KR102286871B1 - Molecular marker for discriminating branchless watermelon cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a molecular marker for distinguishing branchless watermelon varieties and uses thereof, and when using an InDel molecular marker of the present invention for breeding of the branchless watermelon varieties, as branch removal work is not performed in an existing watermelon cultivation process, labor and time required for watermelon cultivation can be reduced. Provided is a primer set for distinguishing the branchless watermelon varieties comprising an oligonucleotide primer set of SEQ ID NOs: 1 and 2.

Description

Molecular marker for discriminating branchless watermelon cultivar and uses thereof

The present invention relates to molecular markers for distinguishing branchless watermelon varieties and their use.

Watermelon ( Citrullus lanatus ) is known to be native to Africa and has been found in Africa, temperate regions and Mediterranean regions. As an annual creeper plant, it is a representative crop in summer. It has a high water content and contains glucose and fructose that are easily absorbed by the body, helping to recover from fatigue. It contains useful ingredients such as lycopene and β-carotene, and contains an amino acid called citrulline, an intermediate metabolite of urea metabolism. It is also known to be effective against high fever.

As watermelons with improved size and taste are developed due to the development of breeding technology, the high-quality watermelon market is revitalized. In the existing watermelon cultivation process, the goal is to produce one watermelon per week, but since geodesic removal requires considerable labor, labor savings can be expected when non-geodesed watermelon varieties are put to practical use.

With the recent radical development in the field of molecular biology, a variety of molecular biological methods are being used to classify plant genus, interspecies, or within species. is being developed Differentiation by DNA analysis at the molecular biology level has the advantage of being able to grasp a number of characteristics along with a quantitative level and excluding the influence of the environment. Through the recently developed next generation sequencing (NGS) technology, DNA information is compared to find differences, and molecular marker development using this technology makes it possible to accurately distinguish watermelon varieties. Among the various types of molecular markers based on genomic information, the InDel (Insertion/Deletion) marker is a method for detecting polymorphisms using PCR (polymerase chain reaction) technology. InDel markers are molecular markers suitable for breed classification because they exist in various genotypes not only between species but also within species by using the difference in the nucleotide sequence inserted or deleted from the nucleotide sequence.

On the other hand, Korea Patent No. 1923647 discloses 'SNP marker for discrimination of Jubilee-type or crimson-type watermelon variety', and Korean Patent Publication No. 2004-0103987 discloses 'A method for developing an apple non-geometric gene-linked scar marker'. However, there is no description of the 'molecular marker and use thereof for distinguishing non-braided watermelon varieties' of the present invention based on the InDel polymorphism.

The present invention was derived from the necessity of distinguishing non-geodesic watermelon varieties, and the present inventors compared the genome sequence of watermelon variety 'CBW5 (variety protection application name: Sunzero)' without geodes and watermelon variety '159' with geodes. Therefore, based on the watermelon standard genome, a molecular marker was developed based on the nucleotide sequence showing the InDel (insertion/deletion) polymorphism present at the Cla97C02G042050 gene position on chromosome 2, and a primer set capable of amplifying the InDel marker was prepared and various As a result of performing PCR on watermelon resources, the present invention was completed by confirming that the InDel marker of the present invention could accurately discriminate non-geodesed watermelon variety CBW5 from 56 geodesic watermelon varieties.

In order to solve the above problems, the present invention provides a primer set for distinguishing a branched watermelon variety, comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2.

In addition, the present invention provides a kit for distinguishing non-braided watermelon varieties including the primer set.

In addition, the present invention comprises the steps of isolating genomic DNA from a watermelon sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set; and detecting the product of the amplification step.

Since the InDel molecular marker of the present invention accurately distinguishes CBW5, a non-geodesed watermelon variety, from various watermelon varieties with geodes, when the InDel molecular marker of the present invention is used for breeding of non-geodesed watermelon varieties, geodesicity is removed in the existing watermelon cultivation process. It will have the effect of reducing the labor and time required for watermelon cultivation by not performing any work.

1 shows the results of confirming the InDel polymorphism between the two varieties by comparing the nucleotide sequences of CBW5(N5), a watermelon variety without geodes, and 159, a watermelon variety with geodes.
2 is a gel electrophoresis result of PCR amplification products of CBW5, a watermelon variety without geodes, and 56 watermelon varieties with geodes, using the Cl-N5-InDel-5 primer set of the present invention. Lane 1: 31226, Lane 2: SP904, Lane 3: 7, Lane 4: 898, Lane 5: 8, Lane 6: 93, Lane 7: SP908, Lane 8: 2, Lane 9: SP909, Lane 10: 98, Lane 11: 3, Lane 12: 96, Lane 13: 4, Lane 14: 885, Lane 15: SP905, Lane 16: 160, Lane 17: 120-1, Lane 18: 6, Lane 19: SP912, Lane 20: Dalcomi Mini, Lane 21: SWM2, Lane 22: 717, Lane 23: 95, Lane 24: SWM3, Lane 25: Apple Mini Nice Shot, Lane 26: SP906, Lane 27: 101, Lane 28: 331, Lane 29: SWM1, Lane 30: 5, Lane 31: 120-2, Lane 32: Sesame Seed, Lane 33: 11602, Lane 34: 10, Lane 35: SP911, Lane 36: Ministar, Lane 37: Dalcomy Pearl, Lane 38: 275, lane 39: 718, lane 40: blueberry honey, lane 41: camean beta, lane 42: 725, lane 43: ginseng honey, lane 44: 274-2, lane 45: 11501, lane 46: 11603, lane 47: SP902, lane 48: SP903, lane 49: 167-2, lane 50: 733, lane 51: mini-me, lane 52: 188, lane 53: 1, lane 54:9, lane 55: SP910, lane 56: 741 ).

In order to achieve the object of the present invention, the present invention provides a primer set for distinguishing a branched watermelon variety comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2.

In the primer set of the present invention, the primer set can distinguish non-braided watermelon varieties, and the non-geodesed watermelon variety refers to a 'pure zero' variety possessed by the Watermelon Research Institute of the Chungcheongbuk-do Agricultural Technology Institute, 'Zero' is a watermelon variety with application number 2019-596 applied for variety protection to the National Seed Management Office.

The primers include oligonucleotides consisting of fragments of 13 or more, 14 or more, 15 or more, 16 or more, 17 or more consecutive nucleotides in the sequences of SEQ ID NOs: 1 and 2 according to the sequence length of each primer. can do. For example, the primer of SEQ ID NO: 1 (18 oligonucleotides) may include an oligonucleotide consisting of fragments of 15 or more, 16 or more, 17 or more consecutive nucleotides in the sequence of SEQ ID NO: 1. In addition, the primer may also include a sequence added, deleted, or substituted in each of the nucleotide sequences of SEQ ID NOs: 1 and 2, capable of binding to the amplification site. The odd-numbered oligonucleotide primers in the SEQ ID NO: are forward primers, and the even-numbered oligonucleotide primers are reverse primers.

The oligonucleotide primer sets of SEQ ID NOs: 1 and 2 according to the present invention are InDel located in the Cla97C02G042050 (http://cucurbitgenomics.org/feature/gene/Cla97C02G042050) gene region of chromosome 2 based on the watermelon standard genome (cultivar 97103). As a primer set capable of amplifying the marker, amplification products of different sizes were identified by deletion of 21 bp bases in the genome sequence of non-braided watermelon cultivar 'CBW5' compared to '159' with geodesic watermelon. Only the non-geodesic watermelon variety 'CBW5' can be specifically discriminated from 56 watermelon varieties.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and can serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

In the present specification, the oligonucleotide used as a primer may contain or insert a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid. It may contain an intercalating agent.

The present invention also provides a kit for distinguishing a branched watermelon variety comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2.

In one embodiment of the present invention, the kit may further include a reagent for performing the amplification reaction, and the reagent for performing the amplification reaction may include DNA polymerase, dNTPs and a buffer, but this not limited

In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

The present invention also

isolating genomic DNA from the watermelon sample;

amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer sets of SEQ ID NOs: 1 and 2; and

It provides a method for discriminating non-geodesian watermelon varieties, comprising the step of detecting the product of the amplification step.

In the method for distinguishing non-geodesed watermelon varieties according to an embodiment of the present invention, the non-geodesed watermelon variety refers to a 'net zero' variety possessed by the Watermelon Research Institute of the Chungcheongbuk-do Agricultural Research and Extension Services, and the 'net zero' is This is a watermelon variety with application number 2019-596 applied for variety protection to the National Seed Management Office.

In addition, in the method for distinguishing the non-braided watermelon variety according to an embodiment of the present invention, the primer set is the same as described above.

The method of the present invention includes isolating genomic DNA from a watermelon sample. The method for isolating the genomic DNA may use a method known in the art, for example, the CTAB method may be used, or a Wizard prep kit (Promega) may be used. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and using the oligonucleotide primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include a polymerase chain reaction (PCR), a ligase chain reaction, a nucleic acid sequence-based amplification, and a transcription-based amplification system. amplification system), strand displacement amplification or amplification via Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a material emitting fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. ^{In addition, when a radioactive isotope such as 32} P or ³⁵ S is added to a PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The oligonucleotide primer sets used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for distinguishing the non-geodesic watermelon variety comprises the step of detecting a product of the amplification step, and the detection of the product of the amplification step is a DNA chip, gel electrophoresis, capillary electrophoresis , radiometric measurements, fluorescence measurements, or phosphorescence measurements, but is not limited thereto. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorometer. can do. In addition, the radioactive measurement method is a ^{radioactive isotope such as 32} P or ³⁵ S is added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

In the method for distinguishing non-braided watermelon varieties according to an embodiment of the present invention, when PCR is performed using the watermelon genomic DNA as a template using the oligonucleotide primer sets of SEQ ID NOs: 1 and 2, a band having a size of 226 bp is detected, it can be discriminated as a watermelon variety with geodes, and when a band with a size of 205 bp is detected, it can be discriminated as a watermelon variety without geodes.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Watermelon resource collection and genomic DNA extraction

56 watermelon varieties with geodesic and non-geodesed watermelon variety 'CBW5' used in the experiment were provided by the Watermelon Research Institute of the Chungcheongbuk-do Agricultural Research and Extension Services, and the non-geodesed watermelon variety 'CBW5' was applied for variety protection to the National Seed Management Office, Application No. 2019 It is a 'pure zero' variety with -596. The leaves of a total of 57 watermelon varieties were collected and genomic DNA was extracted using the CTAB (Cetyl Trimethyl Ammonium Bromide) method. Specifically, liquid nitrogen was added to the leaves of each watermelon variety, and crushed samples were prepared using a mortar, then 700 μl of CTAB (+0.2% Mercaptoethanol) was added, and incubated in a water bath at 65° C. for 30 minutes. After adding 700 μl of chloroform and isoamylalcohol in a ratio of 24:1 and centrifuging to obtain a supernatant, isopropanol was added to the supernatant and centrifuged to remove the supernatant and pellet. obtained. After washing and removing 200 μl of ethanol to the obtained pellet, TE buffer was added to dissolve the pellet, and RNase was added to leave only pure DNA.

2. InDel Marker Development

Among the extracted genomic DNA for each watermelon variety, NGS (Next Generation Sequencing) was performed on the genomic DNA of two varieties 'CBW5' and '159' to obtain DNA sequencing information of the watermelon varieties, and then the bases of the watermelon varieties The sequence was compared and analyzed through the CLC genomics workbench program (version 8.0.1), and the InDel polymorphic region showing the difference in insertion or deletion between the two cultivars 'CBW5' and '159' was searched for. , a primer set for amplifying the InDel polymorphic region was prepared (Table 1). The primer set was prepared by controlling conditions such as the maximum length, the minimum length, the size of the amplification product, the temperature, and the GC ratio.

The InDel marker of the present invention is at the gene location of Cla97C02G042050 (http://cucurbitgenomics.org/feature/gene/Cla97C02G042050) on chromosome 2 based on the watermelon standard genome (cultivar 97103), in the genomic sequence of the watermelon cultivar 'CBW5' It was constructed based on the polymorphic region in which the 21 bp base was deleted (FIG. 1).

Primer set information of the present invention Primer name Sequence (5'→3') (SEQ ID NO:) Cl-N5-InDel-5-F ATACAACAGCGATTCTCC (1) Cl-N5-InDel-5-R GAACACAAGACCTAGACC (2)

3. DNA amplification using polymerase chain reaction (PCR)

The DNA of the watermelon variety extracted using the CTAB method was diluted to a concentration of 10 ng/μl, respectively, and 2 μl (10 ng/μl) of DNA, 2 μl of 2 μM forward primer, 2 μl of 2 μM reverse primer, 2X Taq Mix 10 μl of mix and 4 μl of distilled water were mixed to make a total of 20 μl of a PCR reaction mixture. PCR was pre-denatured at 95° C. for 3 minutes; A total of 35 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 30 seconds; Final extension (final extension) was performed under the condition of 72 ℃ 5 minutes. PCR amplification products were confirmed by electrophoresis on a 3% agarose gel.

Example 1. Identification of non-geodesic watermelon varieties using the InDel marker of the present invention

PCR was performed using DNA samples of various watermelon varieties as templates using the Cl-N5-InDel-5 primer set prepared based on the InDel polymorphism between the watermelon variety with geodes and the watermelon variety without geodesic, and then on an agarose gel. Polymorphism pattern analysis of the Cl-N5-InDel-5 primer set was performed by electrophoresis.

As a result, a 205 bp band was confirmed in 'CBW5', a watermelon cultivar without geodes, and a 226 bp band in 56 geodesic watermelon cultivars. It was confirmed that it is possible to specifically distinguish ' (FIG. 2).

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Molecular marker for discriminating branchless watermelon cultivar and uses thereof <130> PN19168 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atacaacagc gattctcc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaacacaaga cctagacc 18

Claims (4)

A primer set for distinguishing branchless watermelon varieties, comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2. A kit for distinguishing non-braided watermelon varieties comprising the primer set of claim 1. isolating genomic DNA from the watermelon sample;
amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of claim 1; and
A method for discriminating non-geodesian watermelon varieties, comprising the step of detecting the product of the amplification step. The method according to claim 3, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

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