Molecular marker highly linked with amphoteric phenotype of asparagus and application of molecular marker
By developing the asparagus hermaphroditic phenotypic molecular marker BS and using PCR amplification and gel electrophoresis detection, the problem of identifying hermaphroditic plants and ordinary male plants in asparagus was solved, rapid and accurate sex identification was achieved, and breeding efficiency was improved.
Patent Information
- Application Number
- CN202510642598.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-09-23
AI Technical Summary
Existing technologies make it difficult to quickly and accurately distinguish between hermaphroditic plants and ordinary male plants in asparagus. Traditional methods are time-consuming and inefficient, and cannot effectively utilize molecular markers for identification.
A molecular marker BS highly linked to the hermaphroditic phenotype of asparagus was developed, and primer pairs BS-F and BS-R were designed. PCR amplification and agarose gel electrophoresis were performed to detect the presence of a specific band at 1300 bp, indicating male plants; otherwise, hermaphroditic plants were identified.
It achieves rapid and accurate identification of asparagus sex within 1 day, shortens the detection cycle, and has an accuracy rate of ≥98%. It is suitable for large-scale breeding screening and significantly improves breeding efficiency.
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Figure CN120683288A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular genetics, and in particular relates to a molecular marker highly linked to the bisexual phenotype of asparagus and an application thereof. Background Art
[0002] Asparagus (Asparagus officinalis L.) is a typical dioecious plant, serving not only as a model system for studying sex determination and differentiation in dioecious plants (which only account for approximately 6% of seed plants), but also as a model plant for studying chromosomal evolution and the origin of bisexual plants. Molecular marker identification of asparagus sex is primarily based on its sex determination mechanism, which is controlled by an allele (Mm) on the L5 chromosome. Males are M- (including MM supermales and Mm ordinary males), while females are mm. Crossing supermale (MM) plants with female (mm) plants can produce 100% male progeny (Mm), a principle that has become the core of all-male breeding. This technology has already developed several key molecular markers, including the STS dual-marker system (YSM / XSM) based on the Y-chromosome SOFF gene and the X-chromosome WIP2 / NTT gene. This system specifically distinguishes between supermale plants (a 293bp band), female plants (a 245bp band), and normal male plants (two bands appearing simultaneously). Furthermore, the dominant marker combinations Asp1-T7 and Asp1-T7sp can reliably distinguish between males and females and have been successfully used to screen for supermale plants. Compared to traditional methods that require two years of observation during flowering, molecular marker technology can complete identification at the seedling stage, achieving 100% accuracy in screening for supermale plants, significantly shortening the breeding cycle. Recent research has also revealed that the systematic identification of the DUF247 gene family (including the SOFF gene) provides new insights into the mechanisms of female inhibition. Four functional STS markers developed based on the CDS sequences of the SOFF genes have been commercialized and are expected to be applied to other dioecious species in the genus Asparagus.
[0003] Asparagus plants with identical flowers and intact stamens and pistils, commonly known as hermaphroditic plants, occur in only 0-2% of natural asparagus populations and occur only in male plants. Hermaphroditic asparagus plants have the genotype Mm, and therefore, plants with hermaphroditic flowers are also called hermaphroditic plants. Aside from reproductive organs such as flowers and fruit, there are no obvious external morphological differences between male, female, and hermaphroditic asparagus plants. Male Mm and female mm plants can be sexed using traditional methods, which rely on morphological observation during flowering, or by using sex chromosome molecular markers. However, the reproductive organs of hermaphroditic plants are highly similar to those of standard male plants, making traditional methods that rely on phenotypic observation during flowering difficult to distinguish. Determination must be based on fruit set during fruit set, which is time-consuming (taking 2-3 years) and inefficient. Furthermore, because hermaphroditic plants and standard male plants share the same Mm genotype, there are currently no effective molecular markers for distinguishing between hermaphroditic or conventional male phenotypic characteristics. Summary of the Invention
[0004] In response to the above-mentioned problems in the prior art, the present invention aims to provide a molecular marker that is highly linked to the asparagus hermaphroditic phenotype. Another technical problem to be solved by the present invention is to provide the application of a molecular marker that is highly linked to the asparagus hermaphroditic phenotype for distinguishing between hermaphroditic and male asparagus.
[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0006] The nucleotide sequence of a molecular marker highly linked to the bisexual phenotype of asparagus is shown in SEQ ID NO.1.
[0007] Application of the molecular marker in asparagus sex identification.
[0008] The specific sequences of the PCR amplification primers for the molecular markers are as follows:
[0009] BS-F: 5'-TTGAATGTGTCTGGTACGTCG-3',
[0010] BS-R: 5'-TCTACGCTTACAACCCAAGTCA-3'.
[0011] Application of the PCR amplification primers in asparagus sex identification.
[0012] A method for identifying the sex of asparagus, comprising:
[0013] 1) extracting genomic DNA from the asparagus to be identified;
[0014] 2) Using the extracted genomic DNA as a template, PCR amplification was performed using molecularly labeled PCR amplification primers;
[0015] 3) Determine the sex of asparagus based on the results of agarose gel electrophoresis.
[0016] If a specific band appears at 1300 bp in the agarose gel electrophoresis test result, it is determined to be male asparagus, and if there is no band, it is hermaphroditic asparagus.
[0017] Application of the molecular marker in genetic improvement of asparagus.
[0018] Application of the PCR amplification primers in genetic improvement of asparagus.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] 1) This invention discovered for the first time a molecular marker, BS, highly linked to the hermaphroditic phenotype of asparagus. Its nucleotide sequence is shown in SEQ ID NO. 1. A primer pair, BS-F / BS-R, was designed for this marker. Genomic DNA from asparagus was extracted and used as a template for PCR amplification. The agarose gel electrophoresis assay then determined the presence of a specific band at 1300 bp, indicating male asparagus; the absence of a band indicates hermaphroditic asparagus.
[0021] 2) The present invention's method for identifying the sexual phenotype of asparagus based on the molecular marker BS has a detection cycle shortened to one day, with an accuracy rate of ≥98%. It can quickly and accurately identify male and sexual phenotypes at all stages of asparagus growth and development, is suitable for large-scale breeding screening, and significantly improves breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 These are the 7 hermaphroditic and male asparagus plants in the seedling stage to be tested;
[0023] Figure 2 This is the PCR amplification verification result of BS marker in hermaphroditic and male asparagus plants. DETAILED DESCRIPTION
[0024] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.
[0025] The asparagus materials used for the second-generation high-throughput whole-genome sequencing in this application were flower samples of the early-born bisexual and early-born male Emperor plants (http: / / www.springasparagusseed.com / page202), collected at the Base Management Center of Jiangxi Academy of Agricultural Sciences.
[0026] The website of the asparagus reference genome GCF_001876935.1_Aspof.V1 used in this application is https: / / ftp.ncbi.nlm.nih.gov / genomes / all / annotation_releases / 4686 / 100 / GCF_001876935.1_Aspof.V1 / .
[0027] The asparagus materials used in the identification step of Example 3 of this application are seedling asparagus of the Emperor's Early-growing bisexual plants and the Emperor's Early-growing male plants grown in plug trays, which were planted at the Vegetable and Flower Research Institute of Jiangxi Academy of Agricultural Sciences ( Figure 1 ).
[0028] Example 1 BS molecular marker development
[0029] Floral samples from both early-blooming hermaphroditic and early-blooming male Imperial plants (≥30 plants per population) were collected and pooled for next-generation, high-throughput whole-genome sequencing using the Illumina NovaSeq platform. Sequencing data (average depth 30×) were aligned to the asparagus reference genome GCF_001876935.1_Aspof.V1 using the bwa-mem algorithm. Population variation analysis was performed using samtools. A site specific to hermaphroditic plants was identified within the 3614342-3616892 bp interval of chromosome CM007381.1. This site was designated the BS marker (Bisexual), and its nucleotide sequence is shown in SEQ ID NO. 1.
[0030] Example 2 Design of specific primers
[0031] For the identified BS-tagged nucleic acid sequence SEQ ID NO. 1, primer 3 software was used to develop the BS-F and BS-R primer pairs with the following design parameters: TM value 45-65°C, primer length 18 bp-30 bp, and other default parameters. The specific information is as follows:
[0032] BS-F: 5'-TTGAATGTGTCTGGTACGTCG-3' (SEQ ID NO.2),
[0033] BS-R: 5'-TCTACGCTTACAACCCAAGTCA-3' (SEQ ID NO. 3).
[0034] Example 3
[0035] Genomic DNA was extracted from the young leaves of 7 asparagus seedlings (3 male and 4 hermaphroditic). The extraction of asparagus genomic DNA followed conventional molecular biology procedures. PCR amplification was performed using BS-F as the forward primer and BS-R as the rear primer, using the genomic DNA of the test asparagus as the template (the samples to be tested were numbered 1-7, all non-female plants, meaning it was impossible to quickly distinguish between hermaphroditic and male plants). The amplified products were detected by agarose gel electrophoresis. If a specific band appeared at 1300 bp, the asparagus was identified as male; if no band was present, the asparagus was hermaphroditic. The male control was a male early-growing emperor plant, and the hermaphroditic control was a hermaphroditic early-growing emperor plant.
[0036] The PCR reaction system was as follows: 5 μL of 2×PCR Buffer, 1 μL of 2 mM dNTP Mix, 0.5 μL each of 10 μM BS-F and BS-R, 0.2 μL of 5 U / μL Taq enzyme, 1 μL of 100 ng / μL template DNA, and ddH2O to 10 μL.
[0037] The PCR reaction program was as follows: 94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 60 sec, 35 cycles; 72°C for 5 min.
[0038] The results are as follows Figure 2 As shown, BS-F / BS-R successfully amplified a 1338bp PCR product in materials 1, 4, and 7, consistent with the male control. However, this PCR product was absent in materials 2, 3, 5, and 6, consistent with the hermaphroditic control. These results demonstrate that this BS molecular marker can rapidly detect hermaphroditic and male asparagus plants at the seedling stage, with a detection cycle shortened to just one day and 100% accuracy, making it suitable for large-scale breeding and screening.
[0039] The above description is only illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of them will fall within the scope of protection of the present invention.
Claims
1. A molecular marker highly linked to the bisexual phenotype of asparagus, the nucleotide sequence of which is shown in SEQ ID NO.
1.
2. Use of the molecular marker according to claim 1 in sex identification of asparagus.
3. The PCR amplification primer for molecular markers according to claim 1, characterized in that The specific sequence is as follows: BS-F: 5'-TTGAATGTGTCTGGTACGTCG-3', BS-R: 5'-TCTACGCTTACAACCCAAGTCA-3'.
4. Use of the PCR amplification primers according to claim 3 in sex identification of asparagus.
5. The method according to claim 4, characterized in that include: 1) extracting genomic DNA from the asparagus to be identified; 2) Using the extracted genomic DNA as a template, PCR amplification was performed using molecularly labeled PCR amplification primers; 3) Determine the sex of the asparagus based on the results of agarose gel electrophoresis; The specific sequences of the PCR amplification primers for the molecular markers are as follows: BS-F: 5'-TTGAATGTGTCTGGTACGTCG-3', BS-R: 5'-TCTACGCTTACAACCCAAGTCA-3'.
6. The method according to claim 5, characterized in that If a specific band appears at 1300 bp in the agarose gel electrophoresis test result, it is determined to be male asparagus; if there is no band, it is hermaphroditic asparagus.
7. A method for identifying the sex of asparagus, characterized in that: include: 1) extracting genomic DNA from the asparagus to be identified; 2) Using the extracted genomic DNA as a template, PCR amplification was performed using molecularly labeled PCR amplification primers; 3) Determine the sex of the asparagus based on the results of agarose gel electrophoresis; The specific sequences of the PCR amplification primers for the molecular markers are as follows: BS-F: 5'-TTGAATGTGTCTGGTACGTCG-3', BS-R: 5'-TCTACGCTTACAACCCAAGTCA-3'.
8. The method according to claim 7, characterized in that If a specific band appears at 1300 bp in the agarose gel electrophoresis test result, it is determined to be male asparagus; if there is no band, it is hermaphroditic asparagus.
9. Use of the molecular marker according to claim 1 in genetic improvement of asparagus.
10. Use of the PCR amplification primers according to claim 3 in genetic improvement of asparagus.