KR101783347B1 - CAPS marker for discriminating presence or absence of pollen in pear and uses thereof - Google Patents

Abstract

The present invention relates to a CAPS marker for discriminating the presence or absence of a flowerpot and its use. The present invention makes a linkage map and implements a kruskal-wallis test about an F1 mating seedling cultivated by cross-breeding a mini-pear as a pollen parent and a golden-pear as an ovary parent, thereby making a gene map about the pollen presence of the pollen in the pear. Through the present invention, a specific gene locus involved in the presence or absence of the pollen of the pear is checked to check the gene map about the presence or absence of the pollen in the pear for supplying direct information on the genetic background of presence or absence of the pollen in the pear. In addition, the present invention can be very useful in molecular breeding research and breeding industry of the pear by developing a primer for selection of molecular markers of the traits of the presence or absence of the pollen in the pear with reference to a mark close to a location of the high density gene map bout the pollen presence of the pollen in the pear.

Description

CAPS MARKER FOR DETERMINING MARINE PLANT AND USE THEREOF {

The present invention relates to a CAPS marker for discriminating the presence or absence of a flowerpot and a use thereof.

In addition to apples, apples and pears are the world's third largest producer of bananas and citrus fruits, accounting for 80 million tons a year, followed by rosebushes with 25.2 million tons of annual yield, the second highest production volume. As the characteristics of most fruit trees, it has the disadvantage that the breeding cycle is long due to the nature of incompatibility with juveniles and self - irrigation which takes a long time to reach fruit after sowing. Therefore, there is a growing demand for the development of useful trait markers for selection of molecular markers for breeding efficiency.

In 1976, a study of cytoplasmic genetic male sterility was reported, mainly with phenotype as a pollen presence. Commercial farmers are required to rehydrate water or take artificial moisture according to the cultivated variety considering the S genotype. However, most of the domestic pear cultivars, which are cultivated as examples of pear cultivars which make up about 80% of the cultivation area in Korea, have no pollen or small amount. Therefore, the presence or absence of pollen is one of the target traits in the pest breeding program to ensure a stable water content.

GBS (Genotyping-By-Sequencing) was developed and distributed in 2011 as an application technology of NGS (Next-Generation Sequencing), an automation technology for reading DNA genetic information. GBS is a series of analytical processes that enable the development of single nucleotide polymorphisms (SNPs) for a large number of individuals and genotyping based on the NGS technology analysis platform. If you have read the nucleotide sequence information of an individual in the existing NGS, GBS will treat the restriction enzyme for a large number of target objects and use a library with a bar code and an adapter so that a large capacity And the genomic data can be obtained.

Many SNPs from GBS technology have been used in genetic studies of various plant and plant species such as genome-wide association studies, associative mapping and quantitative trait loci mapping. The development of plant genome analysis techniques such as NGS and GBS has enabled the search for genetic mutations associated with target traits and is being explored for use in marker-assisted selection in breeding programs. In particular, mapping of quantitative / qualitative traits based on genetic maps provides insights into the genetic background of agricultural traits and further facilitates the development of trait-related molecular markers for MAS.

Cleaved amplified polymorphic sequences (CAPS), also referred to as restricted amplification polymorphism sequences, are polymorphic fragments generated by restriction enzyme treatment of truncated PCR products. Detection is carried out in advance for the purpose of a gene which knows that there is a difference in nucleotide sequence among varieties. Only the gene of the genome of the species is selected by PCR method and amplified. In most of the markers, there is no difference between the length of the amplified gene and the breed. However, since the internal arrangement of the gene is different, restriction enzymes recognizing the difference in the sequence are selected and processed. It is possible to distinguish the difference between the varieties carrying the restriction enzyme-digested DNA and the varieties having the DNA not digested, and the difference in length is easily detected by electrophoresis.

On the other hand, Korean Patent No. 0882142 discloses' a high-density quantitative trait map related to the enhancement of rice yield, 'and Korean Patent Publication No. 2011-0082973 discloses' a high-density quantitative trait map of rice blast resistance gene And CAPS markers for determining the presence or absence of pots of pear and their uses have not been disclosed at all as in the present invention.

The present invention has been made in view of the above-mentioned needs. The present invention relates to an F1 mating seedling cultivated by crossing a golden pear and a mini pear in a flowerpot, and conducting a kruskal-wallis test Respectively. The gene map of the presence or absence of the blastocyst according to the present invention enables identification of a specific locus involved in the presence or absence of a flower pot. Further, CAPS markers for MAS with or without pollen were prepared based on SNPs selected using GBS-based nucleotide sequence data for F1 breeders of golden and mini pears, and the CAPS markers effectively discriminated pollen presence traits , The present invention has been completed.

In order to accomplish the object of the present invention as described above, the present invention provides a primer set for identifying the presence or absence of pollen of a ship comprising oligonucleotides of SEQ ID NOS: 1 and 2.

In addition, the present invention provides a method for determining the presence or absence of pollen on a stomach using the oligonucleotide primer set.

In addition, the present invention provides the oligonucleotide primer set; A reagent for carrying out an amplification reaction; And a kit for determining the presence or absence of a flower pot in a vessel, including a restriction enzyme.

In addition, the present invention relates to pollen-related gene analysis carried out on an F1 seedling obtained by crossing a golden pear with a pot and a mini-pear with a flower pot, respectively, in the presence or absence of a flower pot The present invention provides a high-density genetic map of the pollen-related relationship shown in Fig.

In the present invention, a mapping map and a kruskal-wallis test were carried out on F1 mating cultivars cultivated by crossing a mini-pear with a flowerpot and a golden pear with a fuller's chin, , Which is a high-density pollen-related gene map that can provide direct information on the genetic background of pollen presence by confirming the specific locus involved in the presence or absence of pollen in the stomach. In addition, by developing a primer for selection of molecular markers for the traits of the traits of the abundance of potted plants, it is very useful in the field of molecular breeding and pearling breeding industry .

1 is a schematic diagram showing information of a genetic analysis group used in the present invention.
FIG. 2 shows photographs showing the separation of the pollen presence (C, D, E) from the golden pear (A) without the pollen, the mini pear with the pollen (B) and the parental F1 mating seedlings.
FIG. 3 shows information on the distribution of pollen presence in the gold embryo and mini embryo F1 hybrid breeding stock in 2016 on the cover of the target site of the association group No. 14.
FIG. 4 is a genetic map showing the positions of chromosomal fragments controlling the presence or absence of pollen in the integrated association map of golden and mini embryos in the group cultivated in FIG.
FIG. 5 is a graph showing the relationship between the primer set (BfuCI-1) and the restriction enzyme Bfu CI (SEQ ID NO: 1 and SEQ ID NO: 2) (D8), 14-071 (D9), 14-075 (D10), 14-064 (D6), 14-068 077 (D11), and 14-079 (D12) were confirmed through a capillary digital gel. H12 is a DNA ladder of 100 bp.
FIG. 6 is a graph showing the fluorescence intensity of 14-084 (A1), 14-086 (A2), 14-088 (A3) using the primer set (BfuCI-1) consisting of SEQ ID No. 1 and SEQ ID No. 2 of the present invention and restriction enzyme Bfu CI, , 14-090 (A4), 14-091 (A5), 14-092 (A6), 14-093 (A7), 14-094 (A8), 14-096 (A9), 14-097 14-098 (A11) and 14-099 (A12) were confirmed through a capillary digital gel. H12 is a DNA ladder of 100 bp.
Figure 7 14-100 (B1), 14-101 (B2 ), 14-104 (B3) by using the primer set (BfuCI-1) with the restriction enzyme Bfu CI consisting of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention , 14-105 (B4), 14-108 (B5), 14-109 (B6), 14-112 (B7), 14-115 (B8), 15-001 (B9), 15-002 (B10) 15-005 (B11), and 15-006 (B12) were confirmed through a capillary digital gel. H12 is a DNA ladder of 100 bp.
8 is 15-008 (C1), 15-014 (C2 ), 15-015 (C3) using a primer set (BfuCI-1) with the restriction enzyme Bfu CI consisting of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention , 15-016 (C4), 15-018 (C5), 15-020 (C6), 15-022 (C7), 15-024 (C8), 15-026 (C9), 15-028 15-029 (C11), 15-032 (C12) were confirmed by capillary digital gel. H12 is a DNA ladder of 100 bp.
FIG. 9 is a graph showing the results of DNA fragmentation of 15-034 (D1), 15-035 (D2) and 15-036 (D3) using a primer set (BfuCI-1) consisting of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention and restriction enzyme Bfu CI. , 15-037 (D4), 15-038 (D5), 15-039 (D6), 15-040 (D7), 15-041 (D8), 15-043 15-045 (D11), 15-046 (D12) were confirmed by capillary digital gel. H12 is a DNA ladder of 100 bp.
FIG. 10 is a graph showing the effect of the primer set (BfuCI-1) and the restriction enzyme Bfu CI (SEQ ID NO: 15-050 (E4), 15-052 (E5), 15-053 (E6), 15-054 (E7), 15-055 (E8) 15-063 (E11), 15-065 (E12) were confirmed by capillary digital gel. H12 is a DNA ladder of 100 bp.

One or more oligonucleotides selected from the group consisting of oligonucleotides consisting of fragments of at least 15 consecutive nucleotides in the sequence of SEQ ID NO: 1 and at least 15 contiguous nucleotides in the sequence of SEQ ID NO: 2 And a set of one or more oligonucleotide primers selected from the group consisting of oligonucleotides consisting of segmented polynucleotides.

The oligonucleotide may preferably be an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 1. In addition, the oligonucleotide may preferably be an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more consecutive nucleotides in the sequence of SEQ ID NO: 2.

An oligonucleotide primer set according to an embodiment of the present invention is a set of primers for discriminating the presence or absence of pollen of a vessel containing oligonucleotides of SEQ ID NOs: 1 and 2. In addition, the primer may also include an added, deleted or substituted sequence of the nucleotide sequence of SEQ ID NO: 1 or 2.

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

As used herein, an oligonucleotide used as a primer may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.

In the primer set according to one embodiment of the present invention, the embryo may be, but is not limited to, a golden embryo and a mini embryo F1 hybrid.

In order to achieve still another object of the present invention,

Isolating genomic DNA from the germplasm;

Amplifying the target sequence by using the separated genomic DNA as a template and carrying out an amplification reaction using the oligonucleotide primer set;

Cleaving the amplification product with a restriction enzyme; And

And separating the cleavage products by size by gel electrophoresis to thereby determine the presence or absence of the pollen of the embryo.

The method of the present invention comprises isolating genomic DNA from a germplasm sample. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. Preferably, a TOYOBO kit is used Can be used. Using the separated genomic DNA as a template, an oligonucleotide primer set according to an embodiment of the present invention may be used as a primer to perform an amplification reaction to amplify the target sequence. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, Strand displacement amplification or amplification with Q [beta] 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 the primer set according to one embodiment of the present invention, the embryo may be, but is not limited to, a golden embryo and a mini embryo F1 hybrid.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material can be, but is not limited to, a fluorescent, phosphorescent or radioactive substance. Preferably, the labeling substance is 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 can be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive. The set of oligonucleotide primers used to amplify the target sequence are as described above.

The method of the present invention comprises detecting said amplification product. The amplification product is detected by digesting the amplified product with a restriction enzyme. The detection of the amplification product can be performed by DNA chip, gel electrophoresis, radioactive 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. In the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When the PCR is performed, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then a radioactive measurement device such as a Geiger counter or liquid scintillation counter The radioactivity can be measured using a liquid scintillation counter.

In the method of the present invention, the restriction enzyme may be Bfu CI, but is not limited thereto.

In order to achieve still another object of the present invention,

An oligonucleotide primer set according to the present invention; A reagent for carrying out an amplification reaction; And a kit for determining the presence or absence of a flower pot in a vessel, including a restriction enzyme.

In the kit of the present invention, the reagent for carrying out the amplification reaction may include a DNA polymerase, dNTPs, a buffer, etc., and the restriction enzyme may preferably be Bfu CI. In addition, the kit of the present invention may further include a user guide describing optimal reaction performing conditions. The manual is a printed document that explains how to use the kit, for example, how to prepare PCR buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

In addition, the present invention relates to pollen-related gene analysis carried out on an F1 seedling obtained by crossing a golden pear with a pot and a mini-pear with a flower pot, respectively, in the presence or absence of a flower pot The present invention provides a high-density genetic map of the pollen-related relationship shown in Fig.

In the high density gene map of the embryo according to an embodiment of the present invention, the abundance pollen related locus of the embryo may be composed of 14 SNP molecular beacons located at 0 to 27.3 cM including the related map SNP marker scaffold162_40781, But is not limited thereto.

In a high density gene map of the embryo according to one embodiment of the present invention, the SNP marker scaffold162_40781 may be amplified using oligonucleotides of SEQ ID NOS: 1 and 2, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. The golden ship Mini boat  F1 with Actual From 87 individuals  DNA extraction

Leaves were collected at the tip of 87 F1 seedlings including golden and mini pears, washed with distilled water, packaged individually and stored at -80 ° C. Genomic DNA was isolated from fresh leaves of fresh pears through CTAB (cetyl trimethyl ammonium bromide). The young leaves of pears were placed in a mortar bowl filled with liquid nitrogen and the ground sample was transferred to a sample tube. 500 μl of CTAB buffer was added, and 2 μl of 2-mercaptoethanol was added, followed by incubation in a constant-temperature water bath at 65 ° C. for 30 minutes. 200 [mu] l of 5M potassium acetate was added and incubated on ice for 30 minutes. 600 페 phenol: chloroform: isoamyl alcohol (P: C: I = 25: 24: 1) was added and the mixture was centrifuged at 13,000 rpm for 10 minutes at 4 캜. The supernatant was transferred to a new sample tube and 600 [mu] l of chloroform: isoamyl alcohol (C: I = 24: 1) was added and centrifuged at 13,000 rpm for 10 minutes at 13,000 rpm.

The supernatant was transferred back to the new sample tube and 600 [mu] l of cold isopropanol was added and incubated at -20 [deg.] C for 30 min. Centrifugation at 13,000 rpm for 5 minutes at 4 < RTI ID = 0.0 > C < / RTI > The isopropanol was discarded and 500 μl of cold 70% EtOH was added to the precipitate, followed by centrifugation at 13,000 rpm for 5 minutes at 4 ° C. EtOH was discarded and dried, then 30 쨉 l of TE buffer was added. Then, RNase A was added, followed by incubation at 37 ° C for 1 hour and storage at -80 ° C. The extracted genomic DNA was quantified as 100 ng / μl for GBS analysis.

Example  2. GBS  Development and analysis of SNP mark using technology

For the sequencing and genotyping, we constructed a library for the GBS implementation of 178 F1 genetic polymorphic DNAs including gold and mini-pear (Elshire et al., 2011, PloS one, 6 (5): e19379. ), The library was sequenced according to the standard protocol of the Illumina Hiseq 2000 sequencer. The resulting sequences were used for bioinformation analysis and the total genome sequence of the Chinese embryo 'Dangshansuli' (Wu et al., 2013, Genome Res, 23 (2): 396-408) Was used as the reference genome. For the development of SNPs for mapping, a large number of SNPs predicted by polymorphism in the reference genome were filtered by the following criteria: (1) biallelic SNPs; (2) 3) The missing value for Full-sib family by SNP should be within 10%.

Example  3. Assessment of pollen presence

All the plant materials were used in F1 experimental seedlings planted at the National Institute of Horticultural Science, Naju Bae Research Institute. In April, 2016, the evaluation of the presence of pollen was referred to the research manual of the genetic resource characteristics of the National Institute of Horticultural Science, Rural Development Administration. 87 petals including gold and mini pears were collected and 5 ~ 10 pre - flowering flowers were collected and all petals were removed. After drying at 25 캜 for 24 hours in a Petri dish, the number of pollen-free individuals was recorded as 1, and the number of pollen-free individuals as 0 was recorded.

The presence or absence of pollen in 2016 of 87 prefectural households was found to be in the ratio of 40:41, and it was 1: 1 by the chi-square test (P < 0.05) (Fig. 3).

Example  4. F1 Typical household  detailed Presence of pollen  Genetic Mapping

High-density gene mapping using GBS-derived SNPs from 87 gold-plated mini-F1 plants was determined based on segregation analysis using the Joinmap 4.1 program (LOD = 6.0 for association groups). On the other hand, the genetic distance between the markers was calculated in units of cM (Kosambi centiMorgans). Based on the generated gene map, Kruskal wallis test with MapQTL 5.0 was applied to estimate the optimal location of the target genes associated with phenotypic recordings. As a result, 14 SNP markers closely related to the presence of pollen were found in association group 14 (Table 1).

No. Association group location
(cM) Signs with or without pots Kruskal-wallis
statistic One 14 0.0 scaffold54.2_403225 16.7 2 14 2.6 scaffold54.2_382812 11.6 3 14 17.1 scaffold244_108187 15.6 4 14 18.1 scaffold244_250422 20.6 5 14 19.1 scaffold202_334156 21.6 6 14 19.6 scaffold202_466883 22.8 7 14 19.9 scaffold545_249739 21.5 8 14 25.4 scaffold162_726978 24.4 9 14 26.3 scaffold638_206795 20.2 10 14 27.1 scaffold1007_123736 26.5 11 14 27.1 scaffold333_243509 24.1 12 14 27.2 scaffold333_218719 22.6 13 14 27.2 scaffold333_174922 22.6 14 14 27.3 scaffold162_40781 20.1

Example  5. GBS  Derived SNP based Presence of pollen  For the development of selective molecular markers PCR  Perform

In order to switch from a gold-pear and mini-pear F1 genetic polymorphism to a PCR-based MAS marker for potted or nonpigmented traits, a primer of 580 bp was prepared by referring to GBS-based sequence data among 14 candidate SNP markers. Are as shown in Table 2 below.

primer The nucleotide sequence (SEQ ID NO) Tm (占 폚) Expected size Restriction enzyme BfuCI-1 F: TCC GGA ACT TAC CAG CTT GA (1) 59 580 Bfu CI R: GGG AAC TCC CTC TGA TGCA (2)

The PCR mixture composition consisted of 6 ㎕ of gDNA of F1 72 individuals in the golden pear and mini pear extracted, 20 Hot of Hot Taq polymerase (GeNetbio, Korea), 8 프 of primer set (4 ㎕ each of forward primer and reverse primer) Lt; / RTI &gt; was added to make a total of 40 &lt; RTI ID = 0.0 &gt; PCR was carried out at 94 ° C for 5 minutes, followed by 30 seconds at 94 ° C, 45 seconds of annealing at the melting temperature (Tm) shown in Table 2, 30 seconds at 72 ° C 35 times PCR was repeatedly performed, and finally, extension at 72 ° C for 7 minutes.

Example  6. For GBS-based SNP-derived CAPS labeling PCR  refine

After the PCR reaction, the PCR product was removed using PCR purification kit (Bioneer, Korea) to increase the activity of the restriction enzyme. Specifically, 40 μl of the PCR product was mixed with 200 μl of a primary buffer (PCR binding buffer), and 240 μl of the mixture was placed in a DNA binding column tube and centrifuged at 13,000 rpm for 1 minute at 4 ° C. Subsequently, the tube was replaced with a 2.0-ml tube, 500 μl of the secondary solution was dispensed, and centrifugation was further performed at 13,000 rpm for 1 minute at 4 ° C. Then, it was dried at room temperature for 5 minutes, centrifuged at 13,000 rpm for 1 minute at 4 ° C, and 30 μl of the tertiary solution was placed in a DNA binding filter column and allowed to stand for 4 minutes and then incubated at 13,000 rpm Gt; min. &Lt; / RTI &gt;

Example  7. GBS  Restriction enzyme treatment for CAPS-based SNP-derived markers

25 μl of the reaction solution containing 21 μl of the purified PCR product, 1.5 μl of restriction enzyme Bfu CI and 2.5 μl of 10 × enzyme buffer (enzyme buffer) was treated at 37 ° C. for 4 hours.

Example  8. Capillary electrophoresis (capillary electrophoresis)

Capillary electrophoresis was performed using a fragment analyzer (Advanced Analytical, Inc., Ames, USA) to confirm the polymorphism of the cleaved fragments of the transformed CAPS label. For analyzing the fragments, a gel was prepared by adding an intercalating dye to a dsDNA 905 separation gel. To the PCR product digested with restriction enzymes, 5 μl of TE (Tris-EDTA) buffer was added Lt; / RTI &gt; Thereafter, capillary electrophoresis was performed on the fragment analyzer.

Example  9. GBS  Based SNP-derived CAPS label validation

In this example, SNP base sequence and SNP polymorphism at the recognition site of restriction enzyme ( Bfu CI) selected through GBS (genotyping-by-sequencing) And capillary electrophoresis using the CAPS label of the present invention showed that the polymorphism band closely related to the presence or absence of the pollen was found in the F1 seedlings as shown in FIGS. 5 to 10 (see Table 3) ).

Specifically, through the BfuCI-1 label, as shown in Table 3, golden pears, mini pears, and arbitrarily selected F1 actual seedlings were classified into two groups for 70 individuals, and a potted golden There were 33 individuals in the abalone and F1 breeders, and 36 mini-boars and F1 breeding birds with pollen characterized by three fragments (Fig. 5 to Fig. 10). The presence or absence of pollen according to the number of fragments (2 to 3) was found to be 32:40 in the separation ratio (Table 3), and the selection ratio was calculated as follows.

Based on the above formula, the selection ratio obtained in the present invention was found to be 55/70 x 100? 78.6%.

Using the CAPS label of the present invention, No. Sample BfuCI-1
(bp) Number of fragments
(/: Missing) Presence of pollen
(1: present, 0: none, /: missing) Selection possible
(○: possible, ×: impossible) One Golden boat 266/294 2 0 ○ 2 Mini boat 267/295 3 One ○ 3 14-059 267/296 2 0 ○ 4 14-060 265/294/561 3 One ○ 5 14-061 266/294/563 3 One ○ 6 14-064 267/296/563 3 0 × 7 14-068 266/295 2 0 ○ 8 14-071 265/294/561 3 0 × 9 14-072 267/296/563 3 One ○ 10 14-075 265/293/561 3 One ○ 11 14-077 270/299/564 3 0 × 12 14-079 266/295/562 3 0 × 13 14-084 275/304/580 3 One ○ 14 14-086 275/305/581 3 One ○ 15 14-088 274/305 2 One × 16 14-090 275/306 2 0 ○ 17 14-091 275/306 2 0 ○ 18 14-092 273/303 2 0 ○ 19 14-093 276/306/581 3 One ○ 20 14-094 275/306 2 0 ○ 21 14-096 275/307 2 0 ○ 22 14-097 273/302/578 3 One ○ 23 14-098 272/299/576 3 One ○ 24 14-099 272/300 2 0 ○ 25 14-100 272/300 2 One × 26 14-101 272/300 2 0 ○ 27 14-104 275/306/579 3 One ○ 28 14-105 277/311/576 2 0 ○ 29 14-108 274/302/578 2 0 ○ 30 14-109 274/304/577 3 One ○ 31 14-112 272/303 2 0 ○ 32 14-115 270/297 3 One ○ 33 15-001 276/309 2 0 ○ 34 15-002 273/302 2 0 ○ 35 15-005 276/306/579 3 One ○ 36 15-006 275/305/578 3 One ○ 37 15-008 277/306/582 3 One ○ 38 15-014 278/311/579 3 One ○ 39 15-015 274/303 2 0 ○ 40 15-016 274/303/579 3 One ○ 41 15-018 272/300/574 3 0 × 42 15-020 274/305 2 0 ○ 43 15-022 275/306 2 0 ○ 44 15-024 273/301/576 3 0 × 45 15-026 274/302 2 0 ○ 46 15-028 272/299 2 0 ○ 47 15-029 273/301 2 0 ○ 48 15-032 270/298 2 0 ○ 49 15-034 270/298 2 0 ○ 50 15-035 275/307 2 0 ○ 51 15-036 273/302/577 3 One ○ 52 15-037 273/302/575 3 One ○ 53 15-038 274/303 2 0 ○ 54 15-039 274/305 2 One × 55 15-040 / / 0 × 56 15-041 272/300 2 0 ○ 57 15-043 276/306/579 3 One ○ 58 15-044 274/304/577 3 One ○ 59 15-045 274/302/579 3 One ○ 60 15-046 272/300 2 One × 61 15-047 273/301 2 One × 62 15-048 272/302/575 3 One ○ 63 15-049 273/301/579 3 One ○ 64 15-050 275/306/579 3 0 × 65 15-052 270/297 2 0 ○ 66 15-053 / / 0 × 67 15-054 / / 0 × 68 15-055 274/304 2 0 ○ 69 15-057 272/300/577 3 0 × 70 15-061 276/308/578 3 0 × 71 15-063 273/302/577 3 One ○ 72 15-065 275/305/578 3 One ○

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> CAPS marker for discriminating presence or absence of pollen in          pear and uses thereof <130> PN17175 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tccggaactt accagcttga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gggaactccc tctgatgaca 20

Claims (9)

A primer set for determining the presence or absence of pollen of a vessel containing oligonucleotides of SEQ ID NOs: 1 and 2. The primer set according to claim 1, wherein the embryo is an F1 hybrid breeding stock of a golden embryo and a mini embryo. Isolating genomic DNA from the germplasm;
Amplifying the target sequence by performing amplification reaction using the separated genomic DNA as a template and using an oligonucleotide primer set according to claim 1 or 2;
Cleaving the amplification product with a restriction enzyme; And
And separating the cleavage products by size by gel electrophoresis of the cleavage products. 4. The method of claim 3, wherein the amplified target sequence is labeled with a fluorescent, phosphorescent or radioactive substance. An oligonucleotide primer set according to claims 1 or 2; A reagent for carrying out an amplification reaction; And a restriction enzyme. 6. The kit according to claim 5, wherein the reagent for carrying out the amplification reaction comprises DNA polymerase, dNTPs and a buffer. delete delete delete

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