT A04 AATCGGGCTG B04 GGACTGGAGT C04 CCGCATCTAC A05 AGGGGTCTTG B05 TGCGCCCTTC C05 GATGACCGCC A06 GGTCCCTGAC B06 TGCTCTGCCC C06 GAACGGACTC A07 GAAACGGGTG B07 GGTGACGCAG C07 GTCCCGACGA A08 GTGACGTAGG B08 GTCCACACGG C08 TGGACCGGTG A09 GGGTAACGCC B09 TGGGGGACTC C09 CTCACCGTCC A10 GTGATCGCAG B10 CTGCTGGGAC C10 TGTCTGGGTG A11 CAATCGCCGT B11 GTAGACCCGT C11 AAAGCTGCGG A12 TCGGCGATAG B12 CCTTGACGCA C12 TGTCATCCCC A13 CAGCACCCAC B13 TTCCCCCGCT C13 AAGCCTCGTC A14 TCTGTGCTGG B14 TCCGCTCTGG C14 TGCGTGCTTG A15 TTCCGAACCC B15 GGAGGGTGTT C15 GACGGATCAG A16 AGCCAGCGAA B16 TTTGCCCGGA C16 CACACTCCAG A17 GACCGCTTGT B17 AGGGAACGAG C17 TTCCCCCCAG A18 AGGTGACCGT B18 CCACAGCAGT C18 TGAGTGGGTG A19 CAAACGTCGG B19 ACCCCCGAAG C19 GTTGCCAGCC A20 GTTGCGATCC B20 GGACCCTTAC C20 ACTTCGCCAC

< Example  2>

Suitable for identification of jujube varieties of the present invention primer  Selection

<2-1> Preparation of jujube variety sample

In order to identify the primer for RAPD suitable for identification of the actual jujube variety, 10 different jujube varieties were provided in the form of leaves in the jujube research institute, which showed a variety of jujube varieties, such as wolves, moochul, Venus, Chuseok, Was used as a sample (material) of the present invention (see Table 2).

No. kind Origin The place of acquisition (or place of purchase) Characteristic One Bo Native species Chungcheongbuk-do Agricultural Research Institute Have normal sugar content in bovine
There is no seed in the nucleus. 2 Wake Improved species Chungcheongbuk-do Agricultural Research Institute High sugar content in the flesh
Used as raw and dried fruits 3 Mud Improved species Chungcheongbuk-do Agricultural Research Institute High sugar content and good yield 4 Venus Improved species Chungcheongbuk-do Agricultural Research Institute It has a high sugar content in the middle and middle, mainly used as dry matter. 5 Thanksgiving Improved species Chungcheongbuk-do Agricultural Research Institute It was developed in 1995 and its exact characteristics are not described. 6 manicure Native species Chungcheongbuk-do Agricultural Research Institute When mature, the surface of the skin is cracked and the sugar content and meat quality are lower than those of other varieties. 7 Demodulation Native species Chungcheongbuk-do Agricultural Research Institute Fruit is large, high sugar content, but uneven fruit 8 Ginkgo New variety Chungcheongbuk-do Agricultural Research Institute High and medium sugar content 9 Good New variety Chungcheongbuk-do Agricultural Research Institute High and medium sugar content 10 King New variety Chungcheongbuk-do Agricultural Research Institute High and medium sugar content

<2-2> DNA extraction

The samples of each of the jujube cultivars prepared in Example <2-1> were frozen using liquid nitrogen, ground in a mortar and equally prepared, and then processed using DNeasy Plant Mini Kit (QIAgen, USA). DNA was extracted and quantified with 10 μg / ml of each DNA with Qubit ^™ fluorometer (Invitrogen, USA).

<2-3> PCR using RAPD primer

In this experiment, PCR analysis was carried out using a total of 60 RAPD primer sets, each of 20 primers A, B and C for discriminating juvenile variety shown in Table 1 above. The PCR conditions were pre-denaturation at 94 ° C for 15 min, denaturation at 94 ° C for 1 min, annealing at 35 ° C for 1 min, extension at 72 ° C for 2 min and amplification for 45 cycles followed by final extension at 72 ° C for 10 min.

To confirm the amplified PCR product, 4ul of Gel loading buffer 6X (Bionix, Korea) was added to the PCR product, and the mixture was subjected to 1.5% agarose gel electrophoresis at 100 V for 30 minutes. Then, a UVP Biospectrum imaging system (UVP , USA) were used to identify the strain-specific bands.

As a result, as shown in Fig. 1, only three primers in a total of 60 sets of RAPD primers showed specific bands according to the cultivars. In detail, a specific band was identified in the primer A08 of the king, a specific band in the primer A09 was confirmed in the Mizogawa king, and a specific band was identified in the primer C08 in the bosom and kingpin. On the other hand, in the B primer set, no specific band was found for each kind.

Specific molecular marker list by RAPD analysis of samples of jujube varieties Sample primer Ginkgo A08 manicure A09 King A09, C08 Bo C08

Therefore, through the above-mentioned results, it was proved through experiments that three RAPD primers suitable for discrimination of the jujube cultivar in a total of 60 RAPD primer sets were proved through experiments. In the present invention, these three RAPD primers are shown in Table 4 As shown in SEQ ID NOS: 1 to 3.

The primer for RAPD for the identification of the jujube variety of the present invention primer The nucleotide sequence (5'-3 ') SEQ ID NO: A08 GTGACGTAGG SEQ ID NO: 1 A09 GGGTAACGCC SEQ ID NO: 2 C08 TGGACCGGTG SEQ ID NO: 3

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Random Amplified Polymorphic DNA primer for discrimination of          Zizyphus jujuba Miller <130> PN1412-366 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> A08 <400> 1 gtgacgtagg 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> A09 <400> 2 gggtaacgcc 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> C08 <400> 3 tggaccggtg 10

Claims (9)

A Random Amplified Polymorphic DNA (RAPD) primer for discriminating jujube varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3. The method according to claim 1,
Wherein the jujube cultivar is one selected from the group consisting of Boeun, Moonrise, Maeil, Venus, Chuseok, Mizo, Demodulation, Kwangwol, The method according to claim 1,
The primer set forth in SEQ ID NO: 1 identifies the local light among the jujube cultivars. The method according to claim 1,
Wherein the sequence number of SEQ ID NO: 2 identifies the taste of the jujube cultivars. The method according to claim 1,
Wherein the sequence number of SEQ ID NO: 2 or SEQ ID NO: 3 identifies the major of the jujube varieties. The method according to claim 1,
Wherein the sequence number of SEQ ID NO: 3 identifies the consensus among the varieties. A composition for discriminating jujube cultivars comprising the primer according to claim 1. A kit for judging a variety of jujube, comprising the composition of claim 7. A method for identifying a variety of jujube cultivars, comprising the step of performing PCR using a primer according to claim 1 for a nucleic acid sample obtained from an individual.

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