KASP molecular markers closely linked to red pine seed yield and application thereof

Abstract

The application relates to a KASP molecular marker closely linked to the fruiting amount of Korean pine and application thereof, and belongs to the technical field of forest tree molecular marker assisted breeding, and relates to a molecular marker of Korean pine and application thereof. Two KASP molecular markers closely linked to the fruiting amount of Korean pine are provided, and the application of the molecular markers in Korean pine breeding is provided. The KASP molecular marker primer in the application can quickly identify the fruiting amount phenotype of Korean pine, and has the advantages of accuracy, rapidness, low cost, short identification period, simple operation and the like, can assist new strain breeding of Korean pine, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding technology for forest trees, and relates to molecular markers for Korean pine and their applications. Background Technology

[0002] Korean pine (Pinus koraiensis) is a national second-class protected wild plant and an important economic tree species in the forest areas of Northeast my country. Korean pine nuts are highly nutritious, containing abundant crude protein, crude fat, polysaccharides, crude fiber, vitamins, minerals, and trace elements, making them a popular pine nut product. Fruit yield is a crucial economic trait affecting Korean pine pine nut production. Therefore, developing molecular markers closely linked to fruit yield and using them for marker-assisted selection of Korean pine fruit yield will effectively save breeding time and land, accelerate the breeding process, and has significant practical implications for the selection and promotion of superior Korean pine varieties. However, currently, there are no molecular markers available for identifying Korean pine fruit yield. Summary of the Invention

[0003] In order to enable the biological identification of the fruit yield of Korean pine, this invention provides a KASP molecular marker closely linked to the fruit yield of Korean pine and its application.

[0004] This invention provides two KASP molecular markers that are closely linked to the fruit yield of Korean pine.

[0005] KASP molecular marker I, which is closely linked to the fruit yield of Korean pine, consists of upstream primer Pk1780727-F1, upstream primer Pk1780727-F2 and downstream primer Pk1780727-R.

[0006] The nucleotide sequence of the upstream primer Pk1780727-F1 is shown in SEQ ID NO.1;

[0007] The nucleotide sequence of the upstream primer Pk1780727-F2 is shown in SEQ ID NO.2;

[0008] The nucleotide sequence of the downstream primer Pk1780727-R is shown in SEQ ID NO.3.

[0009] KASP molecular marker II, which is closely linked to the fruit yield of Korean pine, consists of upstream primers Pk4881799-F1 and Pk4881799-F2 and downstream primer Pk1780727-R.

[0010] The nucleotide sequence of the upstream primer Pk4881799-F1 is shown in SEQ ID NO.4;

[0011] The nucleotide sequence of the upstream primer Pk4881799-F2 is shown in SEQ ID NO.5;

[0012] The nucleotide sequence of the downstream primer Pk4881799-R is shown in SEQ ID NO.6.

[0013] The SNP marker site of KASP molecular marker I, which is closely linked to the fruit yield of Korean pine, is Pk1780727 (a gene pool constructed from 100 Korean pine clones from 7 Korean pine populations; site Pk1780727 is the 1780727th base in this gene pool); this SNP site contains a C / T base mutation. The 3' end of the upstream primer is a variant base. The 5' end of upstream primer Pk1780727-F1 is linked to carboxyfluorescein FAM, and the 5' end of upstream primer Pk1780727-F2 is linked to phosphoramiditin VIC.

[0014] The SNP molecular marker site of KASP molecular marker II, which is closely linked to the fruit yield of Korean pine, is Pk4881799 (a gene pool constructed from 100 Korean pine clones of 7 Korean pine populations; site Pk4881799 is the 4881799th base in this gene pool); this SNP site has a C / T base mutation. The 3' end of the upstream primer is a variant base. The 5' end of the upstream primer Pk4881799-F1 is linked to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk4881799-F2 is linked to phosphoramiditin VIC.

[0015] The application of the aforementioned KASP molecular markers closely linked to the fruit yield of Korean pine, and the application of these molecular markers in Korean pine breeding.

[0016] Furthermore, the molecular markers were used to identify the fruit yield of Korean pine.

[0017] Furthermore, a method for identifying the fruit yield of Korean pine using the aforementioned molecular markers:

[0018] 1. Extract DNA from the red pine tree to be tested;

[0019] 2. PCR amplification was performed using a molecular marker primer set to obtain PCR products;

[0020] 3. After fluorescence detection, the PCR products are genotyped. The fluorescent signal is blue for "T:T" type, green for "C:T" type, and orange for "C:C" type. Among them, T:T and C:T genotypes are genotypes with more fruits, while C:C genotypes are genotypes with fewer fruits.

[0021] In step two, the molecular marker primers used are either Group I or Group II. Group I consists of upstream primers Pk1780727-F1 and Pk1780727-F2, and downstream primer Pk1780727-R. Group II consists of upstream primers Pk4881799-F1 and Pk4881799-F2, and downstream primer Pk1780727-R.

[0022] The nucleotide sequence of the upstream primer Pk1780727-F1 is shown in SEQ ID NO.1;

[0023] The nucleotide sequence of the upstream primer Pk1780727-F2 is shown in SEQ ID NO.2;

[0024] The nucleotide sequence of the downstream primer Pk1780727-R is shown in SEQ ID NO.3;

[0025] The nucleotide sequence of the upstream primer Pk4881799-F1 is shown in SEQ ID NO.4;

[0026] The nucleotide sequence of the upstream primer Pk4881799-F2 is shown in SEQ ID NO.5;

[0027] The nucleotide sequence of the downstream primer Pk4881799-R is shown in SEQ ID NO.6.

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

[0029] Furthermore, the PCR amplification program in step two is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s; annealing temperature starting at 61℃, decreasing by 0.6℃ for each cycle, annealing for 1 min per cycle, annealing at 55℃ after 10 cycles, followed by denaturation at 95℃ for 20 s, extension at 55℃ for 1 min; a total of 27 cycles, running at 25℃ for 1 min.

[0030] The Korean pine seed yield identification kit includes a set of KASP molecular marker I primers and / or a set of KASP molecular marker II primers that are closely linked to the Korean pine seed yield.

[0031] KASP (Kompetitive Allele-Specific PCR) is a competitive allele-specific PCR technique that offers advantages over traditional PCR methods, including high throughput, low cost, high efficiency, and no need for electrophoresis detection.

[0032] In this invention, the two SNP loci are both T in Korean pine with high seed yield, and both C in Korean pine with low seed yield. Korean pine samples with a "T:T" pattern are identified as homozygous materials with high seed yield, those with a "C:T" pattern are identified as heterozygous materials with high seed yield, and those with a "C:C" pattern are identified as homozygous materials with low seed yield. The seed yield phenotype of the present invention is consistent with the primer amplification band pattern of the aforementioned molecular markers in the population and individual tree seed yield phenotypes.

[0033] The KASP molecular marker primers in this invention can rapidly identify the fruit-bearing phenotype of Korean pine and have the advantages of accuracy, speed, low cost, short identification cycle, and simple operation. They can assist in the breeding of new Korean pine varieties and have broad application prospects. Attached Figure Description

[0034] Figure 1 This is the genotyping diagram of the Korean pine population using the molecular marker primer set I in Example 1;

[0035] Figure 2 This is the genotyping diagram of the Korean pine population using the second set of molecular marker primers in Example 1;

[0036] Figure 3 This is a bar graph showing the results of measuring the actual fruit yield of a single tree in the natural population of Korean pine in Example 1. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0039] Specific implementation method one: The KASP molecular marker I closely linked to the fruit yield of red pine in this implementation method consists of upstream primer Pk1780727-F1, upstream primer Pk1780727-F2 and downstream primer Pk1780727-R.

[0040] The nucleotide sequence of the upstream primer Pk1780727-F1 is as follows:

[0041] 5'-GAAGGTGACCAAGTTCATGCTTGAAAACTTGCAATGTTGTAACTGC-3' (SEQ ID NO.1); where the underlined part is the sequence of fluorescein FAM.

[0042] The nucleotide sequence of the upstream primer Pk1780727-F2 is as follows:

[0043] 5'-GAAGGTCGGAGTCAACGGATTCTTGAAAACTTGCAATGTTGTAACTGT-3'

[0044] (SEQ ID NO.2), where the underlined portion is the sequence of fluorescein VIC.

[0045] The nucleotide sequence of the downstream primer Pk1780727-R is as follows:

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

[0047] The SNP marker site of KASP molecular marker I, which is closely linked to the fruit yield of Korean pine, is Pk1780727; this SNP site contains a C / T base mutation. The 3' end of the upstream primer is a variant base. The 5' end of the upstream primer Pk1780727-F1 is linked to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk1780727-F2 is linked to phosphoramiditin VIC. Samples with a "T" base at the SNP site have a higher fruit yield; samples with a "C" base at the SNP site have a lower fruit yield.

[0048] Specific implementation method two: This implementation method uses KASP molecular marker II, which is closely linked to the fruit yield of red pine. The primer set of this molecular marker consists of upstream primer Pk4881799-F1, upstream primer Pk4881799-F2 and downstream primer Pk1780727-R.

[0049] The nucleotide sequence of the upstream primer Pk4881799-F1 is shown in SEQ ID NO.4;

[0050] The nucleotide sequence of the upstream primer Pk4881799-F2 is shown in SEQ ID NO.5;

[0051] The nucleotide sequence of the downstream primer Pk4881799-R is shown in SEQ ID NO.6.

[0052] The nucleotide sequence of the upstream primer Pk4881799-F1 is as follows:

[0053] 5'-GAAGGTGACCAAGTTCATGCTTCAGTTTTACCCCCCGCCGATC-3' (SEQ ID NO.4); where the underlined part is the sequence of fluorescein FAM.

[0054] The nucleotide sequence of the upstream primer Pk4881799-F2 is as follows:

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

[0056] The nucleotide sequence of the downstream primer Pk4881799-R is as follows:

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

[0058] The SNP molecular marker site of KASP molecular marker II, which is closely linked to the fruit yield of Korean pine, is Pk4881799; this SNP site contains a C / T base mutation. The 3' end of the upstream primer is a variant base. The 5' end of the upstream primer Pk4881799-F1 is linked to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk4881799-F2 is linked to phosphoramiditin VIC. Samples with a "T" base at the SNP site have a higher fruit yield; samples with a "C" base at the SNP site have a lower fruit yield.

[0059] Specific Implementation Method 3: The method for determining the amount of red pine seeds in this implementation method:

[0060] 1. Extract DNA from the red pine tree to be tested;

[0061] 2. PCR amplification was performed using a molecular marker primer set to obtain PCR products;

[0062] 3. After fluorescence detection, the PCR products are genotyped. The fluorescent signal is blue for "T:T" type, green for "C:T" type, and orange for "C:C" type. Among them, T:T and C:T genotypes are genotypes with more fruits, while C:C genotypes are genotypes with fewer fruits.

[0063] In step two, the molecular marker primers used are either Group I or Group II. Group I consists of upstream primers Pk1780727-F1 and Pk1780727-F2, and downstream primer Pk1780727-R. Group II consists of upstream primers Pk4881799-F1 and Pk4881799-F2, and downstream primer Pk1780727-R.

[0064] The nucleotide sequence of the upstream primer Pk1780727-F1 is shown in SEQ ID NO.1;

[0065] The nucleotide sequence of the upstream primer Pk1780727-F2 is shown in SEQ ID NO.2;

[0066] The nucleotide sequence of the downstream primer Pk1780727-R is shown in SEQ ID NO.3;

[0067] The nucleotide sequence of the upstream primer Pk4881799-F1 is shown in SEQ ID NO.4;

[0068] The nucleotide sequence of the upstream primer Pk4881799-F2 is shown in SEQ ID NO.5;

[0069] The nucleotide sequence of the downstream primer Pk4881799-R is shown in SEQ ID NO.6;

[0070] In step two, the PCR reaction system is 5 μL, consisting of 1.25 μL of genomic DNA at a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL each of the 10 mM upstream primer and the 10 mM downstream primer.

[0071] The PCR amplification program in step two is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s; annealing temperature starting at 61℃, decreasing by 0.6℃ for each cycle, annealing for 1 min per cycle, annealing at 55℃ after 10 cycles, followed by denaturation at 95℃ for 20 s and extension at 55℃ for 1 min; a total of 27 cycles, running at 25℃ for 1 min.

[0072] This embodiment performs genotyping detection on PCR amplification products and presents the results in a chart. The chart is divided into X and Y axes, with each data point representing an independent Korean pine DNA sample. Samples with the same genotype will cluster together. For example, T:T genotype samples, which show blue fluorescence signals and cluster near the Y axis, are identified as homozygous samples with high seed yield. C:T genotype samples, which show green fluorescence signals and cluster near the center, are identified as heterozygous samples with high seed yield. C:C genotype samples, which show orange fluorescence signals and cluster near the X axis, are identified as homozygous samples with low seed yield.

[0073] Example 1

[0074] The determination of seed yield of individual Korean pine trees in a natural population includes the following steps:

[0075] I. Extraction of DNA from Korean Pine Trees – Using fresh leaves from individual trees in a natural Korean pine population as experimental material, the steps for extracting genomic DNA are as follows:

[0076] ① Take a small amount of fresh leaves and put them into a 2mL centrifuge tube. Add steel balls and grind them on a grinder. Shake 30 times per second for 2 minutes. Add 800μL of 2% CTAB extract and mix well. Place in a 65℃ water bath for 1 hour (shake well every 10 minutes).

[0077] ② After standing to room temperature, add 800 μL of chloroform:isoamyl alcohol (volume ratio 24:1), mix gently for 10 min, centrifuge at 12000 rpm for 15 min, and transfer the supernatant (about 600 μL) to a new 1.5 mL centrifuge tube.

[0078] ③ Add 2 / 3 volume of isopropanol to the supernatant, mix gently, and place at -20℃ for 30 min to 1 h;

[0079] ④ Centrifuge at 12000 rpm for 10 min, then discard the supernatant;

[0080] ⑤ Add anhydrous ethanol to the centrifuge tube to wash the DNA precipitate once, then wash it once with 75% (volume percentage) ethanol, and place it on a clean bench to dry.

[0081] ⑥ Dissolve in 50 μL TE (or ddH2O) and use as genomic DNA for Korean pine.

[0082] 2. Using Korean pine genomic DNA as a template, PCR amplification was performed using a molecular marker primer set to obtain PCR products;

[0083] The molecular marker primer sets used were either Group I or Group II. Group I consisted of upstream primers Pk1780727-F1 and Pk1780727-F2, and downstream primer Pk1780727-R. Group II consisted of upstream primers Pk4881799-F1 and Pk4881799-F2, and downstream primer Pk1780727-R.

[0084] The PCR reaction system consisted of 5 μL: 1.25 μL of genomic DNA at a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL each of the 10 mM upstream primer and the 10 mM downstream primer.

[0085] The PCR amplification program was as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s; annealing temperature starting at 61℃, decreasing by 0.6℃ for each cycle, annealing for 1 min per cycle, annealing at 55℃ after 10 cycles, followed by denaturation at 95℃ for 20 s and extension at 55℃ for 1 min; a total of 27 cycles, with a final run at 25℃ for 1 min.

[0086] III. Genotyping of PCR products after fluorescence detection: Read the fluorescence detection results of PCR products. Homozygous plants with high fruit yield show blue fluorescence clustering near the Y-axis, heterozygous plants show green fluorescence clustering near the middle, and plants with low fruit yield show orange fluorescence clustering near the X-axis.

[0087] In this embodiment, the genotyping diagram of the Korean pine population using the first set of molecular marker primers is as follows: Figure 1 As shown. The T:T genotype, with blue fluorescence signal and clustered near the Y-axis, is identified as a homozygous material with a high yield; the C:T genotype, with green fluorescence signal and clustered near the middle, is identified as a heterozygous material with a high yield; the C:C genotype, with orange fluorescence signal and clustered near the X-axis.

[0088] In this embodiment, the genotyping diagram of the Korean pine population using the second set of molecular marker primers is as follows: Figure 2 As shown. The T:T genotype, with blue fluorescence signal and clustered near the Y-axis, is identified as a homozygous material with a high yield; the C:T genotype, with green fluorescence signal and clustered near the middle, is identified as a heterozygous material with a high yield; the C:C genotype, with orange fluorescence signal and clustered near the X-axis.

[0089] The seed yield of individual Korean pine trees from the natural population tested in this embodiment was determined, and the average seed yield was calculated according to different genotypes (e.g., Figure 3(As shown). Using the first set of molecular marker primers (KASP molecular marker I, closely linked to the fruit yield of Korean pine), the average fruit yield per tree of the C:C genotype was 17.36 seeds, and the average fruit yield per tree of the C:T and T:T genotypes was 25.2 seeds. Using the second set of molecular marker primers (KASP molecular marker II, closely linked to the fruit yield of Korean pine), the average fruit yield per tree of the C:C genotype was 5.8 seeds, and the average fruit yield per tree of the C:T and T:T genotypes was 23.68 seeds. The results of the fruit yield measurements per tree in the natural Korean pine population are consistent with the identification results of this embodiment, indicating that the method of this invention can accurately identify the fruit yield of Korean pine, assist in the selection of Korean pine fruit yield, effectively save breeding time and land, accelerate the breeding process, and has important practical significance for the breeding and promotion of superior Korean pine varieties.

Claims

1. A KASP molecular marker primer set closely linked to the fruit yield of Korean pine, characterized in that, This molecular marker is KASP molecular marker I, which is closely linked to the fruit yield of Korean pine. The primer set of this molecular marker consists of upstream primer Pk1780727-F1, upstream primer Pk1780727-F2 and downstream primer Pk1780727-R. The nucleotide sequence of the upstream primer Pk1780727-F1 is shown in SEQ ID NO.1; The nucleotide sequence of the upstream primer Pk1780727-F2 is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer Pk1780727-R is shown in SEQ ID NO.

3.

2. A KASP molecular marker primer set closely linked to the fruit yield of Korean pine, characterized in that, This molecular marker is KASP molecular marker II, which is closely linked to the fruit yield of Korean pine. The primer set of this molecular marker consists of upstream primer Pk4881799-F1, upstream primer Pk4881799-F2 and downstream primer Pk1780727-R. The nucleotide sequence of the upstream primer Pk4881799-F1 is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer Pk4881799-F2 is shown in SEQ ID NO.5; The nucleotide sequence of the downstream primer Pk4881799-R is shown in SEQ ID NO.

6.

3. The application of the KASP molecular marker primer set closely linked to the fruit yield of Korean pine as described in claim 1 or 2 in the identification of the fruit yield of Korean pine.

4. The application according to claim 3, characterized in that, A method for identifying the fruit yield of Korean pine using the aforementioned molecular marker primer set:

1. Extract DNA from the red pine tree to be tested; 2. PCR amplification was performed using a molecular marker primer set to obtain PCR products; 3. The PCR products were genotyped after fluorescence detection; among them, the T:T and C:T genotypes showed high fruit yield, while the C:C genotype showed low fruit yield. In step two, the molecular marker primer set described in claim 1 or claim 2 is used.

5. The application of the KASP molecular marker closely linked to the fruit yield of Korean pine according to claim 4, characterized in that, In step two, the PCR reaction system is 5 μL, consisting of 1.25 μL of genomic DNA at a concentration of 100 ng / μL, 2.5 μL of HiGeno2×ProbeMixA, 0.25 μL each of the 10 mM upstream primer and the 10 mM downstream primer.

6. The application of the KASP molecular marker closely linked to the fruit yield of Korean pine according to claim 4 or 5, characterized in that, The PCR amplification program in step two is as follows: pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 20 s; annealing temperature starting at 61℃, decreasing by 0.6℃ for each cycle, annealing for 1 min per cycle, annealing at 55℃ after 10 cycles, followed by denaturation at 95℃ for 20 s and extension at 55℃ for 1 min; a total of 27 cycles, running at 25℃ for 1 min.

7. A reagent kit for identifying the fruit yield of Korean pine, characterized in that... The kit includes the KASP molecular marker I primer set and / or the KASP molecular marker II primer set that are closely linked to the fruit yield of Korean pine as described in claim 1 and / or claim 2.

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