KASP molecular markers closely linked to the weight of Korean pine cones and their applications

Abstract

This invention relates to the field of marker-assisted breeding technology for forest trees, specifically to KASP molecular markers and their applications in Korean pine cone weight. The invention provides two KASP molecular markers closely linked to Korean pine cone weight, and their applications in Korean pine breeding. The KASP molecular marker primers in this invention can rapidly identify the Korean pine cone weight phenotype and have advantages such as accuracy, speed, low cost, short identification cycle, and ease of operation. They can assist in the breeding of new Korean pine lines and have broad application prospects.

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 cones are highly nutritious, containing abundant crude protein, crude fat, polysaccharides, crude fiber, vitamins, minerals, and trace elements, making them a popular pine nut product. Cone weight is a crucial economic trait affecting Korean pine cone yield. Therefore, developing molecular markers closely linked to cone weight and using them for marker-assisted selection of Korean pine cone weight will effectively save breeding time and land, accelerate the breeding process, and has significant practical implications for the breeding and promotion of superior Korean pine varieties. However, currently, there are no molecular markers available for identifying Korean pine cone weight. Summary of the Invention

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

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

[0005] KASP molecular marker III, which is closely linked to the weight of Korean pine cones, consists of an upstream primer Pk494448-F1, an upstream primer Pk494448-F2, and a 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, which is closely linked to the weight of Korean pine cones, consists of upstream primers Pk3270935-F1 and 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 KASP molecular marker III, which 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; site Pk494448 is the 494448th base in this gene pool); this SNP site has an A / G base mutation. The 3' end of the upstream primer is a variant base. The 5' end of upstream primer Pk494448-F1 is linked to carboxyfluorescein FAM, and the 5' end of upstream primer Pk494448-F2 is linked to phosphoramiditin VIC.

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

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

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

[0017] Furthermore, a method for identifying the weight of Korean pine cones 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, and the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with large cone weight, and the genotype with orange fluorescence signal is the genotype with small cone weight.

[0021] In step two, the molecular marker primers used are either the Group III or Group IV molecular marker primer sets. The Group III molecular marker primer set consists of upstream primers Pk494448-F1 and Pk494448-F2, and downstream primer Pk494448-R. The Group IV molecular marker primer set consists of upstream primers Pk3270935-F1 and 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 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] Furthermore, in step three, the genotype amplified by the molecular marker primer set III is G:G, with a blue fluorescence signal; the genotype is A:G, with a green fluorescence signal; and the genotype is A:A, with an orange fluorescence signal. In step three, the genotype amplified by the molecular marker primer set IV is T:T, with a blue fluorescence signal; the genotype is T:G, with a green fluorescence signal; and the genotype is G:G, with an orange fluorescence signal.

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

[0032] 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.

[0033] In this invention, the SNP site of molecular marker III in Korean pine with heavy cones is G, and the SNP site of molecular marker III in Korean pine with small cones is A. Korean pine samples with a "G:G" pattern are identified as homozygous materials with heavy cones, those with a "G:A" pattern are identified as heterozygous materials with heavy cones, and those with a "A:A" pattern are identified as homozygous materials with small cones. In this invention, the SNP site of molecular marker IV in Korean pine with heavy cones is T, and the SNP site of molecular marker IV in Korean pine with small cones is G. Korean pine samples with a "T:T" pattern are identified as homozygous materials with heavy cones, those with a "G:T" pattern are identified as heterozygous materials with heavy cones, and those with a "G:G" pattern are identified as homozygous materials with small cones. The method of this invention shows that the phenotype of cone weight in a population is consistent with the primer amplification band pattern of the above molecular markers.

[0034] The KASP molecular marker primers in this invention can rapidly identify the heavy phenotype of Korean pine cones 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

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

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

[0037] Figure 3 This is a bar chart showing the results of the actual cone weight measurement of the natural population of Korean pine in Example 1. Detailed Implementation

[0038] 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.

[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 one: This implementation method uses KASP molecular marker III, which is closely linked to the weight of Korean pine cones. The primer set of this molecular marker consists of upstream primer Pk494448-F1, upstream primer Pk494448-F2 and downstream primer Pk494448-R.

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

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

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

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

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

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

[0047] The SNP molecular marker site of KASP molecular marker III, which is closely linked to the weight of Korean pine cones, is Pk494448; this SNP site contains an A / G base mutation. The 3' end of the upstream primer is a variant base. The 5' end of the upstream primer Pk494448-F1 is linked to carboxyfluorescein FAM, and the 5' end of the upstream primer Pk494448-F2 is linked to phosphoramiditin VIC. Cones tested with a SNP site base of "G" are heavier; cones tested with a SNP site base of "A" are lighter.

[0048] Specific implementation method two: This implementation method uses KASP molecular marker IV, which is closely linked to the weight of Korean pine cones. The primer set of this molecular marker consists of upstream primer Pk3270935-F1, upstream primer Pk3270935-F2 and 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 as follows:

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

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

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

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

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

[0058] The SNP marker site of KASP molecular marker IV, which is closely linked to the weight of Korean pine cones, is Pk3270935; this SNP site contains a G / T base mutation. The 3' end of the upstream primer is a variant base. The 5' end of upstream primer Pk3270935-F1 is linked to carboxyfluorescein FAM, and the 5' end of upstream primer Pk3270935-F2 is linked to phosphoramiditin VIC. Cones tested with a SNP site base of "T" have a larger weight; cones tested with a SNP site base of "G" have a smaller weight.

[0059] Specific Implementation Method 3: This implementation method is used to determine the weight of red pine cones.

[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, and the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with large cone weight, and the genotype with orange fluorescence signal is the genotype with small cone weight.

[0063] In step two, the molecular marker primers used are either group III or group IV. Group III consists of upstream primers Pk494448-F1 and Pk494448-F2, and downstream primer Pk494448-R. Group IV consists of upstream primers Pk3270935-F1 and 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] 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, extension at 55℃ for 1 min; a total of 27 cycles, running at 25℃ for 1 min.

[0072] In step three, the genotype amplified by the molecular marker primer set III is G:G, with a blue fluorescence signal; the genotype is A:G, with a green fluorescence signal; and the genotype is A:A, with an orange fluorescence signal.

[0073] In step three, the genotypes amplified by the molecular marker primer set IV are T:T type, with a blue fluorescence signal; T:G type, with a green fluorescence signal; and G:G type, with an orange fluorescence signal.

[0074] This embodiment performs genotyping detection on PCR amplification products and presents the results in the form of 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 be clustered together.

[0075] The genotypes amplified by the molecular marker primers in group III were G:G, with blue fluorescence signals clustered near the Y-axis, indicating homozygous material with large cone weight; the genotypes were A:G, with green fluorescence signals clustered near the middle, indicating heterozygous material with large cone weight; and the genotypes were A:A, with orange fluorescence signals clustered near the X-axis, indicating homozygous material with small cone weight.

[0076] The genotypes amplified by the molecular marker primers in group IV were T:T, with blue fluorescence signals clustered near the Y-axis, indicating homozygous material with large cone weight; T:G, with green fluorescence signals clustered near the middle, indicating heterozygous material with large cone weight; and G:G, with orange fluorescence signals clustered near the X-axis, indicating homozygous material with small cone weight.

[0077] Example 1

[0078] The following steps are included in the identification of cone weight of individual Korean pine trees in a natural population:

[0079] 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:

[0080] 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).

[0081] 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.

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

[0083] ④ Centrifuge at 12000 rpm for 10 min, then 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 place it on a clean bench to dry.

[0085] ⑥ Dissolve in 50 μL TE (or ddH2O) and use as genomic DNA for Korean pine.

[0086] 2. Using Korean pine genomic DNA as a template, PCR amplification was performed using a molecular marker primer set to obtain PCR products;

[0087] The molecular marker primer sets used were either Group III or Group IV. Group III primer sets consisted of upstream primers Pk494448-F1 and Pk494448-F2, and downstream primer Pk494448-R. Group IV primer sets consisted of upstream primers Pk3270935-F1 and Pk3270935-F2, and downstream primer Pk494448-R.

[0088] The PCR reaction system consisted of 5 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.

[0089] 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.

[0090] 3. After fluorescence detection, the PCR products are genotyped, and the fluorescence signal is blue, green or orange; among them, the genotype with blue or green fluorescence signal is the genotype with large cone weight, and the genotype with orange fluorescence signal is the genotype with small cone weight.

[0091] In step three, the genotype amplified by the molecular marker primer set III is G:G, with a blue fluorescence signal; the genotype is A:G, with a green fluorescence signal; and the genotype is A:A, with an orange fluorescence signal.

[0092] In step three, the genotypes amplified by the molecular marker primer set IV are T:T type, with a blue fluorescence signal; T:G type, with a green fluorescence signal; and G:G type, with an orange fluorescence signal.

[0093] In this embodiment, the genotyping diagram of the Korean pine population using the third set of molecular marker primers is as follows: Figure 1 As shown. Materials with the G:G genotype exhibiting blue fluorescence signals clustered near the Y-axis are identified as homozygous materials with large cones; materials with the A:G genotype exhibiting green fluorescence signals clustered near the center are identified as heterozygous materials with large cones; materials with the A:A genotype exhibiting orange fluorescence signals clustered near the X-axis.

[0094] In this embodiment, the genotyping diagram of the Korean pine population using the molecular marker primer set IV is shown below. Figure 2 As shown. The material with genotype T:T, exhibiting blue fluorescence and clustering near the Y-axis, is identified as a homozygous material with large cones; the material with genotype T:G, exhibiting green fluorescence and clustering near the center, is identified as a heterozygous material with large cones; and the material with genotype G:G, exhibiting orange fluorescence and clustering near the X-axis.

[0095] The cone weight of individual Korean pine trees from the natural population tested in this embodiment was measured, and the average cone weight was calculated according to different genotypes (e.g., Figure 3(As shown). Using the third set of molecular marker primers (KASP molecular marker III, closely linked to Korean pine cone weight), the average cone weight of Korean pine with the A:A genotype was determined to be 0.375 kg, and the average cone weight of Korean pine with the A:G and G:G genotypes was determined to be 0.398 kg. Using the fourth set of molecular marker primers (KASP molecular marker IV, closely linked to Korean pine cone weight), the average cone weight of Korean pine with the G:G genotype was determined to be 0.323 kg, and the average cone weight of Korean pine with the G:T and T:T genotypes was determined to be 0.387 kg. The cone weight results of the natural Korean pine population 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 can assist in the selection of Korean pine cones by cone weight. This will 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 weight of Korean pine cones, characterized in that, This molecular marker is KASP molecular marker III, which is closely linked to the weight of Korean pine cones. The primer set for this molecular marker consists of upstream primer Pk494448-F1, upstream primer Pk494448-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.

2. A KASP molecular marker primer set closely linked to the weight of Korean pine cones, characterized in that, This molecular marker is a KASP molecular marker IV that is closely linked to the weight of Korean pine cones. The primer set for this molecular marker 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. A method for identifying the weight of Korean pine cones using the KASP molecular marker primer set closely linked to the weight of Korean pine cones as described in claim 1 or 2:

1. Extract DNA from the red pine tree to be tested; 2. PCR amplification is performed using the molecular marker primer set described in claim 1 or 2 to obtain PCR products; III. PCR products are then genotyped after fluorescence detection; the fluorescence signal appears as blue, green, or orange. Genotypes with blue or green fluorescence signals are associated with larger cones, while genotypes with orange fluorescence signals are associated with smaller cones. In step two, the molecular marker primers used are either the third group of molecular marker primers or the fourth group of molecular marker primers. Group III of the molecular marker primer set consists of upstream primers Pk494448-F1 and Pk494448-F2, and downstream primer Pk494448-R. The 5' end of upstream primer Pk494448-F1 is linked to carboxyfluorescein FAM, and the 5' end of upstream primer Pk494448-F2 is linked to phosphoramiditin VIC. Cones with a SNP site base of "G" are heavier; cones with a SNP site base of "A" are smaller. A blue fluorescence signal corresponds to genotype G:G; a green fluorescence signal corresponds to genotype A:G; and an orange fluorescence signal corresponds to genotype A:A. Group IV of molecular marker primers consists of upstream primers Pk3270935-F1 and Pk3270935-F2, and downstream primer Pk494448-R. The 5' end of upstream primer Pk3270935-F1 is linked to carboxyfluorescein FAM, and the 5' end of upstream primer Pk3270935-F2 is linked to phosphoramiditin VIC. Cones with a SNP site base of "T" are heavier; cones with a SNP site base of "G" are smaller. A blue fluorescence signal corresponds to genotype T:T; a green fluorescence signal corresponds to genotype T:G; and an orange fluorescence signal corresponds to genotype G:G.

4. The method for determining the weight of Korean pine cones according to claim 3, 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.

5. The method for determining the weight of Korean pine cones according to claim 3 or 4, 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.

6. A reagent kit for identifying the weight of red pine cones, characterized in that, The kit includes the KASP molecular marker primer set as described in claim 1 and / or claim 2.

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