An Indel molecular marker primer for assisting the selection of offspring with long vase life of cut anthurium and its application

Abstract

The present invention discloses an Indel molecular marker primer for assisting in the selection of offspring with long vase life in cut anthuriums and its application. The present invention targets the ACO1 gene of cut anthurium varieties with different vase lives. The developed Indel marker is a molecular marker linked to the long vase life of cut anthuriums. Using the Indel marker primer, it is possible to assist in the selection of cut anthurium plants with different vase lives. This allows for rapid and accurate early selection of plants with long vase life, shortening the breeding cycle and avoiding the drawback of traditional breeding, which only allows selection when offspring plants are flowering and preventing earlier selection. Furthermore, a molecular marker-assisted selection technology system for long vase life in cut anthuriums is established, advancing selection to the seedling stage of hybrid offspring, shortening the breeding cycle by at least half. This also reduces the planting scale and improves the efficiency and benefits of anthurium breeding, thereby achieving targeted, precise, economical, and efficient breeding for vase life in cut anthuriums, laying the foundation for molecular breeding of cut anthuriums for vase life.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers and plant breeding, and more specifically relates to an Indel molecular marker primer for assisting in the selection of offspring of cut anthurium with a long vase life and its application. Background Art

[0002] Anthurium (Anthurium andraeanum Lind.) is a perennial herbaceous plant in the genus Anthurium, Araceae. Its heart-shaped spathes, rich colors, and long flowering period make it suitable for both potted and fresh cut flowers. Its wide range of applications and high economic value make it a fast-growing and highly sought-after high-end tropical cut and potted flower globally. Anthurium breeding efforts began in the 1940s abroad, and by 2000, over 600 varieties had been developed. In contrast, domestic anthurium breeding began later and has focused on potted anthuriums, with relatively little research on cut flower breeding. Therefore, shortening the breeding cycle, improving breeding efficiency and profitability, and rapidly developing breakthrough cut anthurium varieties with independent intellectual property rights, thereby narrowing the gap with foreign countries in cut anthurium breeding, have become key to further promoting the independent and efficient development of my country's anthurium industry.

[0003] Vase life is a key breeding trait for cut anthuriums, with vase life varying over 3.5-fold between different varieties (ranging from 14 to 49 days). Vase life is a quantitative trait influenced by factors such as cultivation practices, harvest season, and postharvest handling, as well as a genetic basis. Traditionally, selection for vase life in cut anthuriums can only be performed when offspring plants are in bloom, preventing the early selection of hybrids with long vase life for seedling propagation. Consequently, the breeding cycle is lengthy. Furthermore, plants with non-target traits cannot be eliminated early in the breeding process, resulting in large offspring populations and high breeding costs. Therefore, developing techniques for selecting for vase life in cut anthuriums early in the breeding process is crucial for shortening the breeding cycle, reducing planting scale, improving breeding efficiency, and narrowing the gap with international breeding methods. However, there are currently few methods or means to shorten the vase life of cut anthurium. In order to establish a fast, convenient and accurate breeding technology system for the vase life of cut anthurium and achieve early selection of the vase life of cut anthurium, it is urgent to develop more efficient, convenient and accurate methods to lay the foundation for the selection and breeding of the vase life trait targets of cut anthurium. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing breeding of cut anthurium vase life, such as the long breeding cycle and the inability to complete the screening and breeding to eliminate plants with non-breeding target traits in advance, and to provide an Indel molecular marker primer and application thereof for assisting in the selection of offspring with long vase life of cut anthurium.

[0005] The first object of the present invention is to provide an Indel molecular marker primer for assisting in selecting offspring of the cut flower Anthurium with a long vase life.

[0006] The second object of the present invention is to provide applications of Indel molecular marker primers.

[0007] The third object of the present invention is to provide a kit for molecular marker-assisted breeding of cut anthurium plants with long vase life.

[0008] The fourth object of the present invention is to provide a method for using Indel molecular markers to assist in breeding cut anthurium plants with long vase life.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The invention provides indel molecular marker primers for assisting in the selection of offspring with long vase life of cut anthurium, comprising an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'. A key gene, the anthurium ACO1 gene, is obtained by cloning two anthurium varieties with different vase lifespans, namely 'Bai Ma Anthurium ' and 'Yan Li Anthurium '. The ACO1 gene consists of two exons and one intron, and the full DNA lengths are 1647 bp and 1641 bp, respectively. The ACO1 gene encodes proteins of 371 and 369 amino acids, respectively, and exhibits differences among anthurium varieties with different vase lifespans. Based on the Anthurium ACO1 gene, Indel markers were further designed and developed, and it was found that the Indel6 marker primer was closely linked to the long vase life of cut Anthurium. Indel markers can assist in the selection of plants with different vase lives of cut Anthurium, and can quickly and accurately select breeding targets for long vase life in the early stages, shortening the breeding cycle and avoiding the defect that traditional breeding can only be carried out when the offspring plants are flowering and cannot be bred early. At the same time, a technical system for molecular marker-assisted selection of vase life of cut Anthurium was established, which realized the early selection of vase life of cut Anthurium and laid the foundation for further molecular breeding of vase life of cut Anthurium.

[0011] Therefore, the present invention provides the use of Indel molecular marker primers in breeding cut anthurium plants with long vase life, in preparing products for assisting breeding cut anthurium plants with long vase life, or in breeding cut anthurium plants with long vase life.

[0012] The invention provides a kit for molecular marker-assisted breeding of cut anthurium plants with long vase life, comprising the above-mentioned Indel molecular marker primers.

[0013] Preferably, the kit contains reagents required for PCR amplification reaction.

[0014] Preferably, the kit further comprises reagents for extracting sample DNA.

[0015] The present invention provides a method for breeding cut anthurium plants with long vase life by using indel molecular markers, comprising the following steps:

[0016] S1. Extract genomic DNA from the parental Anthurium cultivar to be bred and its hybrid progeny;

[0017] S2. PCR amplification was performed using DNA as a template using indel marker primers that assist in selecting long-lived progeny of cut anthurium: upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and downstream primer 5'-AATGATGGTGACGGCGAGTT-3';

[0018] S3. Detect the PCR amplification products by gel electrophoresis and select offspring plants with the same banding pattern as the long-vase-life parent;

[0019] S4. Individual plant identification: Continue to cultivate the selected offspring, and when they bloom, combine the vase-life phenotype identification to select the target vase-life offspring, so that cut anthurium plants with long vase life can be bred.

[0020] Furthermore, in step S1, genomic DNA from Anthurium andraeanum leaves was extracted at a concentration of 50 to 100 ng / μL.

[0021] Furthermore, the PCR amplification system in step S2 is: 1-2 μL DNA template, 5-10 μL 2×M5PAGE TaqPCR Mix, 0.5-1 μL upstream primer, 0.5-1 μL downstream primer, and 3-6 μL ddH2O.

[0022] Furthermore, the PCR amplification program in step S2 is: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55-64°C for 25 s, extension at 72°C for 30 s, 36 cycles, extension at 72°C for 5 min, and storage at 4°C.

[0023] The present invention has the following beneficial effects:

[0024] The present invention targets the ACO1 gene of cut anthurium varieties with different vase longevities. The developed indel marker is a molecular marker linked to the vase longevity of cut anthurium. Using indel markers to assist in the selection of cut anthurium plants with different vase longevities, this allows for faster and more accurate selection of long vase-life plants, shortening the breeding cycle and avoiding the drawback of traditional breeding methods that only allow selection when offspring plants are flowering, preventing earlier selection. Simultaneously, a molecular marker-assisted selection technology system for cut anthurium vase longevity is established, advancing selection to the seedling stage of hybrid offspring, thereby shortening the breeding cycle to approximately half the original, reducing planting scale, and improving the efficiency and benefits of anthurium breeding. Furthermore, based on the anthurium ACO1 gene, the vase longevity of cut anthurium can be improved through genetic modification or gene editing, thereby achieving targeted, precise, economical, and efficient breeding of cut anthurium for vase longevity, laying the foundation for molecular breeding of cut anthurium for vase longevity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the ACO1 gene structure of 'Bai Ma Hong Tong' and 'Guang Hua Yan Li Hong Tong'.

[0026] Figure 2 These are the results of electrophoresis detection of 21 long-vase-life anthurium varieties using marker Indel6.

[0027] Figure 3 These are the results of electrophoresis detection of 21 long-vase-life Anthurium varieties using marker Indel4.

[0028] Figure 4 These are the results of electrophoresis detection of the combined parents of '2021-068' and '2021-057' and 53 F1 strains using marker Indel6 (P1 is 'Royal Mountain Anthurium', P2 is 'Pistachio Anthurium', and the rest are 53 F1 generations, M: DL500 DNA Marker).

[0029] Figure 5 It is the combined parent of '2021-068' and '2021-057' (a: 'Pistachio Anthurium'; b: 'Royal Mountain Anthurium').

[0030] Figure 6These are 13 hybrid offspring plants with long vase life (A:'2021-057-2'; B:'2021-057-6'; C:'2021-057-27'; D:'2021-057-32'; E:'2021-068-6'; F:'2021-068-18'; G:'2021-068-28'; H:'2021-068-34'; I:'2021-068-38'; J:'2021-068-44'; K:'2021-068-66'; L:'2021-068-67'; M:'2021-068-83'). DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0033] The anthurium plants used in the present invention are all planted in the Conghua base of the Guangzhou Flower Research Center and are uniformly managed according to conventional planting methods.

[0034] Example 1 ACO1 gene cloning

[0035] In this example, DNAs of 'Bai Ma Hong Tong' and 'Guang Hua Yan Li Hong Tong' were used as templates to synthesize primers for gene cloning.

[0036] 1. Primer synthesis

[0037] Primers were designed using the genome sequence of 'Anthurium 'Trenza.' The primer length range was 18-22 bp, the GC content was 40-60%, and the annealing temperature of the upstream and downstream primers was preferably controlled within 2°C. The primer sequences are shown in Table 1. The designed primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0038] Table 1 Primer sequences for cloning ACO1 genes in ‘Bai Ma Hong Anthurium’ and ‘Yan Li Hong Anthurium’

[0039]

[0040] 2. DNA Extraction

[0041] Prepare a CTAB extraction solution (1 mL CTAB: 2 μL mercaptoethanol) and preheat in a 60°C oven. Grind 200 mg of clean young leaves of each of 'Bai Ma Hong Anthurium' and 'Guang Hua Yan Li Hong Anthurium' into a powder with liquid nitrogen. Add 650 μL of CTAB extraction solution and mix quickly. Heat in a 60°C oven for 45 minutes, gently shaking every 10 minutes. After cooling to room temperature, add 650 μL of chloroform / isoamyl alcohol (24:1) and gently invert to mix thoroughly. Incubate at 12,000 rpm. Centrifuge for 10 minutes and transfer the supernatant to a new sterilized 1.5 mL centrifuge tube. Do not aspirate the middle protein layer during this process. Add 0.6 times to an equal volume of pre-chilled isopropanol to the supernatant, gently invert to mix evenly, and place in a -20°C refrigerator for more than 1 hour to allow DNA to precipitate and form a flocculent precipitate. Centrifuge at 12,000 rpm for 10 minutes to collect the DNA precipitate, discard the supernatant, wash twice with 75% alcohol, let it dry, add an appropriate amount of ultrapure water to dissolve it, and store it in a -20°C refrigerator.

[0042] 3. PCR amplification

[0043] PCR amplification was performed using DNA from 'Bai Ma Hong Anthurium' and 'Yan Li Hong Anthurium' as templates using synthetic primers. The PCR reaction system consisted of 7.5 μL 2× Phanta Flash Master Mix, 0.75 μL upstream primer, 0.75 μL downstream primer, 1 μL DNA template, and 5 μL ddH₂O. The PCR amplification program was as follows: 95°C denaturation for 3 min; 95°C denaturation for 15 s, 55-64°C annealing for 15 s, 72°C extension for 15 s / kb, 34 cycles, 72°C extension for 5 min, and storage at 4°C.

[0044] 4. Rubber recycling

[0045] The amplified PCR products were detected by agarose gel electrophoresis, and the target fragments were recovered using a common agarose gel DNA recovery kit provided by Nanjing Novozyme Co., Ltd.

[0046] 5. Connect

[0047] To a 0.2mL PCR tube, add the following components in order: 0.5-8μL of DNA fragment, 1μL of pBM23 Vector, 1μL of 10X Topo smart, and fill with ddH2O to a total volume of 10μL. Mix gently and centrifuge at low speed. The PCR reaction program is: incubate at 25°C for 25 minutes.

[0048] 6. Conversion

[0049] Remove the E. coli competent DH5α from the -80℃ refrigerator, add 5μL of the ligation product to the E. coli competent cell, gently pipette to mix, and then place it on ice for 20 minutes; heat shock in a 42℃ water bath for 30 seconds, and quickly place it on ice for 2 minutes; add 900μL of LB without antibiotics to the competent cell, place it in a shaker, and culture it at 37℃ and 200rpm for 1 hour; centrifuge it at 4,000rpm for 1 minute at room temperature, discard part of the supernatant, retain 100μL of the supernatant, and gently blow and mix the bacteria with a pipette to fully suspend the bacteria; spread the suspended bacteria on LB solid culture medium containing 50mg / L Kana, transfer it to an incubator, and culture it at 37℃ overnight.

[0050] 7. Bacteria detection

[0051] Single colonies were selected for bacterial liquid PCR. Single colonies with bright and single bands were selected for shaking and placed in LB liquid medium containing Kana (Kana:LB = 1:1000). The culture was shaken at 37°C and 200 rpm for overnight propagation. The next day, the cells were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the sequences in the transcriptome to select the correct positive colonies.

[0052] Through cloning, sequencing, and splicing comparative analysis of Anthurium genes, we obtained the 1-aminocyclopropane-1-carboxylate oxidase (ACO1) of 'Bai Ma Anthurium' and 'Yan Li Anthurium' respectively. The ACO gene is a key gene in the ethylene biosynthesis process in plants, which can catalyze 1-aminocyclopropane-1-carboxylic acid (ACC) to form ethylene. The structure of the ACO1 gene is shown in the figure below. Figure 1 As shown, the ACO1 gene consists of two exons and one intron. The full length of the ACO1 gene DNA of 'Bai Ma Hong Anthurium' and 'Guang Hua Yan Li Hong Anthurium' are 1647 bp and 1641 bp respectively, encoding proteins of 371 and 369 amino acids respectively.

[0053] Further base sequence analysis of the ACO1 gene of the two obtained cut flower anthurium varieties was performed. The results are shown in Table 2. There are 19 base sequence differences between the two ACO1 genes, of which 15 base sequence differences cause amino acid changes. This shows that there are differences in the ACO1 genes of the two cut flower anthurium varieties, which may be the key gene that causes the different vase life of 'Guanghua Baima Anthurium' and 'Guanghua Yanli Anthurium'.

[0054] Table 2 Differences in base sequence and amino acid changes in the ACO1 gene between ‘Guanghua Baima Honganthus’ and ‘Guanghua Yanli Honganthus’

[0055]

[0056] Note: “-” indicates base deletion.

[0057] Example 2 Development of Indel Markers Tightly Linked to the Vase Life of Cut Anthurium and Molecular Marker Screening

[0058] 1. Indel marker development

[0059] Based on the ACO1 genes of 'Bai Ma Hong Anthurium' and 'Guang Hua Yan Li Hong Anthurium' cloned in Example 1, primers were designed within 200 bp before and after the Indel site using Primer 5.0 software. The primer length ranged from 18 to 22 bp, the amplified product size was between 100 and 250 bp, and the GC content was within the range of 40-60%. The annealing temperature of the upstream and downstream primers was preferably controlled within 2°C. A total of seven Indel-tagged primers were designed. The specific primer sequences are shown in Table 3. The designed primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0060] Table 3 Primer sequences for Indel tagging

[0061]

[0062] 2. Screening of molecular markers closely linked to the vase life of cut anthurium

[0063] The indel-tagged primers developed in Table 3 were used to perform PCR amplification and electrophoresis on the DNA of 21 long-vase-life anthurium varieties to screen for molecular marker primers that were closely linked to the vase-life of cut anthurium. The specific method was as follows: using the DNA of 21 long-vase-life anthurium varieties as templates, PCR amplification was performed using the primers in Table 3. The amplification system was: 1 μL DNA template (50 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer (10 μM / μL), 0.5 μL downstream primer (10 μM / μL), and 3 μL ddH2O; the amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55-64°C for 25 s, extension at 72°C for 30 s, 36 cycles, extension at 72°C for 5 min, and storage at 4°C. The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis under the following conditions: voltage 300 V, current 180 mA, and electrophoresis time 2.5 h. After the electrophoresis, silver staining and color development were performed, and the samples were placed on a white plastic plate and photographed for reading.

[0064] The test results showed that there was an Indel marker linked to the vase life of cut Anthurium. Some marker primers were tested, such as Figure 2 and Figure 3As shown, Indel6 showed two banding patterns in 14 of the 21 long-vase-life Anthurium cultivars and seven showed one banding pattern. Indel4 showed two banding patterns in 11 of the 21 long-vase-life Anthurium cultivars and ten showed one banding pattern. This indicates that Indel6 is tightly linked to long vase life, while the other markers are unlinked.

[0065] Example 3: Using Indel6 Molecular Marker to Assist Breeding of Cut Anthurium Plants with Long Vase Life

[0066] 1. Parent selection and hybrid fruit acquisition

[0067] (1) Parent selection: According to the breeding goal of long vase life, the long vase life of 'Pistachio Anthurium' and the short vase life of 'Royal Mountain Anthurium' were selected as parents. The phenotypes of the parents are as follows: Figure 4 As shown;

[0068] (2) Obtaining hybrid fruits: Collect the pollen from the anthers of the male parent on the same day and place it in a culture dish. Then quickly and gently brush the pollen on the entire inflorescence of the female parent with a brush, cover it with a bag, cultivate the female plant, and harvest the fruit when it turns yellow.

[0069] 2. Seed acquisition and hybrid offspring production

[0070] (1) Seed acquisition: Remove the ripe fruits from the spadix, place them in clean water, pinch open the fruits by hand, wash off the mucus, and obtain the seeds;

[0071] (2) Hybrid offspring production: After the seeds were dried, they were sown directly into a 200-hole seedling screen filled with coarse peat substrate, with one seed per hole. The combination codes were '2021-068' and '2021-057', which were reciprocal crosses.

[0072] 3. Molecular marker-assisted selection of vase life of cut anthurium

[0073] (1) Extraction of genomic DNA from parents and offspring: When the hybrid offspring have 4-5 leaves in the plug tray, 80-120 mg of young leaves from the parents and the later hybrid seedlings are taken to extract genomic DNA from the offspring using the CTAB method;

[0074] (2) PCR amplification and gel electrophoresis detection: Indel-tagged primer Indel6 was used to perform PCR amplification on DNA from different Anthurium andraeanum leaves. The amplification system was as follows: 1 μL DNA template (50 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer (10 uM / μL), 0.5 μL downstream primer (10 uM / μL), 3 μL ddH2O (10 μl or 20 μl can be used for the amplification system). After the PCR system was configured, paraffin oil was added and the system was placed in a PCR amplifier for reaction amplification. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55-64°C for 25 s, extension at 72°C for 30 s, 36 cycles, extension at 72°C for 5 min, and storage at 4°C.

[0075] The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis under the following conditions: voltage 300 V, current 180 mA, and electrophoresis time 2.5 h. After the electrophoresis, silver staining and color development were performed, and the samples were placed on a white plastic plate and photographed for reading.

[0076] (3) Obtaining offspring of long-vase-life cut anthurium: Select offspring with the same morphology as the long-vase-life parent 'Pistachio Anthurium' and continue to cultivate them. When they bloom, combine the vase-life phenotype identification to select the target vase-life offspring.

[0077] Test results such as Figure 5 As shown in the figure, 53 offspring of '2021-068' and '2021-057' were selected for long vase life using Indel6. The results showed that 21 offspring had the same banding pattern as the long vase life parent 'Pistachio Anthurium'; combined with the offspring vase life phenotypic identification results, 13 of the 21 offspring had long vase life, such as Figure 6 As shown, the accuracy of assisted selection using Indel6 was 61.90%; the shortest vase life was 27.00 days and the longest vase life was 35.67 days.

[0078] In summary, the present invention targets the ACO1 gene in cut anthurium varieties with different vase longevities, develops molecular markers linked to long vase longevity in cut anthurium, and establishes a molecular marker-assisted selection technology system for long vase longevity in cut anthurium. This technology uses indel markers to assist in the breeding of cut anthurium plants with long vase longevity. The provided Indel6 marker primers enable faster and more accurate selection of plants with long vase longevity, shortening the breeding cycle and avoiding the drawback of traditional breeding methods that only allow selection when offspring plants are flowering, preventing earlier selection. Furthermore, based on the anthurium ACO1 gene, it is also possible to improve the vase longevity of cut anthurium through genetic modification or gene editing, thereby achieving targeted, precise, economical, and efficient breeding for vase longevity in cut anthurium, laying the foundation for molecular breeding of cut anthurium for vase longevity.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An application of an Indel molecular marker primer for assisting in the selection of offspring of anthurium with long vase life in the breeding of anthurium with long vase life, characterized in that: The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

2. Use of an Indel molecular marker primer for assisting in the selection of offspring of anthurium with long vase life in the preparation of a product for assisting in the breeding of anthurium with long vase life, characterized in that: The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

3. Use of an Indel molecular marker primer for assisting the selection of offspring with long vase life of cut anthurium in the breeding of cut anthurium with long vase life, characterized in that: The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

4. Use of a kit containing indel molecular marker primers in breeding cut anthurium plants with long vase life, characterized in that: The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

5. Use of a kit containing indel molecular marker primers in the breeding of cut flower anthurium with long vase life, characterized in that: The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

6. A method for breeding long-vase-life cut anthurium plants using indel molecular marker primers, characterized in that: The following steps are involved: S1. Extract genomic DNA from the parental Anthurium cultivar to be bred and its hybrid progeny; S2. Using DNA as a template, PCR amplification is performed using the primers; S3. Detect the PCR amplification products by gel electrophoresis and select offspring plants with the same banding pattern as the long-vase-life parent; S4. Individual plant identification: Continue cultivating the selected offspring, and when they bloom, identify the desired offspring based on their vase life phenotypes. This will allow the selection of long-lived cut anthurium plants. The primers include an upstream primer 5'-ACTTACCCAAGATCAGATGGGA-3' and a downstream primer 5'-AATGATGGTGACGGCGAGTT-3'.

7. The method according to claim 6, wherein: In step S1, genomic DNA was extracted from Anthurium andraeanum leaves at a concentration of 50-100 ng / μL.

8. The method according to claim 7, wherein: The PCR amplification reaction system in step S2 is: 1-2 μL DNA template, 5-10 μL 2×M5 PAGE Taq PCR Mix, 0.5-1 μL upstream primer, 0.5-1 μL downstream primer, and 3-6 μL ddH2O.

9. The method according to claim 8, characterized in that The PCR amplification program in step S2 was as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55-64°C for 25 s, extension at 72°C for 30 s, 36 cycles, extension at 72°C for 5 min, and storage at 4°C.

Images