SNP (Single Nucleotide Polymorphism) molecular marker primer for assisting selection of offspring in short vegetative growth period of anthurium andraeanum and application thereof

Abstract

The invention discloses SNP (Single Nucleotide Polymorphism) molecular marker primers for assisting selection of offspring in short vegetative growth period of anthurium andraeanum and application of the SNP molecular marker primers. The CDC73 genes which are differentially expressed in anthurium andraeanum varieties in different vegetative growth periods are taken as targets of SNP loci, the developed SNP marker primer is a molecular marker which is closely linked with the short vegetative growth period, the progeny in the short vegetative growth period can be quickly and accurately selected, and the accuracy rate of auxiliary selection of the progeny in the short vegetative growth period through the SNP marker primer reaches 70.96%. Meanwhile, the invention provides a method for auxiliary selection of anthurium andraeanum short vegetative growth period progeny by utilizing the SNP molecular marker, short vegetative growth period breeding target characters can be quickly and accurately selected in a seedling period, the breeding period is shortened, meanwhile, plants in a non-short vegetative growth period can be directly eliminated, the planting scale is reduced, and anthurium andraeanum breeding efficiency and benefits are improved. And a foundation is laid for molecular breeding of anthurium in the vegetative growth period.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers and plant breeding, and more specifically, relates to a SNP molecular marker primer for assisting the selection of offspring in the short vegetative growth period of anthurium andraeanum and its application. Background Art

[0002] Anthurium andraeanum Lind. is a perennial herbaceous plant belonging to the genus Anthurium in the family Araceae. It has bright colors, unique shapes, and blooms all year round with a long flowering period. It is popular all over the world as a cut flower and potted plant, and has a very broad market prospect. my country has currently formed a large-scale anthurium industry, but the lag in the selection and breeding of new varieties has restricted the efficient and independent development of the anthurium industry. At present, the commercial varieties on the market are still dominated by foreign varieties. Short vegetative growth period is an important target trait for anthurium breeding. Selecting anthurium with a short vegetative growth period for cultivation can significantly shorten the production cycle, save production costs, and improve production efficiency and competitiveness. Therefore, studying the breeding method of new varieties of anthurium with a short vegetative growth period and breeding new varieties of anthurium with a short vegetative growth period are of great significance to further improve the competitiveness of my country's anthurium industry and promote the high-quality development of the anthurium industry.

[0003] Although Anthurium is a plant that blooms all year round, its vegetative growth period is long, which leads to a long conventional hybrid breeding cycle. One offspring cycle is about 3 years. In addition to multiple rounds of improvement, plant line expansion, production testing and other links, the cultivation of new varieties requires even longer time. Therefore, how to further shorten the breeding cycle, improve breeding efficiency and benefits, cultivate breakthrough new Anthurium varieties with independent intellectual property rights as soon as possible, and narrow the gap with foreign countries in Anthurium breeding has become one of the keys to further promote the independent, efficient and high-quality development of my country's Anthurium industry.

[0004] The traditional hybrid breeding method is to select new varieties of anthurium with a short vegetative growth period through phenotypic selection, and this can only be carried out when the offspring plants are flowering. This selection method has a long breeding cycle and cannot eliminate offspring materials with non-breeding target traits at an early stage, increasing breeding costs. At the same time, since the traits during the vegetative growth period are easily affected by seed maturity, seed size, cultivation measures and environmental conditions, the accuracy of phenotypic selection is not high, and there is still a lack of efficient breeding methods.

[0005] Molecular marker-assisted selection breeding is a breeding technology that selects the genotype of the target trait with the help of molecular markers based on the linkage relationship between molecular markers and genes. It has the advantages of early selection, high selection accuracy, accelerated breeding process, and improved breeding efficiency. However, there is no research on molecular marker-assisted short vegetative growth period. Therefore, it is of great significance to carry out research on molecular marker-assisted selection methods for short vegetative growth period offspring and establish a molecular marker-assisted selection technology system for short vegetative growth period offspring of anthurium, which will shorten the breeding cycle of anthurium short vegetative growth period, reduce the planting scale, improve the accuracy and efficiency of breeding, shorten the breeding gap with foreign countries as soon as possible, and further improve the competitiveness and efficiency of my country's anthurium industry. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing anthurium short vegetative growth period breeding, such as the long breeding cycle and the inability to eliminate plants with non-breeding target traits in advance, and to provide a SNP molecular marker primer and its application for assisting the selection of anthurium short vegetative growth period offspring.

[0007] The first object of the present invention is to provide a set of SNP molecular marker primers.

[0008] The second object of the present invention is to provide the application of SNP molecular marker primers.

[0009] The third object of the present invention is to provide a kit.

[0010] The fourth object of the present invention is to provide the use of the kit.

[0011] The fifth object of the present invention is to provide a method for utilizing SNP molecular markers to assist in the selection of anthurium andraeanum offspring with a short vegetative growth period.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The invention provides a set of SNP molecular marker primers, including: upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'.

[0014] The present invention analyzes and obtains differentially expressed CDC73 genes in two anthurium varieties with different vegetative growth periods, 'Guanghua Xiaotianjiao Anthurium' and '2016-035-5', and clones the differentially expressed genes. The anthurium CDC73 gene is used as a target to design and develop SNP markers, and a group of SNP molecular marker primers that are closely linked to the short vegetative growth period of the anthurium are obtained. The molecular marker primers can be used to identify anthuriums with short vegetative growth periods, screen offspring of anthuriums with short vegetative growth periods, and shorten the breeding cycle. At the same time, the SNP marker can assist in the selection of offspring of anthuriums with short vegetative growth periods, achieve the goal of quickly and accurately selecting short vegetative growth periods in the early stages of breeding, shorten the breeding cycle, avoid the defect that traditional breeding can only select when the offspring plants are flowering and cannot be selected in advance, and improve the efficiency and benefit of anthurium breeding.

[0015] The invention provides the use of SNP molecular marker primers in identifying anthurium andraeanum with a short vegetative growth period or in screening offspring of anthurium andraeanum with a short vegetative growth period.

[0016] The invention provides application of SNP molecular marker primers in breeding of anthurium andraeanum during the vegetative growth period.

[0017] The invention provides application of SNP molecular marker primers in preparing a kit for identifying or screening anthuriums with a short vegetative growth period.

[0018] The present invention provides a kit comprising the above-mentioned SNP molecular marker primers.

[0019] Preferably, the kit also contains reagents for extracting sample DNA and reagents required for PCR amplification.

[0020] The present invention provides the use of the kit in identifying anthurium andraeanum in a short vegetative growth period or in screening offspring of anthurium andraeanum in a short vegetative growth period.

[0021] The present invention provides a method for utilizing SNP molecular markers to assist in selecting offspring of anthurium andraeanum in a short vegetative growth period, comprising the following steps:

[0022] S1. Extracting genomic DNA from Anthurium parents and their hybrid offspring to be selected;

[0023] S2. Using the DNA extracted in step S1 as a template, PCR amplification is performed using the above-mentioned SNP molecular marker primers;

[0024] S3. Detect the PCR amplification product by gel electrophoresis, and select the offspring plants with the same band pattern as the parent in the short vegetative growth period;

[0025] S4. Continue to cultivate the selected offspring, and when they bloom, combine the phenotype of the vegetative growth period to obtain Anthurium andraeanum offspring plants with a short vegetative growth period.

[0026] Furthermore, in step S1, genomic DNA from Anthurium andraeanum leaves is extracted at a concentration of 50 to 100 ng / μL.

[0027] 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 1, 0.5-1 μL upstream primer 2, 0.5-1 μL downstream primer, 3-6 μL ddH 2 O.

[0028] 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, 30 cycles, extension at 72°C for 5 min, and storage at 4°C.

[0029] The present invention has the following beneficial effects:

[0030] The present invention takes the CDC73 gene differentially expressed in anthurium in different vegetative growth periods as a target, and develops SNP marker primers that are closely linked to the short vegetative growth period. The selection of anthurium short vegetative growth period offspring is advanced to the hybrid offspring seedling stage through the SNP marker-assisted selection technology, and the offspring without the short vegetative growth period can be directly eliminated, shortening the breeding cycle to half of the original, reducing the planting scale, and improving the efficiency and benefit of anthurium breeding, solving the problem that the existing anthurium hybrid breeding cannot select the short vegetative growth period offspring in advance, eliminate the offspring plants that are not the breeding target, and can only select the short vegetative growth period trait when the hybrid offspring blooms, which is both time-consuming and costly.

[0031] In addition, since the short vegetative growth period trait is easily affected by seed maturity, cultivation measures and environmental conditions, the accuracy of phenotypic selection is not high, and the SNP molecular marker primer provided by the present invention is an indirect selection of genotypes to improve the accuracy of selection. The SNP marker primer provided by the present invention can quickly and accurately select offspring with a short vegetative growth period, shorten the breeding cycle, reduce the scale of planting, and improve breeding efficiency and benefits. It is of great significance to further accelerate the progress of my country's anthurium breeding work, improve breeding and industrial benefits, and promote the independent development, innovative development and high-quality development of the anthurium industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the electrophoresis test result of 21 Anthurium varieties with short vegetative growth period and 22 Anthurium varieties with long vegetative growth period using primers labeled SNP4 (in the figure, 1-21 are short vegetative growth period, 22-43 are long vegetative growth period, and M is DL500 DNA Marker).

[0033] Figure 2These are the results of electrophoresis detection of 21 Anthurium andraeanum varieties with short vegetative growth periods and 22 Anthurium andraeanum varieties with long vegetative growth periods using primers labeled SNP5 (in the figure, 1-21 are short vegetative growth periods, 22-43 are long vegetative growth periods, and M is DL500 DNA Marker).

[0034] Figure 3 The primer pair for marker SNP5 was 2023-110 combined parents and 93 strains F 1 The results of electrophoresis test (in the figure, P1 is 'Guanghua Xiaotianjiao Hongzhang', P2 is '2016-035-5', and the rest are 93 strains of F 1 Generation, M is DL500 DNA Marker).

[0035] Figure 4 It is the hybrid offspring of 10 excellent anthurium germplasm resources with a vegetative growth period shorter than 13 months (A: '2023-110-5'; B: '2023-110-10'; C: '2023-110-14'; D: '2023-110-19'; E: '2023-110-29'; F: '2023-110-38'; G: '2023-110-48'; H: '2023-110-71'; I: '2023-110-81'; J: '2023-110-102').

[0036] Figure 5 It is the statistical result of the vegetative growth period of individual plants of the hybrid offspring of 10 excellent anthurium germplasm resources. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1 Development of SNP markers tightly linked to the short vegetative growth period of Anthurium andraeanum

[0040] After analyzing the differentially expressed genes in the genomes of 'Guanghua Xiaotianjiao Hongzhang' and '2016-035-5' of Anthurium in different vegetative growth stages, the CDC73 (Cell Division Cycle 73) gene of Anthurium that was differentially expressed in different vegetative stages was obtained. After cloning the gene, SNP marker primers were designed based on the CDC73 gene of Anthurium. The base differences (SNP sites) were used for primer design. The differential bases were used as the 3' end of the functionally related primers. Then, two specific primers corresponding to the related alleles were designed using Primer 5.0 software (i.e., there were two upstream primers and one downstream primer). If the length of the amplified target fragment was about 80-200 bp, and the difference in the amplified length of the two alleles was 4-6 bp, in order to increase the specificity of the primers for the two alleles, a mismatch was artificially introduced at the third position of the 3' end of the functionally related primers. Thus, a total of 6 SNP marker primers were designed, and the specific primer sequences are shown in Table 1. The designed primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0041] Table 1 Primer sequences of SNP markers

[0042]

[0043] Example 2 Screening of molecular markers tightly linked to the short vegetative growth period of Anthurium andraeanum

[0044] The CTAB method was used to extract DNA from 21 anthurium varieties with short vegetative growth periods (vegetative growth periods shorter than 13 months) and 22 anthurium varieties with long vegetative growth periods (vegetative growth periods longer than 16 months), all of which were sourced from the Conghua Base of Guangzhou Flower Research Center. PCR amplification and electrophoresis were then performed using the SNP marker primers developed in Example 1 to screen for molecular marker primers that were closely linked to the short vegetative growth period of anthurium.

[0045] The specific method is as follows: DNA of 43 Anthurium varieties in different vegetative growth stages was used as template, and PCR amplification was performed using the primers in Table 1. The amplification system was: 1 μL DNA template (50 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer 1 (10 μM / μL), 0.5 μL upstream primer 2 (10 μM / μL), 0.5 μL downstream primer (10 μM / μL), 3 μL ddH 2 O; the amplification program is: 95℃ pre-denaturation for 3min, 94℃ denaturation for 25s, 55-64℃ annealing for 25s, 72℃ extension for 30s, 30 cycles, 72℃ extension for 5min, and storage at 4℃. The amplified PCR product was subjected to 4-6% polyacrylamide gel electrophoresis under the conditions of voltage 300V, current 180mA, and electrophoresis time 2.5h. After the electrophoresis, silver staining and color development were performed, and finally the band was placed on a white plastic plate and photographed.

[0046] The test results showed that the accuracy of the SNP5 marker primer was higher, while the accuracy of the other markers was lower. Among them, the marker SNP4 had two bands of short vegetative growth period. Among the 43 varieties of anthurium with different vegetative growth periods, 25 had two bands, of which 14 had short vegetative growth period. Figure 1 As shown, the marker accuracy is not high; and the marker SNP5 has 15 consistent band patterns with the short vegetative growth period 'Guanghua Xiaotianjiao Hongzhang' variety among the 43 different vegetative growth period varieties, including 10 with short vegetative growth period, such as Figure 2 As shown, it indicates that the SNP5 marker is linked to a short vegetative growth period.

[0047] Example 3 Using SNP5 molecular marker to assist selection of offspring of anthurium with short vegetative growth period

[0048] 1. Parent selection and hybrid fruit acquisition

[0049] (1) Parent selection: According to the breeding objectives, Anthurium '2016-035-5', which has a long vegetative growth period, was selected as the female parent, and A. 'Xiao Tian Jiao', which has a short vegetative growth period, was selected as the male parent;

[0050] (2) Hybridization and fruit acquisition: Collect 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.

[0051] 2. Seed acquisition and hybrid seedling production

[0052] (1) Obtaining seeds: Remove the ripe fruits from the spadix, put them in clean water, squeeze them open by hand, wash off the mucus, and obtain the seeds;

[0053] (2) Hybrid seedling production: After the seeds are dried, they are directly sown into a 200-hole seedling screen filled with coarse peat substrate, with one seed per hole. The combination code is 2023-110.

[0054] 3. Molecular markers assist in the selection of offspring of anthurium in the short vegetative growth period

[0055] (1) Extraction of genomic DNA from parents and offspring: When the hybrid offspring had 4 to 5 leaves in the plug tray, 80 to 120 mg of young leaves from the parents and the later hybrid seedlings were taken to extract genomic DNA from the offspring using the CTAB method;

[0056] (2) PCR amplification and gel electrophoresis detection: SNP5 marker primers were used to perform PCR amplification on the leaf DNA of the parents and hybrid offspring. The amplification system was as follows: 1 μL DNA template (50 ng / μL), 5 μL PCR-mix, 0.5 μL upstream primer 1 (10 μM / μL), 0.5 μL upstream primer 2 (10 μM / μL), 0.5 μL downstream primer (10 μM / μL), 3 μL ddH 2 O (the amplification system can use 10μL or 20μL); after the PCR system is configured, put it into the PCR gene amplifier for reaction amplification; the PCR amplification program is: 95℃ pre-denaturation for 3min, 94℃ denaturation for 25s, 55-64℃ annealing for 25s, 72℃ extension for 30s, 30 cycles, 72℃ extension for 5min, and storage at 4℃;

[0057] The amplified PCR product was subjected to 4-6% polyacrylamide gel electrophoresis under the conditions of voltage 300V, current 180mA, and electrophoresis time 2.5h. After the electrophoresis, silver staining and color development were performed, and finally the band was placed on a white plastic plate and photographed and read;

[0058] (3) Obtaining offspring of Anthurium with a short vegetative growth period: Select offspring with the same morphology as the short vegetative growth period parent 'Guanghua Xiaotianjiao Anthurium' for further cultivation, and select offspring with a short vegetative growth period based on the short vegetative growth period phenotype identification when they bloom.

[0059] Test results such as Figure 3 As shown in the figure, the SNP5 marker was used to select 93 offspring of 2023-110 for a short vegetative growth period. The results showed that the banding type of 31 offspring was consistent with that of the short vegetative growth period parent 'Guanghua Xiaotianjiao Hongzhang'; combined with the phenotypic identification results of the offspring's vegetative growth period, 22 of the 31 offspring had a short vegetative growth period, indicating that the accuracy of the assisted selection using the SNP5 marker was 70.96%.

[0060] Combined with morphological observation, 10 excellent anthurium germplasm resources with a vegetative growth period of less than 13 months were selected, such as Figure 4 As shown, the vegetative growth period is further counted as follows Figure 5 As shown, Figure 4 -A's offspring had the longest vegetative growth, which was 394 days. Figure 4 -J's offspring had the shortest vegetative growth period, which was 332 days. The above method can quickly screen out excellent anthurium germplasm resources with a short vegetative growth period, reduce the planting scale, and improve the accuracy and efficiency of breeding.

[0061] In summary, the present invention uses the CDC73 gene differentially expressed in anthurium varieties with different vegetative growth periods as the target of the SNP site, develops and screens for the first time SNP markers closely linked to the short vegetative growth period of anthurium, and establishes a molecular marker-assisted selection technology system for anthurium with a short vegetative growth period. The provided SNP5 marker primer pair can quickly and accurately select offspring with a short vegetative growth period, shorten the breeding cycle, reduce the planting scale, and improve breeding efficiency and benefits. It is of great significance to further accelerate the progress of anthurium breeding in my country, improve breeding and industrial benefits, and promote the independent development, innovative development and high-quality development of the anthurium industry.

[0062] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A set of SNP molecular marker primers, characterized in that: The primers include: Upstream primer 1: 5′-TGAGTGTGTAACGGTGGAG-3′; Upstream primer 2: 5′-AAGCCCGAGTGTGTGACGGTGGAA-3′; Downstream primer: 5′-GAAGGATGGTGAAAGGCAAC-3′.

2. Use of the primers according to claim 1 in identifying anthurium andraeanum with a short vegetative growth period or in screening offspring of anthurium andraeanum with a short vegetative growth period.

3. Use of the primers according to claim 1 in breeding of anthurium andraeanum during its vegetative growth period.

4. Use of the primers according to claim 1 in preparing a kit for identifying or screening anthuriums with a short vegetative growth period.

5. A kit, characterized in that: Comprising the primer according to claim 1.

6. The kit according to claim 5, characterized in that: Contains reagents for extracting sample DNA and reagents required for PCR amplification.

7. Use of the kit according to claim 5 or 6 in identifying anthurium andraeanum with a short vegetative growth period or in screening offspring of anthurium andraeanum with a short vegetative growth period.

8. A method for selecting short-vegetative growth period offspring of anthurium andraeanum using SNP molecular markers, characterized in that: The following steps are involved: S1. Extracting genomic DNA from Anthurium parents and their hybrid offspring to be selected; S2. Using the DNA extracted in step S1 as a template, PCR amplification is performed using the primers according to claim 1; S3. Detect the PCR amplification product by gel electrophoresis, and select the offspring plants with the same band pattern as the parent in the short vegetative growth period; S4. Continue to cultivate the selected offspring, and when they bloom, combine the phenotype of the vegetative growth period to obtain Anthurium andraeanum offspring plants with a short vegetative growth period.

9. The method according to claim 8, characterized in that: The PCR amplification 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 1, 0.5-1 μL upstream primer 2, 0.5-1 μL downstream primer, and 3-6 μL ddH2O.

10. The method according to claim 8, characterized in that: 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, 30 cycles, extension at 72°C for 5 min, and storage at 4°C.

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