KR102199731B1 - Molecular marker derived from complete sequencing of chloroplast, mitochondria and nucleus genome of Cucumis sativus for Cucumis sativus F1 hybrid purity checking and uses thereof - Google Patents

Abstract

The present invention relates to a dCAPS molecular marker based on SNPs in a chloroplast genome sequence of Cucumis sativus, and an InDel molecular marker based on insertion or deletion regions in mitochondrial and nuclear genome sequences thereof. By using the molecular markers of the present invention, the purity of Cucumis sativus F_1 can be checked at the whole genome level, so that more accurate and efficient results can be obtained.

Description

Molecular marker derived from complete sequencing of chloroplast, mitochondria and nucleus genome of Cucumis sativus for Cucumis sativus F1 hybrid purity checking and uses thereof, based on complete translation of cucumber chloroplasts, mitochondria and nuclear genome sequences }

The present invention relates to a molecular marker for assaying cucumber F1 purity based on complete translation of the chloroplast, mitochondrial and nuclear genomic sequences of cucumber and its use.

Cucumber is a horticultural crop that is cultivated all over the world, and is produced in large quantities in Northeast Asia, including Korea and China. Cucumber cultivation is being actively carried out in Korea, and cultivation is increasing as it shows high profits per unit area among horticultural crops in domestic facilities. Cucumbers have very diverse characteristics depending on the variety, so a lot of investment is being made to develop a breeding line that combines the advantages of various varieties.Therefore, there are many studies on cucumbers around the world, but cucumbers grown and distributed in Korea There is still a lack of research on it. Existing cucumber breeding has been carried out through the method of crossbreeding through various cucumber breeding lines, but there is a disadvantage that it is difficult to accurately identify the parental copy of offspring individuals. Due to the breeding method in which one breeding line must be repeatedly bred to the repeated parent, it is necessary to accurately identify the parental copy of the individual. Therefore, research on various basic technologies and development of molecular marker technologies are required for low-cost and high-efficiency cucumber breeding.

In general, F _{1, which} is made through crossing of superior parents, has higher quality in terms of production, resistance, and homogeneity compared to parents due to hybrid strength, and seeds obtained from F ₁ plants cannot be reused. We prefer the development of F ₁ seeds with excellent traits, which is also the direction of seed development worldwide. A mass seeding process is performed for commercialization of the developed F ₁ individuals. At this time, the purity of the parents, contamination of other pollen during seeding, or contamination due to self-synthesis of both parents may occur, so the purity test of F ₁ is performed after mass harvesting. In the F ₁ purity test, a certain number of F ₁ is cultivated to check the uniformity of the phenotype or use a molecular marker. The phenotype-dependent discrimination method not only requires a lot of time and labor, but also has disadvantages of poor reproducibility. Also, morphological discrimination does not reflect clear genetic attributes, and early discrimination is impossible. Although some molecular markers are used to test the purity, the molecular marker is not a marker for the purity test of F ₁ , but a purity test is performed by simply selecting a marker that shows the difference between the parents. It is large, and because the purity test is performed only on a specific locus, it is impossible to test at the whole genome level.

In general, both the chloroplast and mitochondrial genomes are known to be inherited from the parent, and due to this unit reproductive genetic feature, the chloroplast and mitochondrial sequences are less mutated and well preserved compared to the nuclear genome, so they are used as barcoding marker regions for different species It is also possible. However, unlike ordinary plants, cucumbers have a unique cytoplasmic genomic inheritance pattern in which the mitochondrial genome is paternal and the chloroplast genome is maternal. This cytoplasmic genetic pattern has the potential to be used in various ways in the breeding of cucumbers. However, since the mitochondria of cucumbers have the largest and most complex mitochondrial genome sequence in the flora, much research has not been conducted so far, and they have not been properly utilized in fields such as molecular breeding.

On the other hand, Korean Patent No. 1174409 discloses a'method for identifying cucumber varieties using supersatellite markers', and Korean Patent No. 1873510 discloses a'marker and primer set for discriminating bitter cucumbers and a method for discriminating using the same'. Although disclosed, there is no description of the molecular marker for the purity assay of cucumber F1 based on the complete translation of the chloroplast, mitochondrial and nuclear genomic sequences of cucumber of the present invention and the use thereof.

The present invention was derived from the above requirements, and the present inventors compared and analyzed the chloroplast genomic sequences of the parent and the parent used for cucumber breeding, and dCAPS based on single nucleotide polymorphism (SNP) between breeding lines. (derived Cleaved Amplified Polymorphic Sequence) molecular markers were produced, and InDel (insertion/deletion/deletion) based on nucleotide sequences showing insertion or deletion polymorphism between breeding lines by comparing and analyzing the mitochondrial genomic sequence or nuclear genomic sequence of the parent and the parent of cucumber deletion) molecular markers were prepared. As a result of performing PCR on various cucumber varieties and their F ₁ individuals using the produced molecular marker, the nuclear genome sequence-based InDel marker can confirm the breeding lineage of the F ₁ individual, and the chloroplast genome sequence-based dCAPS marker By confirming that the parent individual of the F ₁ individual can be distinguished and the InDel marker based on the mitochondrial genome sequence can distinguish the parent individual of the F ₁ individual, the present invention has been completed.

In order to solve the above problems, the present invention provides one or more primer sets selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 1 and 2 and the oligonucleotide primers of SEQ ID NOs: 3 and 4; And the oligonucleotides of SEQ ID NOs: 5 and 6 Primer set, oligonucleotide primer set of SEQ ID NO: 7 and 8, oligonucleotide primer set of SEQ ID NO: 9 and 10, oligonucleotide primer set of SEQ ID NO: 11 and 12, oligonucleotide primer set of SEQ ID NO: 13 and 14, and SEQ ID NO: 15 And one or more primer sets selected from the group consisting of 16 oligonucleotide primer sets; And one or more primer sets selected from the group consisting of an oligonucleotide primer set of SEQ ID NOs: 17 and 18 and an oligonucleotide primer set of SEQ ID NOs: 19 and 20; A primer set for F ₁ purity assay of cucumber ( Cucis sativus ) comprising to provide.

In addition, the present invention is the primer set; And it provides a kit for F ₁ purity assay of cucumber comprising a reagent for carrying out an amplification reaction.

In addition, the present invention comprises the steps of separating genomic DNA from a cucumber sample; Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set to amplify a target sequence; And detecting the product of the amplification step; F ₁ provides a purity test method of cucumber containing.

The primer set of the present invention can obtain accurate and efficient results by verifying the purity of the _first generation F of cucumbers and selection of early fixed lines in the crossbreeding population at the whole genome level, so useful molecules for the production of cucumber seeds and breeding It is expected to be used as a marker.

1 is a chloroplast genomic map of cucumber breeding lines (CS01 and CS02) and their F ₁ (CS01×CS02 and CS02×CS01) individuals on which the present invention is based. The red triangle (type 1) and the blue triangle (type 2) represent the SNP polymorphic regions between the genomic sequences of the cucumber breeding line, and the SNP base of type 1 is A, and the SNP base of type 2 is G.
Figure 2A is a mitochondrial genomic map showing a polymorphic region between the breeding lines of cucumbers (CS01 and CS02) and the mitochondrial sequences of these F ₁ (CS01 x CS02 and CS02 x CS01) individuals, which are the basis of the present invention, and Figure 2B is a breeding line. and a picture showing the polymorphic region between the nucleotide sequence of the F ₁ individual and green will display the same genotype are indicated region and the cucumber mitochondrial reference dielectric red will display the indicated of cucumber mitochondrial reference genome and the other genotype area, The red triangle indicates the region used for molecular marker development among the InDel polymorphic regions in which some bases were inserted or deleted.
3 is a dCAPS molecular marker based on SNP in the chloroplast genomic sequence of cucumber (Cp-SNP-01 and Cp-SNP-02), an InDel molecular marker based on an insertion or deletion region in the mitochondrial genome sequence (Mt-InDel-01, Mt -InDel-02, Mt-InDel-03, Mt-InDel-04, Mt-InDel-05 and Mt-InDel-06) and InDel molecular markers based on insertion or deletion regions in the nuclear genomic sequence (Nu-InDel-01 and Nu-InDel-02) was used to perform PCR on various cucumber breeding lines and their F ₁ individuals.

In order to achieve the object of the present invention, the present invention is one or more primer sets selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 1 and 2 and the oligonucleotide primers of SEQ ID NOs: 3 and 4; And of SEQ ID NOs: 5 and 6 Oligonucleotide primer set, oligonucleotide primer set of SEQ ID NO: 7 and 8, oligonucleotide primer set of SEQ ID NO: 9 and 10, oligonucleotide primer set of SEQ ID NO: 11 and 12, oligonucleotide primer set and sequence of SEQ ID NO: 13 and 14 One or more primer sets selected from the group consisting of oligonucleotide primer sets of numbers 15 and 16; Provides a primer set for F ₁ purity assay of cucumber ( Cucumis sativus ) comprising: one or more primer sets selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 17 and 18 and the oligonucleotide primer sets of SEQ ID NOs: 19 and 20 do.

In the primer set for F ₁ purity assay of cucumber according to the present invention, the primer set of SEQ ID NOs: 1 and 2; And the primer sets of SEQ ID NOs: 3 and 4 are dCAPS (derived Cleaved Amplified Polymorphic Sequence) molecular markers based on single nucleotide polymorphism (SNP) between the parental chloroplast genomic sequences used for cucumber breeding, and the chloroplasts of cucumber plants. According to the maternal inheritance of DNA, the parent individual can be identified among the breeding lines of cucumber.

Specifically, the dCAPS primer set (SEQ ID NO: 1 and 2; and SEQ ID NO: 3 and 4; primer set) of the present invention is a single SNP base identified in the breeding line used as the parental copy [parent / parent: A / G ], the Cp-SNP-01 marker is the forward primer when the SNP position base is A due to the G base located at the 22nd position in the primer sequence of SEQ ID NO: 1 (ie, the parental genotype). If the amplification products GAT ^ ATC the EcoR V restriction while the enzyme spot is generated, SNP position nucleotide is G (that is, if they have the genotype of the counterpart) is an amplification product of the forward primer obtain the nucleotide sequence of GATGTC by EcoR V It will not be cut. In addition, the Cp-SNP-02 marker is the GT base located at the 24th and 25th in the primer sequence of SEQ ID NO: 4, so when the SNP position base is G, the Rsa I restriction enzyme site of GT^AC is in the amplification product of the reverse primer. On the other hand, when the SNP position base is A, the amplification product of the reverse primer has the nucleotide sequence of ATAC and is not cleaved by the Rsa I restriction enzyme. The primer set of SEQ ID NO: 1 and 2; Alternatively, if the amplification product obtained by performing PCR with the primer sets of SEQ ID NOs: 3 and 4 is treated with a restriction enzyme, the size of the band can be checked, thereby distinguishing the parental copies used for cucumber breeding according to the base type of the SNP (see Table 2). ).

In addition, in the primer set for F ₁ purity assay of cucumber according to the present invention, the primer sets of SEQ ID NOs: 5 and 6; Primer sets of SEQ ID NOs: 7 and 8; Primer sets of SEQ ID NOs: 9 and 10; A primer set of SEQ ID NOs: 11 and 12; Primer sets of SEQ ID NOs: 13 and 14; And the primer sets of SEQ ID NOs: 15 and 16 are InDel molecular markers based on insertion or deletion polymorphism between the mitochondrial genomic sequences of the parental used for cucumber breeding (see Table 2).

In addition, in the primer set for F ₁ purity assay of cucumber according to the present invention, the primer sets of SEQ ID NOs: 17 and 18; And the primer sets of SEQ ID NOs: 19 and 20 are InDel molecular markers based on insertion or deletion polymorphism between the nuclear genomic sequence of the parental copy used for cucumber breeding, and the F ₁ individual of the cucumber is derived from the parental parental individual used for actual breeding. It is possible to test if it is correct (see Table 2).

Therefore, the InDel molecular marker based on the nuclear genomic sequence of the present invention enables the identification of cucumber F ₁ individuals and their breeding lineage, and the dCAPS molecular marker based on the chloroplast genome sequence allows the parental identification of the F ₁ individual, and is based on the mitochondrial genome sequence. Since InDel molecular markers are capable of distinguishing copies of F ₁ individuals, the F ₁ purity assay of cucumbers can be performed more accurately and efficiently if all the three primer sets of the present invention are used.

The primers of the present invention include oligonucleotides consisting of 15 or more, 16 or more, 17 or more, 18 or more, 19 or more contiguous nucleotide fragments in the sequence of SEQ ID NOs: 1 to 20, depending on the length of each primer sequence. can do. For example, the primer of SEQ ID NO: 1 (24 oligonucleotides) may include an oligonucleotide consisting of segments of 20 or more, 21 or more, 22 or more, and 23 or more contiguous nucleotides in the sequence of SEQ ID NO: 1. In addition, the primer may include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NO: 1 to 20. Oligonucleotide primers of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 are forward primers, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and The oligonucleotide primer of 20 is a reverse primer.

In the present invention, "primer" refers to a single-stranded oligonucleotide sequence that is complementary to a nucleic acid strand to be copied, and can serve as an initiating point for the synthesis of a primer extension product. The length and sequence of the primers should allow the synthesis of the extension product to begin. The specific length and sequence of the primers 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 also comprise a nucleotide analogue, e.g., phosphorothioate, alkylphosphorothioate or peptide nucleic acid, or It may include an intercalating agent.

The present invention also, the primer set; And it provides a kit for F ₁ purity assay of cucumbers comprising a reagent for performing the amplification reaction.

In the kit of the present invention, the primer set is as described above. In addition, the primer set of SEQ ID NO: 1 and 2; Alternatively, since the primer sets of SEQ ID NOs: 3 and 4 are dCAPS primer sets, when the primer set is included, a restriction enzyme may be additionally included. Preferably, when the primer sets of SEQ ID NOs: 1 and 2 are included, the restriction enzyme Eco RV may be included, and when the primer sets of SEQ ID NOs: 3 and 4 are included, the restriction enzyme Rsa I may be further included.

In addition, in the kit of the present invention, reagents for performing the amplification reaction may include DNA polymerase, dNTPs, and buffers. In addition, the kit of the present invention may further include a user's guide describing the optimum reaction performance conditions. The guide is a printout explaining how to use the kit, e.g., how to prepare PCR buffer, suggested reaction conditions, etc. The guide contains a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and a description on the surface of the package containing the kit. In addition, the guide includes information disclosed or provided through electronic media such as the Internet.

The present invention also,

Separating genomic DNA from the cucumber sample;

Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set to amplify a target sequence; And

It provides a method for assaying the F ₁ purity of cucumbers comprising; detecting the product of the amplification step.

The F ₁ purity assay method of cucumber according to an embodiment of the present invention is specifically,

Separating genomic DNA from the cucumber sample; Using the isolated genomic DNA as a template, a primer set of SEQ ID NOs: 5 and 6, a primer set of SEQ ID NOs: 7 and 8, a primer set of SEQ ID NOs: 9 and 10, a primer set of SEQ ID NOs: 11 and 12, and SEQ ID NO: 13 and Target by performing an amplification reaction using one or more primer sets selected from the group consisting of the primer set of 14, the primer set of SEQ ID NO: 15 and 16, the primer set of SEQ ID NO: 17 and 18, and the primer set of SEQ ID NO: 19 and 20 Amplifying the sequence; And detecting the product of the amplification step.

In addition, separating genomic DNA from the cucumber sample; Using the isolated genomic DNA as a template, performing an amplification reaction using one or more primer sets selected from the group consisting of a primer set of SEQ ID NOs: 1 and 2 and a primer set of SEQ ID NOs: 3 and 4 to amplify a target sequence step; Cutting the product of the amplification step with a restriction enzyme; And separating the cut product by size by gel electrophoresis on the cut product.

In the method according to an embodiment of the present invention, the restriction enzyme may be Eco RV or Rsa I, and specifically, the amplification product of the oligonucleotide primer set of SEQ ID NOs: 1 and 2 contains Eco RV restriction enzyme, and SEQ ID NO: The amplification products of the 3 and 4 oligonucleotide primer sets are treated with Rsa I restriction enzyme to cleave the amplification products.

The method of the present invention includes the step of isolating genomic DNA from a cucumber sample. The cucumber sample may be a parent individual, a secondary individual, or a hybrid individual (F ₁ ) used for cucumber breeding, but is not limited thereto. The method for separating the genomic DNA may use a method known in the art, for example, the CTAB method may be used, or the Wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, an amplification reaction may be performed using the oligonucleotide primer set according to an embodiment of the present invention as a primer to amplify a target sequence. Methods of amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and 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 pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

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

In one embodiment of the present invention, the F ₁ purity assay method of cucumber includes the step of detecting a product of the amplification step, and the detection of the amplification step product includes DNA chips, gel electrophoresis, capillary electrophoresis, and radioactivity. It may be performed through measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, capillary electrophoresis may be performed. For capillary electrophoresis, for example, an ABi sequencer can be used. In addition, gel electrophoresis may be performed, and gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplified product. In addition, when performing PCR by labeling Cy-5 or Cy-3 at the 5'-end of the primer, the fluorescence measurement method is labeled with a detectable fluorescent labeling material, and the thus labeled fluorescence is measured using a fluorescence meter. can do. In addition, the radioactivity measurement method includes labeling the amplified product by adding a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution when performing PCR, and then using a radioactivity measuring instrument such as a Geiger counter or liquid scintillation. Radioactivity can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1. Plant resource collection and DNA extraction

Cucumber breeding systems CS01, CS02, CB01, CI03, CI04 and CI05 used in the experiment were provided by Professor Ki-Hwan Song of Sejong University. Young leaves of cucumber were harvested and DNA was extracted through the CTAB (hexadecyltrimethyl ammonium bromide) method.

2. Cucumber chloroplast, mitochondria and nuclear genome sequence analysis

NGS whole-genome sequencing was performed using DNA extracted from cucumber breeding lines CS01 and CS02 and F ₁ individuals (CS01×CS02 and CS02×CS01) raised by their crossing. A PE library (Paired-end library) having an insert size of about 300bp was generated according to the Standard Illumina PE protocol, and the generated library was sequenced using the HiSeq2000 platform (Illumina, USA). Quality trim was performed using the CLC ASSEMBLY CELL package (ver. 4.6 beta; CLC Inc., Denmark; http://www.clcbio.com/products/clc_assembly-cell/) for the sequenced PE read, and Phred De novo assemble was performed with a CLC genome assembler using only high-level PE reads with scores of 20 or higher. Subsequently, based on the previously reported reference genome of cucumber chloroplast (NCBI accession no. DQ865975), only the contigs of the chloroplast genome were extracted, and a single draft sequence was created through alignment and synthesis. The chloroplast genome sequence of the breeding line and its F ₁ individuals used in the present invention was completely decoded through a manual modification process for the draft sequence (FIG. 1). The Annotation process was performed through processes such as GESEQ (https://chlorobox.mpimp-golm.mpg.de/geseq.html), BLAST search, and comparison with existing sequences. In addition, the mitochondrial and nuclear genome sequences of cucumbers were also decoded in the same manner as above based on the cucumber mitochondrial reference genome (NCBI accession no. NC-016005) and the cucumber nuclear reference genome (NCBI accession no. ACHR00000000.3).

3. Sequence variation search and molecular marker development

Polymorphic regions were identified by mapping the chloroplast genomic sequence, mitochondrial genomic sequence, or nuclear genomic sequence of the parental and secondary individuals used for cucumber breeding to the previously reported cucumber chloroplast, mitochondrial or nuclear reference genome sequence. At this time, the basic condition of the program was used as the condition, and among the mapped reads, the appropriately mapped read was SAMtools ver. 1.1 (http://samtools.sourceforge.net/) was used to select, and the polymorphic region was Picard ver. 1.112 (http://broadinstitute.github.io/picard/) and GATK ver. It was excavated under the basic conditions of 3.1 (https://www.broadinstitute.org/gatk/). Based on the polymorphic regions identified above, the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih/gov/tools/primer-blast) and dCAPS Finder 2.0 (http://helix.wustl.edu/) dcaps/dcaps.html) was used to develop molecular markers. Cucumber mitochondria and mutation region information in the nuclear genome and primer set information prepared based on the information are shown in Tables 1 and 2, respectively.

4. PCR analysis using molecular markers

4-1. Chloroplast-based dCAPS molecular marker

Reagent composition and PCR conditions used for PCR analysis using dCAPS molecular markers (Cp-SNP-01 and Cp-SNP-02) based on polymorphism of chloroplast genome sequence are shown in Table 3 and Table 4, respectively, and restriction enzyme treatment The composition for is shown in Table 5 and Table 6 below. In the enzyme treatment, the product of NEB was used as the restriction enzyme and the buffer, and the agarose gel used to confirm the product of the restriction enzyme treatment was prepared in a 0.5X TBE buffer solution at a concentration of 3%, and the equipment and reagents used were as follows. It is shown in Table 7.

4-2. InDel molecular marker based on mitochondria

Using InDel molecular markers (Mt-InDel-01, Mt-InDel-02, Mt-InDel-03, Mt-InDel-04, Mt-InDel-05 and Mt-InDel-06) based on the polymorphism of the mitochondrial genome sequence The reagent composition and PCR conditions used in the PCR analysis are shown in Table 3 and Table 8, respectively, and the agarose gel used to confirm the PCR product was prepared in a 0.5X TBE buffer solution at a concentration of 3%, and was used to confirm the PCR product. The prepared equipment and reagents are shown in Table 7 above.

4-3. InDel molecular marker based on nuclear sequence polymorphism

The reagent composition and PCR conditions used in the PCR analysis using InDel molecular markers (Nu-InDel-01 and Nu-InDel-02) based on the polymorphism of the nuclear genome sequence are shown in Table 9 and Table 10, respectively, and the PCR product was confirmed. The agarose gel used for this was prepared in a 0.5X TBE buffer solution at a concentration of 3%, and the equipment and reagents used to confirm the PCR product are shown in Table 7 above.

Example 1. F of cucumber using the developed marker set _One Purity black

PCR was performed using the primer set of the present invention for the breeding line of cucumbers and their F ₁ individuals. Cucumber individuals used as a parent or counterpart in cucumber breeding were named CS01, CS02, CB01, CI03, CI04 and CI05, and their F ₁ individuals were CS01×CS02, CS02×CS01, CS01×CB01, CB01×CS01, It was named as CI04××CI03, CI03×CI04, CI05×CI03, and CI03×CI05, and the names of the F ₁ individuals were written according to the rules of parent×copy.

As a result of PCR, dCAPS molecular markers (Cp-SNP-01 and Cp-SNP-02) based on SNPs in the chloroplast genome sequence of cucumbers were able to distinguish the parent individuals of F ₁ individuals, and insertion or deletion in the mitochondrial genome sequence of cucumbers Region-based InDel molecular markers (Mt-InDel-01, Mt-InDel-02, Mt-InDel-03, Mt-InDel-04, Mt-InDel-05 and Mt-InDel-06) are the secondary entities of the F ₁ entity. It was possible to distinguish, and it was confirmed that the InDel molecular marker based on the insertion or deletion region in the nuclear genomic sequence of cucumber can confirm the parental copy used for the growth of the F ₁ individual (FIG. 3 ).

Specifically, as a result of performing PCR on the breeding line CS01 and CS02 and their F ₁ individuals using the Cp-SNP-01 primer set, CS01 and CS02 showed bands of 245 bp and 269 bp, respectively, and their It was confirmed that the F ₁ individuals CS01×CS02 and CS02×CS01 also showed bands of 245bp and 269bp, respectively. In other words, through which the band size of the F ₁ the object is equal to the band size of the cucumber object used mobon, it was found that the Cp-SNP-01 is able to distinguish between the mobon strains of cucumber used in the development of F ₁ individual .

In addition, as a result of performing PCR on the breeding lines CS01 and CS02 and their F ₁ individuals using the Mt-InDel-01 primer set, CS01 and CS02 showed bands of 258 bp and 265 bp, respectively, and their F _{In one} individual, CS01×CS02 and CS02×CS01, it was confirmed that bands of size 265bp and 258bp, respectively, appeared. That is, it was found that the band size of the F ₁ individual that through the same the band size of the cucumber object used as counterpart, Mt-InDel-01 is able to distinguish between a cucumber in the counterpart system used in the development of F ₁ individual .

In addition, as a result of PCR for various F ₁ individuals and their breeding lines using the Nu-InDel-01 primer set, if the size of each band of the F ₁ parental entity is different, a double band appears in the F ₁ entity. When the size of each band of the F ₁ parental subject was the same, it was confirmed that a single band appeared in the F ₁ subject. If PCR is performed on a certain F ₁ individual and a cucumber cultivar that is expected to be its parental copy, if a band of a size different from that of the parental copy appears in the F ₁ individual, the parental copy used in the experiment is the actual parental copy of F ₁ . You will see that it is not a copy.

In summary, by using the nuclear genome sequence based InDel molecular marker of the present invention using the cucumber F ₁ individual and check its breeding lines first, the chloroplast genome sequence based dCAPS molecular markers and mitochondrial genome sequences based InDel molecular markers F ₁ individual It was thought that more accurate F ₁ purity testing of cucumbers could be performed by discriminating between the parent and the copy.

<110> Seoul National University R&DB Foundation <120> Molecular marker derived from complete sequencing of chloroplast, mitochondria and nucleus genome of Cucumis sativus for Cucumis sativus F1 hybrid purity checking and uses thereof <130> PN19394 <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggtggagttt ttaaccggtt tgat 24 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttcgagtccg tatagcccta tatta 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tgaagaagga aagagaagac gacta 25 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtaaattttg ggagtcaaaa tcagta 26 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcggactgct tcggttaat 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acctgtccta cgtgccaaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agaaaggtcc aaagcccttc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcagcgagaa agtcagagga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ttctctccgt accgcgtagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcacctggac ctttctttgg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 accttaggcc ggacctctta 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 catccatatg ggccaaagaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ttagaagcag tccgcaggat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 accgtccgta ggagtccttt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ccgggaaata gcaatgaaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 accttcctgc ttcagggaat 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gcttgaattt gggcaacagg t 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tcttgaagtg gaggtgtccg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ccactgatga caatgcctgt 20 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 acgaaatttt ctgtaactca tgtaa 25

Claims (5)

One or more primer sets selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 1 and 2 and the oligonucleotide primers of SEQ ID NOs: 3 and 4; And the oligonucleotide primer sets of SEQ ID NOs: 5 and 6, and the oligonucleotides of SEQ ID NOs: 7 and 8 The group consisting of a nucleotide primer set, an oligonucleotide primer set of SEQ ID NOs: 9 and 10, an oligonucleotide primer set of SEQ ID NOs: 11 and 12, an oligonucleotide primer set of SEQ ID NOs: 13 and 14, and an oligonucleotide primer set of SEQ ID NOs: 15 and 16 One or more primer sets selected from; And one or more primer sets selected from the group consisting of the oligonucleotide primer sets of SEQ ID NOs: 17 and 18 and the oligonucleotide primer sets of SEQ ID NOs: 19 and 20; Cucumber ( Cucumis sativus ) F ₁ purity assay primer set composition comprising . The oligonucleotide primer set composition of claim 1; And a kit for F ₁ purity assay of cucumber ( Cucumis sativus ) comprising a reagent for performing an amplification reaction. The kit of claim 2, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and buffer. Separating genomic DNA from the cucumber sample;
Using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of claim 1 to amplify a target sequence; And
Detecting the product of the amplification step; containing, Cucumber ( Cucumis sativus ) F ₁ purity assay method. The method of claim 4, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, or phosphorescence measurement.

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