Ilex integra sex identification probe and identification method
By designing specific PCR primer pairs (DQ-F and DQ-R) combined with PCR amplification and gel electrophoresis technology, the problem of gender identification of male and female in all holly plants is solved, and rapid and accurate gender identification is achieved, which is suitable for breeding and gardening applications of all holly plants.
Patent Information
- Application Number
- CN202510667109.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art is difficult to quickly and accurately distinguish the male and male sex of all holly plants in the early stages, especially in young plants or in the floral/fruitless seasons, resulting in time-consuming and labor-intensive breeding and application.
设计特异性PCR引物对(DQ-F和DQ-R)用于全缘冬青性别相关基因的扩增,结合PCR扩增和凝胶电泳技术,实现雌雄性别的快速鉴定。
It achieves rapid and accurate identification of male and female genders within 1 hour, shortens identification time, improves work efficiency, has high sensitivity and specificity, and does not need to wait for flowering and fruiting, and is suitable for identification at any growth stage.
Smart Images
Figure CN120272581A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a probe and a method for identifying the sex of Ilex integra Thunb.. Background Art
[0002] Ilex integra Thunb. is an evergreen small tree of the genus Ilex in the family Aquifoliaceae. It has important horticultural ornamental value due to its beautiful leaf shape and bright fruits. This species is dioecious, that is, female flowers and male flowers grow on different plants. Among them, female plants bear berry-like drupes, while male plants do not bear fruits. Based on this dioecious characteristic, it is necessary to distinguish the sex of Ilex integra Thunb. in reproduction and application.
[0003] In the actual breeding and application of Ilex integra Thunb. plants, accurately identifying the sex of plants is crucial. However, its sexual physiological maturity period is relatively long (5 - 6 years after planting). In addition, young or adult plants have no morphological characteristics to indicate their sex during the season without flowers / fruits, which makes determining the sex of breeding materials a time-consuming task.
[0004] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA sequences and has been widely applied in biological sex identification. By designing PCR primers that specifically recognize sex-related genes or sequences, rapid sex identification of the target organism can be achieved. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a probe and a method for identifying the sex of Ilex integra Thunb., which can quickly and accurately distinguish male and female Ilex integra Thunb. plants at an early stage.
[0006] The present invention adopts the following technical solutions to solve the above technical problems:
[0007] A probe for identifying the sex of Ilex integra Thunb. includes a pair of specific PCR primers (DQ-F and DQ-R). Among them, the sequence of the upstream primer (DQ-F) is 5’-CTCTCTCATCCGATCAAACCG-3’, and the sequence of the downstream primer (DQ-R) is 5’-AGCCTAGCTAGAGAGAAGGGA-3’; the pair of specific PCR primers is designed based on the sex-related gene of Ilex integra Thunb. and is used to amplify the target molecular marker to achieve the identification of the male and female sexes of Ilex integra Thunb. plants.
[0008] As one of the preferred embodiments of the present invention, the female plants of Ilex integra Thunb. contain the molecular marker, while the male plants do not contain the molecular marker.
[0009] A method for identifying the sex of Ilex integra Thunb. uses the above probe for identifying the sex of Ilex integra Thunb. for PCR amplification, and includes the following specific steps:
[0010] (1) DNA extraction of the Ilex integra plant to be identified;
[0011] (2) PCR amplification using the specific PCR primer pair;
[0012] (3) Gel electrophoresis and determination of the plant gender based on the electrophoresis results;
[0013] When a specific band appears after amplification and electrophoresis, it proves that the plant to be identified is female Ilex integra; when no specific band appears after amplification and electrophoresis, it proves that the plant to be identified is male Ilex integra.
[0014] As one of the preferred embodiments of the present invention, in the step (1), the leaves of the Ilex integra plant are used for DNA extraction as the template for subsequent PCR amplification.
[0015] As one of the preferred embodiments of the present invention, in the step (2), the PCR amplification reaction system includes: 12.5 μL of 2×PCR Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 1 μL of 50 ng / μL template DNA, and 9.5 μL of sterile water.
[0016] As one of the preferred embodiments of the present invention, in the step (2), the PCR amplification reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 35 cycles; extension at 72°C for 5 min; preservation at 4°C.
[0017] The advantages of the present invention compared with the prior art are as follows:
[0018] The present invention has successfully designed a pair of specific PCR primers (DQ-F and DQ-R) that can be used for the identification of the male and female genders of Ilex integra plants. During the primer design process, the specificity, efficiency, and stability of the primers were considered to ensure their efficient amplification in the PCR reaction and the accuracy of gender identification. Through experimental verification, this primer pair amplified specific bands in female Ilex integra samples, while no corresponding amplification products were observed in male Ilex integra samples, showing good specificity and sensitivity. At the same time, this identification method has the advantages of being fast, accurate, and not requiring flowering and fruiting, and can be applied to the rapid gender identification of Ilex integra plants, providing a powerful tool for the breeding research, landscape application, and industrial production of Ilex plants, with important application value and broad market prospects. Specifically as follows:
[0019] (1) Fast and accurate: Compared with the traditional method, the method of the present invention can be completed in about 1 h, greatly shortening the identification time;
[0020] (2) No need to flower and bear fruit: Traditional methods require waiting for plants to flower and bear fruit to determine gender, while this method can be used to identify gender at any growth stage of the plant, significantly improving work efficiency;
[0021] (3) High sensitivity: The PCR method adopted in the present invention has the advantage of high sensitivity, and can amplify the target sequence from trace DNA samples, being applicable to various sampling conditions;
[0022] (4) Good specificity: The design of the probe primers of the present invention is based on the specific sequences of Ilex integra plants, having good specificity and avoiding the interference of non-specific amplification;
[0023] (5) Having important application value and broad market prospects:
[0024] Horticultural application: In the landscape greening application of Ilex integra plants, rapid and accurate gender identification can help horticulturists rationally allocate male and female plants, improving the landscape effect and ecological benefits;
[0025] Breeding research: In the breeding research of Ilex plants, this technology can accelerate the selection of gender-related traits and genetic research, improving breeding efficiency;
[0026] Industrial application: This technology can be applied to the industrial production of Ilex plants, helping breeding enterprises and landscaping companies quickly screen and identify the gender of plants, improving production efficiency and economic benefits. Description of the Drawings
[0027] Figure 1 It is the amplification result diagram in Experimental Example 1 (in the figure, "M" represents Trans DNA Marker II, 100 - 1500bp; "♀" represents "female plant"; "♂" represents "male plant"). Detailed Implementation Modes
[0028] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation modes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. For the reagent products and experimental methods used in the following embodiments and experimental examples, unless otherwise specified, they are all conventional reagents or methods in the art and will not be elaborated further.
[0029] Example 1
[0030] A sex identification probe for Ilex integra in this embodiment includes a specific PCR primer pair "DQ-F and DQ-R", wherein the sequence of DQ-F (forward primer) is 5'-CTCTCTCATCCGATCAAACCG-3', and the sequence of DQ-R (reverse primer) is 5'-AGCCTAGCTAGAGAGAAGGGA-3'.
[0031] The specific PCR primer pair "DQ-F and DQ-R" in this embodiment is designed based on the sex-related gene of Ilex integra, and is used to amplify the target molecular marker to achieve the identification of the male and female genders of Ilex integra plants.
[0032] Example 2
[0033] A sex identification method for Ilex integra in this embodiment uses the above sex identification probe for Ilex integra to perform PCR amplification, and includes the following specific steps:
[0034] (1) Extract the DNA of the leaves of the Ilex integra plant to be identified, and adjust the concentration to 50 ng / μL for subsequent PCR amplification reaction.
[0035] (2) Use the specific PCR primer pair (DQ-F, DQ-R) in Example 1 to perform PCR amplification.
[0036] The PCR amplification reaction system includes: 12.5 μL of 2×PCR Master Mix, 1 μL of DQ-F (10 μM), 1 μL of DQ-R (10 μM), 1 μL of 50 ng / μL template DNA, and 9.5 μL of sterile water.
[0037] The PCR amplification reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 5 min; storage at 4°C.
[0038] (3) Perform agarose gel electrophoresis, and judge the gender of the plant according to the electrophoresis result;
[0039] When a specific band appears after amplification and electrophoresis, it proves that the plant to be identified is a female Ilex integra; when no specific band appears after amplification and electrophoresis, it proves that the plant to be identified is a male Ilex integra.
[0040] Experimental Example 1
[0041] This experimental example is used to verify the feasibility of the method of the present invention.
[0042] I. Samples
[0043] Select sixteen Ilex integra plants with known genders (8 female Ilex integra and 8 male Ilex integra) as the identification samples of this experimental example.
[0044] II. Experimental Methods
[0045] Genetic sex identification was performed on sixteen known-sex Ilex integra samples according to the method of Example 2 of the present invention.
[0046] 1. DNA Extraction
[0047] The leaf DNA of eight samples of plants to be identified was extracted using a "Plant Tissue Genomic DNA Kit", and the concentration was adjusted to 50 ng / μL for subsequent PCR amplification reactions.
[0048] 2. PCR Amplification
[0049] The DNA of the plants to be identified was subjected to PCR amplification using a specific PCR primer pair (DQ-F, DQ-R).
[0050] The PCR amplification reaction system included: 12.5 μL of 2×PCR Master Mix, 1 μL of DQ-F (10 μM), 1 μL of DQ-R (10 μM), 1 μL of 50 ng / μL template DNA, and 9.5 μL of sterile water.
[0051] The PCR amplification reaction procedure was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 35 cycles; extension at 72°C for 5 min; storage at 4°C.
[0052] 3. Gel Electrophoresis
[0053] The PCR products were detected by 1% agarose gel electrophoresis.
[0054] III. Experimental Results
[0055] The results were as Figure 1 shown: Specific bands (400 bp in size) appeared in 8 female Ilex samples, while no specific bands appeared in 8 male Ilex samples. This result reflects the specificity and reliability of the primer pair designed by the present invention for the sex identification of Ilex integra, indicating the feasibility of the method of the present invention.
[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
Claims
1. A probe for gender identification of Ilex integra, characterized in that, It includes a specific PCR primer pair, wherein the upstream primer sequence is 5’-CTCTCTCATCCGATCAAACCG-3’ and the downstream primer sequence is 5’-AGCCTAGCTAGAGAGAAGGGA-3’; the specific PCR primer pair is used for amplifying a target molecular marker to achieve the identification of the male and female genders of Ilex integra plants.
2. The probe for identifying the sex of Ilex integra as claimed in claim 1, wherein The molecular marker is contained in the female plants of Ilex integra, but not in the male plants.
3. A method for sex identification of Ilex integra, characterized in that, Performing PCR amplification using the Ilex integra gender identification probe according to Claim 1 or 2 includes the following specific steps: (1) Extracting the DNA of the Ilex integra plant to be identified; (2) Performing PCR amplification using the specific PCR primer pair; (3) Gel electrophoresis and judging the gender of the plant according to the electrophoresis result; When a specific band appears after electrophoresis of the amplification, it proves that the plant to be identified is a female Ilex integra; When no specific band appears after electrophoresis of the amplification, it proves that the plant to be identified is a male Ilex integra.
4. The method for identifying the sex of Ilex integra Thunb. according to claim 3, wherein, In step (1), the leaves of the Ilex integra plant are used for DNA extraction and used as the template for subsequent PCR amplification.
5. The method for sex identification of Ilex integra Thunb. according to claim 3, characterized in that, In step (2), the PCR amplification reaction system includes: 12.5 μL of 2×PCR Master Mix, 1 μL of the 10 μM upstream primer, 1 μL of the 10 μM downstream primer, 1 μL of the 50 ng / μL template DNA, and 9.5 μL of sterile water.
6. The method for sex identification of Ilex integra Thunb. according to claim 3, characterized in that, In step (2), the PCR amplification reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for 35 cycles; extension at 72°C for 5 min; preservation at 4°C.