SNP molecular marker primer for assisting progeny selection in short vegetative growth period of anthurium andraeanum and application thereof

Abstract

The application discloses a SNP molecular marker primer for assisting offspring selection in a short vegetative growth period of an anthurium andraeanum and application thereof. The application takes a CDC73 gene differentially expressed in different vegetative growth periods of anthurium andraeanum varieties as a target of a SNP site, and develops a SNP marker primer which is a molecular marker closely linked to the short vegetative growth period, can quickly and accurately select offspring in the short vegetative growth period, and has an accuracy of 70.96% in the assisted selection of offspring in the short vegetative growth period through the SNP marker primer. Meanwhile, a method for assisting selection of offspring in the short vegetative growth period of anthurium andraeanum by using the SNP molecular marker is provided, the target traits of the short vegetative growth period can be quickly and accurately selected in a seedling period, the breeding cycle is shortened, the plants in the non-short vegetative growth period can be directly eliminated, the planting scale is reduced, the breeding efficiency and benefit of the anthurium andraeanum are improved, and a foundation is laid for carrying out molecular breeding of the vegetative growth period of the anthurium andraeanum.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular markers and plant breeding technology. More specifically, it relates to a SNP molecular marker primer for assisting in the selection of offspring of short vegetative growth period of Anthurium andraeanum and its application. BACKGROUND

[0002] Anthurium andraeanum Lind. is a perennial herb of Araceae and Anthurium, which is popular worldwide as a cut flower and potted plant due to its bright color, unique shape, year-round flowering, and long-lasting flowering. China has formed a large-scale Anthurium industry, but the lagging new variety breeding limits the efficient and independent development of the Anthurium industry, and the commercialized varieties on the market are still mainly from abroad. Short vegetative growth period is an important target trait for Anthurium breeding. Cultivating Anthurium with short vegetative growth period 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 short vegetative growth period and breeding new varieties of Anthurium with short vegetative growth period is of great significance for further improving the competitiveness of China's Anthurium industry and promoting the high-quality development of the Anthurium industry.

[0003] Although Anthurium is a year-round flowering plant, its vegetative growth period is relatively long, resulting in a long breeding cycle of conventional hybridization. It takes about 3 years for a progeny cycle, plus multiple rounds of improvement, strain propagation, and production testing. Therefore, how to further shorten the breeding cycle, improve breeding efficiency and benefit, and cultivate breakthrough new varieties of Anthurium with independent intellectual property rights as soon as possible to narrow the gap with foreign countries in Anthurium breeding has become one of the keys to further promoting the independent, efficient, and high-quality development of China's Anthurium industry.

[0004] Traditional hybrid breeding method for breeding new varieties of Anthurium with short vegetative growth period is through phenotypic selection, and can only be performed when the progeny plants are in flower. This selection method has a long breeding cycle and cannot eliminate non-breeding target trait progeny materials in the early stage, increasing the breeding cost. At the same time, due to the influence of 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 method.

[0005] Molecular marker assisted selection breeding is a breeding technique of selecting the genotype of target traits by means of molecular markers according to the linkage relationship between molecular markers and genes, which 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 at present, so it is of great significance to carry out research on the selection method of molecular marker assisted short vegetative growth period offspring, establish the molecular marker assisted short vegetative growth period offspring selection technology system of anthurium andraeanum, shorten the breeding cycle of anthurium andraeanum short vegetative growth period, reduce the planting scale, improve the accuracy and efficiency of breeding, shorten the gap with foreign breeding as soon as possible, and further improve the competitiveness and efficiency of the anthurium andraeanum industry in China. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and deficiencies of long breeding cycle and inability to eliminate non-breeding target trait plants early in the existing anthurium andraeanum short vegetative growth period breeding, and to provide a SNP molecular marker primer for assisting the selection of anthurium andraeanum short vegetative growth period offspring and application thereof.

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

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

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

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

[0011] The fifth object of the present application is to provide a method for breeding anthurium andraeanum short vegetative growth period offspring by using SNP molecular markers.

[0012] The above objects of the present application are achieved by the following technical solutions:

[0013] The present application provides a group of SNP molecular marker primers, which comprises: upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; and downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'.

[0014] The application obtains the differentially expressed CDC73 gene in two varieties of 'Guanghua Xiaotianjiao Honglan' and '2016-035-5' with different vegetative growth periods, and clones the differentially expressed gene, designs and develops a SNP marker with the red palm CDC73 gene as a target, obtains a group of SNP molecular marker primers closely linked to the short vegetative growth period of the red palm, and the molecular marker primers can be used for identifying the short vegetative growth period of the red palm, screening the red palm short vegetative growth period offspring, and shortening the breeding period. At the same time, the SNP marker can assist in the selection of the red palm short vegetative growth period offspring, realize the rapid and accurate selection of the short vegetative growth period in the early breeding stage, shorten the breeding period, avoid the defects that the traditional breeding can only select when the offspring plants bloom and cannot select early, and improve the efficiency and benefit of the red palm breeding.

[0015] The application provides application of the SNP molecular marker primer in identifying the short vegetative growth period red palm or in screening the red palm short vegetative growth period offspring.

[0016] The application provides application of the SNP molecular marker primer in the red palm vegetative growth period breeding.

[0017] The application provides application of the SNP molecular marker primer in preparing a kit for identifying or screening the short vegetative growth period red palm.

[0018] The application provides a kit containing the above-mentioned SNP molecular marker primer.

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

[0020] The application provides application of the above-mentioned kit in identifying the short vegetative growth period red palm or in screening the red palm short vegetative growth period offspring.

[0021] The application provides a method for assisting in selecting the red palm short vegetative growth period offspring by using the SNP molecular marker, comprising the following steps:

[0022] S1. Extracting genomic DNA of the red palm parent and hybrid offspring to be selected;

[0023] S2. Using the DNA extracted in step S1 as a template, and performing PCR amplification by using the above-mentioned SNP molecular marker primer;

[0024] S3. Performing gel electrophoresis detection on the PCR amplification product, and selecting the offspring plant with the same band type as the short vegetative growth period parent;

[0025] S4. Continuing to cultivate the selected offspring, and obtaining the short vegetative growth period red palm offspring plant by combining the vegetative growth period phenotype when the offspring blooms.

[0026] Further, the genomic DNA of the anthurium leaf in step S1 is extracted, and the concentration is 50-100 ng / μL.

[0027] Further, the PCR amplification system in step S2 is as follows: 1-2 μL of DNA template, 5-10 μL of 2xM5PAGE TaqPCR Mix, 0.5-1 μL of upstream primer 1, 0.5-1 μL of upstream primer 2, 0.5-1 μL of downstream primer, and 3-6 μL of ddH2O.

[0028] Further, the PCR amplification procedure in step S2 is as follows: 95℃ pre-denaturation for 3 min, 94℃ denaturation for 25 s, 55-64℃ annealing for 25 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and 4℃ storage.

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

[0030] The present application takes the CDC73 gene differentially expressed in different vegetative growth periods of anthurium as a target, and develops a SNP marker primer closely linked to the short vegetative growth period. The SNP marker assisted selection technology is used to select the offspring of the short vegetative growth period early in the seedling stage of the hybrid offspring, and the offspring of the non-short vegetative growth period can be directly eliminated, so that the breeding period is shortened to half of the original period, the planting scale is reduced, the efficiency and benefit of anthurium breeding are improved, and the problem that the existing anthurium hybrid breeding cannot select the offspring of the short vegetative growth period early, eliminate the offspring plants of the non-breeding target, and can only select the short vegetative growth period trait when the hybrid offspring flowers is solved.

[0031] In addition, since the short vegetative growth period trait is easily affected by seed maturity, cultivation measures and environmental conditions, the accuracy of phenotype selection is not high, while the SNP molecular marker primer provided by the present application is an indirect selection of genotype, which improves the accuracy of selection. The SNP marker primer provided by the present application can quickly and accurately select the offspring of the short vegetative growth period, shorten the breeding period, reduce the planting scale, improve the breeding efficiency and benefit, and has important significance for further accelerating the process of anthurium breeding in China, improving breeding and industrial benefit, and promoting independent development, innovative development and high-quality development of anthurium industry. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the electrophoresis detection result of the marker SNP4 primer on 21 anthurium varieties with short vegetative growth period and 22 anthurium varieties with long vegetative growth period (1-21 in the figure are short vegetative growth period, 22-43 are long vegetative growth period, and M is DL500 DNA Marker).

[0033] Figure 2is the electrophoresis detection result of 21 short vegetative growth period varieties of Anthurium and 22 long vegetative growth period varieties of Anthurium by marker SNP5 primer pair (1-21 in the figure are short vegetative growth period, 22-43 are long vegetative growth period, and M is DL500 DNA Marker).

[0034] Figure 3 is the electrophoresis detection result of 2023-110 combination parents and 93 F1 generations (P1 in the figure is 'Guanghua Xiaotianjiaohongpian', P2 is '2016-035-5', and the rest are 93 F1 generations, and M is DL500 DNA Marker).

[0035] Figure 4 is a single plant of 10 excellent Anthurium germplasm hybrid offspring with 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'; and J: '2023-110-102').

[0036] Figure 5 is the vegetative growth period statistical result of 10 excellent Anthurium germplasm hybrid offspring. DETAILED DESCRIPTION

[0037] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

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

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

[0040] After differential expression gene analysis of the genomes of 'Guanghua Xiaotianjiaohongpang' and '2016-035-5' red palm in different vegetative growth periods, the red palm CDC73 (Cell Division Cycle 73) gene differentially expressed in different vegetative periods was obtained, and the SNP marker primer was designed based on the red palm CDC73 gene. The difference in the base (SNP site) was used for primer design, and the different base was used as the 3' end of the functional related primer. Then, two specific primers corresponding to the related alleles were designed by using Primer 5.0 software (i.e. two upstream primers and one downstream primer). If the length of the target fragment is about 80-200 bp, the length difference of the two alleles is 4-6 bp. In order to increase the specificity of the two allele primers, a mismatch is artificially introduced at the third position of the 3' end of the functional related primer. Thus, a total of 6 SNP marker primers were designed, and the specific primer sequences are shown in Table 1. The designed primers were synthesized by GenScript Biotech (Shanghai) Co., Ltd.

[0041] Table 1 Primer sequences of SNP markers

[0042]

[0043] Example 2 Screening of molecular markers closely linked to short vegetative growth period of red palm

[0044] The CTAB method was used to extract DNA from 21 red palm varieties with short vegetative growth period (the vegetative growth period is shorter than 13 months) and 22 red palm varieties with long vegetative growth period (the vegetative growth period is longer than 16 months). The above materials were all from the Guangzhou Flower Research Center from the base. Then, the SNP marker primers developed in Example 1 were used for PCR amplification and electrophoresis to screen the molecular marker primers closely linked to the short vegetative growth period of red palm.

[0045] The specific method is as follows: the DNA of 43 red palm varieties with different vegetative growth periods was used as the template, and the primers in Table 1 were used for PCR amplification. The amplification system was as follows: 1 μL of DNA template (50 ng / μL), 5 μL of PCR-mix, 0.5 μL of upstream primer 1 (10 μM / μL), 0.5 μL of upstream primer 2 (10 μM / μL), 0.5 μL of downstream primer (10 μM / μL), and 3 μL of ddH2O. The amplification program was as follows: 95 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 25 s, 55-64 ℃ annealing for 25 s, 72 ℃ extension for 30 s, 30 cycles, 72 ℃ extension for 5 min, and 4 ℃ storage. The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis, and the electrophoresis conditions were as follows: voltage 300 V, current 180 mA, and electrophoresis time 2.5 h. After electrophoresis, silver staining and color development were performed, and finally the white plastic plate was placed for photographing and band reading.

[0046] The detection results show that the accuracy of the SNP5 marker primer is higher, and the accuracy of the remaining markers is low. Among them, the marker SNP4, two band types are short vegetative growth period band types, and among the 43 different vegetative growth period varieties of Anthurium, 25 have two band types, among which there are 14 short vegetative growth period types, as shown in Figure 1 , the marker accuracy is not high; while the marker SNP5 is consistent with the short vegetative growth period 'Guanghua Xiaotianjiao Anthurium' variety in 43 different vegetative growth period varieties of Anthurium, among which there are 10 short vegetative growth period types, as shown in Figure 2 , which shows that the SNP5 marker is linked to the short vegetative growth period.

[0047] Example 3: Utilization of SNP5 molecular marker assisted selection of offspring of short vegetative growth period of Anthurium

[0048] 1. Parental selection and hybrid fruit acquisition

[0049] (1) Parental selection: According to the breeding goal, the Anthurium '2016-035-5' with long vegetative growth period was selected as the female parent, and the 'Guanghua Xiaotianjiao Anthurium' with short vegetative growth period was selected as the male parent;

[0050] (2) Hybridization and fruit acquisition: Collect the pollen on the anther of the male parent and put it into a culture dish, then quickly brush the pollen on the whole inflorescence of the female parent with a brush, cover it with a bag, and cultivate the female parent plant. When the fruit turns yellow, harvest the fruit.

[0051] 2. Seed acquisition and hybrid offspring seedling production

[0052] (1) Seed acquisition: Take the mature fruit on the inflorescence, put it into clean water, and squeeze the fruit with your hands to remove the mucus and obtain the seeds;

[0053] (2) Hybrid offspring seedling production: When the seeds are dried, directly sow them in a 200-hole seedling screen filled with coarse peat substrate, with 1 seed per hole. The combination code is 2023-110.

[0054] 3. Molecular marker assisted selection of short vegetative growth period offspring of Anthurium

[0055] (1) Extraction of genomic DNA of parents and offspring: When the hybrid offspring has 4-5 leaves in the plug, 80-120 mg of tender leaves of the parents and hybrid offspring seedlings were taken to extract the genomic DNA of the offspring by CTAB method;

[0056] (2) PCR amplification and gel electrophoresis detection: SNP5 labeled primers were used to amplify the DNA from the leaves of the parent and hybrid offspring of Anthurium. 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 ddH2O (the amplification system can be 10 μL or 20 μL). After the PCR system was prepared, it was placed in a PCR gene amplification instrument for reaction and amplification. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 94℃ denaturation for 25 s, 55~64℃ annealing for 25 s, 72℃ extension for 30 s, 30 cycles, 72℃ extension for 5 min, and storage at 4℃.

[0057] The amplified PCR products were subjected to 4-6% polyacrylamide gel electrophoresis. The electrophoresis conditions were 300V, 180mA, and 2.5h. After electrophoresis, silver staining and color development were performed. Finally, the products were placed on a white plastic plate for photographing and reading the bands.

[0058] (3) Obtaining offspring of Anthurium with short vegetative growth period: Select offspring with the same zoning pattern as the parent with short vegetative growth period 'Guanghua Xiaotianjiao Anthurium' and continue to cultivate them. When flowering, combine the short vegetative growth period phenotypic identification to select offspring with short vegetative growth period.

[0059] Test results as follows Figure 3 As shown, SNP5 markers were used to assist selection on 93 progeny plants of 2023-110 with a short vegetative growth period. The results showed that the banding pattern of 31 progeny plants was consistent with that of the short vegetative growth period parent 'Guanghua Xiaotianjiao Hongzhen'. Combined with the vegetative growth period phenotypic results of the progeny plants, 22 of the 31 progeny plants had a short vegetative growth period, indicating that the accuracy rate of the SNP5 marker-assisted selection was 70.96%.

[0060] Based on morphological observations, 10 superior Anthurium germplasm resources with a vegetative growth period of less than 13 months were selected, such as... Figure 4 As shown, and further statistics on its vegetative growth period are as follows: Figure 5 As shown, where Figure 4 The offspring of type -A had the longest vegetative growth period, at 394 days. Figure 4 The offspring of the -J variety had the shortest vegetative growth period, at 332 days. Using the methods described above, superior Anthurium germplasm resources with short vegetative growth periods can be quickly screened, reducing planting scale and improving breeding accuracy and efficiency.

[0061] In conclusion, the application takes the CDC73 gene differentially expressed in different vegetative growth periods of the Anthurium varieties as the target of the SNP site, develops and screens the SNP marker closely linked to the short vegetative growth period of the Anthurium for the first time, establishes a molecular marker assisted selection technology system for the short vegetative growth period of the Anthurium, and provides the SNP5 marker primer pair which can quickly and accurately select the offspring with a short vegetative growth period, shorten the breeding cycle, reduce the planting scale, and improve the breeding efficiency and benefits. The application has important significance for further accelerating the Anthurium breeding process in China, improving the breeding and industrial benefits, and promoting the independent development, innovative development and high-quality development of the Anthurium industry.

[0062] The above embodiments are preferred embodiments of the application, but the embodiments of the application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the application shall be equivalent replacement modes and shall be included in the protection scope of the application.

Claims

1. The application of an SNP molecular marker primer for assisting the selection of anthurium offspring with a short vegetative growth period in identifying anthuriums with a short vegetative growth period, or in screening anthurium offspring with a short vegetative growth period, characterized in that, Select offspring plants that have the same zonal pattern as the parents with short vegetative growth periods; The parent with the short vegetative growth period is 'Guanghua Xiaotianjiao Hongzhang'; The primers include: Upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; Upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; Downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'.

2. The application of an SNP molecular marker primer for assisting the selection of short-vegetative-growth-period offspring of Anthurium in the preparation of a kit for identifying or screening short-vegetative-growth-period Anthurium, characterized in that, Select offspring plants with the same zonal pattern as the parent with a short vegetative growth period; the parent with a short vegetative growth period is 'Guanghua Xiaotianjiao Hongan'. The primers include: Upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; Upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; Downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'.

3. A kit containing SNP molecular marker primers for selecting short-vegetative-growth-period progeny of Anthurium, used in the identification of short-vegetative-growth-period Anthuriums or in the screening of short-vegetative-growth-period progeny of Anthuriums, characterized in that, Select offspring plants that have the same zonal pattern as the parents with short vegetative growth periods; The parent with the short vegetative growth period is 'Guanghua Xiaotianjiao Hongzhang'; The primers include: Upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; Upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; Downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'.

4. A method for selecting short-vegetative-growth-period offspring of Anthurium using SNP molecular markers, characterized in that, Includes the following steps: S1. Extract genomic DNA from the parent anthurium and its selected hybrid offspring; S2. Using the DNA extracted in step S1 as a template, perform PCR amplification using primers; S3. Perform gel electrophoresis on the PCR amplification products and select offspring plants with the same banding pattern as the parents with short vegetative growth period; S4. Continue to cultivate the selected offspring, and when they flower, combine the vegetative growth period phenotype to obtain short vegetative growth period Anthurium offspring plants. The primers include: Upstream primer 1: 5'-TGAGTGTGTAACGGTGGAG-3'; Upstream primer 2: 5'-AAGCCCGAGTGTGTGACGGTGGAA-3'; Downstream primer: 5'-GAAGGATGGTGAAAGGCAAC-3'; The parent with the short vegetative growth period is 'Guanghua Xiaotianjiao Hongzhen'.

5. The method according to claim 4, characterized in that, The PCR amplification reaction mixture in step S2 is as follows: 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, 3-6 μL ddH2O.

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

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