KASP molecular marker closely linked with Korean pine cones and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker in heavy close linkage with Korean pine cones and application of the KASP molecular marker, relates to the technical field of forest molecular marker-assisted breeding, and relates to a Korean pine molecular marker and application of the Korean pine molecular marker. The invention provides two KASP molecular markers closely linked with Korean pine cones and application of the molecular markers in Korean pine breeding. The KASP molecular marker primer can rapidly identify the Korean pine cone weight phenotype, has the advantages of being accurate, rapid, low in cost, short in identification period, easy and convenient to operate and the like, can assist in Korean pine new strain breeding, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of forest tree molecular marker-assisted breeding, and relates to Korean pine molecular markers and applications thereof. Background Art

[0002] Korean pine (Pinus koraiensis) is a national second-level protected wild plant and an important economic tree species in the northeastern forest area of ​​my country. The fruit of Korean pine has become the most popular pine nut product because of its very high nutritional value and contains a large amount of crude protein, crude fat, polysaccharides and crude fiber as well as vitamins, minerals and trace elements. Cone weight is an important economic trait that affects the yield of Korean pine nuts. Therefore, the development of molecular markers closely linked to cone weight and the auxiliary selection of Korean pine cone weight through molecular markers will effectively save breeding time and land, accelerate the breeding process, and have important practical significance for the selection and promotion of Korean pine varieties. However, there is currently no molecular marker that can be used to identify Korean pine cone weight. Summary of the invention

[0003] In order to realize biological identification of Korean pine cone weight, the present invention provides a KASP molecular marker closely linked to Korean pine cone weight and application thereof.

[0004] The invention provides two KASP molecular markers which are closely linked to the cone weight of Korean pine.

[0005] KASP molecular marker III tightly linked to Korean pine cone weight, the molecular marker primer set consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R;

[0006] The nucleotide sequence of the upstream primer Pk494448-F1 is shown in SEQ ID NO.1;

[0007] The nucleotide sequence of the upstream primer Pk494448-F2 is shown in SEQ ID NO.2;

[0008] The nucleotide sequence of the downstream primer Pk494448-R is shown in SEQ ID NO.3.

[0009] KASP molecular marker IV tightly linked to Korean pine cone weight, the molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R;

[0010] The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4;

[0011] The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5;

[0012] The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.6.

[0013] The SNP molecular marker site of the KASP molecular marker III that is closely linked to the cone weight of Korean pine is Pk494448 (a gene pool constructed from 100 Korean pine clones from 7 Korean pine populations, and the site Pk494448 is the 494448th base in the gene pool); the SNP site has a base mutation of A / G. The 3' end of the upstream primer is a variant base, the 5' end of the upstream primer Pk494448-F1 is connected to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk494448-F2 is connected to phosphoramidite fluorescein VIC.

[0014] The SNP molecular marker site of KASP molecular marker IV closely linked to the cone weight of Korean pine is Pk3270935 (a gene pool constructed from 100 Korean pine clones of 7 Korean pine populations, and the site Pk3270935 is the 3270935th base in the gene pool); the SNP site has a G / T base mutation. The 3' end of the upstream primer is a variant base, the 5' end of the upstream primer Pk3270935-F1 is connected to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk3270935-F2 is connected to phosphoramidite fluorescein VIC.

[0015] The application of the KASP molecular marker tightly linked to Korean pine cone weight, and the application of the molecular marker in Korean pine breeding.

[0016] Furthermore, the molecular markers are used to identify the weight of Korean pine cones.

[0017] Further, a method for identifying the weight of Korean pine cones using the molecular markers is as follows:

[0018] 1. Extract the DNA of the Korean pine to be tested;

[0019] 2. Perform PCR amplification using the molecular marker primer set to obtain a PCR product;

[0020] 3. Genotyping of PCR products after fluorescence detection, the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with heavy cones, and the genotype with orange fluorescence signal is the genotype with small cones;

[0021] Wherein, the molecular marker primer set primers used in step 2 are group III molecular marker primer set or group IV molecular marker primer set; group III molecular marker primer set consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R; group IV molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R;

[0022] The nucleotide sequence of the upstream primer Pk494448-F1 is shown in SEQ ID NO.1;

[0023] The nucleotide sequence of the upstream primer Pk494448-F2 is shown in SEQ ID NO.2;

[0024] The nucleotide sequence of the downstream primer Pk494448-R is shown in SEQ ID NO.3;

[0025] The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4;

[0026] The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5;

[0027] The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.6.

[0028] Furthermore, the PCR reaction system in step 2 is 5 μL: composed of 1.25 μL of genomic DNA with a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL of each upstream primer with a concentration of 10 mM, and 0.75 μL of a downstream primer with a concentration of 10 mM.

[0029] Furthermore, the program of PCR amplification in step 2 is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing temperature starting from 61°C, annealing temperature decreasing by 0.6°C per cycle, annealing for 1 min per cycle, annealing temperature at 55°C after 10 cycles, then denaturation at 95°C for 20 s, extension at 55°C for 1 min; 27 cycles in total, running at 25°C for 1 min.

[0030] Furthermore, in step three, the genotype amplified by the molecular marker primer group III is G:G, and the fluorescent signal is blue; the genotype is A:G, and the fluorescent signal is green; the genotype is A:A, and the fluorescent signal is orange; in step three, the genotype amplified by the molecular marker primer group IV is T:T, and the fluorescent signal is blue; the genotype is T:G, and the fluorescent signal is green; the genotype is G:G, and the fluorescent signal is orange.

[0031] The Korean pine cone weight identification kit includes a KASP molecular marker III primer set closely linked to the Korean pine cone weight and / or a KASP molecular marker IV primer set closely linked to the Korean pine cone weight.

[0032] KASP (Kompetitive Allele-Specific PCR), that is, competitive allele-specific PCR. Compared with the traditional PCR method, KASP technology has the advantages of high throughput, low cost, high efficiency, and no need for electrophoresis detection.

[0033] In the present invention, the SNP site base of the molecular marker III in the Korean pine with heavy cones is G, and the SNP site of the molecular marker III in the Korean pine with small cone weight is A. The Korean pine of "G:G" type to be tested is determined as a homozygous material with heavy cones, the Korean pine of "G:A" type to be tested is determined as a heterozygous material with heavy cones, and the Korean pine of "A:A" type to be tested is determined as a homozygous material with small cone weight. In the present invention, the SNP site base of the molecular marker IV in the Korean pine with heavy cones is T, and the SNP site of the molecular marker IV in the Korean pine with small cone weight is G. The Korean pine of "T:T" type to be tested is determined as a homozygous material with heavy cones, the Korean pine of "G:T" type to be tested is determined as a heterozygous material with heavy cones, and the Korean pine of "G:G" type to be tested is determined as a homozygous material with small cone weight. The method of the present invention is consistent with the primer amplification band type of the above-mentioned molecular marker in the cone weight phenotype of a single plant in the population.

[0034] The KASP molecular marker primers in the present invention can quickly identify the cone weight phenotype of Korean pine, and have the advantages of accuracy, rapidity, low cost, short identification cycle, simple operation, etc., can assist in the breeding of new Korean pine strains, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the genotyping diagram of the Korean pine population by the molecular marker primer set III in Example 1;

[0036] Figure 2 It is the genotyping diagram of the Korean pine population by the molecular marker primer set IV in Example 1;

[0037] Figure 3 It is a bar graph showing the results of measuring the true cone weight of the natural population of Korean pine in Example 1. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0040] Specific implementation method 1: This implementation method is a KASP molecular marker III that is tightly linked to the weight of Korean pine cones. The molecular marker primer set consists of an upstream primer Pk494448-F1, an upstream primer Pk494448-F2, and a downstream primer Pk494448-R;

[0041] The nucleotide sequence of the upstream primer Pk494448-F1 is:

[0042] 5'-GAAGGTGACCAAGTTCATGCTATCTCTTACAGTCCCAGTTATGAAATTA-3' (SEQ ID NO. 1); wherein the underlined portion is the sequence of fluorescein FAM.

[0043] The nucleotide sequence of the upstream primer Pk494448-F2 is:

[0044] 5'-GAAGGTCGGAGTCAACGGATTATCTCTTACAGTCCCAGTTATGAAATTG-3' (SEQ ID NO. 2), wherein the underlined portion is the sequence of fluorescein VIC.

[0045] The nucleotide sequence of the downstream primer Pk494448-R is:

[0046] 5'-TGCTGAAGGTGGATCATTTGGTT-3' (SEQ ID NO. 3).

[0047] The SNP molecular marker site of KASP molecular marker III that is closely linked to the weight of Korean pine cones is Pk494448; there is a base mutation of A / G at this SNP site. The 3' end of the upstream primer is a variant base, the 5' end of the upstream primer Pk494448-F1 is connected to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk494448-F2 is connected to phosphoramidite fluorescein VIC. The cones of the test samples with the base "G" at the SNP site are heavy; the cones of the test samples with the base "A" at the SNP site are light.

[0048] Specific implementation method 2: This implementation method is a KASP molecular marker IV that is tightly linked to the weight of Korean pine cones. The molecular marker primer set consists of an upstream primer Pk3270935-F1, an upstream primer Pk3270935-F2, and a downstream primer Pk494448-R;

[0049] The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4;

[0050] The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5;

[0051] The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.6.

[0052] The nucleotide sequence of the upstream primer Pk3270935-F1 is:

[0053] 5'-GAAGGTGACCAAGTTCATGCTGAAAGTACAAAGGTTGAAGTGTTTCAG-3' (SEQ ID NO. 4); wherein the underlined portion is the sequence of fluorescein FAM.

[0054] The nucleotide sequence of the upstream primer Pk3270935-F2 is:

[0055] 5'-GAAGGTCGGAGTCAACGGATTGAAAGTACAAAGGTTGAAGTGTTTCAT-3' (SEQ ID NO. 5), wherein the underlined portion is the sequence of fluorescein VIC.

[0056] The nucleotide sequence of the downstream primer Pk3270935-R is:

[0057] 5'-TGAAGGACATATGCACCTCAGAAA-3' (SEQ ID NO. 6).

[0058] The SNP molecular marker site of KASP molecular marker IV, which is closely linked to the weight of Korean pine cones, is Pk3270935; there is a G / T base mutation at this SNP site. The 3' end of the upstream primer is a variant base, the 5' end of the upstream primer Pk3270935-F1 is connected to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk3270935-F2 is connected to phosphoramidite fluorescein VIC. The cones of the test samples with the base "T" at the SNP site are heavy; the cones of the test samples with the base "G" at the SNP site are light.

[0059] Specific implementation method three: the method for identifying the weight of red pine cones in this implementation method:

[0060] 1. Extract the DNA of the Korean pine to be tested;

[0061] 2. Perform PCR amplification using the molecular marker primer set to obtain a PCR product;

[0062] 3. Genotyping of PCR products after fluorescence detection, the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with heavy cones, and the genotype with orange fluorescence signal is the genotype with small cones;

[0063] Wherein, the molecular marker primer set primers used in step 2 are group III molecular marker primer set or group IV molecular marker primer set, group III molecular marker primer set consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R; group IV molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R;

[0064] The nucleotide sequence of the upstream primer Pk494448-F1 is shown in SEQ ID NO.1;

[0065] The nucleotide sequence of the upstream primer Pk494448-F2 is shown in SEQ ID NO.2;

[0066] The nucleotide sequence of the downstream primer Pk494448-R is shown in SEQ ID NO.3;

[0067] The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4;

[0068] The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5;

[0069] The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.6;

[0070] The PCR reaction system in step 2 is 5 μL: 1.25 μL of genomic DNA at a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL of each upstream primer at a concentration of 10 mM, and 0.75 μL of a downstream primer at a concentration of 10 mM;

[0071] The PCR amplification program in step 2 was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing temperature starting from 61°C, annealing temperature decreasing by 0.6°C per cycle, annealing for 1 min per cycle, annealing temperature at 55°C after 10 cycles, then denaturation at 95°C for 20 s, extension at 55°C for 1 min; 27 cycles in total, running at 25°C for 1 min;

[0072] In step 3, the genotype amplified by the molecular marker primer set III is G:G type, and the fluorescent signal is blue; the genotype is A:G type, and the fluorescent signal is green; the genotype is A:A type, and the fluorescent signal is orange;

[0073] In step 3, the genotype amplified by the molecular marker primer set IV is T:T, and the fluorescent signal is blue; the genotype is T:G, and the fluorescent signal is green; the genotype is G:G, and the fluorescent signal is orange.

[0074] This embodiment performs typing detection on PCR amplification products and presents them in the form of a chart; the chart is divided into X and Y axes, each data point represents an independent Korean pine DNA sample, and samples with the same genotype are clustered together.

[0075] The genotype amplified by the molecular marker primer set III was G:G, and the fluorescent signal was blue, which was concentrated near the Y axis, and was determined to be homozygous material with large cones; the genotype was A:G, and the fluorescent signal was green, which was concentrated near the middle position, and was determined to be heterozygous material with large cones; the genotype was A:A, and the fluorescent signal was orange, which was concentrated near the X axis, and was determined to be homozygous material with small cones.

[0076] The genotype amplified by the molecular marker primer set IV was T:T type, the fluorescent signal was blue, and the material was concentrated near the Y axis, which was determined to be homozygous material with large cones; the genotype was T:G type, the fluorescent signal was green, and it was concentrated near the middle position, which was determined to be heterozygous material with large cones; the genotype was G:G type, the fluorescent signal was orange, and it was concentrated near the X axis, which was determined to be homozygous material with small cones.

[0077] Example 1

[0078] The method of identifying the weight of Korean pine cones of individual Korean pine trees in a natural population includes the following steps:

[0079] 1. Extract DNA from the Korean pine to be tested: Using fresh leaves from a single plant of a natural population of Korean pine as experimental materials, the steps for extracting genomic DNA are as follows:

[0080] ① Take a small amount of fresh leaves and put them into a 2mL centrifuge tube, add steel balls, grind them on a grinder, shake 30 times per second for 2 minutes, add 800μL of 2% CTAB extract, mix well, and place in a 65℃ water bath for 1h (shake well every 10 minutes);

[0081] ② After standing to room temperature, add 800 μL of chloroform:isoamyl alcohol (volume ratio 24:1) mixture, mix gently for 10 minutes, and then centrifuge at 12000 rpm for 15 minutes. Take the supernatant (about 600 μL) and transfer it to a new 1.5 mL centrifuge tube;

[0082] ③ Add 2 / 3 volume of isopropanol to the supernatant, mix gently and place at -20℃ for 30min~1h;

[0083] ④12000rmp, centrifuge for 10min, discard the supernatant;

[0084] ⑤ Add anhydrous ethanol to the centrifuge tube to wash the DNA precipitate once, then wash it once with 75% (volume percentage) ethanol, and blow dry it on the clean bench;

[0085] ⑥ Add 50 μL TE (or ddH2O) to dissolve and use as Korean pine genomic DNA.

[0086] 2. Using the Korean pine genomic DNA as a template and the molecular marker primer set to perform PCR amplification to obtain a PCR product;

[0087] The molecular marker primer set primers used are group III molecular marker primer set or group IV molecular marker primer set; group III molecular marker primer set consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R; group IV molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R;

[0088] The PCR reaction system was 5 μL: 1.25 μL of genomic DNA at a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL of each upstream primer at a concentration of 10 mM, and 0.75 μL of a downstream primer at a concentration of 10 mM;

[0089] The PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing temperature starting from 61°C, annealing temperature decreasing by 0.6°C per cycle, annealing for 1 min per cycle, annealing temperature at 55°C after 10 cycles, followed by denaturation at 95°C for 20 s and extension at 55°C for 1 min; a total of 27 cycles, running at 25°C for 1 min.

[0090] 3. Genotyping of PCR products after fluorescence detection, the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with heavy cones, and the genotype with orange fluorescence signal is the genotype with small cones;

[0091] Among them, in step 3, the genotype amplified by the molecular marker primer set III is G:G type, and the fluorescent signal is blue; the genotype is A:G type, and the fluorescent signal is green; the genotype is A:A type, and the fluorescent signal is orange;

[0092] In step 3, the genotype amplified by the molecular marker primer set IV is T:T, and the fluorescent signal is blue; the genotype is T:G, and the fluorescent signal is green; the genotype is G:G, and the fluorescent signal is orange.

[0093] The genotyping diagram of the Korean pine population by the molecular marker primer set III in this example is shown in FIG. Figure 1 The genotype is G:G, the fluorescence signal is blue, and it is concentrated near the Y axis, which is determined to be a homozygous material with a large cone; the genotype is A:G, the fluorescence signal is green, and it is concentrated near the middle position, which is determined to be a heterozygous material with a large cone; the genotype is A:A, the fluorescence signal is orange, and it is concentrated near the X axis.

[0094] The genotyping diagram of the Korean pine population by the molecular marker primer set IV in this example is shown in FIG. Figure 2 The genotype is T:T, the fluorescent signal is blue, and the material is gathered near the Y axis, which is determined to be a homozygous material with a large cone; the genotype is T:G, the fluorescent signal is green, and it is gathered near the middle position, which is determined to be a heterozygous material with a large cone; the genotype is G:G, the fluorescent signal is orange, and it is gathered near the X axis.

[0095] The cone weight of each individual plant of the natural population of Korean pine tested in this example was measured, and the average cone weight was calculated according to different genotypes (eg Figure 3As shown). The third group of molecular marker primers (KASP molecular marker III closely linked to the cone weight of Korean pine) measured the average cone weight of Korean pine of A:A genotype to be 0.375kg, and the average cone weight of Korean pine of A:G and G:G genotypes to be 0.398kg. The fourth group of molecular marker primers (KASP molecular marker IV closely linked to the cone weight of Korean pine) measured the average cone weight of Korean pine of G:G genotype to be 0.323kg, and the average cone weight of Korean pine of G:T and T:T genotypes to be 0.387kg. The results of the determination of the cone weight of the natural population of Korean pine are consistent with the identification results of this embodiment, indicating that the method of the present invention can accurately identify the cone weight of Korean pine, and assist in the selection of Korean pine cone weight, which will effectively save breeding time and land, accelerate the breeding process, and have important practical significance for the selection and promotion of Korean pine varieties.

Claims

1. A KASP molecular marker tightly linked to Korean pine cone weight, characterized in that: The molecular marker is a KASP molecular marker III that is tightly linked to the cone weight of Korean pine. The molecular marker primer set consists of an upstream primer Pk494448-F1, an upstream primer Pk494448-F2, and a downstream primer Pk494448-R. The nucleotide sequence of the upstream primer Pk494448-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the upstream primer Pk494448-F2 is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer Pk494448-R is shown in SEQ ID NO.

3.

2. A KASP molecular marker tightly linked to Korean pine cone weight, characterized in that: The molecular marker is KASP molecular marker IV which is tightly linked to the cone weight of Korean pine. The molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R. The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5; The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.

6.

3. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 1 or 2, characterized in that: The application of the molecular marker in Korean pine breeding.

4. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 3, characterized in that: The molecular markers are used to identify the weight of Korean pine cones.

5. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 3, characterized in that: Method for identifying the weight of Korean pine cones using the molecular markers:

1. Extract the DNA of the Korean pine to be tested; 2. Perform PCR amplification using the molecular marker primer set to obtain a PCR product; 3. Genotyping of PCR products after fluorescence detection, the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with heavy cones, and the genotype with orange fluorescence signal is the genotype with small cones; Wherein, the molecular marker primer set primers used in step 2 are group III molecular marker primer set or group IV molecular marker primer set; group III molecular marker primer set consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R; group IV molecular marker primer set consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and downstream primer Pk494448-R; The nucleotide sequence of the upstream primer Pk494448-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the upstream primer Pk494448-F2 is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer Pk494448-R is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer Pk3270935-F1 is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer Pk3270935-F2 is shown in SEQ ID NO.5; The nucleotide sequence of the downstream primer Pk3270935-R is shown in SEQ ID NO.

6.

6. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 5, characterized in that: The PCR reaction system in step 2 is 5 μL: it consists of 1.25 μL of genomic DNA with a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL of each upstream primer with a concentration of 10 mM, and 0.75 μL of downstream primer with a concentration of 10 mM.

7. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 5 or 6, characterized in that: The PCR amplification program in step 2 is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing temperature starting from 61°C, annealing temperature decreasing by 0.6°C per cycle, annealing for 1 min per cycle, annealing temperature at 55°C after 10 cycles, then denaturation at 95°C for 20 s, extension at 55°C for 1 min; a total of 27 cycles, running at 25°C for 1 min.

8. The use of the KASP molecular marker tightly linked to Korean pine cone weight according to claim 7, characterized in that: In step 3, the genotype amplified by the molecular marker primer set III is G:G type, and the fluorescent signal is blue; the genotype is A:G type, and the fluorescent signal is green; the genotype is A:A type, and the fluorescent signal is orange; In step 3, the genotype amplified by the molecular marker primer set IV is T:T, and the fluorescent signal is blue; the genotype is T:G, and the fluorescent signal is green; the genotype is G:G, and the fluorescent signal is orange.

9. A kit for identifying the weight of Korean pine cones, characterized in that The kit comprises a KASP molecular marker III primer set closely linked to the weight of Korean pine cones and / or a KASP molecular marker IV primer set closely linked to the weight of Korean pine cones.

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