KR102272206B1 - Composition for determining scion root of Ehime Kashi 28 gou Mandarin - Google Patents

Abstract

The present invention is a primer set capable of discriminating whether or not the root of the golden rhizome occurs in the root of the golden rhododendron, a composition for discriminating golden rhizomes comprising the primer set, a kit for discriminating golden rhododendrons including the composition, and the composition or kit Thus, it relates to a method for determining whether or not the root of golden hyang occurs. By using the composition for discriminating the root of the golden rhododendron of the present invention, since it is possible to specifically detect the golden rhododendron in which the rhizome is generated, it will be widely used to effectively suppress the decrease in productivity of the golden rhododendron due to the formation of the ginseng root.

Description

Composition for determining scion root of Ehime Kashi 28 gou Mandarin}

The present invention relates to a composition for discriminating the root root of Hwanggeumhyang, and more specifically, the present invention relates to a primer set capable of determining whether or not the root root is generated in the root of a goldenhyang tree, and a composition for discriminating the root root of a goldenhyang tree comprising the primer set , It relates to a kit for determining the root root of a golden-hyang tree comprising the composition, and a method for determining whether or not the root root occurs in a golden-hyang tree using the composition or kit.

For the propagation of citrus varieties, including Onju mandarin, which are mainly cultivated in farms in Jeju Island, the Taengja variety, which is relatively dwarf and strong in cold resistance, is widely used as the rootstock. The Onju citrus variety does not develop root root at the grafting site, but recently, the frequency of occurrence of scion root, which takes root from the root of the main tree, not the root of the main tree, is increasing in the orchard of Mangamaceae. Unlike normal trees for more than two years, the amount of ignition in the trees with rooted roots tends to decrease significantly compared to normal trees, but it is not easy to identify the difference in the early stages unless farmers who grow persimmons take good care of the trees in their orchards. As a result, as time goes by, the initial response to the birch tree fails, and later, it causes a lot of damage to the farms due to the double failure of the yield decrease and the quality of the fruit due to the root damage.

As a method for confirming whether the magnetic root is generated, approximately five methods are known. The first method is the root cutting method, which cuts the root suspected to be the root and checks the new shoots that occur at the cut site after a time has elapsed. The second method is characterized by the characteristic that the growth of the rootstock usually occurs above the graft site. Through this, if a smooth root is generated from the boundary of the grafting site as the starting point or the boundary is gone, it is a method to judge it as a sub-root. The third method is that the root is thicker and stiffer than that of the root, which is the main root. Through this, it is a method to determine the presence or absence of a rhizome after excavating the root. The fourth method shows that when the bark of the root where the rhizome is not formed is peeled off, the bark color is dark yellow, but the root of the rhizome shows a light green bark It is a method to determine the presence or absence of a root by using the method, and the fifth method is to separate the DNA after collecting the sample tissue and then use the SRAP marker (Journal of Horticultural Science and Technology, Vol. 33 Annex II [Abstract Collection], 2015.10, 164- 164).

However, when the above five methods are used, there are disadvantages in that it takes a long time to determine whether self-rooting occurs or it is not possible to clearly distinguish whether self-rooting occurs. For example, in the case of the root cutting method or the method of observing the graft site, it takes a long time because it is necessary to continuously check whether the root is generated, and in the case of the method of checking the shape of the fine root or the bark color, Since it is performed entirely according to the observer's standards, it is difficult to make a clear distinction due to low objectivity. In the case of the SRAP marker method, since several bands are usually detected, it is difficult to accurately confirm whether the marker is detected. .

In order to overcome this disadvantage, in order to discriminate the rhizomes of the rhizome, a kind of persimmon, a primer set capable of specifically amplifying the marker sequence present in the genomic DNA of the rhizomes was developed (Korean Patent No. 1869781). However, the primer set can easily perform root-root discrimination of sub-flowering, but has a disadvantage in that it cannot be applied to other perennials.

Under this background, the present inventors made diligent research efforts to develop a method for more clearly and concisely checking whether or not the rhizome is generated in the golden rhizome, which is a kind of persimmon tree. As a result, the marker sequence present in the genomic DNA of the rhizome generated from the golden rhododendron tree. A primer set capable of specifically amplifying was developed, and the present invention was completed.

One object of the present invention is to provide a primer set that can determine whether or not root root occurrence of goldenhyang trees.

Another object of the present invention is to provide a composition for determining the root of a golden hyang tree comprising the primer set.

Another object of the present invention is to provide a kit for determining the root of a golden hyang tree comprising the composition.

Another object of the present invention is to provide a method for determining whether or not rooting occurs in a golden hyang tree using the composition or kit.

The present inventors paid attention to a method of discriminating using a genetic marker while conducting various studies to develop a method for more clearly and concisely confirming whether or not the root of the golden hyang tree is generated in the root of the golden hyang tree. In the case of the method using the genetic markers developed so far, the root-specific marker is amplified by using the PCR method, and the amplified result appears not only in the golden-hyang tree in which the root root is generated but also in the golden-hyang tree in which the root root is not generated. Since the amplification products appearing in the obtained golden hyang form multiple bands, there was a disadvantage in that objective discrimination was difficult.

As a result of carrying out research to overcome the disadvantages of the method using these genetic markers, the present inventors do not amplify the genome of the golden hyang tree in which the root root is not formed, but only the genome of the golden hyang tree in which the root root is formed. When amplifying the genome of a tree, a primer set was developed that can obtain only a single band amplification product.

As such, the primer set capable of specifically amplifying the marker nucleotide sequence included in the genome of the golden hyang tree in which the root is formed is not known at all until now, and was developed for the first time by the present inventors.

One embodiment of the present invention for achieving the above object, which can determine whether or not the root generation of goldenhyang trees, SEQ ID NOs: 1 and 4; SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 4; SEQ ID NOs: 2 and 6; And it provides a primer set consisting of a base pair selected from the group consisting of combinations thereof.

As used herein, the term “golden hyang” refers to a type of mandarin citrus produced from a tree in which a tree grown from a seed obtained by crossing Hallabong and Cheonhyehyang, which are a kind of mangam, is grafted to the rootstock of the tangerine tree.

In the present invention, the golden hyang can be expressed as a golden hyang tree, a golden hyang tree grafted to a tangja tree, and the like, and these expressions can all be interpreted as meaning a golden hyang.

As used herein, the term "self root" (self root: scion root), also called a lateral root (側根), means a small root split from the original root of a plant.

In the present invention, the rhizome can be interpreted as meaning a root generated from a citrus tree grafted with a golden-hyang tree to a tangja tree root. Typically, citrus fruits containing golden flavor are grafted onto the rootstock of the citrus tree, and then bear fruit. At this time, the root uses the root of the citrus tree, but in some cases, roots can be produced from the grafted citrus. The root generated from the grafted citrus tree is called the root.

The term "discrimination" of the present invention means that, when the generation of rootlets is suspected in the golden rhododendron grafted to the rootstock, genomic DNA is extracted from the suspected rhizome, and the extracted genomic DNA contains a root-specific marker nucleotide sequence. It refers to a series of processes to determine whether

The specific nucleotide sequence of each primer constituting the primer set is as follows:

SCAR F01: 5'-CAAACCGGAATCCCAAAGGAAAACC-3' (SEQ ID NO: 1)

SCAR F02: 5'-CAAAACAAACCCAAGTACACG-3' (SEQ ID NO: 2)

SCAR R01: 5'-GACTGCGTACGAATTATGGGCAAAC-3' (SEQ ID NO: 4)

SCAR R02: 5'-GCAAACGTGGGATCTTGCGTG-3' (SEQ ID NO: 5)

SCAR R03: 5'-GAGTACTGGCTCTGCTTATAC-3' (SEQ ID NO: 6)

As used herein, the term "specific sequence" refers to a series of nucleotide sequences capable of synthesizing a PCR product by using it as a template only when the gene to be diagnosed is present without synthesizing the PCR product when reacting with other genes.

In the present invention, the primer set may be used for the purpose of discriminating the golden hyang tree in which the root is formed.

As used herein, the term “primer set (a pair of primers)” refers to nucleotide sequences for amplifying a gene to be diagnosed using PCR, and a forward primer and a reverse primer are set to react with one gene for diagnosis to produce a PCR product. to synthesize In this case, when the forward and reverse primer sets are determined, the size of a PCR product synthesized therefrom can be predicted.

Another embodiment of the present invention provides a composition for discriminating the root of a golden rhododendron comprising the primer set and a kit for discriminating the root of a golden rhododendron comprising the same.

The goldenhyang and jageun are the same as described above.

The kit for discrimination of the present invention is specifically SEQ ID NOs: 1 and 4; SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 4; SEQ ID NOs: 2 and 6; And it is characterized in that by using a primer set consisting of a base pair selected from the group consisting of a combination thereof to specifically discriminate the root-formed golden rhododendron tree.

The kit for discrimination of the present invention may further include a DNA polymerase and a buffer solution thereof, and a PCR reaction buffer of the composition described above, for easily performing a PCR reaction, and whether the PCR product is amplified Components or diagnostic criteria necessary for performing electrophoresis that can confirm may be additionally included in the kit of the present invention.

Another embodiment of the present invention provides a method for discriminating a root-formed golden juniper tree using the composition or kit.

Specifically, the method for determining the golden hyang tree in which the root root is formed comprises the steps of (a) obtaining genomic DNA from a sample derived from the golden hyang tree to determine whether the root root is formed; (b) adding the primer set provided in the present invention to the obtained genomic DNA and performing a polymerase chain reaction (PCR); and (c) confirming the result of performing the PCR.

In the above method, the sample obtained in step (a) is obtained from the Golden Hyang tree, and is not particularly limited thereto, as long as genomic DNA can be obtained therefrom. As an example, the sample obtained in step (a) may be leaves, roots, stems, flowers, etc. can In order to more effectively identify genomic DNA from the sample, the method may further include grinding each sample.

In the above method, step (c) may be performed using a conventional electrophoresis method, and when an amplification product is identified through step (c), it can be determined that the root is formed in the golden hyang tree. .

By using the composition for discriminating the root of the golden rhododendron of the present invention, since it is possible to specifically detect the golden rhododendron in which the rhizome is generated, it will be widely used to effectively suppress the decrease in productivity of the golden rhododendron due to the formation of the ginseng root.

1 is a photograph showing the appearance of the root part of the golden hyang tree in which the root is generated.
Figure 2a is an electrophoresis photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the golden-hyang tree in which the root is formed with the P1-SCAR primer base pair provided in the present invention.
Figure 2b is an electrophoresis photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the golden hyang tree in which the root is formed with the P2-SCAR primer base pair provided in the present invention.
Figure 2c is an electrophoresis photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the golden hyang tree in which the root is formed with the P3-SCAR primer base pair provided in the present invention.
Figure 2d is an electrophoresis photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the golden hyang tree in which the root is formed with the P4-SCAR primer base pair provided in the present invention.
Figure 2e is an electrophoresis photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the golden-hyang tree in which the root is formed with the P6-SCAR primer base pair provided in the present invention.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Identification of the root-formed golden juniper tree

By observing the appearance of the golden-hyang tree grafted and cultivated on the Taengja rootstock, the individual having the rooted root was selected (FIG. 1).

1 is a photograph showing the appearance of the root part of the golden hyang tree in which the root is generated.

As shown in Fig. 1, it was possible to distinguish the golden rhododendron trees with rooted roots externally, and comparing the fruiting results from these differentiated golden rhododendron trees, the fruiting level was significantly lowered in the golden rhododendron trees than in the normal golden rhododendrons. Confirmed.

Example 2: Primer for amplification of goldenhyang specific marker gene in which the root is formed

A primer capable of effectively detecting the discovered gene was developed by excavating a gene specific to the genus of the golden rhododendron from the gene of the golden hyang tree.

SCAR F01: 5'-CAAACCGGAATCCCAAAGGAAAACC-3' (SEQ ID NO: 1)

SCAR F02: 5'-CAAAACAAACCCAAGTACACG-3' (SEQ ID NO: 2)

SCAR F03: 5'-CAAAAAGGGGCAACACAGAAC-3' (SEQ ID NO: 3)

SCAR R01: 5'-GACTGCGTACGAATTATGGGCAAAC-3' (SEQ ID NO: 4)

SCAR R02: 5'-GCAAACGTGGGATCTTGCGTG-3' (SEQ ID NO: 5)

SCAR R03: 5'-GAGTACTGGCTCTGCTTATAC-3' (SEQ ID NO: 6)

9 sets of primer base pairs were set by combining the three types of forward and reverse primers developed above (Table 1).

primer base pair designation F (SEQ ID NO:) R (SEQ ID NO:) P1-SCAR
P2-SCAR
P3-SCAR
P4-SCAR
P5-SCAR
P6-SCAR
P7-SCAR
P8-SCAR
P9-SCAR SCAR F01(1)
SCAR F01(1)
SCAR F01(1)
SCAR F02(2)
SCAR F02(2)
SCAR F02(2)
SCAR F03(3)
SCAR F03(3)
SCAR F03(3) SCAR R01(4)
SCAR R02(5)
SCAR R03(6)
SCAR R01(4)
SCAR R02(5)
SCAR R03(6)
SCAR R01(4)
SCAR R02(5)
SCAR R03(6)

Example 3: PCR analysis for goldenhyang trees with root formed

First, genomic DNA was obtained from the roots of the golden hyang tree in which Hoja root was formed. In this case, as a control, genomic DNA obtained from the roots (PR) of the normal taengjae was used.

Then, PCR was performed using each of the obtained genomic DNA as a template, and using the 9 sets of primer base pairs set in Example 2 above. At this time, the PCR conditions were denatured at 94°C for 5 minutes, followed by amplification 35 times at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 40 seconds, followed by extension at 72°C for 5 minutes.

Finally, by electrophoresis of each amplification product obtained by performing the PCR, it was confirmed whether each amplification product was generated ( FIGS. 2a to 2e ).

Figure 2a is an electrophoresis picture showing the result of PCR amplification with the P1-SCAR primer base pair provided in the present invention of the genomic DNA obtained from the root of the control (Taengja) tree and the root of the goldenhyang tree in which the root is formed, Figure 2b is the control (Taengja) It is an electrophoretic photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the tree root and the root of the golden hyang tree in which the root is formed with the P2-SCAR primer base pair provided in the present invention, Figure 2c is a control (Taengja) tree It is an electrophoretic photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the golden hyang tree in which the root and the root of the root of the P3-SCAR primer provided in the present invention are base-paired, and FIG. 2D is the root and the root of the control (Taengja) tree. It is an electrophoretic photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the formed golden hyang tree with the P4-SCAR primer base pair provided in the present invention, and FIG. It is an electrophoretic photograph showing the result of PCR amplification of the genomic DNA obtained from the root of the P6-SCAR primer base pair provided in the present invention.

As shown in FIGS. 2A to 2E , out of the 9 sets of primer base pairs, 5 sets of primer base pairs (P1-SCAR, P2-SCAR, P3-SCAR, P4-SCAR and P6-SCAR) are polynucleotides included in the genomic DNA of the control group. It was confirmed that, without amplifying nucleotides, only polynucleotides contained in the genomic DNA of the golden-hyang tree in which the root was formed.

From the results of Figs. 2a to 2e, it was found that 5 sets of primer base pairs among the 9 sets of primer base pairs can be used to determine the root-formed golden hyang tree.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for determining scion root of Ehime Kashi 28 gou Mandarin <130> KPA191385-KR <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caaaccggaa tcccaaagga aaacc 25 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caaaacaaac ccaagtacac g 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caaaaagggg caacacagaa c 21 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gactgcgtac gaattatggg caaac 25 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcaaacgtgg gatcttgcgt g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gagtactggc tctgcttata c 21

Claims (6)

SEQ ID NO: 1 and 4, which can determine whether or not the root generation of the golden hyang tree; SEQ ID NOs: 1 and 5; SEQ ID NOs: 1 and 6; SEQ ID NOs: 2 and 4; SEQ ID NOs: 2 and 6; and a primer set consisting of a base pair selected from the group consisting of combinations thereof.
A composition for determining the root of a golden hyang tree comprising the primer set of claim 1.
A kit for discriminating the root root of a golden hyang tree, comprising the primer set of claim 1 or the composition for determining the root root of the golden hyang tree of claim 2.
(a) obtaining genomic DNA from a sample derived from the golden hyang tree to determine whether the root is formed;
(b) adding the primer set of claim 1 or the composition for determining the root of claim 2 to the obtained genomic DNA and performing a polymerase chain reaction (PCR); and
(c) a method of determining the golden hyang tree in which the root is formed, comprising the step of confirming the result of performing the PCR.
5. The method of claim 4,
The sample obtained in step (a) is the leaf, root, stem or flower of the golden-hyang tree, a method for determining the golden-hyang tree in which the root is formed.
5. The method of claim 4,
Through the step (c), when the amplification product is confirmed, it is determined that the root is formed in the golden hyang tree, a method for determining the golden hyang tree in which the root is formed.

Images